Cytokine 71 (2015) 101–108
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Betulinic acid attenuates lung injury by modulation of inflammatory cytokine response in experimentally-induced polymicrobial sepsis in mice Madhu Cholenahalli Lingaraju a, Nitya Nand Pathak a, Jubeda Begum b, Venkanna Balaganur a, Rafia Ahmad Bhat a, Harish Darasaguppe Ramachandra c, Anjaneya Ayanur d, Mahendra Ram a, Vishakha Singh a, Dhirendra Kumar a, Dinesh Kumar a, Surendra Kumar Tandan a,⇑ a
Division of Pharmacology and Toxicology, Indian Veterinary Research Institute, Izatnagar, Bareilly, U.P. 243 122, India Division of Bacteriology and Mycology, Indian Veterinary Research Institute, Izatnagar, Bareilly, U.P. 243 122, India c Division of Animal Biotechnology, Indian Veterinary Research Institute, Izatnagar, Bareilly, U.P. 243 122, India d Division of Pathology, Indian Veterinary Research Institute, Izatnagar, Bareilly, U.P. 243 122, India b
a r t i c l e
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Article history: Received 2 July 2014 Received in revised form 7 September 2014 Accepted 9 September 2014
Keywords: Cecal ligation and puncture Triterpenoid Cytokines Matrix metalloproteinase (MMP)-9 Inflammatory pulmonary insult
a b s t r a c t Sepsis commonly progresses to acute lung injury (ALI), an inflammatory lung disease with high morbidity and mortality. Septic ALI is characterized by excessive production of proinflammatory mediators. It remained refractory to present therapies and new therapies need to be developed to improve further clinical outcomes. Betulinic acid (BA), a pentacyclic lupane group triterpenoid has been shown to have anti-inflammatory activities in many studies. However, its therapeutic efficacy in polymicrobial septic ALI is yet unknown. Therefore, we investigated the effects of BA on septic ALI using cecal ligation and puncture (CLP) model in mice. Vehicle or BA (3, 10, and 30 mg/kg) was administered intraperitoneally, 3 times (0, 24 and 48 h) before CLP and CLP was done on 49th h of the study. Survival rate was observed till 120 h post CLP. Lung tissues were collected for analysis by sacrificing mice 18 h post CLP. BA at 10 and 30 mg/kg dose significantly reduced sepsis-induced mortality and lung injury as implied by attenuated lung histopathological changes, decreased protein and neutrophils infiltration. BA also decreased lung NF-jB expression, cytokine, intercellular adhesion molecule-1, monocyte chemoattractant protein-1 and matrix metalloproteinase-9 levels. These evidences suggest that, the protective effects of BA on lungs are associated with defending action against inflammatory response and BA could be a potential modulatory agent of inflammation in sepsis-induced ALI. Ó 2014 Elsevier Ltd. All rights reserved.
1. Introduction Sepsis is a condition characterized by systemic inflammatory response to infection and commonly progresses to acute lung injury (ALI). The highest incidence of ALI occurs in patients with sepsis [1] carries the highest morbidity and mortality rates when associated with sepsis in intensive care units (ICUs) [2,3]. Lipopolysaccharide (LPS) from the cell wall of Gram-negative bacteria that causes sepsis is the most common pulmonary insult leading to ALI by provoking activation of nuclear factor- kappa B (NF-jB) and the release of proinflammatory mediators including tumor necrosis factor-alpha (TNF-a) and interleukin-6 (IL-6) [4,5]. Additionally, ⇑ Corresponding author. Tel.: +91 581 2300291, +91 581 2301670; fax: +91 581 2303284. E-mail addresses: [email protected], [email protected] (S.K. Tandan). http://dx.doi.org/10.1016/j.cyto.2014.09.004 1043-4666/Ó 2014 Elsevier Ltd. All rights reserved.
LPS also stimulate adhesion, activation, and infiltration of polymorphonuclear neutrophils (PMN) and damaging the alveolar-capillary membrane [6]. Intercellular adhesion molecule-1 (ICAM-1) and monocyte chemoattractant protein-1 (MCP-1) which are known to attract neutrophils [7,8] are increased in sepsis and further results in the tissue infiltration of more PMN, which in turn leads to parenchymal cell dysfunction and tissue damage [9]. Increased matrix metalloproteinases (MMPs) activity cause impairment in lung function due to decreased lung collagen content and disorganized pulmonary parenchymal tissue. Specifically, MMP-9 levels have been shown to be elevated above normal in patients with severe sepsis [10]. Animal models play a significant role in sepsis research [11]. Cecal ligation and puncture (CLP) model is in widespread use and is considered by some experts as the gold standard for animal models of sepsis [12]. Sepsis induced by CLP is a clinically relevant
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model in which lung injury is induced via an inflammatory pathway [13]. Triterpenoids are ubiquitous in plants and have long been considered to be the anti-inflammatory principles in several herbal drugs [14]. Betulinic acid (BA), (3b-hydroxy-lup-20(29)-en-28-oic acid is a naturally occurring pentacyclic lupane group triterpenoid. It exhibits a variety of biological and medicinal properties such as anti-cancer, anti-malarial, anti-inflammatory and anti-HIV properties [15]. BA has shown anti-inflammatory activities in various experimental systems [16–18]. However, its pharmacological potential in murine polymicrobial sepsis-induced lung injury, which is mainly inflammatory in nature, needs to be explored. ALI due to sepsis is thought to result in a high mortality rate, warranting treatment and preventative measures [19]. With the knowledge growing, interfering with proinflammatory cytokines to relieve lung injury has been widely applied [5]. In the present study, we investigated whether pretreatment with BA could prevent alterations in the cytokine and chemokine levels and prevent septic lung injury in a clinically more relevant model that mimics sepsis conditions in humans. Our results indicate that BA is a powerful repressor of major cytokines and chemokines in a mouse model of polymicrobial sepsis-induced lung injury indicating its use as potential anti-inflammatory agent in sepsis associated ALI in mice. 2. Materials and methods 2.1. Animals Male Swiss albino mice were procured from Laboratory Animal Resource Section of the Institute. These animals were kept in polypropylene cages, housed on an animal facility 12–12 h light dark cycle at 22 °C, and fed with standard laboratory diet and water ad libitum. The mice were acclimatized for one week before use. All experimental procedures involving animals were approved by the Institute Animal Ethics Committee, IVRI, Izatnagar. 2.2. Drug and chemical reagents Betulinic acid, odianisidine, hydrochloric acid, acrylamide, bisacrylamide, SDS, gelatine, Triton X-100 and hydrogen peroxide were purchased from Sigma Chemicals Co., St. Louis, USA. EIA kits, dimethylsulfoxide (DMSO) from Genetix Biotech Asia Pvt. Ltd. Rabbit-anti-mouse monoclonal antibody for NF-jB, goat-anti-rabbit secondary HRP conjugated antibodies were procured from Abcam antibodies. All the necessary chemicals for conventional biochemical parameter methods were of analytical grade purchased from Sigma, Genetix and Hi Media Laboratories Pvt. Ltd. 2.3. Experimental sepsis induced by CLP Polymicrobial sepsis was induced by CLP surgical procedure according to previously described method [20]. Animals were anesthetized with an intraperitoneal injection of a mixture of 10 mg/kg xylazine and 100 mg/kg ketamine hydrochloride. The abdomen was gently shaved and cleaned with antiseptic sterile swab. Through a 1 cm abdominal midline incision, the caecum was exposed and ligated below the ileocecal valve without causing bowel obstruction. The caecum was then subjected to a through and through perforation twice with a 21-gauge needle with the caecum squeezed to extrude some fecal contents. After repositioning the caecum, the abdominal incision was closed in layers with chromic gut surgical suture 4-0 and all mice were then resuscitated with 1 mL of warmed sterile lactated Ringer’s subcutaneously. Sham-operated animals were subjected to laparotomy,
intestinal manipulation and resuscitation procedures; however, the caecum was neither ligated nor punctured. 2.4. Experimental design The mice were divided into five groups and treatment was given intraperitoneally 3 times at 0, 24 and 48 h before sham or CLP. Group I (sham control) and group II (CLP control), consisting mice pretreated with vehicle; group III, IV and V (drug pretreated), consisting mice pretreated with BA at the dose rate of 3, 10 and 30 mg/kg body weight, respectively and mice of all the groups were submitted to CLP after 1 h of last dose (that is at 49th h) except group I where the mice underwent sham operation. 2.5. Survival study Survival study was carried out as a separate experiment and 15 animals were used in each group. After animal grouping, drug administration and surgical procedures as noted in experimental design, animals of all the groups were observed for their survival period till 120 h from the time of surgery. 2.6. Animal sacrifice and tissue harvest In a separate experiment held for sampling, the mice were sacrificed by cervical dislocation at 18 h after sham or CLP surgery. The lung tissues were removed, weighed, snap-frozen in liquid nitrogen, homogenized in 50 mM/L phosphate buffer with mammalian cocktail protease inhibitor (pH 7.4) containing 1 mM EDTA at 4 °C to make 5% tissue homogenate. The homogenates were centrifuged at 1500g for 10 min in a cooling centrifuge at 4 °C. The resulting supernatants were stored at 80 °C for further analysis. 2.7. Relative lung weight To assess the lung edema fluid content, which is an indicator of pulmonary inflammation we assessed the relative lung weights of the mice of different groups after 18 h of CLP. Lung weights were expressed as mg of lung per 25 g of mouse to obtain relative organ weight [21]. 2.8. Pulmonary cytokine, chemokine, ICAM-1, MCP-1 and MMP-9 levels Pulmonary levels of TNF-a, IL-6, IL-10, ICAM-1, MCP-1 and MMP-9 were quantified using specific ELISA kits for mice according to the manufacturers’ instructions. 2.9. MPO activity assay MPO activity in the lungs was determined as an index of tissue neutrophil infiltration. MPO activity in the supernatant was measured from the absorbance changes at 460 nm resulting from the decomposition of H2O2 in the presence of odianisidine [22]. 2.10. Gelatin zymography for MMP detection MMP-9 (92 kDa gelatinase, gelatinase B) degrades components of the ECM with high specific activity for denatured collagens (gelatin). Gelatin–SDS–polyacrylamide gel was used to detect the presence of gelatinolytic MMP-9. Both active forms and pro-enzymes are revealed by this technique as the exposure of pro-MMPs to SDS during SDS–PAGE leads to activation without proteolytic cleavage. Electrophoresis was carried out at room temperature at a voltage of 110 mV. When the tracking dye reached the bottom of the gel, it was washed twice with 2.5% Triton X-100 for
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60 min to remove the SDS. The gel was subsequently incubated for 48 h at room temperature in renaturation buffer (50 mM Tris–HCl (pH 7.6), 200 mM NaCl, 5 mM CaCl2). The gel was stained with 0.5% Coomassie blue for 2 h and then destained with water containing 10% glacial acetic acid and 40% methanol. Areas of gelatinolytic degradation appeared as transparent bands on the blue stained background of the gel. A wide range molecular weight protein marker from PureGene (PG-PMT0782, 10–180 kDa) was used to estimate the apparent molecular weights for the bands of substrate degradation. 2.11. Western blot analysis Equal amounts (approximately 50 lg) of protein were loaded per well on a 12% sodium dodecyl sulphate polyacrylamide gel (SDS–PAGE). Subsequently, proteins were transferred onto polyvinylidene difluoride membrane. The membranes were washed in Tris-buffered saline with Tween 20 and incubated in 5% BSA (Sigma) at room temperature for 2 h on a rocker shaker, and followed by TBS-T washing. Incubations with rabbit polyclonal antibodies specific for NF-jB in diluents buffer (5% BSA in TBST) were performed overnight at 4 °C. Then the membrane was washed with TBS-T followed by incubation with the peroxidaseconjugated secondary antibody at room temperature for 2 h and subjected DAB reaction to visualize the bands.
Fig. 1. Survival analysis results (% survival) from mice of different groups. The treatment was given (vehicle or BA) on day 1, 2 and 3 by intraperitoneal route before sham or CLP; the mice underwent sham or CLP surgery 1 h after the last dose on day 3. The survival was observed for 120 h after surgical interventions; alphabets on the lines are used to represents significance. Survival lines which do not share any alphabet in common among them differ significantly (p < 0.05); n = 15 mice per group.
2.12. Histopathological analysis For light microscopic investigations, lung tissue specimens were fixed in 10% formaldehyde and dehydrated with alcohol. Paraffin embedded sections were cut and stained with hematoxylin and eosin (H&E) and examined under a photomicroscope (Olympus BH 2, Tokyo, Japan). All tissue sections were examined microscopically for the histopathological changes by experienced histologists who were blind to treatments. 2.13. Statistical analysis All statistical analysis was done by using Prism version 4 (GraphPad Software Inc., USA). Data were expressed as mean ± S.E.M. The level of statistical significance were determined by one-way analysis of variance (ANOVA) followed by Tukey’s multiple comparison test. Survival statistics were compared with a Kaplan–Meier curve and log-rank test. The differences in the data obtained were considered statistically significant when the p value was less than 0.05. 3. Results 3.1. Effect of pretreatment of BA on survival In a separate experiment mice were observed for survival rate for 5 days. In the vehicle-treated CLP group, the survival rate on the first day of observation was 66.66% and reached a stable 6.66% survival on the fifth day after CLP. Log rank analysis of 5 day survival demonstrated that BA at doses of 10 and 30 mg/kg significantly improved survival (46.66%) of septic mice compared with CLP animals. BA at 3 mg/kg did not confer the significant protection against CLP-induced lethality (Fig. 1). No mortality was observed in the sham group. 3.2. Effect of BA pretreatment on relative lung weight in CLP mice As reflected by relative lung weights, the pulmonary inflammatory fluid accumulation was significantly higher in CLP control group as compared to sham control group (Fig. 2). However, BA
Fig. 2. Relative lung weight from mice of different groups after 18 h of surgery. All data are presented as means ± S.E; Alphabets above the bars represents superscript. Bars which do not share any superscript in common among them differ significantly (p < 0.05); n = 6 mice per group.
pretreated mice had significantly lower pulmonary inflammatory fluid accumulation in their lung than did CLP control group mice at 18 h after CLP. 3.3. Effect of BA pretreatment on lung cytokine levels in CLP mice Protein levels of TNF-a and IL-6 were markedly elevated in lung of septic mice and attenuated to sham levels in mice pretreated with BA. In mice undergoing CLP, BA did not reduce elevated levels of IL-10 (Fig. 3). 3.4. Effect of BA pretreatment on lung ICAM-1, MCP-1 and MPO levels in CLP mice CLP caused an increased trafficking of PMN cells into lungs after CLP, which was evidenced by the increased levels of ICAM-1, MCP1 and MPO in lung CLP control group as compared to sham control group (Fig. 4). Pretreatment with BA 3 days before CLP, significantly reduced the ICAM-1, MCP-1 levels and MPO activity in the lung as compared with the CLP control group. 3.5. Effect of BA pretreatment on activity of MMPs in the lung in CLP mice MMP-9 levels in lungs were determined by EIA kit. As shown in Fig. 5, CLP significantly increased MMP-9 levels in lungs of CLP
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Fig. 3. Cytokine levels in lungs from mice of different groups after 18 h of surgery. Lung TNF-a (a), IL-6 (b) and IL-10 (c) levels. All data are presented as means ± S.E; Alphabets above the bars represents superscript. Bars which do not share any superscript in common among them differ significantly (p < 0.05); n = 6 mice per group, assay in triplicate.
control group mice as compared to sham control group. BA pretreatment significantly reduced the MMP-9 levels in lungs of CLP mice when compared to CLP control group values. Additionally, gelatinase activity of MMP-9 (92 kDa) was measured using gelatin zymography and result is presented in Fig. 6. In CLP control group 18 h after induction of CLP, the lung MMP-9 gelatinase activity was elevated to a significantly higher level as evidenced by thicker transparent bands on the blue stained background of the gel when compared to sham control group which showed thinner bands. BA pretreated CLP group showed the band of intermediate thickness. 3.6. Effect of BA pretreatment on NF-jB protein expression in the lung in CLP mice NF-jB (64 kDa) protein expression in lung of CLP control mice was increased compared to sham mice. BA pretreated mice at 3, 10 and 30 mg/kg dose showed minimal expression of NF-jB protein compared to CLP mice (Fig. 7). The b-actin protein expression was constant in all the groups.
Fig. 4. Neutrophil infiltration markers levels in lungs from mice of different groups after 18 h of surgery. Lung ICAM-1 (a), MCP-1 (b) levels and lung MPO (c) activity. All data are presented as means ± S.E; alphabets above the bars represents superscript. Bars which do not share any superscript in common among them differ significantly (p < 0.05); n = 6 mice per group, assay in triplicate.
Fig. 5. Extracellular matrix degrading enzyme, the MMP-9 levels in lungs from mice of different groups after 18 h of surgery. All data are presented as means ± S.E; Alphabets above the bars represents superscript. Bars which do not share any superscript in common among them differ significantly (p < 0.05); n = 6 mice per group, assay in triplicate.
40 and b, 40). However, in CLP control group, lung tissue section showed alveolar edema, alveolar damage and inflammatory cell infiltration (Fig. 8; c, 10; d, 40). BA pretreatment significantly attenuated the histopathological changes in the lungs of CLP mice, especially edema fluid accumulation and inflammatory cells infiltration at 10 (Fig. 8 e, 10 and f, 40) and 30 mg/kg dose (Fig. 8 g, 10 and h, 40).
3.7. Effect of BA pretreatment on lung histopathology in CLP mice 4. Discussion As shown in photomicrograph (H&E stained), lung sections from the sham control group of mice showed a normal alveolar and bronchi structure with no histopathological changes (Fig. 8 a,
The lung is frequently the first failing organ during the sequential development of multiple organ dysfunction in sepsis
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Fig. 6. Zymogram showing MMP-9 (92 kDa) mediated gelatinolytic activities of lung samples from mice of different groups after 18 h of surgery; n = 6 mice per group, assay in triplicate.
[23]. In the present study, we successfully set up a moderate to severe CLP model as indicated by mortality (84%), lung water content (relative lung weight) and lung damage in histological section. However, it has been observed that BA pretreatment before CLP alleviated the lung inflammatory response and improved survival in murine polymicrobial sepsis model by inhibiting transcription factor, NF-jB and associated maladaptive cytokine release. Furthermore, the subcellular biochemical changes that would favor the lung damage like increased PMN trafficking activity as indicated by elevated ICAM-1, MCP-1 and MPO levels and increased degradation of lung ECM as found by elevated MMP-9 levels following sepsis induction were attenuated by BA pretreatment. BA pretreated mice showed higher survival rate than vehicle pretreated mice after CLP (Fig. 1), reflecting its potential to have protective role in polymicrobial sepsis. It remains to be seen how BA affects the inflammatory response in lung and exerts its protective effect in CLP-induced lung injury to improve overall survival of mice. Edema is a typical symptom of inflammation. To quantify the magnitude of pulmonary edema in CLP-induced lung injury model, we evaluated the relative lung weight [24]. CLP control group showed significantly higher relative lung weights as compared to sham control group (Fig. 2). BA pretreated CLP group mice showed significantly lesser relative lung weights when compared to CLP control group mice, which indicates that BA could inhibit the
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leakage of serous fluid into lung tissue and attenuate the development of lung edema and inflammation. In septic mice, cytokines leads to SIRS, subsequent septic shock, and multiorgan dysfunction. Among these cytokines, TNF-a and IL6 are considered to play important role in the pathophysiology of septic lung injury [25]. Studies showed that TNF-a and IL-6 levels were significantly increased in the CLP group compared with the sham control group [26]. In sepsis, IL-10 has been identified as a vital modulator of the lethal overabundant production of inflammatory cytokines. In several animal models of sepsis, neutralization of IL-10 results in exaggerated proinflammatory cytokine expression and death, while administration of recombinant IL-10 confers significant therapeutic protection [27,28]. In the present study, we observed CLP-induced higher lung levels of cytokine, whereas BA pretreatment before CLP significantly decreased lung proinflammatory cytokines including TNF-a and IL-6 (Fig. 3). However, IL-10 levels remained unaltered. These data suggests the protective effects of BA on septic lung injury in mice might be achieved by inhibition of TNF-a and IL-6 induced tissue damage along with maintained levels of IL-10 which protects experimental subject from proinflammatory cytokine damage. This indicates that BA pretreatment had an anti-inflammatory effect in lungs of CLP mice and could be one of the BA’s mechanism involved in offering protection against severe inflammatory response in lungs and thereby improving the survival. Neutrophil infiltration is an insidious feature in septic lung injury [29] and persistence of high numbers of PMN in BALF after the first week of ARDS is associated with mortality, particularly in patients with sepsis [30]. Recruitment of PMNs into lung air spaces occurs in response to a variety of airway stimuli and involves the stepwise process of adhesion molecule expression on PMNs in both the pulmonary capillaries and the bronchial venules [31]. ICAM-1 is a type of adhesion molecule, involved in PMN recruitment and secondary organ damage in response to infection and inflammation [7]. A high level of proinflammatory mediators in sepsis is reported to up-regulate various adhesion molecules [32]. Absence of this molecule impairs the ability of PMN to migrate into organ tissues and reduces consequent secondary organ damage resulting in improved clinical status and overall survival. MCP-1, a chemokine was shown to act as a chemoattractant for neutrophils [7,8]. MCP-1 is known to orchestrate migration of leukocytes during sepsis leading to tissue injury. It has been shown that MCP-1 levels in lung correlates with neutrophil infiltration in the lung [33]. In patients with Gram-positive and Gram negative infections resulting in sepsis, elevated MCP-1 levels were observed [34,35]. MPO is an enzyme that is found predominantly in PMN cells. Since tissue MPO activity correlates significantly with the number of PMN determined histochemically, it is more commonly used to estimate tissue PMN accumulation in inflamed tissues. Consistent with the earlier reports, we found a significant increase
Fig. 7. Representative photograph of Western blot analysis of lung NF-jB protein from mice of different groups. Lung tissues from a representative mouse of each group were isolated after 18 h of surgery. Beta-actin (42 kDa) protein expression was analyzed as a control. Expression analysis was done using antibodies directed against NF-jB (64 kDa); n = 3 mice per group, blotting analysis in triplicate.
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Fig. 8. Photomicrographs (H&E stained; n = 3 mice per group) of representative lung tissue section of sham operated mouse showing normal bronchi (a, 40, black arrow) and lung parenchyma (b, 40, black arrow) with no observed changes. Lung tissue section of CLP operated mouse showing damaged alveoli, edema (c, 10; black arrows) and infiltration of neutrophils and edema (d, 40, black arrows). Lung tissue section of drug (10 mg/kg BA) pretreated CLP operated mouse showing attenuated histopathological changes marked by less edema (pink tinged sections) and ameliorated alveolar damage (black arrows) (e, 10 and f, 40). Lung tissue section of drug (30 mg/kg BA) pretreated CLP operated mouse showing attenuated histopathological changes marked by almost negligible amount of edema (g, 10, black arrow) and ameliorated alveolar damage (g, 10; h, 40, black arrows).
in the lung levels of ICAM-1, MCP-1 and MPO in CLP control as compared to sham-operated mice. Pretreatment with BA lowered the lung levels of ICAM-1, MCP-1 and MPO activity (Fig. 4)
significantly indicating its attenuating effect on neutrophil infiltration network. Thus, BA treatment in our study could have reduced the severity of sepsis by impairing the leukocyte migration.
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Further, this was supported by the histological sections of the lung. BA injection clearly reduced the leukocyte infiltration and edema, the signs of lung injury in sepsis. The MMPs are a group of structurally related zinc-dependent enzymes which together are capable of degrading all the components of the ECM. Remodeling of the lung architecture is a hallmark of many of the lung diseases for example, loss of alveolar walls. This pathological change involves extensive alterations of lung ECM [36]. Excessive or inappropriate expression of MMP is closely associated with pathogenesis of tissue destructive processes in a wide variety of diseases, including lung diseases [37]. Up regulation of MMP-9 is suggested to be involved in the injury progression [38]. MMP-9 is involved in extracellular matrix degradation and leukocyte migration [39]. Additionally, it is involved in processing of cytokines and chemokines to regulate inflammation and thought to be proinflammatory [40] which may further aggravate sepsis [10]. Given that the MMP-9 had been specifically implicated in the pathogenesis of septic lung injury, we specifically examined level of this enzyme activity in lungs using gelatin zymography. We found increased activity of MMP-9 in lung of CLP mice and propose that the increased MMP-9 activity in the lungs caused the development of a larger pulmonary edema due to destabilized alveolar ECM components. Interestingly, induction of MMP-9 coincided with the PMN infiltration and the maximum of histological manifestations in lungs of CLP mice. In contrast, preadministration of BA at 10 and 30 mg/kg dose attenuated MMP-9 levels as detected in ELISA (Fig. 5) and the defect in MMP-9 activity (Fig. 6) as demonstrated by the zymography in the lungs in response to CLP and added ameliorating effect against the development of edema by stabilizing the lung ECM components thereby laying the protective effect on lung in this model. NF-jB is considered to an important transcriptional factor involved in the production of proinflammatory cytokines, adhesion molecules, and other proinflammatory proteins [41]. A greater or more persistent nuclear accumulation of NF-jB is associated with higher mortality and more persistent organ dysfunction, including pulmonary injury [42]. Further, activation of the NF-jB by TNF-a increases endothelial expression of ICAM-1, MCP-1 and MMP-9 which were found to play important role in trafficking of PMN cells to the site of injury/infection [43,44]. NF-jB and TNF-a interplay together setup a severe inflammatory response and ultimately leads to failure of the particular affected organ. Therefore, inhibiting NF-jB activation appears to be a logical therapeutic target for controlling hyper-inflammatory responses, including ALI. As shown in our results (Fig. 7), CLP control mice had a significantly increased lung NF-jB protein expression which was attenuated by BA pretreatment. Previous studies also reported the potency of BA to inhibit NF-jB protein expression in different models [16,17]. From the results of this study, it is shown that the suppressing effects of BA on NF-jB suggested to be involved in the effects related to lowered cytokines, ICAM-1, MCP-1 and MMP-9 levels and unopposed IL-10 levels thereby exhibiting protective effect on septic lung injury. Histopathological results reveal that there was wide damage of alveoli and accumulation of edema fluid and neutrophils after CLP (Fig. 8). However, BA at 10 and 30 mg/kg doses attenuated the histopathological changes especially damage to alveoli and neutrophil influx further sustaining the protective action of BA against the CLP-induced lung injury. The observations of this study indicate that BA has good antiinflammatory activity and further, attenuating action on neutrophil infiltration and MMP-9 mediated lung damage. These mechanisms are proposed to be the possible protective mechanisms of BA on lung in this model. In conclusion, our results suggest that the BA has antiinflammatory effects in murine model of sepsis and particularly
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the beneficial effects are in sepsis-induced lung injury. Protective mechanisms of BA in this model can be explained via inflammatory response attenuation, reduced infiltration of inflammatory cells and attenuation of production of lung ECM degrading enzymes. These findings possibly provide a rationale basis for the ability of BA to suppress lung inflammatory response. Thus, it is expected that, BA may serve as a potential anti-inflammatory tool for septic ALI. However, further studies employing post-treatment trials are required to support the use of BA as an excellent therapeutic approach to treat sepsis associated ALI conditions. Acknowledgments The authors thank the Director and the Joint Director (Academic), Indian Veterinary Research Institute, Izatnagar, Bareilly, U.P., India for financial support. References [1] Hudson LD, Milberg JA, Anardi D, Maunder RJ. Clinical risks for development of the acute respiratory distress syndrome. Am J Respir Crit Care Med 1995;151:293–301. [2] Rubenfeld GD, Caldwell E, Peabody E, Weaver J, Martin DP, Neff M, et al. Incidence and outcomes of acute lung injury. N Engl J Med 2005;353:1685–93. [3] Johnson ER, Matthay MA. Acute lung injury: epidemiology, pathogenesis, and treatment. J Aerosol Med Pulm Drug Deliv 2010;23:243–52. [4] Carter AB, Monick MM, Hunninghake GW. Lipopolysaccharide-induced NFkappaB activation and cytokine release in human alveolar macrophages is PKC-independent and TK- and PC-PLC-dependent. Am J Respir Cell Mol Biol 1998;18:384–91. [5] Gong Q, Yin H, Fang M, Xiang Y, Yuan CL, Zheng GY, et al. Heme oxygenase-1 upregulation significantly inhibits TNF-a and Hmgb1 releasing and attenuates lipopolysaccharide-induced acute lung injury in mice. Int Immunopharmacol 2008;8:792–8. [6] Erickson SE, Martin GS, Davis JL, Matthay MA, Eisner MD. Recent trends in acute lung injury mortality: 1996–2005. Crit Care Med 2009;37:1574–9. [7] Hildebrand F, Pape HC, Harwood P, Müller K, Hoevel P, Pütz C, et al. Role of adhesion molecule ICAM in the pathogenesis of polymicrobial sepsis. Exp Toxicol Pathol 2005;56:281–90. [8] Zhao X, Dib M, Andersson E, Shi C, Widegren B, Wang X, et al. Alterations of adhesion molecule expression and inflammatory mediators in acute lung injury induced by septic and non-septic challenges. Lung 2005;183:87–100. [9] Neviere RR, Cepinskas G, Madorin WS, Hoque N, Karmazyn M, Sibbald WJ, et al. LPS pretreatment ameliorates peritonitis-induced myocardial inflammation and dysfunction: role of myocytes. Am J Physiol 1999;277:885–92. [10] Hoffmann U, Bertsch T, Dvortsak E, Liebetrau C, Lang S, Liebe V, et al. Matrix-metalloproteinases and their inhibitors are elevated in severe sepsis: prognostic value of TIMP-1 in severe sepsis. Scand J Infect Dis 2006;38: 867–72. [11] Cotroneo TM, Hugunin KM, Shuster KA, Hwang HJ, Kakaraparthi BN, NemzekHamlin JA. Effects of buprenorphine on a cecal ligation and puncture model in C57BL/6 mice. J Am Assoc Lab Anim Sci 2012;51:357–65. [12] Dyson A, Singer M. Animal models of sepsis: why does preclinical efficacy fail to translate to the clinical setting? Crit Care Med 2009;37:30–7. [13] Rasaiah VP, Malloy JL, Lewis JF, Veldhuizen RA. Early surfactant administration protects against lung dysfunction in a mouse model of ARDS. Am J Physiol Lung Cell Mol Physiol 2003;284:783–90. [14] Eksßiog˘lu-Demiralp E, Kardasß ER, Ozgül S, Yag˘ci T, Bilgin H, Sehirli O, et al. Betulinic acid protects against ischemia/reperfusion-induced renal damage and inhibits leukocyte apoptosis. Phytother Res 2010;24:325–32. [15] Zhao H, Holmes SS, Baker GA, Challa S, Bose HS, Song Z. Ionic derivatives of betulinic acid as novel HIV-1 protease inhibitors. J Enzyme Inhib Med Chem 2012;27:715–21. [16] Takada Y, Aggarwal BB. Betulinic acid suppresses carcinogen-induced NFkappa B activation through inhibition of I kappa B alpha kinase and p65 phosphorylation: abrogation of cyclooxygenase-2 and matrix metalloprotease-9. J Immunol 2003;171:3278–86. [17] Viji V, Helen A, Luxmi VR. Betulinic acid inhibits endotoxin-stimulated phosphorylation cascade and pro-inflammatory prostaglandin E2 production in human peripheral blood mononuclear cells. Br J Pharmacol 2011;162: 1291–303. [18] Nader MA, Baraka HN. Effect of betulinic acid on neutrophil recruitment and inflammatory mediator expression in lipopolysaccharide-induced lung inflammation in rats. Eur J Pharm Sci 2012;46:106–13. [19] Zambon M, Vincent JL. Mortality rates for patients with acute lung injury/ ARDS have decreased over time. Chest 2008;133:1120–7. [20] Wichmann MW, Haisken JM, Ayala A, Chaudry IH. Melatonin administration following hemorrhagic shock decreases mortality from subsequent septic challenge. J Surg Res 1996;65:109–14.
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[21] van Zoelen MA, Florquin S, Meijers JC, de Beer R, de Vos AF, de Boer OJ, et al. Platelet-activating factor receptor contributes to host defense against Pseudomonas aeruginosa pneumonia but is not essential for the accompanying inflammatory and procoagulant response. J Immunol 2008;180:3357–65. [22] Asaduzzaman M, Zhang S, Lavasani S, Wang Y, Thorlacius H. LFA-1 and MAC-1 mediate pulmonary recruitment of neutrophils and tissue damage in abdominal sepsis. Shock 2008;30:254–9. [23] Angus DC, Linde-Zwirble WT, Lidicker J, Clermont G, Carcillo J, Pinsky MR. Epidemiology of severe sepsis in the United States: analysis of incidence, outcome, and associated costs of care. Crit Care Med 2001;29:1303–10. [24] Remick DG, Newcomb DE, Bolgos GL, Call DR. Comparison of the mortality and inflammatory response of two models of sepsis: lipopolysaccharide versus cecal ligation and puncture. Shock 2000;13:110–6. [25] Simons RK, Junger WG, Loomis WH, Hoyt DB. Acute lung injury in endotoxemic rats is associated with sustained circulating IL-6 levels and intrapulmonary CINC activity and neutrophil recruitment–role of circulating TNF-alpha and IL-beta? Shock 1996;6:39–45. [26] Rongione AJ, Kusske AM, Kwan K, Ashley SW, Reber HA, McFadden DW. Interleukin-10 protects against lethality of intra-abdominal infection and sepsis. J Gastrointest Surg 2000;4:70–6. [27] Standiford TJ, Strieter RM, Lukacs NW, Kunkel SL. Neutralization of IL-10 increases lethality in endotoxemia: cooperative effects of macrophage inflammatory protein-2 and tumor necrosis factor. J Immunol 1995;155:2222–9. [28] Walley KR, Lukacs NW, Standiford TJ, Strieter RM, Kunkel SL. Balance of inflammatory cytokines related to severity and mortality of murine sepsis. Infect Immun 1996;64:4733–8. [29] Hasan Z, Palani K, Rahman M, Thorlacius H. Targeting CD44 expressed on neutrophils inhibits lung damage in abdominal sepsis. Shock 2011;35:567–72. [30] Steinberg KP, Milberg JA, Martin TR, Maunder RJ, Cockrill BA, Hudson LD. Evolution of bronchoalveolar cell populations in the adult respiratory distress syndrome. Am J Respir Crit Care Med 1994;150:113–22. [31] Wagner JG, Roth AR. Neutrophil migration mechanisms, with an emphasis on the pulmonary vasculature1. Pharmacol Rev 2000;52:349–74. [32] Parent C, Eichacker PQ. Neutrophil and endothelial cell interactions in sepsis. The role of adhesion molecules. Infect Dis Clin North Am 1999;13:427–47.
[33] Puneet P, Hegde A, Ng SW, Lau HY, Lu J, Moochhala SM, et al. Preprotachykinin-A gene products are key mediators of lung injury in polymicrobial sepsis. J Immunol 2006;176:3813–20. [34] Wagner JG, Birmingham NP, Jackson-Humbles D, Jiang Q, Harkema JR, Peden DB. Supplementation with c-tocopherol attenuates endotoxin-induced airway neutrophil and mucous cell responses in rats. Free Radical Biol Med 2013;68:101–9. [35] Bossink AW, Paemen L, Jansen PM, Hack CE, Thijs LG, van Damme J. Plasma levels of the chemokines monocyte chemotactic proteins-1 and -2 are elevated in human sepsis. Blood 1995;86:3841–7. [36] Atkinson JJ, Senior RM. Matrix metalloproteinase-9 in lung remodeling. Am J Respir Cell Mol Biol 2003;8:12–24. [37] Ohbayashi H. Matrix metalloproteinases in lung diseases. Curr Protein Pept Sci 2002;3:409–21. [38] Warner RL, Beltran L, Younkin EM, et al. Role of stromelysin 1 and gelatinase B in experimental acute lung injury. Am J Respir Cell Mol Biol 2001;24:537–44. [39] Renckens R, Roelofs JJ, Florquin S, de Vos AF, Lijnen HR, van’t Veer C, et al. Matrix metalloproteinase-9 deficiency impairs host defense against abdominal sepsis. J Immunol 2006;176:3735–41. [40] McQuibban GA, Gong JH, Wong JP, Wallace JL, Clark-Lewis I, Overall CM. Matrix metalloproteinase processing of monocyte chemoattractant proteins generates CC chemokine receptor antagonists with anti-inflammatory properties in vivo. Blood 2002;100:1160–7. [41] Bureau F, Delhalle S, Bonizzi G, Fievez L, Dogne S, Kirschvink N, et al. Mechanisms of persistent NF-kappa B activity in the bronchi of an animal model of asthma. J Immunol 2000;165:5822–30. [42] Arnalich F, Garcia-Palomero E, López J, Jiménez M, Madero R, Renart J, et al. Predictive value of nuclear factor jB activity and plasma cytokine levels in patients with sepsis. Infect Immun 2000;68:1942–5. [43] Ichiki T, Jougasaki M, Setoguchi M, Imamura J, Nakashima H, Matsuoka T, et al. Cardiotrophin-1 stimulates intercellular adhesion molecule-1 and monocyte chemoattractant protein-1 in human aortic endothelial cells. Am J Physiol Heart Circ Physiol 2008;294:750–63. [44] Collins T, Read MA, Neish AS, Whitley MZ, Thanos D, Maniatis T. Transcriptional regulation of endothelial cell adhesion molecules: NF-kappa B and cytokine-inducible enhancers, FASEB J 1995;9:899–909.
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Cytokine journal homepage: www.journals.elsevier.com/cytokine
Betulinic acid attenuates lung injury by modulation of inflammatory cytokine response in experimentally-induced polymicrobial sepsis in mice Madhu Cholenahalli Lingaraju a, Nitya Nand Pathak a, Jubeda Begum b, Venkanna Balaganur a, Rafia Ahmad Bhat a, Harish Darasaguppe Ramachandra c, Anjaneya Ayanur d, Mahendra Ram a, Vishakha Singh a, Dhirendra Kumar a, Dinesh Kumar a, Surendra Kumar Tandan a,⇑ a
Division of Pharmacology and Toxicology, Indian Veterinary Research Institute, Izatnagar, Bareilly, U.P. 243 122, India Division of Bacteriology and Mycology, Indian Veterinary Research Institute, Izatnagar, Bareilly, U.P. 243 122, India c Division of Animal Biotechnology, Indian Veterinary Research Institute, Izatnagar, Bareilly, U.P. 243 122, India d Division of Pathology, Indian Veterinary Research Institute, Izatnagar, Bareilly, U.P. 243 122, India b
a r t i c l e
i n f o
Article history: Received 2 July 2014 Received in revised form 7 September 2014 Accepted 9 September 2014
Keywords: Cecal ligation and puncture Triterpenoid Cytokines Matrix metalloproteinase (MMP)-9 Inflammatory pulmonary insult
a b s t r a c t Sepsis commonly progresses to acute lung injury (ALI), an inflammatory lung disease with high morbidity and mortality. Septic ALI is characterized by excessive production of proinflammatory mediators. It remained refractory to present therapies and new therapies need to be developed to improve further clinical outcomes. Betulinic acid (BA), a pentacyclic lupane group triterpenoid has been shown to have anti-inflammatory activities in many studies. However, its therapeutic efficacy in polymicrobial septic ALI is yet unknown. Therefore, we investigated the effects of BA on septic ALI using cecal ligation and puncture (CLP) model in mice. Vehicle or BA (3, 10, and 30 mg/kg) was administered intraperitoneally, 3 times (0, 24 and 48 h) before CLP and CLP was done on 49th h of the study. Survival rate was observed till 120 h post CLP. Lung tissues were collected for analysis by sacrificing mice 18 h post CLP. BA at 10 and 30 mg/kg dose significantly reduced sepsis-induced mortality and lung injury as implied by attenuated lung histopathological changes, decreased protein and neutrophils infiltration. BA also decreased lung NF-jB expression, cytokine, intercellular adhesion molecule-1, monocyte chemoattractant protein-1 and matrix metalloproteinase-9 levels. These evidences suggest that, the protective effects of BA on lungs are associated with defending action against inflammatory response and BA could be a potential modulatory agent of inflammation in sepsis-induced ALI. Ó 2014 Elsevier Ltd. All rights reserved.
1. Introduction Sepsis is a condition characterized by systemic inflammatory response to infection and commonly progresses to acute lung injury (ALI). The highest incidence of ALI occurs in patients with sepsis [1] carries the highest morbidity and mortality rates when associated with sepsis in intensive care units (ICUs) [2,3]. Lipopolysaccharide (LPS) from the cell wall of Gram-negative bacteria that causes sepsis is the most common pulmonary insult leading to ALI by provoking activation of nuclear factor- kappa B (NF-jB) and the release of proinflammatory mediators including tumor necrosis factor-alpha (TNF-a) and interleukin-6 (IL-6) [4,5]. Additionally, ⇑ Corresponding author. Tel.: +91 581 2300291, +91 581 2301670; fax: +91 581 2303284. E-mail addresses: [email protected], [email protected] (S.K. Tandan). http://dx.doi.org/10.1016/j.cyto.2014.09.004 1043-4666/Ó 2014 Elsevier Ltd. All rights reserved.
LPS also stimulate adhesion, activation, and infiltration of polymorphonuclear neutrophils (PMN) and damaging the alveolar-capillary membrane [6]. Intercellular adhesion molecule-1 (ICAM-1) and monocyte chemoattractant protein-1 (MCP-1) which are known to attract neutrophils [7,8] are increased in sepsis and further results in the tissue infiltration of more PMN, which in turn leads to parenchymal cell dysfunction and tissue damage [9]. Increased matrix metalloproteinases (MMPs) activity cause impairment in lung function due to decreased lung collagen content and disorganized pulmonary parenchymal tissue. Specifically, MMP-9 levels have been shown to be elevated above normal in patients with severe sepsis [10]. Animal models play a significant role in sepsis research [11]. Cecal ligation and puncture (CLP) model is in widespread use and is considered by some experts as the gold standard for animal models of sepsis [12]. Sepsis induced by CLP is a clinically relevant
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model in which lung injury is induced via an inflammatory pathway [13]. Triterpenoids are ubiquitous in plants and have long been considered to be the anti-inflammatory principles in several herbal drugs [14]. Betulinic acid (BA), (3b-hydroxy-lup-20(29)-en-28-oic acid is a naturally occurring pentacyclic lupane group triterpenoid. It exhibits a variety of biological and medicinal properties such as anti-cancer, anti-malarial, anti-inflammatory and anti-HIV properties [15]. BA has shown anti-inflammatory activities in various experimental systems [16–18]. However, its pharmacological potential in murine polymicrobial sepsis-induced lung injury, which is mainly inflammatory in nature, needs to be explored. ALI due to sepsis is thought to result in a high mortality rate, warranting treatment and preventative measures [19]. With the knowledge growing, interfering with proinflammatory cytokines to relieve lung injury has been widely applied [5]. In the present study, we investigated whether pretreatment with BA could prevent alterations in the cytokine and chemokine levels and prevent septic lung injury in a clinically more relevant model that mimics sepsis conditions in humans. Our results indicate that BA is a powerful repressor of major cytokines and chemokines in a mouse model of polymicrobial sepsis-induced lung injury indicating its use as potential anti-inflammatory agent in sepsis associated ALI in mice. 2. Materials and methods 2.1. Animals Male Swiss albino mice were procured from Laboratory Animal Resource Section of the Institute. These animals were kept in polypropylene cages, housed on an animal facility 12–12 h light dark cycle at 22 °C, and fed with standard laboratory diet and water ad libitum. The mice were acclimatized for one week before use. All experimental procedures involving animals were approved by the Institute Animal Ethics Committee, IVRI, Izatnagar. 2.2. Drug and chemical reagents Betulinic acid, odianisidine, hydrochloric acid, acrylamide, bisacrylamide, SDS, gelatine, Triton X-100 and hydrogen peroxide were purchased from Sigma Chemicals Co., St. Louis, USA. EIA kits, dimethylsulfoxide (DMSO) from Genetix Biotech Asia Pvt. Ltd. Rabbit-anti-mouse monoclonal antibody for NF-jB, goat-anti-rabbit secondary HRP conjugated antibodies were procured from Abcam antibodies. All the necessary chemicals for conventional biochemical parameter methods were of analytical grade purchased from Sigma, Genetix and Hi Media Laboratories Pvt. Ltd. 2.3. Experimental sepsis induced by CLP Polymicrobial sepsis was induced by CLP surgical procedure according to previously described method [20]. Animals were anesthetized with an intraperitoneal injection of a mixture of 10 mg/kg xylazine and 100 mg/kg ketamine hydrochloride. The abdomen was gently shaved and cleaned with antiseptic sterile swab. Through a 1 cm abdominal midline incision, the caecum was exposed and ligated below the ileocecal valve without causing bowel obstruction. The caecum was then subjected to a through and through perforation twice with a 21-gauge needle with the caecum squeezed to extrude some fecal contents. After repositioning the caecum, the abdominal incision was closed in layers with chromic gut surgical suture 4-0 and all mice were then resuscitated with 1 mL of warmed sterile lactated Ringer’s subcutaneously. Sham-operated animals were subjected to laparotomy,
intestinal manipulation and resuscitation procedures; however, the caecum was neither ligated nor punctured. 2.4. Experimental design The mice were divided into five groups and treatment was given intraperitoneally 3 times at 0, 24 and 48 h before sham or CLP. Group I (sham control) and group II (CLP control), consisting mice pretreated with vehicle; group III, IV and V (drug pretreated), consisting mice pretreated with BA at the dose rate of 3, 10 and 30 mg/kg body weight, respectively and mice of all the groups were submitted to CLP after 1 h of last dose (that is at 49th h) except group I where the mice underwent sham operation. 2.5. Survival study Survival study was carried out as a separate experiment and 15 animals were used in each group. After animal grouping, drug administration and surgical procedures as noted in experimental design, animals of all the groups were observed for their survival period till 120 h from the time of surgery. 2.6. Animal sacrifice and tissue harvest In a separate experiment held for sampling, the mice were sacrificed by cervical dislocation at 18 h after sham or CLP surgery. The lung tissues were removed, weighed, snap-frozen in liquid nitrogen, homogenized in 50 mM/L phosphate buffer with mammalian cocktail protease inhibitor (pH 7.4) containing 1 mM EDTA at 4 °C to make 5% tissue homogenate. The homogenates were centrifuged at 1500g for 10 min in a cooling centrifuge at 4 °C. The resulting supernatants were stored at 80 °C for further analysis. 2.7. Relative lung weight To assess the lung edema fluid content, which is an indicator of pulmonary inflammation we assessed the relative lung weights of the mice of different groups after 18 h of CLP. Lung weights were expressed as mg of lung per 25 g of mouse to obtain relative organ weight [21]. 2.8. Pulmonary cytokine, chemokine, ICAM-1, MCP-1 and MMP-9 levels Pulmonary levels of TNF-a, IL-6, IL-10, ICAM-1, MCP-1 and MMP-9 were quantified using specific ELISA kits for mice according to the manufacturers’ instructions. 2.9. MPO activity assay MPO activity in the lungs was determined as an index of tissue neutrophil infiltration. MPO activity in the supernatant was measured from the absorbance changes at 460 nm resulting from the decomposition of H2O2 in the presence of odianisidine [22]. 2.10. Gelatin zymography for MMP detection MMP-9 (92 kDa gelatinase, gelatinase B) degrades components of the ECM with high specific activity for denatured collagens (gelatin). Gelatin–SDS–polyacrylamide gel was used to detect the presence of gelatinolytic MMP-9. Both active forms and pro-enzymes are revealed by this technique as the exposure of pro-MMPs to SDS during SDS–PAGE leads to activation without proteolytic cleavage. Electrophoresis was carried out at room temperature at a voltage of 110 mV. When the tracking dye reached the bottom of the gel, it was washed twice with 2.5% Triton X-100 for
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60 min to remove the SDS. The gel was subsequently incubated for 48 h at room temperature in renaturation buffer (50 mM Tris–HCl (pH 7.6), 200 mM NaCl, 5 mM CaCl2). The gel was stained with 0.5% Coomassie blue for 2 h and then destained with water containing 10% glacial acetic acid and 40% methanol. Areas of gelatinolytic degradation appeared as transparent bands on the blue stained background of the gel. A wide range molecular weight protein marker from PureGene (PG-PMT0782, 10–180 kDa) was used to estimate the apparent molecular weights for the bands of substrate degradation. 2.11. Western blot analysis Equal amounts (approximately 50 lg) of protein were loaded per well on a 12% sodium dodecyl sulphate polyacrylamide gel (SDS–PAGE). Subsequently, proteins were transferred onto polyvinylidene difluoride membrane. The membranes were washed in Tris-buffered saline with Tween 20 and incubated in 5% BSA (Sigma) at room temperature for 2 h on a rocker shaker, and followed by TBS-T washing. Incubations with rabbit polyclonal antibodies specific for NF-jB in diluents buffer (5% BSA in TBST) were performed overnight at 4 °C. Then the membrane was washed with TBS-T followed by incubation with the peroxidaseconjugated secondary antibody at room temperature for 2 h and subjected DAB reaction to visualize the bands.
Fig. 1. Survival analysis results (% survival) from mice of different groups. The treatment was given (vehicle or BA) on day 1, 2 and 3 by intraperitoneal route before sham or CLP; the mice underwent sham or CLP surgery 1 h after the last dose on day 3. The survival was observed for 120 h after surgical interventions; alphabets on the lines are used to represents significance. Survival lines which do not share any alphabet in common among them differ significantly (p < 0.05); n = 15 mice per group.
2.12. Histopathological analysis For light microscopic investigations, lung tissue specimens were fixed in 10% formaldehyde and dehydrated with alcohol. Paraffin embedded sections were cut and stained with hematoxylin and eosin (H&E) and examined under a photomicroscope (Olympus BH 2, Tokyo, Japan). All tissue sections were examined microscopically for the histopathological changes by experienced histologists who were blind to treatments. 2.13. Statistical analysis All statistical analysis was done by using Prism version 4 (GraphPad Software Inc., USA). Data were expressed as mean ± S.E.M. The level of statistical significance were determined by one-way analysis of variance (ANOVA) followed by Tukey’s multiple comparison test. Survival statistics were compared with a Kaplan–Meier curve and log-rank test. The differences in the data obtained were considered statistically significant when the p value was less than 0.05. 3. Results 3.1. Effect of pretreatment of BA on survival In a separate experiment mice were observed for survival rate for 5 days. In the vehicle-treated CLP group, the survival rate on the first day of observation was 66.66% and reached a stable 6.66% survival on the fifth day after CLP. Log rank analysis of 5 day survival demonstrated that BA at doses of 10 and 30 mg/kg significantly improved survival (46.66%) of septic mice compared with CLP animals. BA at 3 mg/kg did not confer the significant protection against CLP-induced lethality (Fig. 1). No mortality was observed in the sham group. 3.2. Effect of BA pretreatment on relative lung weight in CLP mice As reflected by relative lung weights, the pulmonary inflammatory fluid accumulation was significantly higher in CLP control group as compared to sham control group (Fig. 2). However, BA
Fig. 2. Relative lung weight from mice of different groups after 18 h of surgery. All data are presented as means ± S.E; Alphabets above the bars represents superscript. Bars which do not share any superscript in common among them differ significantly (p < 0.05); n = 6 mice per group.
pretreated mice had significantly lower pulmonary inflammatory fluid accumulation in their lung than did CLP control group mice at 18 h after CLP. 3.3. Effect of BA pretreatment on lung cytokine levels in CLP mice Protein levels of TNF-a and IL-6 were markedly elevated in lung of septic mice and attenuated to sham levels in mice pretreated with BA. In mice undergoing CLP, BA did not reduce elevated levels of IL-10 (Fig. 3). 3.4. Effect of BA pretreatment on lung ICAM-1, MCP-1 and MPO levels in CLP mice CLP caused an increased trafficking of PMN cells into lungs after CLP, which was evidenced by the increased levels of ICAM-1, MCP1 and MPO in lung CLP control group as compared to sham control group (Fig. 4). Pretreatment with BA 3 days before CLP, significantly reduced the ICAM-1, MCP-1 levels and MPO activity in the lung as compared with the CLP control group. 3.5. Effect of BA pretreatment on activity of MMPs in the lung in CLP mice MMP-9 levels in lungs were determined by EIA kit. As shown in Fig. 5, CLP significantly increased MMP-9 levels in lungs of CLP
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Fig. 3. Cytokine levels in lungs from mice of different groups after 18 h of surgery. Lung TNF-a (a), IL-6 (b) and IL-10 (c) levels. All data are presented as means ± S.E; Alphabets above the bars represents superscript. Bars which do not share any superscript in common among them differ significantly (p < 0.05); n = 6 mice per group, assay in triplicate.
control group mice as compared to sham control group. BA pretreatment significantly reduced the MMP-9 levels in lungs of CLP mice when compared to CLP control group values. Additionally, gelatinase activity of MMP-9 (92 kDa) was measured using gelatin zymography and result is presented in Fig. 6. In CLP control group 18 h after induction of CLP, the lung MMP-9 gelatinase activity was elevated to a significantly higher level as evidenced by thicker transparent bands on the blue stained background of the gel when compared to sham control group which showed thinner bands. BA pretreated CLP group showed the band of intermediate thickness. 3.6. Effect of BA pretreatment on NF-jB protein expression in the lung in CLP mice NF-jB (64 kDa) protein expression in lung of CLP control mice was increased compared to sham mice. BA pretreated mice at 3, 10 and 30 mg/kg dose showed minimal expression of NF-jB protein compared to CLP mice (Fig. 7). The b-actin protein expression was constant in all the groups.
Fig. 4. Neutrophil infiltration markers levels in lungs from mice of different groups after 18 h of surgery. Lung ICAM-1 (a), MCP-1 (b) levels and lung MPO (c) activity. All data are presented as means ± S.E; alphabets above the bars represents superscript. Bars which do not share any superscript in common among them differ significantly (p < 0.05); n = 6 mice per group, assay in triplicate.
Fig. 5. Extracellular matrix degrading enzyme, the MMP-9 levels in lungs from mice of different groups after 18 h of surgery. All data are presented as means ± S.E; Alphabets above the bars represents superscript. Bars which do not share any superscript in common among them differ significantly (p < 0.05); n = 6 mice per group, assay in triplicate.
40 and b, 40). However, in CLP control group, lung tissue section showed alveolar edema, alveolar damage and inflammatory cell infiltration (Fig. 8; c, 10; d, 40). BA pretreatment significantly attenuated the histopathological changes in the lungs of CLP mice, especially edema fluid accumulation and inflammatory cells infiltration at 10 (Fig. 8 e, 10 and f, 40) and 30 mg/kg dose (Fig. 8 g, 10 and h, 40).
3.7. Effect of BA pretreatment on lung histopathology in CLP mice 4. Discussion As shown in photomicrograph (H&E stained), lung sections from the sham control group of mice showed a normal alveolar and bronchi structure with no histopathological changes (Fig. 8 a,
The lung is frequently the first failing organ during the sequential development of multiple organ dysfunction in sepsis
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Fig. 6. Zymogram showing MMP-9 (92 kDa) mediated gelatinolytic activities of lung samples from mice of different groups after 18 h of surgery; n = 6 mice per group, assay in triplicate.
[23]. In the present study, we successfully set up a moderate to severe CLP model as indicated by mortality (84%), lung water content (relative lung weight) and lung damage in histological section. However, it has been observed that BA pretreatment before CLP alleviated the lung inflammatory response and improved survival in murine polymicrobial sepsis model by inhibiting transcription factor, NF-jB and associated maladaptive cytokine release. Furthermore, the subcellular biochemical changes that would favor the lung damage like increased PMN trafficking activity as indicated by elevated ICAM-1, MCP-1 and MPO levels and increased degradation of lung ECM as found by elevated MMP-9 levels following sepsis induction were attenuated by BA pretreatment. BA pretreated mice showed higher survival rate than vehicle pretreated mice after CLP (Fig. 1), reflecting its potential to have protective role in polymicrobial sepsis. It remains to be seen how BA affects the inflammatory response in lung and exerts its protective effect in CLP-induced lung injury to improve overall survival of mice. Edema is a typical symptom of inflammation. To quantify the magnitude of pulmonary edema in CLP-induced lung injury model, we evaluated the relative lung weight [24]. CLP control group showed significantly higher relative lung weights as compared to sham control group (Fig. 2). BA pretreated CLP group mice showed significantly lesser relative lung weights when compared to CLP control group mice, which indicates that BA could inhibit the
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leakage of serous fluid into lung tissue and attenuate the development of lung edema and inflammation. In septic mice, cytokines leads to SIRS, subsequent septic shock, and multiorgan dysfunction. Among these cytokines, TNF-a and IL6 are considered to play important role in the pathophysiology of septic lung injury [25]. Studies showed that TNF-a and IL-6 levels were significantly increased in the CLP group compared with the sham control group [26]. In sepsis, IL-10 has been identified as a vital modulator of the lethal overabundant production of inflammatory cytokines. In several animal models of sepsis, neutralization of IL-10 results in exaggerated proinflammatory cytokine expression and death, while administration of recombinant IL-10 confers significant therapeutic protection [27,28]. In the present study, we observed CLP-induced higher lung levels of cytokine, whereas BA pretreatment before CLP significantly decreased lung proinflammatory cytokines including TNF-a and IL-6 (Fig. 3). However, IL-10 levels remained unaltered. These data suggests the protective effects of BA on septic lung injury in mice might be achieved by inhibition of TNF-a and IL-6 induced tissue damage along with maintained levels of IL-10 which protects experimental subject from proinflammatory cytokine damage. This indicates that BA pretreatment had an anti-inflammatory effect in lungs of CLP mice and could be one of the BA’s mechanism involved in offering protection against severe inflammatory response in lungs and thereby improving the survival. Neutrophil infiltration is an insidious feature in septic lung injury [29] and persistence of high numbers of PMN in BALF after the first week of ARDS is associated with mortality, particularly in patients with sepsis [30]. Recruitment of PMNs into lung air spaces occurs in response to a variety of airway stimuli and involves the stepwise process of adhesion molecule expression on PMNs in both the pulmonary capillaries and the bronchial venules [31]. ICAM-1 is a type of adhesion molecule, involved in PMN recruitment and secondary organ damage in response to infection and inflammation [7]. A high level of proinflammatory mediators in sepsis is reported to up-regulate various adhesion molecules [32]. Absence of this molecule impairs the ability of PMN to migrate into organ tissues and reduces consequent secondary organ damage resulting in improved clinical status and overall survival. MCP-1, a chemokine was shown to act as a chemoattractant for neutrophils [7,8]. MCP-1 is known to orchestrate migration of leukocytes during sepsis leading to tissue injury. It has been shown that MCP-1 levels in lung correlates with neutrophil infiltration in the lung [33]. In patients with Gram-positive and Gram negative infections resulting in sepsis, elevated MCP-1 levels were observed [34,35]. MPO is an enzyme that is found predominantly in PMN cells. Since tissue MPO activity correlates significantly with the number of PMN determined histochemically, it is more commonly used to estimate tissue PMN accumulation in inflamed tissues. Consistent with the earlier reports, we found a significant increase
Fig. 7. Representative photograph of Western blot analysis of lung NF-jB protein from mice of different groups. Lung tissues from a representative mouse of each group were isolated after 18 h of surgery. Beta-actin (42 kDa) protein expression was analyzed as a control. Expression analysis was done using antibodies directed against NF-jB (64 kDa); n = 3 mice per group, blotting analysis in triplicate.
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Fig. 8. Photomicrographs (H&E stained; n = 3 mice per group) of representative lung tissue section of sham operated mouse showing normal bronchi (a, 40, black arrow) and lung parenchyma (b, 40, black arrow) with no observed changes. Lung tissue section of CLP operated mouse showing damaged alveoli, edema (c, 10; black arrows) and infiltration of neutrophils and edema (d, 40, black arrows). Lung tissue section of drug (10 mg/kg BA) pretreated CLP operated mouse showing attenuated histopathological changes marked by less edema (pink tinged sections) and ameliorated alveolar damage (black arrows) (e, 10 and f, 40). Lung tissue section of drug (30 mg/kg BA) pretreated CLP operated mouse showing attenuated histopathological changes marked by almost negligible amount of edema (g, 10, black arrow) and ameliorated alveolar damage (g, 10; h, 40, black arrows).
in the lung levels of ICAM-1, MCP-1 and MPO in CLP control as compared to sham-operated mice. Pretreatment with BA lowered the lung levels of ICAM-1, MCP-1 and MPO activity (Fig. 4)
significantly indicating its attenuating effect on neutrophil infiltration network. Thus, BA treatment in our study could have reduced the severity of sepsis by impairing the leukocyte migration.
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Further, this was supported by the histological sections of the lung. BA injection clearly reduced the leukocyte infiltration and edema, the signs of lung injury in sepsis. The MMPs are a group of structurally related zinc-dependent enzymes which together are capable of degrading all the components of the ECM. Remodeling of the lung architecture is a hallmark of many of the lung diseases for example, loss of alveolar walls. This pathological change involves extensive alterations of lung ECM [36]. Excessive or inappropriate expression of MMP is closely associated with pathogenesis of tissue destructive processes in a wide variety of diseases, including lung diseases [37]. Up regulation of MMP-9 is suggested to be involved in the injury progression [38]. MMP-9 is involved in extracellular matrix degradation and leukocyte migration [39]. Additionally, it is involved in processing of cytokines and chemokines to regulate inflammation and thought to be proinflammatory [40] which may further aggravate sepsis [10]. Given that the MMP-9 had been specifically implicated in the pathogenesis of septic lung injury, we specifically examined level of this enzyme activity in lungs using gelatin zymography. We found increased activity of MMP-9 in lung of CLP mice and propose that the increased MMP-9 activity in the lungs caused the development of a larger pulmonary edema due to destabilized alveolar ECM components. Interestingly, induction of MMP-9 coincided with the PMN infiltration and the maximum of histological manifestations in lungs of CLP mice. In contrast, preadministration of BA at 10 and 30 mg/kg dose attenuated MMP-9 levels as detected in ELISA (Fig. 5) and the defect in MMP-9 activity (Fig. 6) as demonstrated by the zymography in the lungs in response to CLP and added ameliorating effect against the development of edema by stabilizing the lung ECM components thereby laying the protective effect on lung in this model. NF-jB is considered to an important transcriptional factor involved in the production of proinflammatory cytokines, adhesion molecules, and other proinflammatory proteins [41]. A greater or more persistent nuclear accumulation of NF-jB is associated with higher mortality and more persistent organ dysfunction, including pulmonary injury [42]. Further, activation of the NF-jB by TNF-a increases endothelial expression of ICAM-1, MCP-1 and MMP-9 which were found to play important role in trafficking of PMN cells to the site of injury/infection [43,44]. NF-jB and TNF-a interplay together setup a severe inflammatory response and ultimately leads to failure of the particular affected organ. Therefore, inhibiting NF-jB activation appears to be a logical therapeutic target for controlling hyper-inflammatory responses, including ALI. As shown in our results (Fig. 7), CLP control mice had a significantly increased lung NF-jB protein expression which was attenuated by BA pretreatment. Previous studies also reported the potency of BA to inhibit NF-jB protein expression in different models [16,17]. From the results of this study, it is shown that the suppressing effects of BA on NF-jB suggested to be involved in the effects related to lowered cytokines, ICAM-1, MCP-1 and MMP-9 levels and unopposed IL-10 levels thereby exhibiting protective effect on septic lung injury. Histopathological results reveal that there was wide damage of alveoli and accumulation of edema fluid and neutrophils after CLP (Fig. 8). However, BA at 10 and 30 mg/kg doses attenuated the histopathological changes especially damage to alveoli and neutrophil influx further sustaining the protective action of BA against the CLP-induced lung injury. The observations of this study indicate that BA has good antiinflammatory activity and further, attenuating action on neutrophil infiltration and MMP-9 mediated lung damage. These mechanisms are proposed to be the possible protective mechanisms of BA on lung in this model. In conclusion, our results suggest that the BA has antiinflammatory effects in murine model of sepsis and particularly
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the beneficial effects are in sepsis-induced lung injury. Protective mechanisms of BA in this model can be explained via inflammatory response attenuation, reduced infiltration of inflammatory cells and attenuation of production of lung ECM degrading enzymes. These findings possibly provide a rationale basis for the ability of BA to suppress lung inflammatory response. Thus, it is expected that, BA may serve as a potential anti-inflammatory tool for septic ALI. However, further studies employing post-treatment trials are required to support the use of BA as an excellent therapeutic approach to treat sepsis associated ALI conditions. Acknowledgments The authors thank the Director and the Joint Director (Academic), Indian Veterinary Research Institute, Izatnagar, Bareilly, U.P., India for financial support. References [1] Hudson LD, Milberg JA, Anardi D, Maunder RJ. Clinical risks for development of the acute respiratory distress syndrome. Am J Respir Crit Care Med 1995;151:293–301. [2] Rubenfeld GD, Caldwell E, Peabody E, Weaver J, Martin DP, Neff M, et al. Incidence and outcomes of acute lung injury. N Engl J Med 2005;353:1685–93. [3] Johnson ER, Matthay MA. Acute lung injury: epidemiology, pathogenesis, and treatment. J Aerosol Med Pulm Drug Deliv 2010;23:243–52. [4] Carter AB, Monick MM, Hunninghake GW. Lipopolysaccharide-induced NFkappaB activation and cytokine release in human alveolar macrophages is PKC-independent and TK- and PC-PLC-dependent. Am J Respir Cell Mol Biol 1998;18:384–91. [5] Gong Q, Yin H, Fang M, Xiang Y, Yuan CL, Zheng GY, et al. Heme oxygenase-1 upregulation significantly inhibits TNF-a and Hmgb1 releasing and attenuates lipopolysaccharide-induced acute lung injury in mice. Int Immunopharmacol 2008;8:792–8. [6] Erickson SE, Martin GS, Davis JL, Matthay MA, Eisner MD. Recent trends in acute lung injury mortality: 1996–2005. Crit Care Med 2009;37:1574–9. [7] Hildebrand F, Pape HC, Harwood P, Müller K, Hoevel P, Pütz C, et al. Role of adhesion molecule ICAM in the pathogenesis of polymicrobial sepsis. Exp Toxicol Pathol 2005;56:281–90. [8] Zhao X, Dib M, Andersson E, Shi C, Widegren B, Wang X, et al. Alterations of adhesion molecule expression and inflammatory mediators in acute lung injury induced by septic and non-septic challenges. Lung 2005;183:87–100. [9] Neviere RR, Cepinskas G, Madorin WS, Hoque N, Karmazyn M, Sibbald WJ, et al. LPS pretreatment ameliorates peritonitis-induced myocardial inflammation and dysfunction: role of myocytes. Am J Physiol 1999;277:885–92. [10] Hoffmann U, Bertsch T, Dvortsak E, Liebetrau C, Lang S, Liebe V, et al. Matrix-metalloproteinases and their inhibitors are elevated in severe sepsis: prognostic value of TIMP-1 in severe sepsis. Scand J Infect Dis 2006;38: 867–72. [11] Cotroneo TM, Hugunin KM, Shuster KA, Hwang HJ, Kakaraparthi BN, NemzekHamlin JA. Effects of buprenorphine on a cecal ligation and puncture model in C57BL/6 mice. J Am Assoc Lab Anim Sci 2012;51:357–65. [12] Dyson A, Singer M. Animal models of sepsis: why does preclinical efficacy fail to translate to the clinical setting? Crit Care Med 2009;37:30–7. [13] Rasaiah VP, Malloy JL, Lewis JF, Veldhuizen RA. Early surfactant administration protects against lung dysfunction in a mouse model of ARDS. Am J Physiol Lung Cell Mol Physiol 2003;284:783–90. [14] Eksßiog˘lu-Demiralp E, Kardasß ER, Ozgül S, Yag˘ci T, Bilgin H, Sehirli O, et al. Betulinic acid protects against ischemia/reperfusion-induced renal damage and inhibits leukocyte apoptosis. Phytother Res 2010;24:325–32. [15] Zhao H, Holmes SS, Baker GA, Challa S, Bose HS, Song Z. Ionic derivatives of betulinic acid as novel HIV-1 protease inhibitors. J Enzyme Inhib Med Chem 2012;27:715–21. [16] Takada Y, Aggarwal BB. Betulinic acid suppresses carcinogen-induced NFkappa B activation through inhibition of I kappa B alpha kinase and p65 phosphorylation: abrogation of cyclooxygenase-2 and matrix metalloprotease-9. J Immunol 2003;171:3278–86. [17] Viji V, Helen A, Luxmi VR. Betulinic acid inhibits endotoxin-stimulated phosphorylation cascade and pro-inflammatory prostaglandin E2 production in human peripheral blood mononuclear cells. Br J Pharmacol 2011;162: 1291–303. [18] Nader MA, Baraka HN. Effect of betulinic acid on neutrophil recruitment and inflammatory mediator expression in lipopolysaccharide-induced lung inflammation in rats. Eur J Pharm Sci 2012;46:106–13. [19] Zambon M, Vincent JL. Mortality rates for patients with acute lung injury/ ARDS have decreased over time. Chest 2008;133:1120–7. [20] Wichmann MW, Haisken JM, Ayala A, Chaudry IH. Melatonin administration following hemorrhagic shock decreases mortality from subsequent septic challenge. J Surg Res 1996;65:109–14.
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M.C. Lingaraju et al. / Cytokine 71 (2015) 101–108
[21] van Zoelen MA, Florquin S, Meijers JC, de Beer R, de Vos AF, de Boer OJ, et al. Platelet-activating factor receptor contributes to host defense against Pseudomonas aeruginosa pneumonia but is not essential for the accompanying inflammatory and procoagulant response. J Immunol 2008;180:3357–65. [22] Asaduzzaman M, Zhang S, Lavasani S, Wang Y, Thorlacius H. LFA-1 and MAC-1 mediate pulmonary recruitment of neutrophils and tissue damage in abdominal sepsis. Shock 2008;30:254–9. [23] Angus DC, Linde-Zwirble WT, Lidicker J, Clermont G, Carcillo J, Pinsky MR. Epidemiology of severe sepsis in the United States: analysis of incidence, outcome, and associated costs of care. Crit Care Med 2001;29:1303–10. [24] Remick DG, Newcomb DE, Bolgos GL, Call DR. Comparison of the mortality and inflammatory response of two models of sepsis: lipopolysaccharide versus cecal ligation and puncture. Shock 2000;13:110–6. [25] Simons RK, Junger WG, Loomis WH, Hoyt DB. Acute lung injury in endotoxemic rats is associated with sustained circulating IL-6 levels and intrapulmonary CINC activity and neutrophil recruitment–role of circulating TNF-alpha and IL-beta? Shock 1996;6:39–45. [26] Rongione AJ, Kusske AM, Kwan K, Ashley SW, Reber HA, McFadden DW. Interleukin-10 protects against lethality of intra-abdominal infection and sepsis. J Gastrointest Surg 2000;4:70–6. [27] Standiford TJ, Strieter RM, Lukacs NW, Kunkel SL. Neutralization of IL-10 increases lethality in endotoxemia: cooperative effects of macrophage inflammatory protein-2 and tumor necrosis factor. J Immunol 1995;155:2222–9. [28] Walley KR, Lukacs NW, Standiford TJ, Strieter RM, Kunkel SL. Balance of inflammatory cytokines related to severity and mortality of murine sepsis. Infect Immun 1996;64:4733–8. [29] Hasan Z, Palani K, Rahman M, Thorlacius H. Targeting CD44 expressed on neutrophils inhibits lung damage in abdominal sepsis. Shock 2011;35:567–72. [30] Steinberg KP, Milberg JA, Martin TR, Maunder RJ, Cockrill BA, Hudson LD. Evolution of bronchoalveolar cell populations in the adult respiratory distress syndrome. Am J Respir Crit Care Med 1994;150:113–22. [31] Wagner JG, Roth AR. Neutrophil migration mechanisms, with an emphasis on the pulmonary vasculature1. Pharmacol Rev 2000;52:349–74. [32] Parent C, Eichacker PQ. Neutrophil and endothelial cell interactions in sepsis. The role of adhesion molecules. Infect Dis Clin North Am 1999;13:427–47.
[33] Puneet P, Hegde A, Ng SW, Lau HY, Lu J, Moochhala SM, et al. Preprotachykinin-A gene products are key mediators of lung injury in polymicrobial sepsis. J Immunol 2006;176:3813–20. [34] Wagner JG, Birmingham NP, Jackson-Humbles D, Jiang Q, Harkema JR, Peden DB. Supplementation with c-tocopherol attenuates endotoxin-induced airway neutrophil and mucous cell responses in rats. Free Radical Biol Med 2013;68:101–9. [35] Bossink AW, Paemen L, Jansen PM, Hack CE, Thijs LG, van Damme J. Plasma levels of the chemokines monocyte chemotactic proteins-1 and -2 are elevated in human sepsis. Blood 1995;86:3841–7. [36] Atkinson JJ, Senior RM. Matrix metalloproteinase-9 in lung remodeling. Am J Respir Cell Mol Biol 2003;8:12–24. [37] Ohbayashi H. Matrix metalloproteinases in lung diseases. Curr Protein Pept Sci 2002;3:409–21. [38] Warner RL, Beltran L, Younkin EM, et al. Role of stromelysin 1 and gelatinase B in experimental acute lung injury. Am J Respir Cell Mol Biol 2001;24:537–44. [39] Renckens R, Roelofs JJ, Florquin S, de Vos AF, Lijnen HR, van’t Veer C, et al. Matrix metalloproteinase-9 deficiency impairs host defense against abdominal sepsis. J Immunol 2006;176:3735–41. [40] McQuibban GA, Gong JH, Wong JP, Wallace JL, Clark-Lewis I, Overall CM. Matrix metalloproteinase processing of monocyte chemoattractant proteins generates CC chemokine receptor antagonists with anti-inflammatory properties in vivo. Blood 2002;100:1160–7. [41] Bureau F, Delhalle S, Bonizzi G, Fievez L, Dogne S, Kirschvink N, et al. Mechanisms of persistent NF-kappa B activity in the bronchi of an animal model of asthma. J Immunol 2000;165:5822–30. [42] Arnalich F, Garcia-Palomero E, López J, Jiménez M, Madero R, Renart J, et al. Predictive value of nuclear factor jB activity and plasma cytokine levels in patients with sepsis. Infect Immun 2000;68:1942–5. [43] Ichiki T, Jougasaki M, Setoguchi M, Imamura J, Nakashima H, Matsuoka T, et al. Cardiotrophin-1 stimulates intercellular adhesion molecule-1 and monocyte chemoattractant protein-1 in human aortic endothelial cells. Am J Physiol Heart Circ Physiol 2008;294:750–63. [44] Collins T, Read MA, Neish AS, Whitley MZ, Thanos D, Maniatis T. Transcriptional regulation of endothelial cell adhesion molecules: NF-kappa B and cytokine-inducible enhancers, FASEB J 1995;9:899–909.
