http://informahealthcare.com/ipi ISSN: 0892-3973 (print), 1532-2513 (electronic) Immunopharmacol Immunotoxicol, 2014; 36(1): 11–16 ! 2014 Informa Healthcare USA, Inc. DOI: 10.3109/08923973.2013.861482
RESEARCH ARTICLE
Protective effect of taraxasterol against LPS-induced endotoxic shock by modulating inflammatory responses in mice 1
Department of Animal Medicine, Agricultural College of Yanbian University, Yanji, Jilin, PR China and 2Experimental Base of Agriculture, Jilin University, Changchun, Jilin, PR China Abstract
Keywords
Taraxasterol, a pentacyclic-triterpene, was isolated from the Chinese medicinal herb Taraxacum officinale. In the present study, we investigated the protective effect of taraxasterol on murine model of endotoxic shock and the mechanism of its action. Mice were treated with 2.5, 5 and 10 mg/kg of taraxasterol prior to a lethal dose of lipopolysaccharide (LPS) challenge. Survival of mice was monitored twice a day for 7 days. To further understand the mechanism, the serum levels of inflammatory cytokine tumor necrosis factor-a (TNF-a), interferon-g (IFN-g), interleukin1b (IL-1b), interleukin-6 (IL-6) and mediator nitric oxide (NO), prostaglandin E2 (PGE2) as well as histology of lungs were examined. The results showed that taraxasterol significantly improved mouse survival and attenuated tissue injury of the lungs in LPS-induced endotoxemic mice. Further studies revealed that taraxasterol significantly reduced TNF-a, IFN-g, IL-1b, IL-6, NO and PGE2 levels in sera from mice with endotoxic shock. These results indicate that taraxasterol has a protective effect on murine endotoxic shock induced by LPS through modulating inflammatory cytokine and mediator secretion. This finding might provide a new strategy for the treatment of endotoxic shock and associated inflammation.
Endotoxic shock, inflammatory responses, LPS, survival, taraxasterol
Introduction Endotoxic shock, which results from Gram-negative bacterial infections, is a major cause of death among patients and animals1,2. One of the major reasons is that endotoxin or lipopolysaccharide (LPS), a component of the Gram-negative bacterial cell wall, induces the disturbance of immune and inflammatory responses, causes extensive tissue damage and is considered to play a central role in mediating diseases caused by Gram-negative bacteria3–5. The inflammatory response after challenge with LPS is associated with the release of proinflammatory cytokines and other inflammatory mediators, including tumor necrosis factor-a (TNF-a), interleukin-1 (IL1), interleukin-6 (IL-6), interferon-g (IFN-g), nitric oxide (NO) and prostaglandin E2 (PGE2). The progressive production of these inflammatory cytokines and mediators may result in the systemic inflammatory response syndrome, severe tissue damage, septic shock or even death6–8. In spite of rapid progress in developing antibiotics and other therapeutic methods in clinical practice, sepsis remains a leading cause of morbidity and mortality in the intensive care unit9,10. Thus, there is an urgent need to develop new therapeutic tools for the treatment of septic shock.
*These authors contributed equally to this work. Address for correspondence: Xuemei Zhang, Department of Animal Medicine, Agricultural College of Yanbian University, Gongyuan Street, Yanji, Jilin 133002, PR China. E-mail: [email protected]
History Received 30 August 2013 Accepted 29 October 2013 Published online 29 November 2013
Taraxacum officinale has long been used in traditional oriental medicine for its lactating, choleretic, diuretic, antirheumatic and anti-inflammatory properties11,12. It is widely used for treating various inflammatory or infectious diseases clinically such as hepatitis, upper respiratory tract infections, bronchitis, pneumonia and as a compress for its anti-mastopathy activity13,14. Pharmacological activities of Taraxacum officinale have been in part evaluated so far15,16. Recently, Taraxacum officinale extracts have been shown to inhibit LPS-induced inflammatory responses by reducing NO, PGE2 and pro-inflammatory cytokines production via inactivation of nuclear factor-B (NF-B) and mitogen-activated protein kinase signal pathway in RAW 264.7 cells17,18. Moreover, the aqueous extract of Taraxacum officinale was assessed to contain acute anti-inflammatory activity by showing its protective effects against cholecystokinin-induced acute pancreatitis in rats19, and we also reported its protective effects against LPS-induced acute lung injury in mice20. Taraxasterol (Figure 1) isolated from Taraxacum officinale is one of its main active constituents. Recently, our study has shown that taraxasterol has the in vitro anti-inflammatory activity by suppressing the production of various cytokines and inflammatory mediators in LPS-induced murine RAW 264.7 macrophages21, and the in vivo protective effect on chronic inflammatory lung disease asthma in mice22. However, no study thus far has addressed whether taraxasterol has the in vivo protective effect on endotoxic shock and what the underlying mechanisms. Therefore, as a part
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Xuemei Zhang1*, Huanzhang Xiong1*, Hongyu Li2, and Yao Cheng1
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Immunopharmacol Immunotoxicol, 2014; 36(1): 11–16
intraperitoneally with LPS at a lethal dose of 32 mg/kg as our previous study23, mice in control and taraxasterol alone groups were given normal saline. The survival of mice in each group was monitored for 7 days twice a day.
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Serum preparation
Figure 1. Chemical structure of taraxasterol.
of our on-going screening program to evaluate the anti-inflammatory potentials of natural compounds, we studied the effects of taraxasterol on survival in the experimental murine model of endotoxic shock induced by lethal LPS challenge and further clarified the mechanism involved.
Materials and methods Reagents Taraxasterol was obtained from Chengdu Fenruisi Biotechnology Co. (Chengdu, Sichuan, China), and its purity was 99.5% based on HPLC analysis. LPS (Escherichia coli 055:B5) and Griess reagent were purchased from Sigma Chemical Co. (St. Louis, MO). Mouse TNF-a, IFN-g, IL-1b, IL-6 and PGE2 ELISA kits were purchased from R&D Systems (Minneapolis, MN). Animals Male Kunming mice, weighing 20–22 g, were purchased from the Center of Experimental Animals of Medical College of Yanbian University (Yanji, Jilin, China). The mice were kept in microisolator cages and received food and water ad libitum. Before experimentation, the mice were allowed to adapt to the experimental environment for a minimum of 1 week. All animal experimental procedures were performed in accordance with the guidelines of the Ethical Committee for the Experimental Use of Animals at Yanbian University (Yanji, Jilin, China). Survival study To investigate the protective effect of taraxasterol on LPSinduced endotoxic shock, mice were randomly divided into six groups: control group, LPS group, taraxasterol alone group and taraxasterol (at doses of 2.5, 5 and 10 mg/kg, respectively) þ LPS groups. Mice from taraxasterol þ LPS groups were administered intragastrically with taraxasterol (2.5, 5 and 10 mg/kg) in 0.5% Sodium Tvlose once per day for 5 days consecutively. Mice from the control and LPS groups were administered intragastrically with 0.5% Sodium Tvlose. Mice from taraxasterol alone were administered intragastrically with taraxasterol at 10 mg/kg once per day for 5 days consecutively. One hour after taraxasterol or 0.5% Sodium Tvlose treatment on day 5, mice were injected
The mice were randomly divided into four groups: control group, LPS group, taraxasterol alone group and taraxasterol þ LPS group. The mice were administered intragastrically with 10 mg/kg taraxasterol or equal volume 0.5% Sodium Tvlose once per day for 5 days consecutively. One hour after taraxasterol or 0.5% Sodium Tvlose treatment on day 5, mice were given LPS (32 mg/kg) or normal saline by intraperitoneal injection. Blood samples were collected from the brachial plexus and sera were separated by centrifugation at 1, 3, 6 and 12 h following LPS administration. Sera were stored at 80 C until they were assayed for cytokines and mediators. Serum cytokine and mediator assays The concentrations of TNF-a, IFN-g, IL-1b, IL-6 and PGE2 in sera were measured by sandwich ELISA using commercially available reagents according to the manufacturer’s instructions. Briefly, microwell plates were coated overnight at 4 C with mouse TNF-a, IFN-g, IL-1b, IL-6 or PGE2 capture antibody and blocked at room temperature for 1 h with 1% BSA in phosphate-buffered saline with shaking. Samples from the sera of LPS-induced mice and internal standard were incubated at room temperature for 2 h with shaking, followed by mouse TNF-a, IFN-g, IL-1b, IL-6 or PGE2 biotinylated detection antibody for 1 h and an avidin horseradish peroxidase (Av-HRP) conjugate for 30 min. TMB substrate solution was added and incubated in the dark for 15 min. The reaction was stopped by addition of 1 M H2SO4 and absorbance was measured at 450 nm on a microplate reader (TECAN-GENious, Austria). The levels of TNF-a, IFN-g, IL-1b, IL-6 and PGE2 were expressed as picograms or nanograms per milliliter of sera based on the appropriate standard curve. The concentration of NO in sera was measured using Griess reagent. Briefly, the samples were mixed with equal volume of Griess reagent and incubated at room temperature for 15 min. The absorbance was measured at 540 nm on a microplate reader. Nitrite concentration was determined using a sodium nitrite serial dilution standard curve. Histopathologic examination of lung At 24 h after LPS challenge, survival mice were euthanized, the lungs were harvested and fixed in 10% buffered formalin for 24 h, dehydrated, embedded in paraffin and then stained with hematoxylin-eosin (H&E) and observed by light microscopy. Statistical analysis Survival data were analyzed with the Kaplan–Meier test. Other data were expressed as mean SEM. Differences between mean values of normally distributed data were assessed with one-way ANOVA (Dunnett’s t-test) and two-tailed
DOI: 10.3109/08923973.2013.861482
Protective effect of taraxasterol against LPS-induced endotoxic shock
Student’s t-test. Statistical significance was accepted at p50.05.
Effect of taraxasterol on serum inflammatory mediator NO and PGE2 production in LPS-induced mice
Results
NO and PGE2 levels in sera were determined with the Griess assay and ELISA kit. As shown in Figure 4, treatment with LPS alone resulted in significant increases in NO and PGE2 production as compared to that in control group (p50.05 or p50.01). However, taraxasterol markedly inhibited NO (Figure 4a) and PGE2 (Figure 4b) production at different time points after LPS injection as compared to the LPS group (p50.05 or p50.01). The serum levels for NO and PGE2 in taraxasterol alone group were near unchangeable as compared to that in control group.
Effect of taraxasterol on survival rate in mice with LPS-induced endotoxic shock
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The protective effect of different doses of taraxasterol against endotoxic shock was assessed by observing survival rate of mice induced with LPS. As shown in Figure 2, the administration of 32 mg/kg LPS alone resulted in a survival rate of 10% after 36 h in mice. In contrast, in the groups that mice received taraxasterol at doses of 2.5, 5 and 10 mg/kg, survival rates were up to 30%, 40% and 70% after 36 h, respectively. The survival rates (5 mg/kg and 10 mg/kg groups) were significantly increased compared to that of the group that only received LPS (p50.05 or p50.01). No toxic effects of taraxasterol were observed in mice only given taraxasterol and without LPS challenge even in the group received doses as high as 10 mg/kg of taraxasterol. Effect of taraxasterol on serum inflammatory cytokine TNF-a, IFN-g, IL-1b and IL-6 production in LPS-induced mice To define the effects of taraxasterol on cytokine responses in vivo, which associated with fatal outcome, we collected sera from mice at different time points after LPS injection, and determined cytokine concentrations with ELISA. As shown in Figure 3, treatment with LPS alone resulted in significant increases in serum cytokine production as compared to that in control group (p50.05 or p50.01). In contrast, treatment with taraxasterol consistently and significantly reduced serum TNF-a (Figure 3a), IFN-g (Figure 3b), IL-1b (Figure 3c) and IL-6 (Figure 3d) production at different time points as compared to the LPS group (p50.05 or p50.01). The serum levels for TNF-a, IFN-g, IL-1b and IL-6 in taraxasterol alone group were near unchangeable as compared to that in control group or close to baseline.
Effect of taraxasterol on lung histopathological change in LPS-induced mice As the lung is the primary target organ of endotoxic shock, the effect of taraxasterol on the lungs of mice was determined at 24 h after LPS challenge by histochemical staining with H&E. Pulmonary histology was normal in the control group (Figure 5a). The lungs of mice exposed to LPS showed significant pro-inflammatory alterations characterized by lung edema, alveolar hemorrhage, inflammatory cell influx and destruction of epithelial and endothelial cell structure (Figure 5c). In contrast, treatment with taraxasterol remarkably ameliorated the above histological changes (Figure 5d). There was no abnormal histological change in taraxasterol alone group (Figure 5b).
Discussion Sepsis and endotoxic shock can be caused by Gram-positive and -negative bacteria and other microorganisms. In the case of Gram-negative bacteria, endotoxin, a normal constituent of the bacterial wall, also known as LPS, has been considered as one of the principal agents causing the undesirable effects in this critical illness. Experimental models of endotoxic shock have demonstrated that a single injection of LPS into animals can produce pathological changes that are characteristic of the septic shock syndrome24. So LPS-induced murine
Figure 2. Effect of different doses of taraxasterol (Tara) on survival rate of mice induced with LPS. Mice were divided into control, LPS, taraxasterol alone and Tara þ LPS groups (n ¼ 10 for each group). Mice were administered intragastrically with 2.5, 5 and 10 mg/kg of Tara or 0.5% Sodium Tvlose prior to intraperitoneal injection of LPS or normal saline as described in the section titled ‘‘Materials and methods’’. The survival rate was assessed every 12 h for 7 days throughout the experiment. ##p50.01 versus control group; *p50.05, **p50.01 versus LPS group.
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Figure 3. Effect of taraxasterol (Tara) on serum TNF-a, IFN-g, IL-1b and IL-6 induced by LPS. Mice were divided into control, LPS, taraxasterol alone and Tara þ LPS groups (n ¼ 20 for each group). Mice were administered intragastrically with 10 mg/kg Tara or 0.5% Sodium Tvlose prior to intraperitoneal injection of LPS or normal saline as described in the section titled ‘‘Materials and methods’’. Serum concentrations of TNF-a, IFN-g, IL-1b and IL-6 in each group were measured at 1, 3, 6 and 12 h following LPS challenge. Data represent means SEMs. #p50.05, ##p50.01 versus control group; *p50.05, **p50.01 versus LPS group.
Figure 4. Effect of taraxasterol (Tara) on serum NO and PGE2 induced by LPS. Mice were divided into control, LPS, taraxasterol alone and Tara þ LPS groups (n ¼ 20 for each group). Mice were administered intragastrically with 10 mg/kg Tara or 0.5% Sodium Tvlose prior to intraperitoneal injection of LPS or normal saline as described in the section titled ‘‘Materials and methods’’. Serum concentrations of NO and PGE2 in each group were measured at 1, 3, 6 and 12 h following LPS challenge. Data represent means SEMs. #p50.05, ##p50.01 versus control group; *p50.05, **p50.01 versus LPS group.
endotoxic shock model is widely used in the search for drugs and treatment tools for sepsis caused by Gram-negative bacterial infections in vivo. In the present study, we used a murine endotoxic shock model and studied the effect of
taraxasterol on survival of mice induced with a lethal dose of LPS and the mechanism involved. Taraxasterol improved the survival rate of mice and attenuated tissue injury of the lungs of endotoxic shock. Furthermore, taraxasterol significantly decreased serum inflammatory cytokine and mediator levels from mice with endotoxic shock, indicating that inflammatory cytokine and mediator regulation by taraxasterol could be useful as part of a novel strategy prevention of endotoxic shock. It is well established that LPS binds to mammalian cell membrane molecules, such as CD14 and Toll-like receptors (TLR). LPS is recognized and interacted with TLR4 on cell membrane, and leads to progressive production of host pro-inflammatory cytokines and mediators including TNF-a, IL-1b, IL-6, IFN, PGE2 and NO through TLR4 signaling pathway25. Septic shock is induced through a complex cascade triggered by these inflammatory cytokines and mediators, and higher mortality correlates with higher serum levels of these cytokines and mediators26. So inhibition of the pro-inflammatory cytokines and mediators is regarded as effective methods to manage septic shock27. Especially, there is an increasing interest in compounds isolated from herbal medications. Additionally to their pleiotropic immunomodulatory properties, they are nontoxic and pharmacologically safe28. In order to explore the mechanisms underlying the protective action of taraxasterol against endotoxic shock, we further investigated the regulatory effects of taraxasterol on production of serum proinflammatory cytokines and mediators in LPS-induced murine endotoxic shock. Early cytokine responses after LPS challenge have been well characterized and are known to occur within hours of LPS challenge in vivo. Specifically, TNF-a, IFN-g, IL-1b and IL-6 have been shown to be produced early in the response and suggested to play critical roles in driving physiological/
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DOI: 10.3109/08923973.2013.861482
Protective effect of taraxasterol against LPS-induced endotoxic shock
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Figure 5. Effect of taraxasterol on lung histopathological change in mice with LPS-induced endotoxic shock (400, H&E staining). Mice were divided into control, LPS, taraxasterol alone and taraxasterol þ LPS groups. Mice were administered intragastrically with 10 mg/kg taraxasterol or 0.5% Sodium Tvlose prior to intraperitoneal injection of LPS or normal saline as described in the section titled ‘‘Materials and methods’’. Lungs (n ¼ 3) were processed for histological changes at 24 h following LPS challenge. (a) Control group, (b) Taraxasterol alone group, (c) LPS group, (d) Taraxasterol þ LPS group.
pathological responses that lead to septic shock29,30. TNF-a has been identified as the predominant cytokine that mediates LPS-induced lethality31. It mediates early-stage responses of inflammation by inducing the release of other cytokines and mediators, including IL-1, IL-6 and NO. NO can promote TNF-a production in turn. The data demonstrated the significance of TNF-a in the pathological damage of inflammation and septic shock32, and its abnormally high serum levels are often correlated with poor prognosis33. IFN-g and TNF-a have relevant roles in the lethality of experimental models of endotoxic shock. Increased levels of IFN-g were associated to systemic release of TNF-a. IFN-g could contribute to the extent of LPS-induced macrophage activation. This activation is accompanied by an increased systemic release of TNF-a, which is ultimately responsible for the endotoxic shock34. Likewise, IL-1b is one of the most important inflammatory cytokines induced by LPS. During inflammation, increases in the release of IL-1b lead to cell or tissue damage35,36. IL-6 is also pivotal pro-inflammatory cytokine, regarded as an endogenous mediator of LPSinduced fever37. Recently, our study has demonstrated that taraxasterol could inhibit TNF-a, IL-1b and IL-6 production in LPS-induced RAW 264.7 macrophages in vitro21. In the present study, the results demonstrate that pretreatment with taraxasterol significantly inhibited serum TNF-a,
IFN-g, IL-1b and IL-6 production in LPS-induced endotoxemic mice, and it is consistent with our previous study in vitro. It indicates that the protective effect of taraxasterol on endotoxic shock mice may be attributable, at least in part, to inhibition of pro-inflammatory cytokine production. A variety of studies have shown that the pleiotropic proinflammatory mediators NO and PGE2 are involved in the pathogenesis of inflammation and infection, and play critical roles in inflammatory diseases38–40. NO is produced from L -arginine by nitric oxide synthases (NOSs) and involved in various pathophysiological processes including inflammation and septic shock41, its uncontrolled release can cause inflammatory destruction of target tissue during an infection42. PGE2 is generated by the sequential metabolism of arachidonic acid by cyclooxygenases (COXs)43. COXs and PGE2 are still attractive drug targets for the relief of pain and inflammation44. Therefore, a compound capable of preventing the release of NO and PGE2 could potentially possess anti-inflammatory activities. Recently, our study has demonstrated that taraxasterol could inhibit NO and PGE2 production in LPS-induced RAW 264.7 macrophages in vitro21. In this study, we found taraxasterol inhibited the production of serum NO and PGE2 in LPS-induced endotoxemic mice, it is also consistent with our previous result in vitro. It indicates that the protective effect of
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taraxasterol on endotoxic shock mice may be attributable, at least in part, to inhibition of pro-inflammatory mediator production. In conclusion, the present study showed that pretreatment with taraxasterol isolated from Taraxacum officinale could improve survival and attenuate lung injury of mice in endotoxic shock by suppressing the production of serum TNF-a, IFN-g, IL-1b, IL-6, NO and PGE2. It suggests that taraxasterol may be a potential protective agent against endotoxic shock.
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19. 20. 21. 22.
Declaration of interest
23.
This work was supported by a grant from the National Natural Science Foundation of China (No. 31060349). The authors declare no conflict of interest.
24.
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RESEARCH ARTICLE
Protective effect of taraxasterol against LPS-induced endotoxic shock by modulating inflammatory responses in mice 1
Department of Animal Medicine, Agricultural College of Yanbian University, Yanji, Jilin, PR China and 2Experimental Base of Agriculture, Jilin University, Changchun, Jilin, PR China Abstract
Keywords
Taraxasterol, a pentacyclic-triterpene, was isolated from the Chinese medicinal herb Taraxacum officinale. In the present study, we investigated the protective effect of taraxasterol on murine model of endotoxic shock and the mechanism of its action. Mice were treated with 2.5, 5 and 10 mg/kg of taraxasterol prior to a lethal dose of lipopolysaccharide (LPS) challenge. Survival of mice was monitored twice a day for 7 days. To further understand the mechanism, the serum levels of inflammatory cytokine tumor necrosis factor-a (TNF-a), interferon-g (IFN-g), interleukin1b (IL-1b), interleukin-6 (IL-6) and mediator nitric oxide (NO), prostaglandin E2 (PGE2) as well as histology of lungs were examined. The results showed that taraxasterol significantly improved mouse survival and attenuated tissue injury of the lungs in LPS-induced endotoxemic mice. Further studies revealed that taraxasterol significantly reduced TNF-a, IFN-g, IL-1b, IL-6, NO and PGE2 levels in sera from mice with endotoxic shock. These results indicate that taraxasterol has a protective effect on murine endotoxic shock induced by LPS through modulating inflammatory cytokine and mediator secretion. This finding might provide a new strategy for the treatment of endotoxic shock and associated inflammation.
Endotoxic shock, inflammatory responses, LPS, survival, taraxasterol
Introduction Endotoxic shock, which results from Gram-negative bacterial infections, is a major cause of death among patients and animals1,2. One of the major reasons is that endotoxin or lipopolysaccharide (LPS), a component of the Gram-negative bacterial cell wall, induces the disturbance of immune and inflammatory responses, causes extensive tissue damage and is considered to play a central role in mediating diseases caused by Gram-negative bacteria3–5. The inflammatory response after challenge with LPS is associated with the release of proinflammatory cytokines and other inflammatory mediators, including tumor necrosis factor-a (TNF-a), interleukin-1 (IL1), interleukin-6 (IL-6), interferon-g (IFN-g), nitric oxide (NO) and prostaglandin E2 (PGE2). The progressive production of these inflammatory cytokines and mediators may result in the systemic inflammatory response syndrome, severe tissue damage, septic shock or even death6–8. In spite of rapid progress in developing antibiotics and other therapeutic methods in clinical practice, sepsis remains a leading cause of morbidity and mortality in the intensive care unit9,10. Thus, there is an urgent need to develop new therapeutic tools for the treatment of septic shock.
*These authors contributed equally to this work. Address for correspondence: Xuemei Zhang, Department of Animal Medicine, Agricultural College of Yanbian University, Gongyuan Street, Yanji, Jilin 133002, PR China. E-mail: [email protected]
History Received 30 August 2013 Accepted 29 October 2013 Published online 29 November 2013
Taraxacum officinale has long been used in traditional oriental medicine for its lactating, choleretic, diuretic, antirheumatic and anti-inflammatory properties11,12. It is widely used for treating various inflammatory or infectious diseases clinically such as hepatitis, upper respiratory tract infections, bronchitis, pneumonia and as a compress for its anti-mastopathy activity13,14. Pharmacological activities of Taraxacum officinale have been in part evaluated so far15,16. Recently, Taraxacum officinale extracts have been shown to inhibit LPS-induced inflammatory responses by reducing NO, PGE2 and pro-inflammatory cytokines production via inactivation of nuclear factor-B (NF-B) and mitogen-activated protein kinase signal pathway in RAW 264.7 cells17,18. Moreover, the aqueous extract of Taraxacum officinale was assessed to contain acute anti-inflammatory activity by showing its protective effects against cholecystokinin-induced acute pancreatitis in rats19, and we also reported its protective effects against LPS-induced acute lung injury in mice20. Taraxasterol (Figure 1) isolated from Taraxacum officinale is one of its main active constituents. Recently, our study has shown that taraxasterol has the in vitro anti-inflammatory activity by suppressing the production of various cytokines and inflammatory mediators in LPS-induced murine RAW 264.7 macrophages21, and the in vivo protective effect on chronic inflammatory lung disease asthma in mice22. However, no study thus far has addressed whether taraxasterol has the in vivo protective effect on endotoxic shock and what the underlying mechanisms. Therefore, as a part
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Xuemei Zhang1*, Huanzhang Xiong1*, Hongyu Li2, and Yao Cheng1
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Immunopharmacol Immunotoxicol, 2014; 36(1): 11–16
intraperitoneally with LPS at a lethal dose of 32 mg/kg as our previous study23, mice in control and taraxasterol alone groups were given normal saline. The survival of mice in each group was monitored for 7 days twice a day.
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Serum preparation
Figure 1. Chemical structure of taraxasterol.
of our on-going screening program to evaluate the anti-inflammatory potentials of natural compounds, we studied the effects of taraxasterol on survival in the experimental murine model of endotoxic shock induced by lethal LPS challenge and further clarified the mechanism involved.
Materials and methods Reagents Taraxasterol was obtained from Chengdu Fenruisi Biotechnology Co. (Chengdu, Sichuan, China), and its purity was 99.5% based on HPLC analysis. LPS (Escherichia coli 055:B5) and Griess reagent were purchased from Sigma Chemical Co. (St. Louis, MO). Mouse TNF-a, IFN-g, IL-1b, IL-6 and PGE2 ELISA kits were purchased from R&D Systems (Minneapolis, MN). Animals Male Kunming mice, weighing 20–22 g, were purchased from the Center of Experimental Animals of Medical College of Yanbian University (Yanji, Jilin, China). The mice were kept in microisolator cages and received food and water ad libitum. Before experimentation, the mice were allowed to adapt to the experimental environment for a minimum of 1 week. All animal experimental procedures were performed in accordance with the guidelines of the Ethical Committee for the Experimental Use of Animals at Yanbian University (Yanji, Jilin, China). Survival study To investigate the protective effect of taraxasterol on LPSinduced endotoxic shock, mice were randomly divided into six groups: control group, LPS group, taraxasterol alone group and taraxasterol (at doses of 2.5, 5 and 10 mg/kg, respectively) þ LPS groups. Mice from taraxasterol þ LPS groups were administered intragastrically with taraxasterol (2.5, 5 and 10 mg/kg) in 0.5% Sodium Tvlose once per day for 5 days consecutively. Mice from the control and LPS groups were administered intragastrically with 0.5% Sodium Tvlose. Mice from taraxasterol alone were administered intragastrically with taraxasterol at 10 mg/kg once per day for 5 days consecutively. One hour after taraxasterol or 0.5% Sodium Tvlose treatment on day 5, mice were injected
The mice were randomly divided into four groups: control group, LPS group, taraxasterol alone group and taraxasterol þ LPS group. The mice were administered intragastrically with 10 mg/kg taraxasterol or equal volume 0.5% Sodium Tvlose once per day for 5 days consecutively. One hour after taraxasterol or 0.5% Sodium Tvlose treatment on day 5, mice were given LPS (32 mg/kg) or normal saline by intraperitoneal injection. Blood samples were collected from the brachial plexus and sera were separated by centrifugation at 1, 3, 6 and 12 h following LPS administration. Sera were stored at 80 C until they were assayed for cytokines and mediators. Serum cytokine and mediator assays The concentrations of TNF-a, IFN-g, IL-1b, IL-6 and PGE2 in sera were measured by sandwich ELISA using commercially available reagents according to the manufacturer’s instructions. Briefly, microwell plates were coated overnight at 4 C with mouse TNF-a, IFN-g, IL-1b, IL-6 or PGE2 capture antibody and blocked at room temperature for 1 h with 1% BSA in phosphate-buffered saline with shaking. Samples from the sera of LPS-induced mice and internal standard were incubated at room temperature for 2 h with shaking, followed by mouse TNF-a, IFN-g, IL-1b, IL-6 or PGE2 biotinylated detection antibody for 1 h and an avidin horseradish peroxidase (Av-HRP) conjugate for 30 min. TMB substrate solution was added and incubated in the dark for 15 min. The reaction was stopped by addition of 1 M H2SO4 and absorbance was measured at 450 nm on a microplate reader (TECAN-GENious, Austria). The levels of TNF-a, IFN-g, IL-1b, IL-6 and PGE2 were expressed as picograms or nanograms per milliliter of sera based on the appropriate standard curve. The concentration of NO in sera was measured using Griess reagent. Briefly, the samples were mixed with equal volume of Griess reagent and incubated at room temperature for 15 min. The absorbance was measured at 540 nm on a microplate reader. Nitrite concentration was determined using a sodium nitrite serial dilution standard curve. Histopathologic examination of lung At 24 h after LPS challenge, survival mice were euthanized, the lungs were harvested and fixed in 10% buffered formalin for 24 h, dehydrated, embedded in paraffin and then stained with hematoxylin-eosin (H&E) and observed by light microscopy. Statistical analysis Survival data were analyzed with the Kaplan–Meier test. Other data were expressed as mean SEM. Differences between mean values of normally distributed data were assessed with one-way ANOVA (Dunnett’s t-test) and two-tailed
DOI: 10.3109/08923973.2013.861482
Protective effect of taraxasterol against LPS-induced endotoxic shock
Student’s t-test. Statistical significance was accepted at p50.05.
Effect of taraxasterol on serum inflammatory mediator NO and PGE2 production in LPS-induced mice
Results
NO and PGE2 levels in sera were determined with the Griess assay and ELISA kit. As shown in Figure 4, treatment with LPS alone resulted in significant increases in NO and PGE2 production as compared to that in control group (p50.05 or p50.01). However, taraxasterol markedly inhibited NO (Figure 4a) and PGE2 (Figure 4b) production at different time points after LPS injection as compared to the LPS group (p50.05 or p50.01). The serum levels for NO and PGE2 in taraxasterol alone group were near unchangeable as compared to that in control group.
Effect of taraxasterol on survival rate in mice with LPS-induced endotoxic shock
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The protective effect of different doses of taraxasterol against endotoxic shock was assessed by observing survival rate of mice induced with LPS. As shown in Figure 2, the administration of 32 mg/kg LPS alone resulted in a survival rate of 10% after 36 h in mice. In contrast, in the groups that mice received taraxasterol at doses of 2.5, 5 and 10 mg/kg, survival rates were up to 30%, 40% and 70% after 36 h, respectively. The survival rates (5 mg/kg and 10 mg/kg groups) were significantly increased compared to that of the group that only received LPS (p50.05 or p50.01). No toxic effects of taraxasterol were observed in mice only given taraxasterol and without LPS challenge even in the group received doses as high as 10 mg/kg of taraxasterol. Effect of taraxasterol on serum inflammatory cytokine TNF-a, IFN-g, IL-1b and IL-6 production in LPS-induced mice To define the effects of taraxasterol on cytokine responses in vivo, which associated with fatal outcome, we collected sera from mice at different time points after LPS injection, and determined cytokine concentrations with ELISA. As shown in Figure 3, treatment with LPS alone resulted in significant increases in serum cytokine production as compared to that in control group (p50.05 or p50.01). In contrast, treatment with taraxasterol consistently and significantly reduced serum TNF-a (Figure 3a), IFN-g (Figure 3b), IL-1b (Figure 3c) and IL-6 (Figure 3d) production at different time points as compared to the LPS group (p50.05 or p50.01). The serum levels for TNF-a, IFN-g, IL-1b and IL-6 in taraxasterol alone group were near unchangeable as compared to that in control group or close to baseline.
Effect of taraxasterol on lung histopathological change in LPS-induced mice As the lung is the primary target organ of endotoxic shock, the effect of taraxasterol on the lungs of mice was determined at 24 h after LPS challenge by histochemical staining with H&E. Pulmonary histology was normal in the control group (Figure 5a). The lungs of mice exposed to LPS showed significant pro-inflammatory alterations characterized by lung edema, alveolar hemorrhage, inflammatory cell influx and destruction of epithelial and endothelial cell structure (Figure 5c). In contrast, treatment with taraxasterol remarkably ameliorated the above histological changes (Figure 5d). There was no abnormal histological change in taraxasterol alone group (Figure 5b).
Discussion Sepsis and endotoxic shock can be caused by Gram-positive and -negative bacteria and other microorganisms. In the case of Gram-negative bacteria, endotoxin, a normal constituent of the bacterial wall, also known as LPS, has been considered as one of the principal agents causing the undesirable effects in this critical illness. Experimental models of endotoxic shock have demonstrated that a single injection of LPS into animals can produce pathological changes that are characteristic of the septic shock syndrome24. So LPS-induced murine
Figure 2. Effect of different doses of taraxasterol (Tara) on survival rate of mice induced with LPS. Mice were divided into control, LPS, taraxasterol alone and Tara þ LPS groups (n ¼ 10 for each group). Mice were administered intragastrically with 2.5, 5 and 10 mg/kg of Tara or 0.5% Sodium Tvlose prior to intraperitoneal injection of LPS or normal saline as described in the section titled ‘‘Materials and methods’’. The survival rate was assessed every 12 h for 7 days throughout the experiment. ##p50.01 versus control group; *p50.05, **p50.01 versus LPS group.
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Figure 3. Effect of taraxasterol (Tara) on serum TNF-a, IFN-g, IL-1b and IL-6 induced by LPS. Mice were divided into control, LPS, taraxasterol alone and Tara þ LPS groups (n ¼ 20 for each group). Mice were administered intragastrically with 10 mg/kg Tara or 0.5% Sodium Tvlose prior to intraperitoneal injection of LPS or normal saline as described in the section titled ‘‘Materials and methods’’. Serum concentrations of TNF-a, IFN-g, IL-1b and IL-6 in each group were measured at 1, 3, 6 and 12 h following LPS challenge. Data represent means SEMs. #p50.05, ##p50.01 versus control group; *p50.05, **p50.01 versus LPS group.
Figure 4. Effect of taraxasterol (Tara) on serum NO and PGE2 induced by LPS. Mice were divided into control, LPS, taraxasterol alone and Tara þ LPS groups (n ¼ 20 for each group). Mice were administered intragastrically with 10 mg/kg Tara or 0.5% Sodium Tvlose prior to intraperitoneal injection of LPS or normal saline as described in the section titled ‘‘Materials and methods’’. Serum concentrations of NO and PGE2 in each group were measured at 1, 3, 6 and 12 h following LPS challenge. Data represent means SEMs. #p50.05, ##p50.01 versus control group; *p50.05, **p50.01 versus LPS group.
endotoxic shock model is widely used in the search for drugs and treatment tools for sepsis caused by Gram-negative bacterial infections in vivo. In the present study, we used a murine endotoxic shock model and studied the effect of
taraxasterol on survival of mice induced with a lethal dose of LPS and the mechanism involved. Taraxasterol improved the survival rate of mice and attenuated tissue injury of the lungs of endotoxic shock. Furthermore, taraxasterol significantly decreased serum inflammatory cytokine and mediator levels from mice with endotoxic shock, indicating that inflammatory cytokine and mediator regulation by taraxasterol could be useful as part of a novel strategy prevention of endotoxic shock. It is well established that LPS binds to mammalian cell membrane molecules, such as CD14 and Toll-like receptors (TLR). LPS is recognized and interacted with TLR4 on cell membrane, and leads to progressive production of host pro-inflammatory cytokines and mediators including TNF-a, IL-1b, IL-6, IFN, PGE2 and NO through TLR4 signaling pathway25. Septic shock is induced through a complex cascade triggered by these inflammatory cytokines and mediators, and higher mortality correlates with higher serum levels of these cytokines and mediators26. So inhibition of the pro-inflammatory cytokines and mediators is regarded as effective methods to manage septic shock27. Especially, there is an increasing interest in compounds isolated from herbal medications. Additionally to their pleiotropic immunomodulatory properties, they are nontoxic and pharmacologically safe28. In order to explore the mechanisms underlying the protective action of taraxasterol against endotoxic shock, we further investigated the regulatory effects of taraxasterol on production of serum proinflammatory cytokines and mediators in LPS-induced murine endotoxic shock. Early cytokine responses after LPS challenge have been well characterized and are known to occur within hours of LPS challenge in vivo. Specifically, TNF-a, IFN-g, IL-1b and IL-6 have been shown to be produced early in the response and suggested to play critical roles in driving physiological/
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DOI: 10.3109/08923973.2013.861482
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Figure 5. Effect of taraxasterol on lung histopathological change in mice with LPS-induced endotoxic shock (400, H&E staining). Mice were divided into control, LPS, taraxasterol alone and taraxasterol þ LPS groups. Mice were administered intragastrically with 10 mg/kg taraxasterol or 0.5% Sodium Tvlose prior to intraperitoneal injection of LPS or normal saline as described in the section titled ‘‘Materials and methods’’. Lungs (n ¼ 3) were processed for histological changes at 24 h following LPS challenge. (a) Control group, (b) Taraxasterol alone group, (c) LPS group, (d) Taraxasterol þ LPS group.
pathological responses that lead to septic shock29,30. TNF-a has been identified as the predominant cytokine that mediates LPS-induced lethality31. It mediates early-stage responses of inflammation by inducing the release of other cytokines and mediators, including IL-1, IL-6 and NO. NO can promote TNF-a production in turn. The data demonstrated the significance of TNF-a in the pathological damage of inflammation and septic shock32, and its abnormally high serum levels are often correlated with poor prognosis33. IFN-g and TNF-a have relevant roles in the lethality of experimental models of endotoxic shock. Increased levels of IFN-g were associated to systemic release of TNF-a. IFN-g could contribute to the extent of LPS-induced macrophage activation. This activation is accompanied by an increased systemic release of TNF-a, which is ultimately responsible for the endotoxic shock34. Likewise, IL-1b is one of the most important inflammatory cytokines induced by LPS. During inflammation, increases in the release of IL-1b lead to cell or tissue damage35,36. IL-6 is also pivotal pro-inflammatory cytokine, regarded as an endogenous mediator of LPSinduced fever37. Recently, our study has demonstrated that taraxasterol could inhibit TNF-a, IL-1b and IL-6 production in LPS-induced RAW 264.7 macrophages in vitro21. In the present study, the results demonstrate that pretreatment with taraxasterol significantly inhibited serum TNF-a,
IFN-g, IL-1b and IL-6 production in LPS-induced endotoxemic mice, and it is consistent with our previous study in vitro. It indicates that the protective effect of taraxasterol on endotoxic shock mice may be attributable, at least in part, to inhibition of pro-inflammatory cytokine production. A variety of studies have shown that the pleiotropic proinflammatory mediators NO and PGE2 are involved in the pathogenesis of inflammation and infection, and play critical roles in inflammatory diseases38–40. NO is produced from L -arginine by nitric oxide synthases (NOSs) and involved in various pathophysiological processes including inflammation and septic shock41, its uncontrolled release can cause inflammatory destruction of target tissue during an infection42. PGE2 is generated by the sequential metabolism of arachidonic acid by cyclooxygenases (COXs)43. COXs and PGE2 are still attractive drug targets for the relief of pain and inflammation44. Therefore, a compound capable of preventing the release of NO and PGE2 could potentially possess anti-inflammatory activities. Recently, our study has demonstrated that taraxasterol could inhibit NO and PGE2 production in LPS-induced RAW 264.7 macrophages in vitro21. In this study, we found taraxasterol inhibited the production of serum NO and PGE2 in LPS-induced endotoxemic mice, it is also consistent with our previous result in vitro. It indicates that the protective effect of
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taraxasterol on endotoxic shock mice may be attributable, at least in part, to inhibition of pro-inflammatory mediator production. In conclusion, the present study showed that pretreatment with taraxasterol isolated from Taraxacum officinale could improve survival and attenuate lung injury of mice in endotoxic shock by suppressing the production of serum TNF-a, IFN-g, IL-1b, IL-6, NO and PGE2. It suggests that taraxasterol may be a potential protective agent against endotoxic shock.
Immunopharmacol Immunotoxicol, 2014; 36(1): 11–16
19. 20. 21. 22.
Declaration of interest
23.
This work was supported by a grant from the National Natural Science Foundation of China (No. 31060349). The authors declare no conflict of interest.
24.
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