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The effects of porcine pulmonary surfactant on smoke inhalation injury Yu Sun, MD,a,1 Xiaochen Qiu, MS,b,1 Guosheng Wu, MD,a,1 Junjie Wang, MS,a Jiahui Li, MS,a Hao Tang, MD,c and Zhaofan Xia, MDa,* a
Department of Burn Surgery, Changhai Hospital, Second Military Medical University, Shanghai, P. R. China Department of General Surgery, 309th Hospital of PLA, Beijing, P.R. China c Department of Respiratory Medicine, Changzheng Hospital, Second Military Medical University, Shanghai, P. R. China b
article info
abstract
Article history:
Background: Our previous study, consistent with others, demonstrated that administering
Received 11 February 2015
an exogenous surfactant was a potential therapy for acute lung injury and acute respira-
Received in revised form
tory distress syndrome. However, the underlying mechanisms remain largely unknown. In
4 May 2015
the present study, we investigated the effect of instilled porcine pulmonary surfactant
Accepted 12 May 2015
(PPS) on rat inhalation injury model induced by smoke and the possible mechanism.
Available online xxx
Materials and methods: Fifteen SpragueeDawley rats were equally randomized to three groups as follows (n ¼ 5 in each group): sham control group (C group), inhalation injury
Keywords:
group (II group), and inhalation injury þ PPS treatment group (PPS group). Lung tissues
Inhalation injury
were assayed for wet/dry ratio, histologic, terminal dUTP nick-end labeling staining, and
Porcine pulmonary surfactant
Western blotting examinations. The myeloperoxidase activity was tested in lung tissues as
Surfactant protein A
well. Bronchoalveolar lavage fluid was collected to determine the total protein concen-
MPO activity
trations, inflammatory cytokines, surfactant protein A (SP-A), and SP-D.
Interleukin 8
Results: Our present work exhibited that PPS had therapeutic effects on smoke inhalation injury reflected by significant increase of PaO2 values, improved edema status, decreased vascular permeability, amelioration of lung histopathology, and reduction of inflammatory response. In addition, PPS treatment could increase endogenous SP-A levels both in lung tissue and bronchoalveolar lavage fluid. Further correlation analysis showed that SP-A was negatively correlated with both myeloperoxidase activity and interleukin 8 levels. Conclusions: These results indicate that PPS can attenuate smoke-induced inhalation injury at least partly through stimulating production of endogenous SP-A and inhibiting the release of proinflammatory cytokines such as interleukin 8. The increasing production of endogenous SP-A may be due to the antioxidant effect of PPS, which contains no SP-A. ª 2015 Elsevier Inc. All rights reserved.
1.
Background
Smoke inhalation injury is common in burn victims and significantly contributes to the morbidity and mortality of
burn injuries [1,2]. About 22% of burns combine with smoke inhalation injury, which increases burn-related mortality by 20% [3]. Previous studies have described that smoke inhalation injury is linked with an inflammatory response of the lung
* Corresponding author. Department of Burn Surgery, Changhai Hospital, Second Military Medical University, 168 Changhai Road, Shanghai, P. R. China. Tel.: þ86 021 31161821; fax: þ86 021 65589829. E-mail address: [email protected] (Z. Xia). 1 These authors contributed equally to this work. 0022-4804/$ e see front matter ª 2015 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.jss.2015.05.019
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[2,4]. Over the past years, major advances have been made in the treatment of burns, but the advances in the medication treatment of smoke inhalation injury were scarce [5e8]. Pulmonary surfactant (PS) is the product of type II alveolar cells and mainly composed of phospholipids and surfactantspecific proteins [9]. It is the phospholipids that are responsible for the critical reduction in surface tension, the main function of PS [10], whereas the surfactant proteins (SP) mainly participate in regulating host immune defense and modulating inflammatory responses [11]. There are four SPs that have been identified, including SP-A, SP-B, SP-C, and SPD. SP-B and SP-C are hydrophobic and interact with surfactant lipids to help reduce surface tension, whereas hydrophilic SP-A and SP-D are members of the collectin family involved in pulmonary immunity [12]. Previous studies have showed that expression of SP-A was downregulated in lung tissue after smoke-induced acute and chronic lung injury [13,14]. Surfactants are routinely used to treat preterm infants with neonatal respiratory distress syndrome and also used in the treatment of infants and adults with acute lung injury (ALI) and/or acute respiratory distress syndrome (ARDS). Our previous experiments have showed obvious therapeutic effects of PS on adult rabbits with ARDS [15] and adult rats with ALI induced by lipopolysaccharides or oleic acid [16,17]. Treatment with PS was found to increase PaO2, decrease breath rate, raise survival rate, and improve histologic appearance of the lung. Xie [18] and Chi [19] et al. have also reported PS is effective in alleviating smoking-induced lung injury and smoke inhalation injury. However, the underlying mechanisms remain largely unknown. Therefore, we built a rat model of smoke inhalation injury to investigate its therapeutic effect and the potential mechanism.
2.
Materials and methods
2.1.
Surfactant preparation
Porcine pulmonary surfactant (PPS) was isolated from pig bronchoalveolar lavage fluid (BALF) using a protocol modified from the one used by Enhorning et al. [20]. Briefly, PPS was extracted from BALF using sequential centrifugation, chloroform-methanol extraction, and acetone precipitation, which contains more than 90% phospholipids and about 1% hydrophobic protein, mainly SP-B and -C, but no hydrophilic proteins (SP-A and SP-D). It has been approved for use in clinical trials of neonatal respiratory distress syndrome treatments by the State Food and Drug Administration of China [21].
2.2.
Animals and reagents
Studies were performed on male SpragueeDawley rats (aged 8e10 wk, weighing 180e200 g, Experimental Animal Center of the Second Military Medical University, Shanghai, China). They were housed in individual cages in a temperaturecontrolled room with a 12 h lightedark cycle and free access to food and water. All animal experiments were approved by the Second Military Medical University in accordance with the
Guide for Care and Use of Laboratory Animals published by the US NIH (publication no. 96-01). Total protein extraction kit was purchased from Cell Signaling Technology (Beverly, MA). Enzyme-linked immunosorbent assay (ELISA) kits of interleukin 8 (IL-8), tumor necrosis factor alpha (TNF-a), SP-A, and SP-D were obtained from R&D Corporation (Minneapolis, MN). The following primary antibodies were used: antieSP-A, antieSP-D, and antieb-actin (SigmaeAldrich, St. Louis, MO). The anti-goat or anti-mouse secondary antibody was provided by the Sigma Chemical Company. The enhanced chemiluminescence (ECL) western blotting substract and the bicinchoninic acid protein assay were purchased from Thermo Fisher Scientific Inc (Rockford, IL). Polyvinylidene fluoride membranes were provided by Millipore (Bedford, MA).
2.3.
Smoke inhalation injury model
A rat model of smoke inhalation injury has been established as previously described [22]. Smoke was produced by a selfmade smoke generator. In our present experiment, five rats were exposed each time. Smoke was generated by slowly smoldering wood shavings (120 g/kg body weight). The data presented in this work are derived from rats exposed to successive 9-min periods of smoke, which were separated into three times by 30-s exposures to ambient air.
2.4.
Experiment design
A total of 15 rats were equally randomized to three groups (n ¼ 5 in each group) as follows: sham control group (C group, ambient air inhalation), inhalation injury group (II group), and inhalation injury þ PPS treatment group (PS group). Specifically, the first 5 smoke-exposing rats were mixed with the second 5 rats and then went on to randomize to the II group and PS group in a blinded fashion. The dosage and optimal administration time in our present work were chosen on the basis of our previous experiments [16,17,21,23,24]. Thirty minutes after exposing, 150 mg/kg of PPS (the concentration was 50 mg/mL) and the same volume of normal saline were instilled intratracheally the in PS and II groups, respectively. PPS or saline was intratracheally instilled into the lungs by the following procedure: a suction catheter was advanced down the trachea to a point near the carina. The suction catheter was modified by occluding the main lumen and cutting multiple small holes in the catheter tip so that PPS or saline was delivered in numerous directions. PPS or saline was administered to the animal’s lungs while the animal’s body was rotated. Two-milliliter syringes connected to the suction catheter were used to administer PPS or saline first, followed by administration of air. The rats were sacrificed 12 h later, and then airways were dissected 0.3 cm away from tracheal bifurcation to collect the lung.
2.5.
Arterial blood analysis
A total of 0.2-mL arterial blood was obtained from the carotid artery, and PaO2, PaCO2, and pH were measured at 0, 2, 4, 6, and 12 h after smoke inhalation using an automatic analyzer (ABL90 FLEX analyzer, Radiometer, Denmark).
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2.6.
Lung wet/dry ratio
The right upper lobe was weighed soon after excision and then dried in an oven at 60 C for 72 h. The lung tissue wet-todry (W/D) weight ratio was calculated to assess lung tissue edema.
2.7.
BALF analysis
The hilum of the right lung was ligated to perform alveolar lavage of the left lung by instilling 3-mL 4 C potassium phosphate-buffered solution and withdrawing 30 s later. This procedure was repeated three times, and the BALF was collected and centrifuged (4 C, 1000 r/min, 10 min). The total protein concentration of BALF was determined using Lowry’s method.
2.8.
Morphologic evaluation
Postmortem, the right lower lobe of lung, was fixed by 15% formaldehyde and embedded in paraffin to cut into 5-mm sections. The sections were stained with hematoxylin and eosin, and visualized using an inverted microscope (Olympus, Japan). The lung sections were stained with hematoxylin and eosin and examined with light microscopy. Lung injury was scored in a blinded fashion. Hyperemia, atelectasis, and neutrophil infiltration were scored as follows: 0 ¼ minimal; 1 ¼ mild; 2 ¼ moderate; 3 ¼ severe; and 4 ¼ maximal. Intraalveolar edema was scored as follows: 0 ¼ absent; 1 ¼ present [21]. The paraffin-embedded slides were stained with the terminal dUTP nick-end labeling (TUNEL) technique to detect the apoptosis of pulmonary cells. A TUNEL Apoptosis Detection Kit (Millipore) was applied according to the manufacturer’s protocol. From each biopsy, five fields were evaluated under a light microscope at a 200 magnification for TUNEL-positive cells. The number of positive cells is calculated for analysis.
2.9. ELISA detection of inflammatory cytokines, SP-A, and SP-D Concentrations of IL-8 as well as the levels of TNF-a, SP-A, and SP-D in BALF were detected using ELISA kits in accordance to the manufacturer’s construction (R&D systems).
2.10.
3
Myeloperoxidase activity assay
Frozen lung tissue was homogenized and processed for measurement of myeloperoxidase (MPO) activity. The MPO Colorimetric Activity Assay kit (BioVision, CA) was used for MPO determination, according to the instructions provided with the kit.
2.11.
Western blot detection of SP-A and SP-D
Lung tissues were smashed and centrifuged (4 C, 1500 r/min) for 10 min. Then, the supernatant was collected. The concentration of protein was determined using bicinchoninic acid methods. After that, the proteins were separated with 10% sodium dodecyl sulfate-polyacrylamide gelelectrophoresis (SDS-PAGE) and transferred to a polyvinylidene fluoride membrane. The membrane was blocked by incubation in TBST (10 mM Tris-HCL, pH 7.5, 150 mM NaCl, 0.1% Tween 20) containing 5% milk for 1 h. Next, primary goat antieSP-A antibody, mouse antieSP-D antibody (Santa Cruz, Dallas, TX), and secondary peroxidase antibody were added for incubation. b-actin (Bioworld, Louis Park, MN) was used as the internal standards at a dilution of 1:1000. Finally, the membranes were incubated with a luminescence-producing enzyme and exposed briefly to x-ray film.
2.12.
Statistical analysis
Each experiment was conducted in three independent times. Data were represented as mean standard deviation. All the statistical tests were performed using SPSS 17.0 (IBM, Armonk, NY). Between-groups comparison was calculated with one way analysis of variance and Student t-test. Correlations between continuous variables were calculated by Pearson correlation coefficients. P < 0.05 was considered statistically significant.
3.
Results
3.1.
PPS instillation improved pulmonary oxygenation
As shown in Figure 1, compared with the II group, PaO2 value of arterial blood was significantly increased in the PS group both at 6 and 12 h, respectively (6 h, P < 0.05; 12 h, P < 0.01). PaCO2 value at 2 h in PS group was significantly lower than that in II group (P < 0.05), and it was still a little bit lower at 6
Fig. 1 e Effects of PPS on pulmonary oxygenation in rats with smoke-induced inhalation injury. (A) PaO2. (B) PaCO2. (C) pH. Data are presented as mean ± standard deviation, with n [ 5 in each group. *P < 0.05 PS group compared with the II group, **P < 0.01 PS group compared with the II group.
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and 12 h. Besides, pH values at 2, 6, and 12 h were also significantly increased in the PS group compared with those in the II group (2 h, P < 0.01; 6, 12 h, P < 0.05). The PaO2, PaCO2, and pH values in the C group did not show any marked change and were kept at nearly normal levels over the time.
3.2. PPS instillation ameliorated lung histopathologic changes At 12 h after smoke inhalation, the lung tissues of rats in the II group exhibited marked infiltration of inflammatory cells, diffuse alveolar hemorrhage, large amount of inflammatory exudates, and increased apoptotic cells (Fig. 2A first panel, II). There were increased apoptotic cells verified by the TUNEL staining (black arrows, Fig. 2A second panel, II). However, the symptoms of those histopathologic changes were markedly
lessened by PPS treatment, as assessed by lung injury scores and TUNEL-positive cells (Fig. 2B and C).
3.3.
PPS instillation ameliorated lung tissue edema
The lung W/D ratio was calculated as a parameter of lung edema. The ratio increased in the II group compared with that in C group (Fig. 3A). PPS treatment attenuated the increase of the W/D ratio.
3.4.
PPS instillation reduced lung vascular permeability
The vascular permeability was analyzed by proteins of BALF in different groups. As shown in Figure 3B, the protein concentration of BALF in the II group was significantly increased when compared with that in the C group, and it was
Fig. 2 e Effects of PPS on lung histopathologic changes in rats with smoke-induced inhalation injury. (A) First panel: Lung tissue slides were stained with hematoxylin and eosin. Second panel: Lung tissue slides were subjected to TUNEL assay, TUNEL-positive or apoptotic cells (arrows). Shown are representative photographs of histology slides. (B) Lung injury scores: Smoke inhalation resulted in a marked increase of lung injury scores, and treatment with PS significantly blocked this increase. Data are presented as mean ± standard deviation (n [ 25 fields in each group; *P < 0.01 versus C group, **P < 0.01 versus II group). (C) Analysis of the positive cells. Data revealed that sharply increasing apoptosis of the lung induced by smoke was attenuated by PS. Data are presented as mean ± standard deviation (n [ 25 fields in each group; *P < 0.01 versus C group, **P < 0.01 versus II group). (Color version of figure is available online.)
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Fig. 3 e Effects of PPS on lung tissue edema and vascular permeability in rats with smoke-induced inhalation injury. (A) Lung W/D ratio. (B) Total protein concentration in BALF. Data are presented as mean ± standard deviation, with n [ 5 in each group. **P < 0.05 compared with C group, ##P < 0.05 compared with II group. significantly decreased after PPS treatment, indicating that the vascular permeability was reduced by PPS.
3.5.
PPS instillation reduced MPO activity and IL-8 levels
The lung tissue MPO activity in the PS group was markedly lower than that of the II group (Fig. 4A). When compared with the C group, IL-8 levels in BALF increased significantly in the II group (P < 0.05, Fig. 4B). After the PPS treatment, IL-8 levels remarkably decreased compared with II group. And there was a significant positive correlation between IL-8 and MPO (P < 0.05, r ¼ 0.824, Fig. 4C). In terms of TNF-a, no difference was found in the three groups (data not shown).
3.6.
PPS instillation increased the production of SP-A
Both SP-A and SP-D levels in BALF were significantly decreased after smoke inhalation (P < 0.05). Treatment with PPS increased SP-A levels but not SP-D in BALF (Fig. 5A and B). Furthermore, the Western blotting experiment showed that SP-A expression did increase after instillation of PPS, and SP-D expression in the PS group increased a little bit when
compared with the II group and further decreased compared with the C group (Fig. 5C). Correlation analysis showed that the SP-A level was negatively correlated with IL-8 level (r ¼ 0.8, P ¼ 0.005; Fig. 6).
4.
Discussion
Similar to previous studies, our experiments demonstrate that the PPS used in our research has the therapeutic effect on inhalation injury induced by smoke, which was confirmed by the increased PaO2 values, improved edema status, decreased vascular permeability and proinflammatory cytokine release, and less lung morphologic injury. Smoke inhalation-induced lung injury was characterized with a large number of neutrophils accumulation [2]. In our work, we found that PPS treatment clearly attenuated the accumulation of neutrophils in the rat model of smoke inhalation, which was proved by two independent evidences as follows: decreased MPO activity of lung tissues and histologic reduction of neutrophils accumulation in and around the alveoli. We also found increased levels of IL-8 in BALF, the major chemotactic factor playing an important role in
Fig. 4 e Effects of PPS on lung tissues MPO activity and IL-8 levels in BALF in rats with smoke-induced inhalation injury. (A) Activity of MPO in the lung, *P < 0.05 compared with C group, **P < 0.05 compared with II group. (B) IL-8 levels in BALF, #P < 0.05 compared with C group, ##P < 0.05 compared with II group. (C) Correlation between MPO and IL-8. The circles represent five rats of II group and five rats of PS group. Data are presented as mean ± standard deviation, with n [ 5 in each group.
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Fig. 5 e Effects of PPS on SP-A and SP-D production and release at 12 h after smoke inhalation. SP-A (A) and SP-D (B) levels in BALF were detected by ELISA, and expressions of SP-A and SP-D (C) were determined by Western blotting at 12 h after smoke inhalation. Data are presented as mean ± standard deviation (n [ 5 in each group), *P < 0.05 compared with C group, #P < 0.05 compared with II group.
recruiting neutrophils into the distal air spaces of the lung after smoke inhalation [25], which was significantly downregulated by PPS treatment. In addition, MPO was positively correlated with IL-8. These data indicate that the attenuation effect of PPS on neutrophils accumulation in smoke inhalation injury is partially by suppressing release of IL-8. The mechanism through which exogenous PPS modulates IL-8 in smoke-induced inhalation injury is not known. By literature, there remains some controversies about whether native SP-A is pro- or anti-inflammatory. Previous studies reported that SP-A could stimulate production of several cytokines, whereas other studies reported that SP-A inhibited the production of inflammatory factor by alveolar macrophages and other inflammatory cells [26e30]. The reasons may be due to differences in SP-A purification methods, the oligomeric state of the SP-A, the cell types studied, the assays used, or other subtle technical differences. In our study, we found that SP-A expression in lungs and levels in BALF decreased after smoke inhalation and were significantly increased by exogenous PPS (contains no SP-A), while the decreased SP-D was not
Fig. 6 e Correlation between levels of SP-A and IL-8 in BALF in rats with smoke-induced inhalation injury. The circles represent five rats of II group and five rats of PS group.
markedly restored by PPS. Furthermore, the SP-A and IL-8 levels in BALF were closely negatively correlated. Based on these data and previous report, we provide the first evidence that exogenous PPS could stimulate the production of endogenous SP-A, which may play an important role in regulating the proinflammatory cytokine IL-8. To determine the potential regulatory role of SP-A on IL-8 production and release in smoke-induced inhalation injury, further experiments including genetically engineered mice that have no ability to produce SP-A and another set of mice that overproduce SP-A are needed. Because the PPS used in our experiment contains no hydrophilic proteins (SP-A and SP-D) like most natural PS or synthetic surfactants [9,31e33], the detected SP-A and SP-D in our experiment were probably endogenous. A previous study reported that the Spa/ mice were associated with increased lavage TNF-a levels after infection of various pathogens, consistent with another two studies showing that SP-A attenuated TNF secretion elicited by lipopolysaccharides in alveolar macrophages [29,34]. In contrast, Koptides reported SP-A could stimulate TNF-a release from monocytes via Nuclear Factor-kB (NF-kB)edependent pathways [35]. However, no difference was found of TNF-a in the present work. The most reasonable explanation for this is that smoke-induced inhalation injury may not involve TNF-a production, at least not in early stage (our observation period) [36]. Although the available data here do not allow for a complete understanding of the different alterations of SP-A and SP-D after PPS treatment, some factors need to be considered. First, SP-A and SP-D perform distinct host defense functions in lung, such as showing differences in binding preferences to apoptotic cells and opposite effects on regulation of oxidant response from alveolar macrophages [37,38]. Second, the mechanism of degradation of SP-A and SP-D is different. SP-A declines as a consequence of inflammatory response in the lung, and the decline of SP-D mainly results from a major injury occurring to type II alveolar epithelial cells [39]. It is reported that smoke inhalation, independent of burn injury, could induce an oxidant stress that persists for at least the first 48 h after smoke exposure, and inhibition of thyroid transcription factor 1 (a critical regulator of transcription for SP-A) activity by oxidative stress may be the mechanism of decreased SP-A expression after smoke inhalation injury [13,40]. In our present work, we found the PPS was able to
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inhibit MPO activity, indicating that exogenous surfactant with no SP-A and SP-D has antioxidant effect. Thus, the antioxidant effect of PPS may be a potential contribution to the increase of endogenous SP-A expression in the rat model of smoke inhalation. Despite the encouraging experimental evidence that exogenous surfactant administration was a potential adjunctive therapy in ALI and/or ARDS, the beneficial effects and safety issues of exogenous surfactant supplement in treatment of ALI and/or ARDS patients except neonatal respiratory distress syndrome is controversial [41,42]. These differences of therapeutic effects were dependent on the doses of PPS, approach of medication administration, the severity and stage of ALI and/or ARDS, and the causes of ALI. However, the response to exogenous surfactant in experimental smoke inhalation injury models has generally been positive, suggesting that surfactant will find a place in the medication for the treatment of ALI and/or ARDS. Further clinical studies are warranted based on pathophysiological understanding and animal research.
5.
Conclusions
In summary, exogenous PPS could stimulate the production of endogenous SP-A and inhibit the release of IL-8, thus attenuating the neutrophils accumulation in lungs of smoke-induced inhalation injury rat. The increasing production of endogenous SP-A is due to the antioxidant effect of PPS, which contains no SP-A. Further studies will be required to clarify the precise mechanism of the stimulation of exogenous PPS on SP-A production and determine the potential regulatory role of SP-A on proinflammatory cytokine IL-8. We speculate that PPS administration through the trachea can be developed as an ideal treatment for smoke-induced lung injury, as those patients usually require intubation and mechanical ventilation. However, randomized controlled clinical trials of surfactant therapy in patients are needed before clinical application. A clinical trial of instillation of surfactant to treat patients with inhalation injury by smoke is underway at our medical center.
Acknowledgment The authors thank Miss Alison Hagemeister (a PhD student in Institute of Biochemistry & Molecular Immunology, RWTH Aachen University) to critically correct and proof-read our article. This work was funded by Young Talents Training Program of Shanghai Health System (XYQ2013079 and XYQ2013075), National Natural Science Foundation of China (81120108015 and 81370137), National Basic Research Program of China (973 Program, 2012CB518100), and “Twelfth Five-Year” Scientific Program of China (AWS11J008). This work was also supported by Shanghai “priority” for clinical key discipline project and Joint Research Program of important diseases of Shanghai Health System (2013ZYJB0008). Authors’ contributions: Y.S., X.Q., and G.W. performed the animal experiments. Y.S., X.Q., G.W. and H.T. wrote the
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article. J.W. assisted in the animal experiments. J.L. performed statistical analysis. Z.X. was responsible for the design of the experiments, analysis of experiment results, and the final revision of the article. All authors have read and approved the article.
Disclosure The authors report no proprietary or commercial interest in any product mentioned or concept discussed in this article. Conflicts of interest: None.
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[30] Cheng G, Ueda T, Nakajima H, et al. Suppressive effects of SP-A on ionomycin-induced IL-8 production and release by eosinophils. Int Arch Allergy Immunol 1998;117:59. [31] Weg JG, Balk RA, Tharratt RS, et al. Safety and potential efficacy of an aerosolized surfactant in human sepsisinduced adult respiratory distress syndrome. JAMA 1994;272: 1433. [32] Anzueto A, Baughman RP, Guntupalli KK, et al. Aerosolized surfactant in adults with sepsis-induced acute respiratory distress syndrome. Exosurf Acute Respiratory Distress Syndrome Sepsis Study Group. N Engl J Med 1996;334:1417. [33] Gregory TJ, Steinberg KP, Spragg R, et al. Bovine surfactant therapy for patients with acute respiratory distress syndrome. Am J Respir Crit Care Med 1997;155:1309. [34] Sano H, Sohma H, Muta T, Nomura S, Voelker DR, Kuroki Y. Pulmonary surfactant protein A modulates the cellular response to smooth and rough lipopolysaccharides by interaction with CD14. J Immunol 1999;163:387. [35] Koptides M, Umstead TM, Floros J, Phelps DS. Surfactant protein A activates NF-kappa B in the THP-1 monocytic cell line. Am J Phys 1997;273:L382. [36] Hales CA, Elsasser TH, Ocampo P, Efimova O. TNF-alpha in smoke inhalation lung injury. J Appl Phys 1997;82:1433. [37] Ja¨kel A, Clark H, Reid KB, Sim RB. The human lung surfactant proteins A (SP-A) and D (SP-D) interact with apoptotic target cells by different binding mechanisms. Immunobiology 2010; 215:551. [38] McCormack FX, Whitsett JA. The pulmonary collectins, SP-A and SP-D, orchestrate innate immunity in the lung. J Clin Invest 2002;109:707. [39] Greene KE, Wright JR, Steinberg KP, et al. Serial changes in surfactant-associated proteins in lung and serum before and after onset of ARDS. Am J Respir Crit Care Med 1999; 160:1843. [40] Dubick MA, Carden SC, Jordan BS, Langlinais PC, Mozingo DW. Indices of antioxidant status in rats subjected to wood smoke inhalation and/or thermal injury. Toxicology 2002;176:145. [41] Schultz MJ, Kesecioglu J. Is surfactant a promising additive drug in ALI/ARDS-patients? Curr Drug Targets 2004;5:445. [42] Willson DF, Notter RH. The future of exogenous surfactant therapy. Respir Care 2011;56:1369.
Available online at www.sciencedirect.com
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The effects of porcine pulmonary surfactant on smoke inhalation injury Yu Sun, MD,a,1 Xiaochen Qiu, MS,b,1 Guosheng Wu, MD,a,1 Junjie Wang, MS,a Jiahui Li, MS,a Hao Tang, MD,c and Zhaofan Xia, MDa,* a
Department of Burn Surgery, Changhai Hospital, Second Military Medical University, Shanghai, P. R. China Department of General Surgery, 309th Hospital of PLA, Beijing, P.R. China c Department of Respiratory Medicine, Changzheng Hospital, Second Military Medical University, Shanghai, P. R. China b
article info
abstract
Article history:
Background: Our previous study, consistent with others, demonstrated that administering
Received 11 February 2015
an exogenous surfactant was a potential therapy for acute lung injury and acute respira-
Received in revised form
tory distress syndrome. However, the underlying mechanisms remain largely unknown. In
4 May 2015
the present study, we investigated the effect of instilled porcine pulmonary surfactant
Accepted 12 May 2015
(PPS) on rat inhalation injury model induced by smoke and the possible mechanism.
Available online xxx
Materials and methods: Fifteen SpragueeDawley rats were equally randomized to three groups as follows (n ¼ 5 in each group): sham control group (C group), inhalation injury
Keywords:
group (II group), and inhalation injury þ PPS treatment group (PPS group). Lung tissues
Inhalation injury
were assayed for wet/dry ratio, histologic, terminal dUTP nick-end labeling staining, and
Porcine pulmonary surfactant
Western blotting examinations. The myeloperoxidase activity was tested in lung tissues as
Surfactant protein A
well. Bronchoalveolar lavage fluid was collected to determine the total protein concen-
MPO activity
trations, inflammatory cytokines, surfactant protein A (SP-A), and SP-D.
Interleukin 8
Results: Our present work exhibited that PPS had therapeutic effects on smoke inhalation injury reflected by significant increase of PaO2 values, improved edema status, decreased vascular permeability, amelioration of lung histopathology, and reduction of inflammatory response. In addition, PPS treatment could increase endogenous SP-A levels both in lung tissue and bronchoalveolar lavage fluid. Further correlation analysis showed that SP-A was negatively correlated with both myeloperoxidase activity and interleukin 8 levels. Conclusions: These results indicate that PPS can attenuate smoke-induced inhalation injury at least partly through stimulating production of endogenous SP-A and inhibiting the release of proinflammatory cytokines such as interleukin 8. The increasing production of endogenous SP-A may be due to the antioxidant effect of PPS, which contains no SP-A. ª 2015 Elsevier Inc. All rights reserved.
1.
Background
Smoke inhalation injury is common in burn victims and significantly contributes to the morbidity and mortality of
burn injuries [1,2]. About 22% of burns combine with smoke inhalation injury, which increases burn-related mortality by 20% [3]. Previous studies have described that smoke inhalation injury is linked with an inflammatory response of the lung
* Corresponding author. Department of Burn Surgery, Changhai Hospital, Second Military Medical University, 168 Changhai Road, Shanghai, P. R. China. Tel.: þ86 021 31161821; fax: þ86 021 65589829. E-mail address: [email protected] (Z. Xia). 1 These authors contributed equally to this work. 0022-4804/$ e see front matter ª 2015 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.jss.2015.05.019
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[2,4]. Over the past years, major advances have been made in the treatment of burns, but the advances in the medication treatment of smoke inhalation injury were scarce [5e8]. Pulmonary surfactant (PS) is the product of type II alveolar cells and mainly composed of phospholipids and surfactantspecific proteins [9]. It is the phospholipids that are responsible for the critical reduction in surface tension, the main function of PS [10], whereas the surfactant proteins (SP) mainly participate in regulating host immune defense and modulating inflammatory responses [11]. There are four SPs that have been identified, including SP-A, SP-B, SP-C, and SPD. SP-B and SP-C are hydrophobic and interact with surfactant lipids to help reduce surface tension, whereas hydrophilic SP-A and SP-D are members of the collectin family involved in pulmonary immunity [12]. Previous studies have showed that expression of SP-A was downregulated in lung tissue after smoke-induced acute and chronic lung injury [13,14]. Surfactants are routinely used to treat preterm infants with neonatal respiratory distress syndrome and also used in the treatment of infants and adults with acute lung injury (ALI) and/or acute respiratory distress syndrome (ARDS). Our previous experiments have showed obvious therapeutic effects of PS on adult rabbits with ARDS [15] and adult rats with ALI induced by lipopolysaccharides or oleic acid [16,17]. Treatment with PS was found to increase PaO2, decrease breath rate, raise survival rate, and improve histologic appearance of the lung. Xie [18] and Chi [19] et al. have also reported PS is effective in alleviating smoking-induced lung injury and smoke inhalation injury. However, the underlying mechanisms remain largely unknown. Therefore, we built a rat model of smoke inhalation injury to investigate its therapeutic effect and the potential mechanism.
2.
Materials and methods
2.1.
Surfactant preparation
Porcine pulmonary surfactant (PPS) was isolated from pig bronchoalveolar lavage fluid (BALF) using a protocol modified from the one used by Enhorning et al. [20]. Briefly, PPS was extracted from BALF using sequential centrifugation, chloroform-methanol extraction, and acetone precipitation, which contains more than 90% phospholipids and about 1% hydrophobic protein, mainly SP-B and -C, but no hydrophilic proteins (SP-A and SP-D). It has been approved for use in clinical trials of neonatal respiratory distress syndrome treatments by the State Food and Drug Administration of China [21].
2.2.
Animals and reagents
Studies were performed on male SpragueeDawley rats (aged 8e10 wk, weighing 180e200 g, Experimental Animal Center of the Second Military Medical University, Shanghai, China). They were housed in individual cages in a temperaturecontrolled room with a 12 h lightedark cycle and free access to food and water. All animal experiments were approved by the Second Military Medical University in accordance with the
Guide for Care and Use of Laboratory Animals published by the US NIH (publication no. 96-01). Total protein extraction kit was purchased from Cell Signaling Technology (Beverly, MA). Enzyme-linked immunosorbent assay (ELISA) kits of interleukin 8 (IL-8), tumor necrosis factor alpha (TNF-a), SP-A, and SP-D were obtained from R&D Corporation (Minneapolis, MN). The following primary antibodies were used: antieSP-A, antieSP-D, and antieb-actin (SigmaeAldrich, St. Louis, MO). The anti-goat or anti-mouse secondary antibody was provided by the Sigma Chemical Company. The enhanced chemiluminescence (ECL) western blotting substract and the bicinchoninic acid protein assay were purchased from Thermo Fisher Scientific Inc (Rockford, IL). Polyvinylidene fluoride membranes were provided by Millipore (Bedford, MA).
2.3.
Smoke inhalation injury model
A rat model of smoke inhalation injury has been established as previously described [22]. Smoke was produced by a selfmade smoke generator. In our present experiment, five rats were exposed each time. Smoke was generated by slowly smoldering wood shavings (120 g/kg body weight). The data presented in this work are derived from rats exposed to successive 9-min periods of smoke, which were separated into three times by 30-s exposures to ambient air.
2.4.
Experiment design
A total of 15 rats were equally randomized to three groups (n ¼ 5 in each group) as follows: sham control group (C group, ambient air inhalation), inhalation injury group (II group), and inhalation injury þ PPS treatment group (PS group). Specifically, the first 5 smoke-exposing rats were mixed with the second 5 rats and then went on to randomize to the II group and PS group in a blinded fashion. The dosage and optimal administration time in our present work were chosen on the basis of our previous experiments [16,17,21,23,24]. Thirty minutes after exposing, 150 mg/kg of PPS (the concentration was 50 mg/mL) and the same volume of normal saline were instilled intratracheally the in PS and II groups, respectively. PPS or saline was intratracheally instilled into the lungs by the following procedure: a suction catheter was advanced down the trachea to a point near the carina. The suction catheter was modified by occluding the main lumen and cutting multiple small holes in the catheter tip so that PPS or saline was delivered in numerous directions. PPS or saline was administered to the animal’s lungs while the animal’s body was rotated. Two-milliliter syringes connected to the suction catheter were used to administer PPS or saline first, followed by administration of air. The rats were sacrificed 12 h later, and then airways were dissected 0.3 cm away from tracheal bifurcation to collect the lung.
2.5.
Arterial blood analysis
A total of 0.2-mL arterial blood was obtained from the carotid artery, and PaO2, PaCO2, and pH were measured at 0, 2, 4, 6, and 12 h after smoke inhalation using an automatic analyzer (ABL90 FLEX analyzer, Radiometer, Denmark).
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2.6.
Lung wet/dry ratio
The right upper lobe was weighed soon after excision and then dried in an oven at 60 C for 72 h. The lung tissue wet-todry (W/D) weight ratio was calculated to assess lung tissue edema.
2.7.
BALF analysis
The hilum of the right lung was ligated to perform alveolar lavage of the left lung by instilling 3-mL 4 C potassium phosphate-buffered solution and withdrawing 30 s later. This procedure was repeated three times, and the BALF was collected and centrifuged (4 C, 1000 r/min, 10 min). The total protein concentration of BALF was determined using Lowry’s method.
2.8.
Morphologic evaluation
Postmortem, the right lower lobe of lung, was fixed by 15% formaldehyde and embedded in paraffin to cut into 5-mm sections. The sections were stained with hematoxylin and eosin, and visualized using an inverted microscope (Olympus, Japan). The lung sections were stained with hematoxylin and eosin and examined with light microscopy. Lung injury was scored in a blinded fashion. Hyperemia, atelectasis, and neutrophil infiltration were scored as follows: 0 ¼ minimal; 1 ¼ mild; 2 ¼ moderate; 3 ¼ severe; and 4 ¼ maximal. Intraalveolar edema was scored as follows: 0 ¼ absent; 1 ¼ present [21]. The paraffin-embedded slides were stained with the terminal dUTP nick-end labeling (TUNEL) technique to detect the apoptosis of pulmonary cells. A TUNEL Apoptosis Detection Kit (Millipore) was applied according to the manufacturer’s protocol. From each biopsy, five fields were evaluated under a light microscope at a 200 magnification for TUNEL-positive cells. The number of positive cells is calculated for analysis.
2.9. ELISA detection of inflammatory cytokines, SP-A, and SP-D Concentrations of IL-8 as well as the levels of TNF-a, SP-A, and SP-D in BALF were detected using ELISA kits in accordance to the manufacturer’s construction (R&D systems).
2.10.
3
Myeloperoxidase activity assay
Frozen lung tissue was homogenized and processed for measurement of myeloperoxidase (MPO) activity. The MPO Colorimetric Activity Assay kit (BioVision, CA) was used for MPO determination, according to the instructions provided with the kit.
2.11.
Western blot detection of SP-A and SP-D
Lung tissues were smashed and centrifuged (4 C, 1500 r/min) for 10 min. Then, the supernatant was collected. The concentration of protein was determined using bicinchoninic acid methods. After that, the proteins were separated with 10% sodium dodecyl sulfate-polyacrylamide gelelectrophoresis (SDS-PAGE) and transferred to a polyvinylidene fluoride membrane. The membrane was blocked by incubation in TBST (10 mM Tris-HCL, pH 7.5, 150 mM NaCl, 0.1% Tween 20) containing 5% milk for 1 h. Next, primary goat antieSP-A antibody, mouse antieSP-D antibody (Santa Cruz, Dallas, TX), and secondary peroxidase antibody were added for incubation. b-actin (Bioworld, Louis Park, MN) was used as the internal standards at a dilution of 1:1000. Finally, the membranes were incubated with a luminescence-producing enzyme and exposed briefly to x-ray film.
2.12.
Statistical analysis
Each experiment was conducted in three independent times. Data were represented as mean standard deviation. All the statistical tests were performed using SPSS 17.0 (IBM, Armonk, NY). Between-groups comparison was calculated with one way analysis of variance and Student t-test. Correlations between continuous variables were calculated by Pearson correlation coefficients. P < 0.05 was considered statistically significant.
3.
Results
3.1.
PPS instillation improved pulmonary oxygenation
As shown in Figure 1, compared with the II group, PaO2 value of arterial blood was significantly increased in the PS group both at 6 and 12 h, respectively (6 h, P < 0.05; 12 h, P < 0.01). PaCO2 value at 2 h in PS group was significantly lower than that in II group (P < 0.05), and it was still a little bit lower at 6
Fig. 1 e Effects of PPS on pulmonary oxygenation in rats with smoke-induced inhalation injury. (A) PaO2. (B) PaCO2. (C) pH. Data are presented as mean ± standard deviation, with n [ 5 in each group. *P < 0.05 PS group compared with the II group, **P < 0.01 PS group compared with the II group.
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and 12 h. Besides, pH values at 2, 6, and 12 h were also significantly increased in the PS group compared with those in the II group (2 h, P < 0.01; 6, 12 h, P < 0.05). The PaO2, PaCO2, and pH values in the C group did not show any marked change and were kept at nearly normal levels over the time.
3.2. PPS instillation ameliorated lung histopathologic changes At 12 h after smoke inhalation, the lung tissues of rats in the II group exhibited marked infiltration of inflammatory cells, diffuse alveolar hemorrhage, large amount of inflammatory exudates, and increased apoptotic cells (Fig. 2A first panel, II). There were increased apoptotic cells verified by the TUNEL staining (black arrows, Fig. 2A second panel, II). However, the symptoms of those histopathologic changes were markedly
lessened by PPS treatment, as assessed by lung injury scores and TUNEL-positive cells (Fig. 2B and C).
3.3.
PPS instillation ameliorated lung tissue edema
The lung W/D ratio was calculated as a parameter of lung edema. The ratio increased in the II group compared with that in C group (Fig. 3A). PPS treatment attenuated the increase of the W/D ratio.
3.4.
PPS instillation reduced lung vascular permeability
The vascular permeability was analyzed by proteins of BALF in different groups. As shown in Figure 3B, the protein concentration of BALF in the II group was significantly increased when compared with that in the C group, and it was
Fig. 2 e Effects of PPS on lung histopathologic changes in rats with smoke-induced inhalation injury. (A) First panel: Lung tissue slides were stained with hematoxylin and eosin. Second panel: Lung tissue slides were subjected to TUNEL assay, TUNEL-positive or apoptotic cells (arrows). Shown are representative photographs of histology slides. (B) Lung injury scores: Smoke inhalation resulted in a marked increase of lung injury scores, and treatment with PS significantly blocked this increase. Data are presented as mean ± standard deviation (n [ 25 fields in each group; *P < 0.01 versus C group, **P < 0.01 versus II group). (C) Analysis of the positive cells. Data revealed that sharply increasing apoptosis of the lung induced by smoke was attenuated by PS. Data are presented as mean ± standard deviation (n [ 25 fields in each group; *P < 0.01 versus C group, **P < 0.01 versus II group). (Color version of figure is available online.)
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Fig. 3 e Effects of PPS on lung tissue edema and vascular permeability in rats with smoke-induced inhalation injury. (A) Lung W/D ratio. (B) Total protein concentration in BALF. Data are presented as mean ± standard deviation, with n [ 5 in each group. **P < 0.05 compared with C group, ##P < 0.05 compared with II group. significantly decreased after PPS treatment, indicating that the vascular permeability was reduced by PPS.
3.5.
PPS instillation reduced MPO activity and IL-8 levels
The lung tissue MPO activity in the PS group was markedly lower than that of the II group (Fig. 4A). When compared with the C group, IL-8 levels in BALF increased significantly in the II group (P < 0.05, Fig. 4B). After the PPS treatment, IL-8 levels remarkably decreased compared with II group. And there was a significant positive correlation between IL-8 and MPO (P < 0.05, r ¼ 0.824, Fig. 4C). In terms of TNF-a, no difference was found in the three groups (data not shown).
3.6.
PPS instillation increased the production of SP-A
Both SP-A and SP-D levels in BALF were significantly decreased after smoke inhalation (P < 0.05). Treatment with PPS increased SP-A levels but not SP-D in BALF (Fig. 5A and B). Furthermore, the Western blotting experiment showed that SP-A expression did increase after instillation of PPS, and SP-D expression in the PS group increased a little bit when
compared with the II group and further decreased compared with the C group (Fig. 5C). Correlation analysis showed that the SP-A level was negatively correlated with IL-8 level (r ¼ 0.8, P ¼ 0.005; Fig. 6).
4.
Discussion
Similar to previous studies, our experiments demonstrate that the PPS used in our research has the therapeutic effect on inhalation injury induced by smoke, which was confirmed by the increased PaO2 values, improved edema status, decreased vascular permeability and proinflammatory cytokine release, and less lung morphologic injury. Smoke inhalation-induced lung injury was characterized with a large number of neutrophils accumulation [2]. In our work, we found that PPS treatment clearly attenuated the accumulation of neutrophils in the rat model of smoke inhalation, which was proved by two independent evidences as follows: decreased MPO activity of lung tissues and histologic reduction of neutrophils accumulation in and around the alveoli. We also found increased levels of IL-8 in BALF, the major chemotactic factor playing an important role in
Fig. 4 e Effects of PPS on lung tissues MPO activity and IL-8 levels in BALF in rats with smoke-induced inhalation injury. (A) Activity of MPO in the lung, *P < 0.05 compared with C group, **P < 0.05 compared with II group. (B) IL-8 levels in BALF, #P < 0.05 compared with C group, ##P < 0.05 compared with II group. (C) Correlation between MPO and IL-8. The circles represent five rats of II group and five rats of PS group. Data are presented as mean ± standard deviation, with n [ 5 in each group.
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Fig. 5 e Effects of PPS on SP-A and SP-D production and release at 12 h after smoke inhalation. SP-A (A) and SP-D (B) levels in BALF were detected by ELISA, and expressions of SP-A and SP-D (C) were determined by Western blotting at 12 h after smoke inhalation. Data are presented as mean ± standard deviation (n [ 5 in each group), *P < 0.05 compared with C group, #P < 0.05 compared with II group.
recruiting neutrophils into the distal air spaces of the lung after smoke inhalation [25], which was significantly downregulated by PPS treatment. In addition, MPO was positively correlated with IL-8. These data indicate that the attenuation effect of PPS on neutrophils accumulation in smoke inhalation injury is partially by suppressing release of IL-8. The mechanism through which exogenous PPS modulates IL-8 in smoke-induced inhalation injury is not known. By literature, there remains some controversies about whether native SP-A is pro- or anti-inflammatory. Previous studies reported that SP-A could stimulate production of several cytokines, whereas other studies reported that SP-A inhibited the production of inflammatory factor by alveolar macrophages and other inflammatory cells [26e30]. The reasons may be due to differences in SP-A purification methods, the oligomeric state of the SP-A, the cell types studied, the assays used, or other subtle technical differences. In our study, we found that SP-A expression in lungs and levels in BALF decreased after smoke inhalation and were significantly increased by exogenous PPS (contains no SP-A), while the decreased SP-D was not
Fig. 6 e Correlation between levels of SP-A and IL-8 in BALF in rats with smoke-induced inhalation injury. The circles represent five rats of II group and five rats of PS group.
markedly restored by PPS. Furthermore, the SP-A and IL-8 levels in BALF were closely negatively correlated. Based on these data and previous report, we provide the first evidence that exogenous PPS could stimulate the production of endogenous SP-A, which may play an important role in regulating the proinflammatory cytokine IL-8. To determine the potential regulatory role of SP-A on IL-8 production and release in smoke-induced inhalation injury, further experiments including genetically engineered mice that have no ability to produce SP-A and another set of mice that overproduce SP-A are needed. Because the PPS used in our experiment contains no hydrophilic proteins (SP-A and SP-D) like most natural PS or synthetic surfactants [9,31e33], the detected SP-A and SP-D in our experiment were probably endogenous. A previous study reported that the Spa/ mice were associated with increased lavage TNF-a levels after infection of various pathogens, consistent with another two studies showing that SP-A attenuated TNF secretion elicited by lipopolysaccharides in alveolar macrophages [29,34]. In contrast, Koptides reported SP-A could stimulate TNF-a release from monocytes via Nuclear Factor-kB (NF-kB)edependent pathways [35]. However, no difference was found of TNF-a in the present work. The most reasonable explanation for this is that smoke-induced inhalation injury may not involve TNF-a production, at least not in early stage (our observation period) [36]. Although the available data here do not allow for a complete understanding of the different alterations of SP-A and SP-D after PPS treatment, some factors need to be considered. First, SP-A and SP-D perform distinct host defense functions in lung, such as showing differences in binding preferences to apoptotic cells and opposite effects on regulation of oxidant response from alveolar macrophages [37,38]. Second, the mechanism of degradation of SP-A and SP-D is different. SP-A declines as a consequence of inflammatory response in the lung, and the decline of SP-D mainly results from a major injury occurring to type II alveolar epithelial cells [39]. It is reported that smoke inhalation, independent of burn injury, could induce an oxidant stress that persists for at least the first 48 h after smoke exposure, and inhibition of thyroid transcription factor 1 (a critical regulator of transcription for SP-A) activity by oxidative stress may be the mechanism of decreased SP-A expression after smoke inhalation injury [13,40]. In our present work, we found the PPS was able to
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inhibit MPO activity, indicating that exogenous surfactant with no SP-A and SP-D has antioxidant effect. Thus, the antioxidant effect of PPS may be a potential contribution to the increase of endogenous SP-A expression in the rat model of smoke inhalation. Despite the encouraging experimental evidence that exogenous surfactant administration was a potential adjunctive therapy in ALI and/or ARDS, the beneficial effects and safety issues of exogenous surfactant supplement in treatment of ALI and/or ARDS patients except neonatal respiratory distress syndrome is controversial [41,42]. These differences of therapeutic effects were dependent on the doses of PPS, approach of medication administration, the severity and stage of ALI and/or ARDS, and the causes of ALI. However, the response to exogenous surfactant in experimental smoke inhalation injury models has generally been positive, suggesting that surfactant will find a place in the medication for the treatment of ALI and/or ARDS. Further clinical studies are warranted based on pathophysiological understanding and animal research.
5.
Conclusions
In summary, exogenous PPS could stimulate the production of endogenous SP-A and inhibit the release of IL-8, thus attenuating the neutrophils accumulation in lungs of smoke-induced inhalation injury rat. The increasing production of endogenous SP-A is due to the antioxidant effect of PPS, which contains no SP-A. Further studies will be required to clarify the precise mechanism of the stimulation of exogenous PPS on SP-A production and determine the potential regulatory role of SP-A on proinflammatory cytokine IL-8. We speculate that PPS administration through the trachea can be developed as an ideal treatment for smoke-induced lung injury, as those patients usually require intubation and mechanical ventilation. However, randomized controlled clinical trials of surfactant therapy in patients are needed before clinical application. A clinical trial of instillation of surfactant to treat patients with inhalation injury by smoke is underway at our medical center.
Acknowledgment The authors thank Miss Alison Hagemeister (a PhD student in Institute of Biochemistry & Molecular Immunology, RWTH Aachen University) to critically correct and proof-read our article. This work was funded by Young Talents Training Program of Shanghai Health System (XYQ2013079 and XYQ2013075), National Natural Science Foundation of China (81120108015 and 81370137), National Basic Research Program of China (973 Program, 2012CB518100), and “Twelfth Five-Year” Scientific Program of China (AWS11J008). This work was also supported by Shanghai “priority” for clinical key discipline project and Joint Research Program of important diseases of Shanghai Health System (2013ZYJB0008). Authors’ contributions: Y.S., X.Q., and G.W. performed the animal experiments. Y.S., X.Q., G.W. and H.T. wrote the
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article. J.W. assisted in the animal experiments. J.L. performed statistical analysis. Z.X. was responsible for the design of the experiments, analysis of experiment results, and the final revision of the article. All authors have read and approved the article.
Disclosure The authors report no proprietary or commercial interest in any product mentioned or concept discussed in this article. Conflicts of interest: None.
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