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Betulin attenuates lung and liver injuries in sepsis
International Immunopharmacology 30 (2016) 50–56
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Betulin attenuates lung and liver injuries in sepsis Hongyu Zhao ⁎, Zhenning Liu, Wei Liu, Xinfei Han, Min Zhao Department of Emergency Medicine, Shengjing Hospital of China Medical University, Shenyang 110004, People's Republic of China
a r t i c l e
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Article history: Received 21 July 2015 Received in revised form 13 November 2015 Accepted 22 November 2015 Available online 28 November 2015 Keywords: Sepsis Betulin Inflammation NF-κB MAPK
a b s t r a c t Sepsis is a complex condition with unacceptable mortality. Betulin is a natural extract with multiple bioactivities. This study aims to evaluate the potential effects of betulin on lung and liver injury in sepsis. Cecal ligation and puncture was used to establish the rat model of sepsis. A single dose of 4 mg/kg or 8 mg/kg betulin was injected intraperitoneally immediately after the model establishment. The survival rate was recorded every 12 h for 96 h. The organ injury was examined using hematoxylin and eosin staining and serum biochemical test. The levels of proinflammatory cytokines and high mobility group box 1 in the serum were measured using ELISA. Western blotting was used to detect the expression of proteins in NF-κB and MAPK signaling pathways. Betulin treatment significantly improved the survival rate of septic rats, and attenuated lung and liver injury in sepsis, including the reduction of lung wet/dry weight ratio and activities of alanine aminotransferase and aspartate aminotransferase in the serum. In addition, levels of tumor necrosis factor-α, interleukin-1β, interleukin-6 and high mobility group box 1 in the serum were also lowered by betulin treatment. Moreover, sepsis-induced activation of the NF-κB and MAPK signaling pathway was inhibited by betulin as well. Our findings demonstrate the protective effect of betulin in lung and liver injury in sepsis. This protection may be mediated by its anti-inflammatory and NF-κB and MAPK inhibitory effects. © 2015 Elsevier B.V. All rights reserved.
1. Introduction Sepsis, a serious condition with a very high mortality in intensive care, is characterized by a systemic uncontrolled hyper-inflammatory response, disseminated intravascular coagulation and multiple organ dysfunctions [1–5]. The pathogenesis of sepsis is complicated. Till now, the physiopathological mechanisms of sepsis have been studied well. Many mediators such as proinflammatory cytokines, chemokines and free radicals were found to be involved in the processes of sepsis [6,7]. However, despite the investigation of various therapeutic strategies to improve sepsis care, an effective compound has seldom been found. Antibiotics are still the main strategy in clinical care and the mortality of sepsis is still as high as 30% to 40% [8,9]. Multiple organ failure is one of the important reasons that cause death in septic patients [10]. Multiple-organ dysfunction syndrome (MODS) frequently occurs in severe sepsis and sepsis shock. The severity of organ dysfunction often determines the prognosis of sepsis [11]. As a systemic inflammatory response syndrome, in the process of sepsis, an inflammatory cascade was activated and excess proinflammatory cytokines were secreted into periphery blood and organs. Uncontrolled inflammation causes capillary permeability, endothelial damage, interstitial edema and hyperdynamic circulation, which subsequently lead to tissue hypoxia, metabolic failure and finally MODS [10]. ⁎ Corresponding author at: Department of Emergency Medicine, Shengjing Hospital of China Medical University, 36 Sanhao Street, Shenyang, 110004, People's Republic of China. E-mail address: [email protected] (H. Zhao).
http://dx.doi.org/10.1016/j.intimp.2015.11.028 1567-5769/© 2015 Elsevier B.V. All rights reserved.
Therefore, compounds that have an effective anti-inflammatory profile and target multiple-organs would be beneficial in sepsis therapy. Betulin (lup-20(29)-ene-3β, 28-diol) is a triterpene extracted from birch tree bark. It can be converted to betulinic acid by chemosynthesis or biotransformation. The three active positions in their structure empower these two compounds with various pharmacological activities, such as antitumor, anti-HIV, anti-inflammatory, antiviral and antibacterial activities [12]. Moreover, previous studies have shown that betulin could decrease LPS-induced inflammation by preventing nuclear factor kappa-B (NF-κB) activation [13]. Betulin also inhibited the activation of p38 and c-Jun N-terminal kinase (JNK) transduction pathways in ethanol-induced liver stellate cells [14]. These two signaling pathways have high correlations of inflammation and cell stress. In addition, they were found to be activated in sepsis, and pharmacological inhibition of these two pathways was involved in the anti-inflammatory effects [15,16]. In the present study, a sepsis model was established by cecal ligation and puncture (CLP) and the potential effect of betulin on lung and liver injury in sepsis was evaluated. 2. Materials and methods 2.1. Animals A total of 92 male Sprague–Dawley rats (8 weeks old) were supplied by the Experimental Animal Centre of China Medical University (Shenyang, China). The animals were housed in cages located in a room with stable temperature (22–24 °C) and a 12/12 h light/dark cycle and
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received water and food ad libitum. All animal protocols were approved by the ethics committee of China Medical University (Shenyang, China).
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high mobility group box 1 (HMGB-1) were determined using commercial enzyme-linked immunosorbent assay (ELISA) kits specific for rat following the manufacturer's instructions (USCN, Wuhan, China).
2.2. Induction of sepsis 2.7. Protein preparation Sepsis was induced by CLP according to a method described previously [17]. Briefly, after fasting for 12 h, rats were anesthetized with 3.5 ml/kg chloral hydrate. A 1.5 cm midline incision was performed and the cecum was exposed and ligated at the root. The cecum was punctured three times using a 5 ml syringe needle. Light pressure was applied to expel a small amount of fecal material. The cecum was then returned and the wound was closed. All the animals received 3 ml/100 g saline subcutaneously immediately after the operation for fluid resuscitation. Rats in the sham group received only cecum exposure but not ligation or puncture on the cecum.
Lung and liver tissues were homogenized in icy NP-40 lysis buffer supplement with 1% Triton X-100 and 1 mM phenylmethanesulfonylfluoride. Tissue homogenate was centrifuged at 12,000 g for 10 min and the supernatants were collected. Nuclear and cytosolic proteins were separated using a Nuclear and Cytoplasmic Protein Extraction Kit (Beyotime Institute of Biotechnology, Haimen, China) following the manufacturer's protocol. Protein concentration was determined using a bicinchoninic acid protein assay kit (Beyotime). 2.8. Western blot analysis
2.3. Betulin treatment and mortality experimental protocols Rats were randomly divided into 5 groups: (1) sham group (n = 22); (2) CLP group (n = 24); (3) CLP + dexamethasone 2 mg/kg group (DEX) (n = 24); (4) CLP + betulin 4 mg/kg group (Betulin L) (n = 24); and (5) CLP + betulin 8 mg/kg group (Betulin H) (n = 22). Rats were administrated intraperitoneally with DEX (Tianjinxinzheng, Tianjin, China) or betulin (Meilun, Dalian, China, purity N 98%) dissolved in saline immediately after the model establishment. Rats in the sham and CLP groups received only saline. The choice of administration time and dosage was based on our primary experiments. Ten rats in each group were randomly selected to record the survival rate. Rats were monitored every 12 h for 96 h and the numbers of surviving rats were recorded. In the remaining 52 rats, one in the CLP group and one in the Betulin L group died at 24 h after the operation. One rat in the CLP group and one in the Betulin L group were randomly selected to perform primary experiments. Six rats in each group were randomly selected to measure the wet and dry weight of lungs at 24 h after treatment. The last six rats in each group were used to perform plasmic, biochemical and molecular biological assays.
An equal amount of protein (40 μg) for each sample was separated in the SDS-PAGE and transferred onto the polyvinylidene fluoride membranes (Millipore, Billerica, MA, USA) using the wet transfer method. Membranes were blocked in 5% non-fat milk for 1 h at room temperature and incubated in primary antibodies at 4 °C overnight. After a wash stage using Tris buffered saline, with Tween-20 (TBST), membranes were incubated in horseradish peroxidase conjugated goat anti-rabbit or goat anti-mouse IgG (1:5000, Beyotime) for 45 min at room temperature. Finally, membranes were immersed in enhanced electrochemiluminescence reagent (7 Sea Pharmtech, Shanghai, China) and exposed using X-ray film. Primary antibodies against extracellular regulated protein kinases (ERK) (bs-2637R), p-ERK (bs-1522R), JNK (bs-10562R), p-JNK (bs-1640R), p38 (bs-0637R), p-p38 (bs-5477R), inhibitors of NF-κBα (IκBα) (bs-1287R), and p-p65 (bs-0982R) from Bioss, Beijing, China, and p65 (BA0610) from Boster, Wuhan, China were used and the protein levels were standardized to β-actin (sc-47,778, Santa Cruz Biotechnology, Santa Cruz, CA, USA) for whole cell lysate and Histone H3 (bs-17422R, Bioss) for the nuclear fraction. 2.9. Statistical analysis
2.4. Measurement of the lung wet/dry weight ratio Lungs were weighed immediately after being isolated and dried in a microwave oven at 80 °C to constant weight. The wet/dry weight ratio was calculated.
Data were expressed as mean ± standard deviation (SD). Survival data were analyzed by the Kaplan–Meier curve and log-rank test. Other data were analyzed by one-way analysis of variance, and the Fisher's least significant difference test was used for post hoc comparisons. P value less than 0.05 was considered statistically significant.
2.5. Histological analysis 3. Results Twenty-four hours after betulin treatment, tissues of lung and liver were harvested and fixed with 4% paraformaldehyde overnight. The samples were then dehydrated in ascending grades of alcohols, embedded in paraffin, and cut into 5-μm sections. To perform hematoxylin and eosin (H&E) staining, sections were immersed in xylene for 15 min and hydrated through graded ethanol. Then sections were stained with hematoxylin (Solarbio, Beijing, China) for 5 min and with eosin (Solarbio) for 3 min. After being dehydrated with increasing concentrations of ethanol and cleared in xylene, sections were mounted in Permount and observed under an optic microscope (DP73, Olympus, Tokyo, Japan). 2.6. Serum analysis Blood sample was obtained 24 h after betulin treatment. Whole blood was centrifuged at 7700 g for 10 min at 4 °C. Serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were determined using commercial assay kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, China) according to the manufacturer's instructions. Serum tumor necrosis factor-α (TNF-α), interleukin (IL)-1β, IL-6 and
3.1. Effect of betulin on survival rate in CLP rats All the rats in the sham group survived at the end of monitoring. In the vehicle-treated CLP group, the survival rate for 48 h was 40% and reached 0% at 96 h. No rat survived at the end of the experiment in this group. Betulin 4 mg/kg reduced the mortality to some extent but was not significant. The survival rate was 70% at 48 h and 30% at 96 h. DEX 2 mg/kg and betulin 8 mg/kg significantly improved the survival of septic rats, compared with the CLP group (P b 0.01). In the DEX group, the survival rate was 90% at 48 h and 80% at 96 h. In the betulin H group, the survival rate was 80% at 48 h and 60% at 96 h (Fig. 1). Although the survival rate in the DEX group was higher than that in the betulin H group, there is no significant difference (P N 0.05). 3.2. Effect of betulin on CLP-induced lung and liver injuries H&E staining illustrated the normal cell structures of the lung and liver in the sham operated rats (Fig. 1A). After CLP operation for 24 h, lung tissue showed marked inflammatory cell infiltration, edema,
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were markedly attenuated by DEX 2 mg/kg or betulin 8 mg/kg administration. In line with the histological observation, the dramatically increased lung wet/dry weight ratio showed that pulmonary edema occurred in the CLP rats (Fig. 2B). DEX 2 mg/kg and betulin 8 mg/kg evidently reduced the wet/dry weight ration of CLP rats (P b 0.01 vs. CLP group). Serum ALT and AST activities, representative markers of liver injury, were determined in this study. Twenty-four hours after CLP, activities of ALT and AST in serum were dramatically increased (Fig. 2C and D). DEX 2 mg/kg and betulin 8 mg/kg attenuated elevations in serum ALT and AST activities that occurred at 24 h after CLP (P b 0.01 vs. CLP group). The effects of DEX 2 mg/kg and betulin 8 mg/kg were similar.
3.3. Effect of betulin on CLP-induced inflammatory cytokine production and HMGB1 release
Fig. 1. Effect of betulin on the survival rate of septic rats. DEX 2 mg/kg and betulin 8 mg/kg significantly increased the survival rate of septic rats. n = 10. **P b 0.01 compared with the sham group. ##P b 0.01 compared with the CLP group. DEX: dexamethasone, L: low-dosage, 4 mg/kg, H: high-dosage, 8 mg/kg.
pulmonary interstitial hyperemia, hemorrhage, and alveolar collapse. In the liver tissue, cell boundary was blunted and inflammation and hepatocellular necrosis was found. These pathological alterations
Twenty-four hours after CLP, marked elevations of TNF-α, IL-1β and IL-6 levels were found in the serum compared with that in the sham group (Fig. 3A–C, P b 0.01). Betulin 8 mg/kg significantly suppressed these increases. Betulin 4 mg/kg slightly decreased the levels of cytokines but did not reach significant levels. The serum level of HMGB1 increased dramatically at 24 h after CLP as well (Fig. 3D, P b 0.01 vs. sham group). DEX 2 mg/kg and betulin 8 mg/kg restored this change. The effect of DEX 2 mg/kg was equivalent to that of betulin 8 mg/kg.
Fig. 2. Effect of betulin on lung and liver injury in septic rats. (A) Hematoxylin–eosine stained lung and liver sections of septic rats. CLP induced inflammatory cell infiltration, edema, pulmonary interstitial hyperemia, hemorrhage, and alveolar collapse in the lung and hepatocellular necrosis in the liver. Betulin treatment restored these changes. Betulin administration also decreased wet/dry weight ratio of lungs (B) and reduced ALT (C) and AST (D) level in the serum of septic rats. The effect of betulin 8 mg/kg was equivalent to DEX 2 mg/kg. Scale bar: 100 μm. Data are expressed as mean ± standard deviation (SD). **P b 0.01 compared with the sham group. ##P b 0.01 compared with the CLP group. DEX: dexamethasone, L: low-dosage, 4 mg/kg, H: high-dosage, 8 mg/kg.
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Fig. 3. Effect of betulin on serum levels of proinflammatory-cytokines and HMGB-1 in septic rats. DEX 2 mg/kg and betulin 8 mg/kg significantly reduced the release of TNF-α (A), IL-1β (B), IL-6 (C) and HMGB-1 (D) into the serum. Data are expressed as mean ± standard deviation (SD). **P b 0.01 compared with the sham group. ##P b 0.01 compared with the CLP group. DEX: dexamethasone, L: low-dosage, 4 mg/kg, H: high-dosage, 8 mg/kg.
3.4. Effect of betulin on CLP-induced NF-κB signaling activation in the lung and liver As shown in Figs. 4 and 5, a marked reduction of cytosolic IκBα protein expression in both lung and liver was found at 24 h after CLP (A), thereby, NF-κB/p65 was phosphorylated and NF-κB/p65 nuclear accumulation was increased (B and C). After betulin treatment, rats in the 8 mg/kg group effectively suppressed these changes. In the 4 mg/kg group, except on the NF-κB nuclear expression, the effect of betulin did not show any significance.
3.5. Effect of betulin on CLP-induced mitogen-activated protein kinase (MAPK) activation in the lung and the liver In the lung, three components of MAPK, ERK, JNK and p38 were over-phosphorylated at 24 h after CLP compared with the sham group (Fig. 6, P b 0.01). Betulin 4 mg/kg reduced phosphor-ERK and p38 expressions but not JNK. Betulin 8 mg/kg suppressed phosphorylation of all the three MAPK proteins. In the liver, consistent with that in the lung, all the three proteins were highly phosphorylated 24 h after CLP (Fig. 7). Betulin 4 and 8 mg/kg dose-dependently suppressed these phosphorylations.
4. Discussion Sepsis is a severe clinical syndrome with complicated pathological mechanisms. The systemic inflammatory and immune responses during the development of sepsis involve numerous factors. Natural extracts which have various bioactivities and multiple target organs may be more effective in sepsis treatment than synthetic drugs that generally have specific targets. In the present study, we demonstrated that betulin showed a markedly protective effect against sepsis, as represented by the increasing survival rate of septic rats induced by CLP and attenuating lung and liver injury. The effect was equivalent to that of positive drug DEX. This protective effect of betulin was mediated by its antiinflammatory effect and HMGB-1 inhibition. In addition, the NF-κB and MAPK suppression was involved in the protective mechanisms of betulin as well. When the infection develops beyond the compensatory ability of the body, the function of distinct organ systems are compromised. In general, the respiratory system is the first system where failure happens, followed by hepatic, renal, cardiac and other systems. The number of failing organs is closely related with the mortality of septic patients. In the present study, we examined lung and liver injury by H&E staining. Consistent with previous studies [18,19], these two organs were injured 24 h after model establishment. In addition, increased activities of
Fig. 4. Effect of betulin on NF-κB activation in the lung of septic rats. Betulin markedly upregulated expression of IκBα (A), inhibited the phosphorylation of p65 (B), and prevented p65 nuclear accumulation (C) in the lung. Data are expressed as mean ± standard deviation (SD). **P b 0.01 compared with the sham group. ##P b 0.01 compared with the CLP group. L: low-dosage, 4 mg/kg, H: high-dosage, 8 mg/kg.
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Fig. 5. Effect of betulin on NF-κB activation in the liver of septic rats. Betulin markedly upregulated expression of IκBα (A), inhibited the phosphorylation of p65 (B), and prevented p65 nuclear accumulation (C) in the liver. Data are expressed as mean ± standard deviation (SD). **P b 0.01 compared with the sham group. ##P b 0.01 compared with the CLP group. L: low-dosage, 4 mg/kg, H: high-dosage, 8 mg/kg.
ALT and AST also reflected the pathological change of liver. These pathological alterations were significantly attenuated by betulin treatment. This multi-organ functional protective effect of betulin was very likely to contribute to the survival improvement. Runaway inflammatory response plays a key role in the development of sepsis. Therefore, effective inflammatory control is an important part in sepsis treatment. Inflammatory response in sepsis is mainly represented by the over-production of proinflammatory cytokines such as TNF-α, IL-1β and IL-6, which are considered to be the characteristic of early sepsis. In addition, reducing the secretion of these cytokines delayed the development of sepsis and decreased the mortality of septic patients or animal models [20–22]. In the present study, in agreement with previous studies, markedly elevated TNF-α, IL-1β and IL-6 levels were found in the serum of septic rats. Although we have not observed the time-dependent changes of these cytokines, and according to the previous reports, the peak of secretion does not occur at 24 h after CLP, we still can use these cytokines to evaluate the anti-inflammatory effect of betulin. Betulin has been reported to have the ability to attenuate secretion of inflammatory cytokines [13,23,24]. In line with these studies, our results demonstrated that betulin treatment significantly reduced the levels of the three cytokines
in the serum. In addition, we compared DEX treatment, a typical anti-inflammatory drug, with betulin intervention. We found that these two compounds have the same anti-inflammatory effect in sepsis. These results suggest that the anti-inflammation properties of betulin are involved in its anti-sepsis effect. In addition to these proinflammatory cytokines, some other cytokines have also been found to play important roles in sepsis. HMGB1 was originally considered as a highly conserved structural DNA-binding protein that is responsible for DNA replication [25–27]. In 1999, Kevin Tracey and colleagues reported the cytokine-like properties of HMGB1. This and the following studies found that the serum level of HMGB1 is increased at late time points after endotoxin exposure. Overproduced HMGB1 can trigger the release of the above proinflammatory cytokines and exacerbate the inflammation. Targeting HMGB1 may be effective in treating related diseases [28–30]. In sepsis, HMGB1 is well accepted as a late mediator of inflammation. It has been found to be elevated in patients with severe sepsis [31,32]. In a rodent experimental model of abdominal sepsis, anti-HMGB1 antibody treatment significantly increased the survival rate of septic animals and attenuated CLP-induced diaphragmatic dysfunction [33]. In the present study, the elevated HMGB1 level was lowered by betulin
Fig. 6. Effect of betulin on MAPK activation in the lung of septic rats. (A) Typical blots of proteins of MAPK. Betulin evidently inhibited the phosphorylations of ERK (B), JNK (C) and p38 (D) in the lung. Data are expressed as mean ± standard deviation (SD). **P b 0.01 compared with the sham group. ##P b 0.01 compared with the CLP group. L: low-dosage, 4 mg/kg, H: high-dosage, 8 mg/kg.
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Fig. 7. Effect of betulin on MAPK activation in the liver of septic rats. (A) Typical blots of proteins of MAPK. Betulin evidently inhibited the phosphorylations of ERK (B), JNK (C) and p38 (D) in the liver. Data are expressed as mean ± standard deviation (SD). **P b 0.01 compared with the sham group. ##P b 0.01 compared with the CLP group. L: low-dosage, 4 mg/kg, H: high-dosage, 8 mg/kg.
administration, which may cause, at least partly, the decrease of proinflammatory cytokines and contribute to the organ protection from sepsis. NF-κB is a ubiquitous transcription factor which is responsible for the transcription of a number of genes, including proinflammatory cytokines, chemokines, adhesion molecules, and other inducible enzymes [34,35]. In the NF-κB family there are five proteins—p65 (RelA), RelB, c-Rel, NF-κB1 (p105/p50), and NF-κB2 (p100/p52). Under a normal state, the inactive form of these proteins were inhibited by IκB-α, β, γ, and ε in the cytosol, and can be activated by various stimuli such as TNF-α, which establishes a vicious cycle. Importantly, early activation of NF-κB in organs such as the lung, liver and spleen positively correlates with the mortality in CLP-induced sepsis [36,37]. Inhibition of NF-κB showed to be beneficial for sepsis therapy [38,39]. In our observations, activated NF-κB in both pulmonary and hepatic tissues was suppressed by betulin administration. Although whether this effect is directly or indirectly unknown, it should be involved in the pharmacological mechanisms of the protective effect of betulin. In MAPK proteins, JNK and p38 are considered to be the stressactivated protein kinases. Although ERK has been implicated as a growth factor-activated kinase, it is still activated by some stresses [40,41]. Inhibition of MAPK activation was demonstrated to be beneficial in the anti-inflammatory effect and sepsis treatment of drugs [15,42,43]. In the present study, the over-activation of ERK, JNK and p38 was evidently inhibited by betulin treatment, which may be involved in the pharmacological mechanisms of betulin as well. In this study, we only found the effects of betulin on anti-inflammation, HMGB-1 secretion, and NF-κB and MAPK inhibition. The relationship between them has not been investigated further. NF-κB is known to be activated by HMGB-1 via ERK [44], through p38 mediated TNF-α release in the heart with ischemia/reperfusion [45], and these cross-talk mechanisms may also be involved in the process of sepsis. The detailed mechanisms of the protective effect against sepsis of betulin need to be studied in the future. In conclusion, betulin exerts effective protective effects on CLPinduced lung and liver injury and improved the survival of septic rats. This effect may be acted through decreasing proinflammatory cytokines and HMGB-1 productions. The suppression of the NF-κB and MAPK signaling pathway may be related with this protective effect as well. The present study provides a novel therapeutic strategy for sepsis treatment. Author contributions The experiment was designed by Hongyu Zhao and Min Zhao. The manuscript was written by Hongyu Zhao. The experiment was
performed by Hongyu Zhao, Zhenning Liu, Wei Liu and Xinfei Han. The data were analyzed by Hongyu Zhao and Zhenning Liu. Disclosure The authors reported no proprietary or commercial interest in any product mentioned or concept discussed in the article. Acknowledgements This study was supported by a grant from the National Key Clinical Specialties of the Ministry of Health of China (No.: 2012-649) and the Department of Science and Technology, Liaoning Province (No.: 2015020502). References [1] P. Damas, A. Reuter, P. Gysen, J. Demonty, M. Lamy, P. Franchimont, Tumor necrosis factor and interleukin-1 serum levels during severe sepsis in humans, Crit. Care Med. 17 (1989) 975–978. [2] C. Marty, B. Misset, F. Tamion, C. Fitting, J. Carlet, J.M. Cavaillon, Circulating interleukin-8 concentrations in patients with multiple organ failure of septic and nonseptic origin, Crit. Care Med. 22 (1994) 673–679. [3] L.C. Casey, R.A. Balk, R.C. Bone, Plasma cytokine and endotoxin levels correlate with survival in patients with the sepsis syndrome, Ann. Intern. Med. 119 (1993) 771–778. [4] G. Wakabayashi, J.A. Gelfand, W.K. Jung, R.J. Connolly, J.F. Burke, C.A. Dinarello, Staphylococcus epidermidis induces complement activation, tumor necrosis factor and interleukin-1, a shock-like state and tissue injury in rabbits without endotoxemia. Comparison to Escherichia coli, J. Clin. Invest. 87 (1991) 1925–1935. [5] J. Gu, P. Sun, H. Zhao, H.R. Watts, R.D. Sanders, N. Terrando, et al., Dexmedetomidine provides renoprotection against ischemia–reperfusion injury in mice, Crit. Care 15 (2011) R153. [6] O.O. Nduka, J.E. Parrillo, The pathophysiology of septic shock, Crit. Care Clin. 25 (2009) 677–702 vii. [7] I. Cinel, S.M. Opal, Molecular biology of inflammation and sepsis: a primer, Crit. Care Med. 37 (2009) 291–304. [8] C.I. Pro, D.M. Yealy, J.A. Kellum, D.T. Huang, A.E. Barnato, L.A. Weissfeld, et al., A randomized trial of protocol-based care for early septic shock, N. Engl. J. Med. 370 (2014) 1683–1693. [9] J. Zhou, C. Qian, M. Zhao, X. Yu, Y. Kang, X. Ma, et al., Epidemiology and outcome of severe sepsis and septic shock in intensive care units in mainland China, PLoS ONE 9 (2014), e107181. [10] S.D. Krau, Making sense of multiple organ dysfunction syndrome, Crit. Care Nurs. Clin. North Am. 19 (2007) 87–97. [11] J.L. Vincent, R. Moreno, J. Takala, S. Willatts, A. De Mendonca, H. Bruining, et al., The SOFA (Sepsis-related Organ Failure Assessment) score to describe organ dysfunction/failure. On behalf of the Working Group on Sepsis-Related Problems of the European Society of Intensive Care Medicine, Intensive Care Med. 22 (1996) 707–710. [12] S. Alakurtti, T. Makela, S. Koskimies, J. Yli-Kauhaluoma, Pharmacological properties of the ubiquitous natural product betulin, Eur. J. Pharm. Sci. 29 (2006) 1–13. [13] Q. Wu, H. Li, J. Qiu, H. Feng, Betulin protects mice from bacterial pneumonia and acute lung injury, Microb. Pathog. 75 (2014) 21–28.
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International Immunopharmacology journal homepage: www.elsevier.com/locate/intimp
Betulin attenuates lung and liver injuries in sepsis Hongyu Zhao ⁎, Zhenning Liu, Wei Liu, Xinfei Han, Min Zhao Department of Emergency Medicine, Shengjing Hospital of China Medical University, Shenyang 110004, People's Republic of China
a r t i c l e
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Article history: Received 21 July 2015 Received in revised form 13 November 2015 Accepted 22 November 2015 Available online 28 November 2015 Keywords: Sepsis Betulin Inflammation NF-κB MAPK
a b s t r a c t Sepsis is a complex condition with unacceptable mortality. Betulin is a natural extract with multiple bioactivities. This study aims to evaluate the potential effects of betulin on lung and liver injury in sepsis. Cecal ligation and puncture was used to establish the rat model of sepsis. A single dose of 4 mg/kg or 8 mg/kg betulin was injected intraperitoneally immediately after the model establishment. The survival rate was recorded every 12 h for 96 h. The organ injury was examined using hematoxylin and eosin staining and serum biochemical test. The levels of proinflammatory cytokines and high mobility group box 1 in the serum were measured using ELISA. Western blotting was used to detect the expression of proteins in NF-κB and MAPK signaling pathways. Betulin treatment significantly improved the survival rate of septic rats, and attenuated lung and liver injury in sepsis, including the reduction of lung wet/dry weight ratio and activities of alanine aminotransferase and aspartate aminotransferase in the serum. In addition, levels of tumor necrosis factor-α, interleukin-1β, interleukin-6 and high mobility group box 1 in the serum were also lowered by betulin treatment. Moreover, sepsis-induced activation of the NF-κB and MAPK signaling pathway was inhibited by betulin as well. Our findings demonstrate the protective effect of betulin in lung and liver injury in sepsis. This protection may be mediated by its anti-inflammatory and NF-κB and MAPK inhibitory effects. © 2015 Elsevier B.V. All rights reserved.
1. Introduction Sepsis, a serious condition with a very high mortality in intensive care, is characterized by a systemic uncontrolled hyper-inflammatory response, disseminated intravascular coagulation and multiple organ dysfunctions [1–5]. The pathogenesis of sepsis is complicated. Till now, the physiopathological mechanisms of sepsis have been studied well. Many mediators such as proinflammatory cytokines, chemokines and free radicals were found to be involved in the processes of sepsis [6,7]. However, despite the investigation of various therapeutic strategies to improve sepsis care, an effective compound has seldom been found. Antibiotics are still the main strategy in clinical care and the mortality of sepsis is still as high as 30% to 40% [8,9]. Multiple organ failure is one of the important reasons that cause death in septic patients [10]. Multiple-organ dysfunction syndrome (MODS) frequently occurs in severe sepsis and sepsis shock. The severity of organ dysfunction often determines the prognosis of sepsis [11]. As a systemic inflammatory response syndrome, in the process of sepsis, an inflammatory cascade was activated and excess proinflammatory cytokines were secreted into periphery blood and organs. Uncontrolled inflammation causes capillary permeability, endothelial damage, interstitial edema and hyperdynamic circulation, which subsequently lead to tissue hypoxia, metabolic failure and finally MODS [10]. ⁎ Corresponding author at: Department of Emergency Medicine, Shengjing Hospital of China Medical University, 36 Sanhao Street, Shenyang, 110004, People's Republic of China. E-mail address: [email protected] (H. Zhao).
http://dx.doi.org/10.1016/j.intimp.2015.11.028 1567-5769/© 2015 Elsevier B.V. All rights reserved.
Therefore, compounds that have an effective anti-inflammatory profile and target multiple-organs would be beneficial in sepsis therapy. Betulin (lup-20(29)-ene-3β, 28-diol) is a triterpene extracted from birch tree bark. It can be converted to betulinic acid by chemosynthesis or biotransformation. The three active positions in their structure empower these two compounds with various pharmacological activities, such as antitumor, anti-HIV, anti-inflammatory, antiviral and antibacterial activities [12]. Moreover, previous studies have shown that betulin could decrease LPS-induced inflammation by preventing nuclear factor kappa-B (NF-κB) activation [13]. Betulin also inhibited the activation of p38 and c-Jun N-terminal kinase (JNK) transduction pathways in ethanol-induced liver stellate cells [14]. These two signaling pathways have high correlations of inflammation and cell stress. In addition, they were found to be activated in sepsis, and pharmacological inhibition of these two pathways was involved in the anti-inflammatory effects [15,16]. In the present study, a sepsis model was established by cecal ligation and puncture (CLP) and the potential effect of betulin on lung and liver injury in sepsis was evaluated. 2. Materials and methods 2.1. Animals A total of 92 male Sprague–Dawley rats (8 weeks old) were supplied by the Experimental Animal Centre of China Medical University (Shenyang, China). The animals were housed in cages located in a room with stable temperature (22–24 °C) and a 12/12 h light/dark cycle and
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received water and food ad libitum. All animal protocols were approved by the ethics committee of China Medical University (Shenyang, China).
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high mobility group box 1 (HMGB-1) were determined using commercial enzyme-linked immunosorbent assay (ELISA) kits specific for rat following the manufacturer's instructions (USCN, Wuhan, China).
2.2. Induction of sepsis 2.7. Protein preparation Sepsis was induced by CLP according to a method described previously [17]. Briefly, after fasting for 12 h, rats were anesthetized with 3.5 ml/kg chloral hydrate. A 1.5 cm midline incision was performed and the cecum was exposed and ligated at the root. The cecum was punctured three times using a 5 ml syringe needle. Light pressure was applied to expel a small amount of fecal material. The cecum was then returned and the wound was closed. All the animals received 3 ml/100 g saline subcutaneously immediately after the operation for fluid resuscitation. Rats in the sham group received only cecum exposure but not ligation or puncture on the cecum.
Lung and liver tissues were homogenized in icy NP-40 lysis buffer supplement with 1% Triton X-100 and 1 mM phenylmethanesulfonylfluoride. Tissue homogenate was centrifuged at 12,000 g for 10 min and the supernatants were collected. Nuclear and cytosolic proteins were separated using a Nuclear and Cytoplasmic Protein Extraction Kit (Beyotime Institute of Biotechnology, Haimen, China) following the manufacturer's protocol. Protein concentration was determined using a bicinchoninic acid protein assay kit (Beyotime). 2.8. Western blot analysis
2.3. Betulin treatment and mortality experimental protocols Rats were randomly divided into 5 groups: (1) sham group (n = 22); (2) CLP group (n = 24); (3) CLP + dexamethasone 2 mg/kg group (DEX) (n = 24); (4) CLP + betulin 4 mg/kg group (Betulin L) (n = 24); and (5) CLP + betulin 8 mg/kg group (Betulin H) (n = 22). Rats were administrated intraperitoneally with DEX (Tianjinxinzheng, Tianjin, China) or betulin (Meilun, Dalian, China, purity N 98%) dissolved in saline immediately after the model establishment. Rats in the sham and CLP groups received only saline. The choice of administration time and dosage was based on our primary experiments. Ten rats in each group were randomly selected to record the survival rate. Rats were monitored every 12 h for 96 h and the numbers of surviving rats were recorded. In the remaining 52 rats, one in the CLP group and one in the Betulin L group died at 24 h after the operation. One rat in the CLP group and one in the Betulin L group were randomly selected to perform primary experiments. Six rats in each group were randomly selected to measure the wet and dry weight of lungs at 24 h after treatment. The last six rats in each group were used to perform plasmic, biochemical and molecular biological assays.
An equal amount of protein (40 μg) for each sample was separated in the SDS-PAGE and transferred onto the polyvinylidene fluoride membranes (Millipore, Billerica, MA, USA) using the wet transfer method. Membranes were blocked in 5% non-fat milk for 1 h at room temperature and incubated in primary antibodies at 4 °C overnight. After a wash stage using Tris buffered saline, with Tween-20 (TBST), membranes were incubated in horseradish peroxidase conjugated goat anti-rabbit or goat anti-mouse IgG (1:5000, Beyotime) for 45 min at room temperature. Finally, membranes were immersed in enhanced electrochemiluminescence reagent (7 Sea Pharmtech, Shanghai, China) and exposed using X-ray film. Primary antibodies against extracellular regulated protein kinases (ERK) (bs-2637R), p-ERK (bs-1522R), JNK (bs-10562R), p-JNK (bs-1640R), p38 (bs-0637R), p-p38 (bs-5477R), inhibitors of NF-κBα (IκBα) (bs-1287R), and p-p65 (bs-0982R) from Bioss, Beijing, China, and p65 (BA0610) from Boster, Wuhan, China were used and the protein levels were standardized to β-actin (sc-47,778, Santa Cruz Biotechnology, Santa Cruz, CA, USA) for whole cell lysate and Histone H3 (bs-17422R, Bioss) for the nuclear fraction. 2.9. Statistical analysis
2.4. Measurement of the lung wet/dry weight ratio Lungs were weighed immediately after being isolated and dried in a microwave oven at 80 °C to constant weight. The wet/dry weight ratio was calculated.
Data were expressed as mean ± standard deviation (SD). Survival data were analyzed by the Kaplan–Meier curve and log-rank test. Other data were analyzed by one-way analysis of variance, and the Fisher's least significant difference test was used for post hoc comparisons. P value less than 0.05 was considered statistically significant.
2.5. Histological analysis 3. Results Twenty-four hours after betulin treatment, tissues of lung and liver were harvested and fixed with 4% paraformaldehyde overnight. The samples were then dehydrated in ascending grades of alcohols, embedded in paraffin, and cut into 5-μm sections. To perform hematoxylin and eosin (H&E) staining, sections were immersed in xylene for 15 min and hydrated through graded ethanol. Then sections were stained with hematoxylin (Solarbio, Beijing, China) for 5 min and with eosin (Solarbio) for 3 min. After being dehydrated with increasing concentrations of ethanol and cleared in xylene, sections were mounted in Permount and observed under an optic microscope (DP73, Olympus, Tokyo, Japan). 2.6. Serum analysis Blood sample was obtained 24 h after betulin treatment. Whole blood was centrifuged at 7700 g for 10 min at 4 °C. Serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were determined using commercial assay kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, China) according to the manufacturer's instructions. Serum tumor necrosis factor-α (TNF-α), interleukin (IL)-1β, IL-6 and
3.1. Effect of betulin on survival rate in CLP rats All the rats in the sham group survived at the end of monitoring. In the vehicle-treated CLP group, the survival rate for 48 h was 40% and reached 0% at 96 h. No rat survived at the end of the experiment in this group. Betulin 4 mg/kg reduced the mortality to some extent but was not significant. The survival rate was 70% at 48 h and 30% at 96 h. DEX 2 mg/kg and betulin 8 mg/kg significantly improved the survival of septic rats, compared with the CLP group (P b 0.01). In the DEX group, the survival rate was 90% at 48 h and 80% at 96 h. In the betulin H group, the survival rate was 80% at 48 h and 60% at 96 h (Fig. 1). Although the survival rate in the DEX group was higher than that in the betulin H group, there is no significant difference (P N 0.05). 3.2. Effect of betulin on CLP-induced lung and liver injuries H&E staining illustrated the normal cell structures of the lung and liver in the sham operated rats (Fig. 1A). After CLP operation for 24 h, lung tissue showed marked inflammatory cell infiltration, edema,
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were markedly attenuated by DEX 2 mg/kg or betulin 8 mg/kg administration. In line with the histological observation, the dramatically increased lung wet/dry weight ratio showed that pulmonary edema occurred in the CLP rats (Fig. 2B). DEX 2 mg/kg and betulin 8 mg/kg evidently reduced the wet/dry weight ration of CLP rats (P b 0.01 vs. CLP group). Serum ALT and AST activities, representative markers of liver injury, were determined in this study. Twenty-four hours after CLP, activities of ALT and AST in serum were dramatically increased (Fig. 2C and D). DEX 2 mg/kg and betulin 8 mg/kg attenuated elevations in serum ALT and AST activities that occurred at 24 h after CLP (P b 0.01 vs. CLP group). The effects of DEX 2 mg/kg and betulin 8 mg/kg were similar.
3.3. Effect of betulin on CLP-induced inflammatory cytokine production and HMGB1 release
Fig. 1. Effect of betulin on the survival rate of septic rats. DEX 2 mg/kg and betulin 8 mg/kg significantly increased the survival rate of septic rats. n = 10. **P b 0.01 compared with the sham group. ##P b 0.01 compared with the CLP group. DEX: dexamethasone, L: low-dosage, 4 mg/kg, H: high-dosage, 8 mg/kg.
pulmonary interstitial hyperemia, hemorrhage, and alveolar collapse. In the liver tissue, cell boundary was blunted and inflammation and hepatocellular necrosis was found. These pathological alterations
Twenty-four hours after CLP, marked elevations of TNF-α, IL-1β and IL-6 levels were found in the serum compared with that in the sham group (Fig. 3A–C, P b 0.01). Betulin 8 mg/kg significantly suppressed these increases. Betulin 4 mg/kg slightly decreased the levels of cytokines but did not reach significant levels. The serum level of HMGB1 increased dramatically at 24 h after CLP as well (Fig. 3D, P b 0.01 vs. sham group). DEX 2 mg/kg and betulin 8 mg/kg restored this change. The effect of DEX 2 mg/kg was equivalent to that of betulin 8 mg/kg.
Fig. 2. Effect of betulin on lung and liver injury in septic rats. (A) Hematoxylin–eosine stained lung and liver sections of septic rats. CLP induced inflammatory cell infiltration, edema, pulmonary interstitial hyperemia, hemorrhage, and alveolar collapse in the lung and hepatocellular necrosis in the liver. Betulin treatment restored these changes. Betulin administration also decreased wet/dry weight ratio of lungs (B) and reduced ALT (C) and AST (D) level in the serum of septic rats. The effect of betulin 8 mg/kg was equivalent to DEX 2 mg/kg. Scale bar: 100 μm. Data are expressed as mean ± standard deviation (SD). **P b 0.01 compared with the sham group. ##P b 0.01 compared with the CLP group. DEX: dexamethasone, L: low-dosage, 4 mg/kg, H: high-dosage, 8 mg/kg.
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Fig. 3. Effect of betulin on serum levels of proinflammatory-cytokines and HMGB-1 in septic rats. DEX 2 mg/kg and betulin 8 mg/kg significantly reduced the release of TNF-α (A), IL-1β (B), IL-6 (C) and HMGB-1 (D) into the serum. Data are expressed as mean ± standard deviation (SD). **P b 0.01 compared with the sham group. ##P b 0.01 compared with the CLP group. DEX: dexamethasone, L: low-dosage, 4 mg/kg, H: high-dosage, 8 mg/kg.
3.4. Effect of betulin on CLP-induced NF-κB signaling activation in the lung and liver As shown in Figs. 4 and 5, a marked reduction of cytosolic IκBα protein expression in both lung and liver was found at 24 h after CLP (A), thereby, NF-κB/p65 was phosphorylated and NF-κB/p65 nuclear accumulation was increased (B and C). After betulin treatment, rats in the 8 mg/kg group effectively suppressed these changes. In the 4 mg/kg group, except on the NF-κB nuclear expression, the effect of betulin did not show any significance.
3.5. Effect of betulin on CLP-induced mitogen-activated protein kinase (MAPK) activation in the lung and the liver In the lung, three components of MAPK, ERK, JNK and p38 were over-phosphorylated at 24 h after CLP compared with the sham group (Fig. 6, P b 0.01). Betulin 4 mg/kg reduced phosphor-ERK and p38 expressions but not JNK. Betulin 8 mg/kg suppressed phosphorylation of all the three MAPK proteins. In the liver, consistent with that in the lung, all the three proteins were highly phosphorylated 24 h after CLP (Fig. 7). Betulin 4 and 8 mg/kg dose-dependently suppressed these phosphorylations.
4. Discussion Sepsis is a severe clinical syndrome with complicated pathological mechanisms. The systemic inflammatory and immune responses during the development of sepsis involve numerous factors. Natural extracts which have various bioactivities and multiple target organs may be more effective in sepsis treatment than synthetic drugs that generally have specific targets. In the present study, we demonstrated that betulin showed a markedly protective effect against sepsis, as represented by the increasing survival rate of septic rats induced by CLP and attenuating lung and liver injury. The effect was equivalent to that of positive drug DEX. This protective effect of betulin was mediated by its antiinflammatory effect and HMGB-1 inhibition. In addition, the NF-κB and MAPK suppression was involved in the protective mechanisms of betulin as well. When the infection develops beyond the compensatory ability of the body, the function of distinct organ systems are compromised. In general, the respiratory system is the first system where failure happens, followed by hepatic, renal, cardiac and other systems. The number of failing organs is closely related with the mortality of septic patients. In the present study, we examined lung and liver injury by H&E staining. Consistent with previous studies [18,19], these two organs were injured 24 h after model establishment. In addition, increased activities of
Fig. 4. Effect of betulin on NF-κB activation in the lung of septic rats. Betulin markedly upregulated expression of IκBα (A), inhibited the phosphorylation of p65 (B), and prevented p65 nuclear accumulation (C) in the lung. Data are expressed as mean ± standard deviation (SD). **P b 0.01 compared with the sham group. ##P b 0.01 compared with the CLP group. L: low-dosage, 4 mg/kg, H: high-dosage, 8 mg/kg.
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Fig. 5. Effect of betulin on NF-κB activation in the liver of septic rats. Betulin markedly upregulated expression of IκBα (A), inhibited the phosphorylation of p65 (B), and prevented p65 nuclear accumulation (C) in the liver. Data are expressed as mean ± standard deviation (SD). **P b 0.01 compared with the sham group. ##P b 0.01 compared with the CLP group. L: low-dosage, 4 mg/kg, H: high-dosage, 8 mg/kg.
ALT and AST also reflected the pathological change of liver. These pathological alterations were significantly attenuated by betulin treatment. This multi-organ functional protective effect of betulin was very likely to contribute to the survival improvement. Runaway inflammatory response plays a key role in the development of sepsis. Therefore, effective inflammatory control is an important part in sepsis treatment. Inflammatory response in sepsis is mainly represented by the over-production of proinflammatory cytokines such as TNF-α, IL-1β and IL-6, which are considered to be the characteristic of early sepsis. In addition, reducing the secretion of these cytokines delayed the development of sepsis and decreased the mortality of septic patients or animal models [20–22]. In the present study, in agreement with previous studies, markedly elevated TNF-α, IL-1β and IL-6 levels were found in the serum of septic rats. Although we have not observed the time-dependent changes of these cytokines, and according to the previous reports, the peak of secretion does not occur at 24 h after CLP, we still can use these cytokines to evaluate the anti-inflammatory effect of betulin. Betulin has been reported to have the ability to attenuate secretion of inflammatory cytokines [13,23,24]. In line with these studies, our results demonstrated that betulin treatment significantly reduced the levels of the three cytokines
in the serum. In addition, we compared DEX treatment, a typical anti-inflammatory drug, with betulin intervention. We found that these two compounds have the same anti-inflammatory effect in sepsis. These results suggest that the anti-inflammation properties of betulin are involved in its anti-sepsis effect. In addition to these proinflammatory cytokines, some other cytokines have also been found to play important roles in sepsis. HMGB1 was originally considered as a highly conserved structural DNA-binding protein that is responsible for DNA replication [25–27]. In 1999, Kevin Tracey and colleagues reported the cytokine-like properties of HMGB1. This and the following studies found that the serum level of HMGB1 is increased at late time points after endotoxin exposure. Overproduced HMGB1 can trigger the release of the above proinflammatory cytokines and exacerbate the inflammation. Targeting HMGB1 may be effective in treating related diseases [28–30]. In sepsis, HMGB1 is well accepted as a late mediator of inflammation. It has been found to be elevated in patients with severe sepsis [31,32]. In a rodent experimental model of abdominal sepsis, anti-HMGB1 antibody treatment significantly increased the survival rate of septic animals and attenuated CLP-induced diaphragmatic dysfunction [33]. In the present study, the elevated HMGB1 level was lowered by betulin
Fig. 6. Effect of betulin on MAPK activation in the lung of septic rats. (A) Typical blots of proteins of MAPK. Betulin evidently inhibited the phosphorylations of ERK (B), JNK (C) and p38 (D) in the lung. Data are expressed as mean ± standard deviation (SD). **P b 0.01 compared with the sham group. ##P b 0.01 compared with the CLP group. L: low-dosage, 4 mg/kg, H: high-dosage, 8 mg/kg.
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Fig. 7. Effect of betulin on MAPK activation in the liver of septic rats. (A) Typical blots of proteins of MAPK. Betulin evidently inhibited the phosphorylations of ERK (B), JNK (C) and p38 (D) in the liver. Data are expressed as mean ± standard deviation (SD). **P b 0.01 compared with the sham group. ##P b 0.01 compared with the CLP group. L: low-dosage, 4 mg/kg, H: high-dosage, 8 mg/kg.
administration, which may cause, at least partly, the decrease of proinflammatory cytokines and contribute to the organ protection from sepsis. NF-κB is a ubiquitous transcription factor which is responsible for the transcription of a number of genes, including proinflammatory cytokines, chemokines, adhesion molecules, and other inducible enzymes [34,35]. In the NF-κB family there are five proteins—p65 (RelA), RelB, c-Rel, NF-κB1 (p105/p50), and NF-κB2 (p100/p52). Under a normal state, the inactive form of these proteins were inhibited by IκB-α, β, γ, and ε in the cytosol, and can be activated by various stimuli such as TNF-α, which establishes a vicious cycle. Importantly, early activation of NF-κB in organs such as the lung, liver and spleen positively correlates with the mortality in CLP-induced sepsis [36,37]. Inhibition of NF-κB showed to be beneficial for sepsis therapy [38,39]. In our observations, activated NF-κB in both pulmonary and hepatic tissues was suppressed by betulin administration. Although whether this effect is directly or indirectly unknown, it should be involved in the pharmacological mechanisms of the protective effect of betulin. In MAPK proteins, JNK and p38 are considered to be the stressactivated protein kinases. Although ERK has been implicated as a growth factor-activated kinase, it is still activated by some stresses [40,41]. Inhibition of MAPK activation was demonstrated to be beneficial in the anti-inflammatory effect and sepsis treatment of drugs [15,42,43]. In the present study, the over-activation of ERK, JNK and p38 was evidently inhibited by betulin treatment, which may be involved in the pharmacological mechanisms of betulin as well. In this study, we only found the effects of betulin on anti-inflammation, HMGB-1 secretion, and NF-κB and MAPK inhibition. The relationship between them has not been investigated further. NF-κB is known to be activated by HMGB-1 via ERK [44], through p38 mediated TNF-α release in the heart with ischemia/reperfusion [45], and these cross-talk mechanisms may also be involved in the process of sepsis. The detailed mechanisms of the protective effect against sepsis of betulin need to be studied in the future. In conclusion, betulin exerts effective protective effects on CLPinduced lung and liver injury and improved the survival of septic rats. This effect may be acted through decreasing proinflammatory cytokines and HMGB-1 productions. The suppression of the NF-κB and MAPK signaling pathway may be related with this protective effect as well. The present study provides a novel therapeutic strategy for sepsis treatment. Author contributions The experiment was designed by Hongyu Zhao and Min Zhao. The manuscript was written by Hongyu Zhao. The experiment was
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