Microbial Pathogenesis 75 (2014) 21e28

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Betulin protects mice from bacterial pneumonia and acute lung injury Qianchao Wu a, 1, Hongyu Li b, 1, Jiaming Qiu a, Haihua Feng a, * a b

Key Laboratory of Zoonosis, Ministry of Education, College of Veterinary Medicine, Jilin University, Changchun, Jilin 130062, PR China Experimental Base of Agriculture, Jilin University, Changchun, Jilin 130062, PR China

a r t i c l e i n f o

a b s t r a c t

Article history: Received 2 July 2014 Received in revised form 15 August 2014 Accepted 18 August 2014 Available online 27 August 2014

Betulin, a naturally occurring triterpene, has shown anti-HIV activity, but details on the antiinflammatory activity are scanty. In this study, we sought to investigate the effect of Betulin on LPSinduced activation of cell lines with relevance for lung inflammation in vitro and on lung inflammation elicited by either LPS or viable Escherichia coli (E. coli) in vivo. In vitro, Betulin inhibited LPS-induced tumor necrosis factor a (TNF-a) and (interleukin) IL-6 levels and up-regulated the level of IL-10. Also Betulin suppressed the phosphorylation of nuclear factor-kB (NF-kB) p65 protein in LPS-stimulated RAW 264.7 cells. In vivo, Betulin alleviated LPS-induced acute lung injury. Treatment with Betulin diminished pro-inflammatory cytokines, myeloperoxidase activity and bacterial loads in lung tissue during gramnegative pneumonia. Our findings demonstrated that Betulin inhibits pro-inflammatory responses induced by the gram-negative stimuli LPS and E. coli, suggesting that Betulin may represent a novel strategy for the treatment of lung inflammation. © 2014 Elsevier Ltd. All rights reserved.

Keywords: Betulin Macrophages Cytokines Bacterial pneumonia Acute lung injury

1. Introduction Gram-negative bacterial pneumonia is a leading cause of mortality in the United States and worldwide [1,2]. When antibiotics became available, mortality rates decreased from 77% to 28% [3]. However, since then, mortality has not decreased dramatically, despite increasing medical aptitude in the following decades [4]. One explanation for the persistent adverse outcomes for pneumonia is the gram-negative pathogen-initiated inflammatory response that can spiral out of control and lead to acute lung injury (ALI) or the acute respiratory distress (ARDS) [5]. Lipopolysaccharide (LPS), a major constituent of the outer cell wall of gramnegative bacteria, is the predominant mediator of inflammatory responses to these microorganisms [6]. Inflammation can be mediated by inflammatory mediators, which includes the reactive oxygen species, pro-inflammatory cytokines, and chemotactic factors [7,8]. The recruitment of proinflammatory cytokines to the bronchoalveolar space is a key element of the acute inflammatory response in the lung. Among these pro-inflammatory cytokines, such as (interleukin) IL-6, IL-1b and (tumor necrosis factor a) TNF-a, are regulated by a nuclear

* Corresponding author. Institute of Zoonosis, Department of Veterinary Pharmacology, College of Veterinary Medicine, Jilin University, Changchun, Jilin 130062, PR China. Tel.: þ86 431 87836161; fax: þ86 431 87836160. E-mail address: [email protected] (H. Feng). 1 These authors contributed equally to this work. http://dx.doi.org/10.1016/j.micpath.2014.08.005 0882-4010/© 2014 Elsevier Ltd. All rights reserved.

factor-kB (NF-kB), but they also activate NF-kB [9,10]. Thus, these mediators arouse the innate immune response, but their overexpression leads to organ failure, tissue injury and even death [11]. Regulating expression of these inflammatory mediators can, therefore, be beneficial in decreasing inflammatory diseases. Thus, for successful resolution of infection, the application of Chinese herbal medicine has recently become a focus of interest [12,13]. Betulin (Fig. 1), is an abundant naturally occurring triterpene in the bark of Betula platyphylla Suk. Betulin has three active positions in its structure, secondary hydroxyl group at C-3, primary hydroxyl group at C-28 and a double CeC bonding at C-20, where chemical modulations can be performed in order to obtain different types of derivatives [14]. As a precursor, Betulin could be easily converted to Betulinic acid by chemosynthesis or biotransformation [15]. Both of them have a wide spectrum of biological and pharmacological activities such as: anti-HIV, and cytotoxicity against a variety of tumor cell lines [15e17]. The protective effect of Betulin against TPA-induced model of inflammation in mice has been noted [18]. However, the detailed mechanism of antiinflammatory activities of Betulin is unclear. As proof of principle, we sought to investigate the protective effect of Betulin against lung inflammation induced by LPS and Escherichia coli (E. coli). In the present study, we demonstrate that Betulin has potent antiinflammatory effects on cells which are important for innate immune response of the pulmonary compartment. In vivo, Betulin treatment reduced acute lung inflammation induced by purified LPS and E. coli.

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Fig. 1. Molecular structure of Betulin.

2. Materials and methods 2.1. Chemicals and reagents Betulin was purchased from the National Institute for the Control of Pharmaceutical and Biological Products (Jilin, China). Dimethyl sulfoxide (DMSO), LPS (E. coli 055:B5), and 3-(4, 5dimethylthiazol-2-y1)-2, 5-diphenyltetrazolium bromide (MTT) were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Dulbecco's modified Eagle's medium (DMEM), Fetal bovine serum (FBS), penicillin and streptomycin for cell culture use were obtained from Invitrogen-Gibco (Grand Island, NY). Mouse TNFea, IL-6, IL-1b and IL-10 enzyme-linked immunosorbent assay (ELISA) kits were purchased from Biolegend (CA, USA). The myeloperoxidase (MPO) determination kit was provided by the Jiancheng Bioengineering Institute of Nanjing (Jiangsu, China). Antibodies against phospoIkB, IkB, phospo-p65 and p65 were obtained from Cell Signaling Technologies (Beverly, MA). Secondary antibodies and b-actin were obtained from Tianjin Sungene Biotech Co., Ltd (Tianjin, China). All other chemicals were of reagent grade. 2.2. Animals C57BL/6J male mice aged 8 weeks were obtained from the Experimental Animal Center of Jilin University. Animal experiments were approved by and conducted in accordance with the guidelines of the Animal Care and Use Committee of Jilin University. 2.3. Cell line experiments The RAW 264.7 mouse macrophage cell line was obtained from the China Cell Line Bank (Beijing China). Cells were cultured in DMEM supplemented with 3 mM glutamine, antibiotics (100 U/mL penicillin and 100 U/mL streptomycin), and 10% heat-inactivated FBS and maintained at 37  C in a humidified incubator containing 5% CO2. To investigate the effect of Betulin on cytokine responses from LPS-treated cells, RAW 264.7 cells (4  105) were seeded into 24well plates and pretreated with 2, 4, or 8 mg/ml Betulin for 1 h prior to treatment with 1 mg/ml of LPS for 6, 12, or 24 h. Cell free supernatants were harvested for ELISA. Cell viability was assessed by the MTT assay. RAW 264.7 cells were plated at a density of 4  105 cells/mL onto 96-well plates containing 100 mL of DMEM, and incubated in a 37  C, 5% CO2 incubator for 1 h. Then the cells were treated with 50 mL of Betulin of different concentrations (2e8 mg/mL) for 1 h, followed by stimulation with 50 mL LPS (1 mg/ mL). After 18 h of LPS stimulation, 20 mL MTT (5 mg/mL) was added to each well, and the cells were further incubated for an additional 4 h. The supernatant was removed and cells were lysed with 150 mL/well DMSO. The optical density was measured at 570 nm on a microplate reader (TECAN, Austria).

To determine effects of Betulin on LPS-induced NF-kB pathway, we examined the effect of Betulin on translocation of NF-kB (p65) into the nucleus by Western blotting. RAW 264.7 cells were plated onto 6-well plates and incubated for 24 h, then pretreated with Betulin for 1 h. After LPS (1 mg/ml) stimulation for 30 min, the cells were collected and washed twice with cold PBS. The cells were lysed in lysis buffer containing 50 mM Tris (pH 7.6), 150 mM NaCl, 5 mM EDTA (pH 8.0), 0.6% NP-40, 1 mM Na3VO4, 20 mM b-glycerophosphate, 1 mM phenylmethylsulfonyl fluoride, 2 mM p-nitrophenyl phosphate, and 1:25 Complete Mini Protease Inhibitor cocktail (Boehringer, Mannheim, Germany) and maintained on ice for 30 min. The cell lysates were washed via dilution and centrifugal concentration. The total protein of RAW 264.7 cells was extracted by Thermo Scientific MPER Mammalian Protein Extraction Reagent. The protein concentrations were determined using a BCA protein assay kit (Beyotime, China). Aliquots of the lysates were separated on 12% SDS-polyacrylamide gel and transferred onto a polyvinylidene fluoride (PVDF) membrane (BIO-RAD) with a glycine transfer buffer [192 mM glycine, 25 mM TriseHCl (pH 8.8), 20% methanol (v/v)]. After blocking the nonspecific site with blocking solution (5% (w/v) nonfat dry milk), the membrane was incubated overnight with the indicated antibodies against NF-kB p65, b-actin, and IkB proteins, phospho-specific antibodies to NF-kB p65 and IkB proteins at 4  C. The membrane was then washed three times for 5 min each with TBST and a 1:7000 (v/v) dilution of horseradish peroxidase-labeled IgG was added at room temperature for 1 h. Antibody binding was visualized with an enhanced chemiluminescence (ECL) Western blotting detection system. The b-actin western blot was performed as an internal control of protein loading. 2.4. Induction of acute lung injury and pneumonia The mice were randomly divided into four groups: control; LPS; LPS þ Betulin (4 or 8 mg/kg) and there are 8 mice in each group. LPS-induced ALI was induced as previously described [19]. Briefly, mice were anesthetized and challenged with intranasal 10 mg of LPS in 50 ml PBS. Control mice were given 50 ml PBS i.n. instillation without LPS. Betulin at 4 and 8 mg/kg was i.p. injected 1 h prior to LPS administration. For lung infection, E. coli (American Type Culture Collection 25922) was grown in tryptone soy broth (TSB) at 37  C overnight under conditions of constant agitation. Bacteria were harvested by centrifugation at 2900g for 15 min and washed twice in sterile isotonic saline solution, and resuspended in sterile 0.9% saline solution at a concentration of 20  106 CFU(colonyforming units)/50 mL. Mice were anesthetized intraperitoneally with ketamine and xylazine and 20  106 CFUs were instilled intranasally. To investigate the effects of Betulin treatment, mice were administered 200 mL of Betulin (8 mg/kg) subcutaneously 2 h after infection with E. coli and then at 12-h intervals. The control mice were treated with 200 mL of sterile PBS on the same schedule. 2.5. Bronchoalveolar lavage fluid (BALF) collection and analysis The collection of BALF was performed three times through a tracheal cannula with 0.5 ml of autoclaved PBS, instilled up to a total volume of 1.3 ml. The fluid recovered from each sample was centrifuged (4  C, 3000 rpm, 10 min) to pellet the cells. The cell pellets were re-suspended in PBS for total cell counts using a hemacytometer, and cytospins were prepared for differential cell counts by staining with the WrightGiemsa staining method. At least 200 cells were counted per slide. The concentrations of cytokine TNF-a, IL-6 and IL-10 in the supernatants of the BALF were measured by ELISA using

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commercially available reagents according to the manufacturer's instructions.

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3. Results 3.1. Effect of Betulin on the cytokines and NF-kB activation in vitro

2.6. Lung wet-to-dry weight (W/D) ratio 24 h after intranasal instillation of LPS, the lungs were excised. Each lung was blotted dry, weighed, and then placed in an oven at 80  C for 48 h to obtain the “dry” weight. The ratio of the wet lung to the dry lung was calculated to assess tissue edema. 2.7. Determination of bacterial load After mice were euthanized, whole lungs were harvested and homogenized in four volumes of sterile saline with a tissue homogenizer (ProScience, Oxford, CT, USA). CFUs were determined from serial dilutions of the samples, plated on tryptic soy agar (TSA) plates and incubated at 37  C for 16 h before colonies were counted. 2.8. Preparation of homogenates For cytokine measurements, right lungs were excised, weighed and diluted 1:4 in sterile saline. After homogenization, samples were diluted with one volume of lysis buffer containing 300 mmol/ L NaCl, 30 mmol/L Tris, 2 mmol/L MgCl2, 2 mmol/L CaCl2, 2% Triton X-100 and AEBSF [4-(2- aminoethyl)benzeensulfonyl fluoride, EDTA-Na2, pepstatin and leupeptin] (all 8 mg/mL, pH 7.4; SigmaeAldrich) and incubated at 4  C for 30 min. Homogenates were centrifuged at 2900g at 4  C for 15 min, and supernatants were stored at 20  C until assays were performed. 2.9. Myeloperoxidase (MPO) activity assay Neutrophil and macrophage parenchymal infiltration, as reflected by MPO activity, was measured as described previously [20]. Whole lungs were weighed, frozen at 70  C, and then homogenized in hydroxyethyl piperazine ethanesulfonic acid (HEPES) (pH 8.0) containing 0.5% cetyltrimethyl ammonium bromide (CTAB) and subjected to three freezeethaw cycles. The homogenate was centrifuged (4  C, 13,000  g, 30 min) and the cell-free extracts were stored at 20  C until further use. The MPO activity was assayed using a mouse MPO ELISA kit. Samples were diluted in phosphate citrate buffer (pH 5.0) and the absorbance of the sample was measured at 460 nm using a microplate reader. The specific activity of MPO in the lung is expressed as U/g of the tissue. 2.10. Histological examination For histological examination, lungs were harvested at 24 h of bacterial infection. These samples were fixed with 10% buffered formalin, embedded in paraffin, sliced and then stained with hematoxylin and eosin (H&E). A quantitative morphometric analysis of interstitial damage, endothelialitis, peribronchitis, edema, thrombus formation and pleuritis was performed, as has been described previously [21]. Each item was scored 0 (absent) to 4 (very severe) and then calculated for a total score of lung injury [21].

Our in vitro studies in RAW 264.7 cells demonstrated that, in the absence of Betulin, LPS challenge (1 mg/mL) resulted in a burst of TNF-a (Fig. 2A) and a modest peak in IL-6 (Fig. 2B) and IL-10 (Fig. 2C) concentration, both occurring at 24 h. The concentrations of IL-6 (Fig. 2B) in the culture supernatants of RAW 264.7 cells increased steadily over the 12 h period and decreased by 24 h. Pretreatment with Betulin prior to LPS induction attenuated TNF-a (Fig. 2A) and IL-6 (Fig. 2B) levels in a concentration-dependent manner. The concentration of IL-10 was markedly increased at 24 h in groups treated with Betulin and showed significant change as compared to the LPS group (Fig. 2C). We also evaluated the effect of Betulin on LPS-induced activation of the NF-kB pathway and found that the phosphorylation of p65 NF-kB in RAW 264.7 cells increased after LPS administration but was significantly inhibited by Betulin in a concentration dependent manner (Fig. 2D). As shown in Fig. 2E, LPS induced phosphorylation of IkB was also strongly attenuated by Betulin. Viability was determined by MTT assay and results showed that Betulin did not display any cytotoxicity against RAW 264.7 cells at concentrations ranging from 2 to 8 mg/ml. These results demonstrated that the viability of these cells was not affected by Betulin (Fig. 2F). Thus, the effects of Betulin could not be attributed to cytotoxic effects. 3.2. Betulin reduces LPS-induced ALI After demonstrating that Betulin reduced inflammatory mediator production in vitro, we applied Betulin in vivo in LPS-induced lung inflammation. After 24 h of LPS inflammation, the number of PMNs and macrophages in BALF significantly increased as compared to the control group. Betulin treatment for 1 h prior LPS administration markedly blocked PMN infiltration and led to a significant lowering of the number of macrophages (Fig. 3A, B, C). To measure pulmonary edema, lung W/D Ratio was measured. After 24 h, treatment with Betulin significantly inhibited edema of the lung by 7.8%e10.6% (Fig. 3G). A strong effect of treatment was observed on cytokines levels in the lung. Levels of the cytokines TNF-a and IL-6 were dramatically increased, while IL-10 was only slightly increased compared with control group. Pretreatment with Betulin (4 or 8 mg/kg) upregulated the IL-10 level and down-regulated TNF-a and IL-6 levels in a dose-dependent manner (Fig. 3D, E, F). 3.3. Betulin enhances bacterial clearance in a model of E. coli pneumonia The experiments described above revealed that pretreatment with Betulin attenuates acute lung injury induced by intranasal delivery of LPS, as reflected by reductions in PMN counts, W/D ratio and cytokine levels in BAL fluid or in lung. We were interested in determining the effect of Betulin on bacterial burden in this model of E. coli pneumonia. Lungs were harvested 24 h after E. coli instillation and CFUs were determined. The CFUs 24 h postinfection from mice that were treated with 8 mg/kg Betulin were significantly lower than the CFUS in the E. coli group (P ¼ 0.013; Fig. 4A).

2.11. Statistical analysis All values were expressed as means ± SEM. Differences between mean values of normally distributed data were assessed with one-way ANOVA ((Dunnett's t-test) and two-tailed Student's t-test. Statistical significance was accepted at P < 0.05 or P < 0.01.

3.4. Betulin blocks leukocyte recruitment to the lung in E. coli pneumonia To assess PMN accumulation in lung tissue, MPO levels were determined. E. coli instillation increased lung MPO levels from

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Fig. 2. Betulin regulates pro-inflammatory cytokines production and inhibits NF-kB activation in vitro. The cells were pretreated with different concentrations (2, 4 or 8 mg/ml) of Betulin for 1 h prior to stimulation with 1 mg/ml of LPS for 24 h or 30 min to separately determine effect of Betulin on the production of TNF-a (A), IL-6 (B), IL-10 (C) and on the detection of the NF-kB p65 activation (D) and phosphorylated IkB (E) in LPS-stimulated RAW 264.7 cells. Protein samples were analyzed by Western blot with phospho-specific antibodies as described in Materials and methods. Cell viability was determined by an MTT assay (F). The values are means ± SEM of three independent experiments. ## P < 0.01 vs. control group; *P < 0.05 and **P < 0.01 vs. LPS group.

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Fig. 3. Betulin reduces LPS-induced ALI. Mice were given an intraperitoneal injection of Betulin (4 or 8 mg/kg) 1 h prior to administration of LPS. BALF was collected at 24 h following LPS challenge to analyze the number of total cells (A), neutrophils (B), macrophages (C) and inflammatory cytokines TNF-a (D), IL-6 (E), and IL-10 (F). The lung W/D ratio (G) was determined at 24 h after LPS given. The values are means ± SEM (n ¼ 8). ##P < 0.01 vs. control group; *P < 0.05 and **P < 0.01 vs. LPS group.

0.39 ± 0.01 U/g (PBS control) to 0.81 ± 0.09 U/g (p < 0.01; Fig. 4B). Betulin treatment efficiently inhibited the release of MPO activity. This biochemical evidence for Betulin-mediated decreases in PMN infiltration was confirmed by histologic examination of lung tissue (Fig. 4C). The numbers of PMNs in large and small airways were markedly decreased with Betulin, and decrements in interstitial edema formation in the presence of Betulin were noted (Fig. 4C). 3.5. Betulin decreases pro-inflammatory mediators in E. coli pneumonia To investigate potential anti-inflammatory effect of Betulin in experimental E. coli pneumonia, the levels of several inflammatory cytokines that have been assigned important roles in the pathogenesis of ALI/ARDS [22] were determined in the supernatants of lung homogenates. Administration of E. coli increased TNF-a, IL-6, IL-1b and IL-10 (Fig. 4E, F, G, H). Betulin markedly decreased TNFa, IL-6 and IL-1b with evident increases in IL-10 (Fig. 4E, F, G, H). 4. Discussion Pneumonia is associated with a profound inflammatory response within the pulmonary compartment. Inflammation induced by gram-negative pathogens is predominantly elicited by bacterial cell wall component LPS [23]. Whereas pathogenmediated inflammation is essential for host defense, unrestrained activation of leukocytes and lung tissue resident cells can lead to excess tissue injury [24] and in the case of pneumonia, subsequent hypoxemia. Phytochemical and pharmacological studies have identified many potential anti-inflammatory substances, especially those derived from plants used in folk medicine, so natural products are becoming increasingly important as sources of pharmacotherapeutics, either for the treatment of infectious lung diseases or the treatment of

noninfectious lung diseases [25e27]. In this study, we sought to determine the effect of Betulin on acute lung injury induced by instillation of LPS and on already-established lung inflammation induced by E. coli. Our data indicated that Betulin both dampened LPS-initiated ALI and enhanced pneumonia host defense in mice. We set out to assess the effects of Betulin on inflammatory responses in RAW 264.7 macrophages. Activated macrophagederived pro-inflammatory cytokines play critical roles in inflammatory diseases [28], and evidences from several clinical studies indicated that a lot of pro-inflammatory cytokines, notably TNF-a and IL-6 participated in the early development of inflammation [22]. This present study demonstrates that Betulin inhibits production of TNF-a and IL-6 in LPS-stimulated RAW 264.7 cells in a dose-dependent manner. LPS induces TNF-a and other inflammatory gene expression by activating the transcription factors NFkB in macrophages [29,30]. To further characterize the nature of the inhibitory effect of Betulin on cytokine production, we examined the effects of Betulin on the activation of the transcription factor NF-kB, which regulates the expression of a large number of genes in response to infection, inflammation and other endogenous and exogenous stressors. In unstimulated cells, NF-kB is localized to the cytosol due to its binding with IkB. However, NFkB can be activated by some stimulation of various receptors including TNF receptor, Toll-like receptors (TLRs) and T-cell receptor (TCR). Persistent activation of NF-kB is central to the pathogenesis of many inflammatory lung disorders including chronic obstructive pulmonary, pneumonia, and acute lung injury. We found that LPS-induced NF-kB p65 translocation from the cytoplasm to the nucleus was strongly inhibited by Betulin treatment (Fig. 2D, E). Our results suggest that Betulin suppresses LPSinduced pro-inflammatory cytokine production maybe by inhibiting the activation of the transcription factor NF-kB in RAW 264.7 cells.

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Fig. 4. Betulin exerts anti-inflammatory effects during E. coli pneumonia. Gram-negative pneumonia was induced by intranasal inoculation of 20  106 CFU E. coli (ATCC 25922). 200 mL of Betulin (8 mg/kg) was injected intraperitoneally subcutaneously 2 h after infection with E. coli and then at 12-h intervals. CFUs were determined in lung homogenates (A). MPO levels were determined to assess PMN accumulation in lung tissue (B). Representative lung histology of Betulin treatment (C) with corresponding inflammation scores (D) is shown (H&E staining, magnification 100, scale bars ¼ 100 mm). Impact of Betulin on inflammatory cytokines TNF-a (E), IL-6 (F), IL-1b (G) and IL-10 (H) levels were determined in whole lung homogenates. The values presented are mean ± SEM (n ¼ 8). ##P < 0.01 vs. control group; *P < 0.05 and **P < 0.01 vs. LPS group.

These strong in vitro effects prompted us to investigate effects of Betulin during in vivo pulmonary inflammation. To this end, mice were intranasally instilled with LPS and treated with Betulin. Betulin treatment especially attenuated inflammation at 24 h after LPS administration. Recent studies have shown that neutrophils are key cells in the inflammatory response that

characterizes ALI [31], which are the earliest immune cells to be recruited to the site of injury or inflammation. Numerous evidence linking PMNs and lung injury and the critical involvement of cytokines in the recruitment of PMNs into tissues, it was hoped that the study of cytokines in BAL would provide clues about the mechanisms that regulate injury in the lungs [32e34]. It is likely

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that pro-inflammatory cytokines, notably TNF-a and IL-6, participate in the early development of inflammation; they have been shown to play a crucial role in ALI and ARDS [22]. Antiinflammatory cytokines like IL-10 are also produced during endotoxemia. They down-regulate production of proinflammatory cytokines and provide a key mechanism for limiting the inflammatory response in the lungs [35]. We found that, the pretreatment of Betulin (4 or 8 mg/kg) significantly lowered LPS-induced pro-inflammatory cytokines TNF-a and IL-6 production, and increased IL-10 concentration compared to the LPS group. Furthermore, intratracheal LPS instillation induced PMN recruitment that was blocked by Betulin. Moreover, edema is a typical symptom of inflammation not only in systemic inflammation but also in local inflammation. Our experiments showed that Betulin may significantly inhibit edema of the lung, as shown by a W/D ratio in the Betulin group that was significantly lower than the LPS group. To expand our data on Betulin effects on LPS-induced pulmonary inflammation, we determined the impact of Betulin on an inflammatory response in the lung elicited by the clinically relevant gram-negative pathogen E. coli. Betulin treatment diminished PMN numbers in lung tissue at 24 h after infection; this result was associated with lower lung inflammation scores at 24 h. Different from results in vitro and in LPS-induced ALI, Betulin not only decreased production of pro-inflammatory mediators, including IL6 and TNF-a, but also suppressed the level of IL-1b in lung homogenates harvested from E. coli infected mice. Further studies should focus on the basic mechanisms governing Betulin inhibition of pro-inflammatory cytokines and PMN production. And the concentration of IL-10 was slightly increased in groups treated with Betulin. Of interest, data from clinical trials in the early phase of ARDS have identified significantly higher BALF levels of IL-1b and IL-6 in non-survivors [36]. These cytokines can bind to receptors on the surface of gram-negative bacteria to favor growth of the bacteria [37,38]. Although Betulin did not carry direct antimicrobial actions, it enhanced microbial clearance of a modest dose of E. coli from the lung, perhaps in part by regulating levels of these cytokines. Future studies should also address the on clinical relevance of our studies. In summary, we show that Betulin strongly reduces acute lung injury induced by LPS. Postponed administration of this compound after induction of gram-negative pneumonia similarly reduced PMNs and pro-inflammatory cytokines in lungs, and promoted the clearance of E. coli pneumonia. However, from a clinical standpoint, the anti-inflammatory effects of Betulin should be further studied and clinical use of Betulin to reduce lung injury is still far away. Further research is needed to address the effects of Betulin inhibition on pulmonary inflammation in the setting of infectious and noninfectious disease.

Acknowledgments The manuscript was written by Qianchao Wu and Haihua Feng. All of the experiments were performed by Qianchao Wu and the help of Hongyu Li and Jiaming Qiu. This work was supported by the: 1) National Natural Science Foundation of China (no. 31372478).

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Betulin protects mice from bacterial pneumonia and acute lung injury.

Betulin, a naturally occurring triterpene, has shown anti-HIV activity, but details on the anti-inflammatory activity are scanty. In this study, we so...
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