大黄■虫丸含药血清调控大鼠原代肝星状细胞BAMBI表达的研究
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摘要
目的:研究大黄■虫丸含药血清对LPS诱导大鼠肝星状细胞(HSC)骨成形蛋白-激活素膜结合阻断因子(BAMBI)表达的影响及其作用机制。方法:体外培养并鉴定Wistar大鼠原代HSC,提取并制备大黄■虫丸含药血清,分别予空白对照血清、大黄■虫丸中药含药血清培养细胞,LPS刺激后,应用Western Blot法和免疫组化检测HSC中TLR4、MyD88、BAMBI蛋白变化;EMSA检测NF-κB结合蛋白的活性;结果:Western Blot法和免疫组化结果:与空白对照组比较,LPS处理组TLR4、MYD88表达升高,BAMBI表达降低。与LPS处理组比较,含药血清和LPS共处理组TLR4、MYD88降低,BAMBI表达升高。EMSA结果显示,LPS处理组NF-κB结合蛋白的活性增强,LPS与含药血清共处理组NF-κB结合蛋白的活性降低。结论:大黄■虫丸含药血清可通过抑制LPS诱导的HSCs中TLR4-MYD88的表达,进而减少NF-κB活化,促进TGF-β假受体BAMBI的表达,这可能是发挥其抗纤维化作用的机制之一。
Objective: To investigate the effect of serum containing Dahuang Zhechong Pills(DHZC) on the expression of bone morphogenetic protein-activator membrane-blocking factor(BAMBI) induced by LPS in rats hepatic stellate cells(HSC) and its mechanism. Methods: The primary HSCs of Wistar rats were cultured and identified in vitro, and the DHZC containing serum was extracted and prepared. The cells were cultured with control serum and DHZC containing serum respectively. After LPS stimulation, Western blot and immunohistochemistry were used to detect TLR4, MyD88 and BAMBI protein in HSCs. EMSA was used to detect NF-κB binding protein activity. Results: Western blot and immunohistochemistry results: Compared with the control group, the expressions of TLR4 and MYD88 wereincreased and the expression of BAMBI was decreased in the LPS treatment group. Compared with the LPS treatment group, TLR4 and MYD88 were decreased and BAMBI expression was increased in the DHZC+LPS group. EMSA results showed that the activity of NF-κB binding protein was increased in LPS treatment group, and the activity of NF-κB binding protein was decreased in DHZC+LPS group. Conclusion: Serum containing Dahuang Zhechong Pills can inhibit the expression of TLR4-MYD88 in LPS-induced HSCs, and further reduce NF-κB activation and promote the expression of TGF-β pseudo-receptor BAMBI, which may be one of the mechanisms of its anti-fibrosis.
Objective: To investigate the effect of serum containing Dahuang Zhechong Pills(DHZC) on the expression of bone morphogenetic protein-activator membrane-blocking factor(BAMBI) induced by LPS in rats hepatic stellate cells(HSC) and its mechanism. Methods: The primary HSCs of Wistar rats were cultured and identified in vitro, and the DHZC containing serum was extracted and prepared. The cells were cultured with control serum and DHZC containing serum respectively. After LPS stimulation, Western blot and immunohistochemistry were used to detect TLR4, MyD88 and BAMBI protein in HSCs. EMSA was used to detect NF-κB binding protein activity. Results: Western blot and immunohistochemistry results: Compared with the control group, the expressions of TLR4 and MYD88 wereincreased and the expression of BAMBI was decreased in the LPS treatment group. Compared with the LPS treatment group, TLR4 and MYD88 were decreased and BAMBI expression was increased in the DHZC+LPS group. EMSA results showed that the activity of NF-κB binding protein was increased in LPS treatment group, and the activity of NF-κB binding protein was decreased in DHZC+LPS group. Conclusion: Serum containing Dahuang Zhechong Pills can inhibit the expression of TLR4-MYD88 in LPS-induced HSCs, and further reduce NF-κB activation and promote the expression of TGF-β pseudo-receptor BAMBI, which may be one of the mechanisms of its anti-fibrosis.
引文
[1] Puche JE,Saiman Y,Friedman SL.Hepatic stellate cells and liver fibrosis[J].Compr Physiol,2013,3(4):1473-1492.
[2] Cassiman D,Libbrecht L,Desmet V,et al.Hepatic stellate cell/myofibroblast subpopulations in fibrotic human and rat livers[J].J Hepatol,2002,36(2):200-209.
[3] Tahashi Y,Matsuzaki K,Date M,et al.Differential regulation of TGF-βeta signal in hepatic stellate cells between acute and chronic rat liver injury[J].Hepatology,2002,35(1):49-61.
[4] Onichtchouk D,Chen YG,Dosch R,et al.Silencing of TGF-βsignalling by the pseudoreceptor BAMBI[J].Nature,1999,401(6752):480-485.
[5] 陈云,杜建,王玉梅.慢性乙型肝炎血浆内毒素与肝纤维化指标及肝脏病的关系[J].临床肝胆病杂志,2005,21(1):3-4.
[6] 中国中西医结合学会肝病专业委员会.肝纤维化中西医结合诊疗指南[S].中华肝脏病杂志,2006,14(11):866-870.
[7] 李仪奎.中药药理实验方法学[M].上海:上海科学技术出版社,2006:50-53.
[8] 赵彤,朱梅刚,黄宗义,等.肺癌癌基因蛋白产物同步检测的对比分析[J].癌症,1995(1):13-15.
[9] Weiskirchen R,Tacke F.Liver fibrosis:From pathogenesis to novel therapies[J].Digestive diseases,2016,34:410-422.
[10] Xiao L,Christopher B,Sven H,et al.The Mechanism of hepatic stellate cell inactivation during reversal of liver fibrosis[J].Gastroenterology,2016,150(4):S1020-S1021.
[11] Bilzer M,Roggel F,Gerbes AL.Role of Kupffer cells in host defense and liver disease[J].Liver Int,26(10):1175-1186.
[12] Puche JE,Saiman Y,Friedman SL.Hepatic stellate cells and liver fibrosis[J].Compr Physiol,2013,3(4):1473-1492.
[13] Friedman SL.A deer in the headlights:BAMBI meets liver fibrosis[J].Nat Med,2007,13(11):1281-1282.
[14] Shi H,Dong L,Dang X,et al.Effect of chlorogenic acid on LPS-induced proinflammatory signalling in hepatic stellate cells[J].Inflamm Res,2013,62(6):581-587.
[15] Aoyama T,Paik YH,Seki E.Toll-like receptor signaling and liver fibrosis[J].Gastroenterol Res Pract,2010,2010:1687-6121.
[16] Brun P,Castagliuolo I,Pinzani M,et al.Exposure to bacterial cell wall products triggers an inflammatory phenotype in hepatic stellate cells[J].Am J Physiol Gastrointest Liver Physiol,2005,289(3):G571-G578.
[17] Muhlbauer M,Weiss TS,Thasler WE,et al.LPS-mediated NF-B activation varies between activated human hepatic stellate cells from different donors[J].Biochem Biophys Res Commun,2004,325(1):191-197.
[18] Guarner C,Soriano.Bacterial translocation and its consequences in patients with cirrhosis[J].Eur J Gastroenterol Hepatol,2005,17(1):27-31.
[19] Lu YC,Yeh WC,Ohashi PS.LPS/TLR4 signal transduction pathway[J].Cytokine,2008,42(2):145-151.
[20] Akira S,Takeda K,Kaisho T.Toll-like receptors:critical proteins linking innate and acquired immunity[J].Nat Immunol,2001,2(8):675-680.
[21] 张红星,刘旭东,王朝阳.大黄虫丸联合恩替卡韦治疗慢性乙型肝炎肝硬化疗效观察[J].中国中西医结合消化杂志,2016(8):575-577.
[22] 潘志恒,程木华,李林,等.大黄虫丸对慢性肝病核素肝脏功能显像定量分析结果的影响[J].第四军医大学学报,2006(23):2193-2196.
[23] 李文新,车念聪,王金光,等.大黄虫丸加黄芪、水红花子对拮抗大鼠肝纤维化作用的影响[J].中华中医药杂志,2016,31(12):5320-5322.
[2] Cassiman D,Libbrecht L,Desmet V,et al.Hepatic stellate cell/myofibroblast subpopulations in fibrotic human and rat livers[J].J Hepatol,2002,36(2):200-209.
[3] Tahashi Y,Matsuzaki K,Date M,et al.Differential regulation of TGF-βeta signal in hepatic stellate cells between acute and chronic rat liver injury[J].Hepatology,2002,35(1):49-61.
[4] Onichtchouk D,Chen YG,Dosch R,et al.Silencing of TGF-βsignalling by the pseudoreceptor BAMBI[J].Nature,1999,401(6752):480-485.
[5] 陈云,杜建,王玉梅.慢性乙型肝炎血浆内毒素与肝纤维化指标及肝脏病的关系[J].临床肝胆病杂志,2005,21(1):3-4.
[6] 中国中西医结合学会肝病专业委员会.肝纤维化中西医结合诊疗指南[S].中华肝脏病杂志,2006,14(11):866-870.
[7] 李仪奎.中药药理实验方法学[M].上海:上海科学技术出版社,2006:50-53.
[8] 赵彤,朱梅刚,黄宗义,等.肺癌癌基因蛋白产物同步检测的对比分析[J].癌症,1995(1):13-15.
[9] Weiskirchen R,Tacke F.Liver fibrosis:From pathogenesis to novel therapies[J].Digestive diseases,2016,34:410-422.
[10] Xiao L,Christopher B,Sven H,et al.The Mechanism of hepatic stellate cell inactivation during reversal of liver fibrosis[J].Gastroenterology,2016,150(4):S1020-S1021.
[11] Bilzer M,Roggel F,Gerbes AL.Role of Kupffer cells in host defense and liver disease[J].Liver Int,26(10):1175-1186.
[12] Puche JE,Saiman Y,Friedman SL.Hepatic stellate cells and liver fibrosis[J].Compr Physiol,2013,3(4):1473-1492.
[13] Friedman SL.A deer in the headlights:BAMBI meets liver fibrosis[J].Nat Med,2007,13(11):1281-1282.
[14] Shi H,Dong L,Dang X,et al.Effect of chlorogenic acid on LPS-induced proinflammatory signalling in hepatic stellate cells[J].Inflamm Res,2013,62(6):581-587.
[15] Aoyama T,Paik YH,Seki E.Toll-like receptor signaling and liver fibrosis[J].Gastroenterol Res Pract,2010,2010:1687-6121.
[16] Brun P,Castagliuolo I,Pinzani M,et al.Exposure to bacterial cell wall products triggers an inflammatory phenotype in hepatic stellate cells[J].Am J Physiol Gastrointest Liver Physiol,2005,289(3):G571-G578.
[17] Muhlbauer M,Weiss TS,Thasler WE,et al.LPS-mediated NF-B activation varies between activated human hepatic stellate cells from different donors[J].Biochem Biophys Res Commun,2004,325(1):191-197.
[18] Guarner C,Soriano.Bacterial translocation and its consequences in patients with cirrhosis[J].Eur J Gastroenterol Hepatol,2005,17(1):27-31.
[19] Lu YC,Yeh WC,Ohashi PS.LPS/TLR4 signal transduction pathway[J].Cytokine,2008,42(2):145-151.
[20] Akira S,Takeda K,Kaisho T.Toll-like receptors:critical proteins linking innate and acquired immunity[J].Nat Immunol,2001,2(8):675-680.
[21] 张红星,刘旭东,王朝阳.大黄虫丸联合恩替卡韦治疗慢性乙型肝炎肝硬化疗效观察[J].中国中西医结合消化杂志,2016(8):575-577.
[22] 潘志恒,程木华,李林,等.大黄虫丸对慢性肝病核素肝脏功能显像定量分析结果的影响[J].第四军医大学学报,2006(23):2193-2196.
[23] 李文新,车念聪,王金光,等.大黄虫丸加黄芪、水红花子对拮抗大鼠肝纤维化作用的影响[J].中华中医药杂志,2016,31(12):5320-5322.