核因子-κB信号通路在胆汁淤积性肝损伤中的作用及其机制

The role of nuclear factor-κB signaling in bile acid-induced liver injury in cholestasis

目的:探讨核因子-κB(NF-κB)信号通路在胆汁淤积性肝损伤中的作用机制。方法:构建小鼠胆总管结扎模型,将小鼠随机分为Sham组、BDL14D组与BDL14D+BAY 11-7085组,每组10只,术后14d取小鼠血清检测转氨酶、胆汁酸及胆红素水平,取肝组织行苏木精-伊红(HE)染色观察肝脏损伤,行蛋白印迹实验检测NF-κB信号通路分子及炎症因子的变化。体外培养L-O2细胞,用胆汁酸甘氨鹅脱氧胆酸钠(GCDCA)刺激,并用NF-κB信号通路特异性抑制剂BAY 11-7085进行干预,观察NF-κB信号通路及炎症因子的变化。结果:胆总管结扎14 d后,BDL14D组小鼠血清中谷草转氨酶(AST)、谷丙转氨酶(ALT)、总胆汁酸(TBA)水平、总胆红素(TBIL)和直接胆红素(DBIL)(1104.022±253.524、622.896±79.863、73.602±17.233、393.522±95.625、199.940±85.811)均明显高于Sham组(84.313±37.254、39.778±21.463、19.342±16.112、4.136±1.728、2.104±1.022);HE染色示BDL14D组小鼠肝组织坏死灶形成;蛋白印迹分析显示BDL14D组小鼠肝组织磷酸化NF-κB抑制因子(p-IκB)、磷酸化NF-κB转录因子p65(p-p65)、白细胞介素(IL)-1β和IL-6的蛋白水平明显升高。使用抑制剂BAY 11-7085干预的BDL14D+BAY 11-7085组小鼠血清中AST和ALT(837.304±197.912、399.160±68.821)明显低于BDL14D组,p-IκB、p-p65、IL-1β和IL-6的蛋白水平也显著减低,肝组织坏死灶面积也明显减少,结果提示用BAY 11-7085通过阻断NF-κB信号通路能有效减轻炎症反应和肝组织坏死,显著改善肝功能状态。体外细胞实验亦证实,给予BAY 11-7085预处理可有效阻断GCDCA诱导的NF-κB信号通路激活,明显抑制肝细胞炎症因子的蛋白表达。结论:肝脏胆汁淤积通过激活NF-κB信号通路引起炎症反应,从而造成肝组织坏死和肝功能损伤。

Objective To explore the role of nuclear factor-κB(NF-κB)signaling in bile acid-induced liver injury in cholestasis.Methods Obstructive cholestasis was induced by surgical ligation of the common bile duct in C57BL/6J mice,which were randomly divided into Sham group,BDL14D group,and BDL14D+BAY 11-7085 group.Plasma and liver were collected from mice at 14 days after surgery.The plasma levels of liver enzymes,total bile acid,and bilirubin were measured.Liver sections were prepared and analyzed by hematoxylin and eosin(HE)staining.L-O2 cells were cultured and stimulated by Sodium glycochenodeoxycholate(GCDCA)with or without BAY 11-7085 pretreatment.Western blotting was used to detect the activation of NF-κB signaling pathway and the protein expression of interleukin(IL)-1βand IL-6.Results BDL mice displayed significantly higher levels of plasma aspartate aminotransferase(AST),alanine aminotransferase(ALT),TBA,total bilirubin(TBIL),and direct bilirubin(DBIL)(1104.022±253.524,622.896±79.863,73.602±17.233,393.522±95.625,199.940±85.811)after 14 days of surgery compared to the mice in Sham group(84.313±37.254,39.778±21.463,19.342±16.112,4.136±1.728,2.104±1.022).Histological examination revealed large areas of necrosis in livers from BDL mice but not Sham-operated livers.BDL also led to the activation of NF-κB signaling,reflected by a marked increase in the protein levels of p-IκB,p-p65,IL-1β,and IL-6.In contrast,intervention with BAY 11-7085 led to the blockage of NF-κB signaling and inflammatory response induced by BDL,resulting in a significant reduce of liver necrosis and plasma AST and ALT levels(837.304±197.912,399.160±68.821).Moreover,our in vitro studies confirmed that BAY 11-7085 pretreatment was sufficient to block bile acid-induced activation of NF-κB signaling and suppress the expression of proinflammatory factors in liver cells.Conclusion These findings suggest that NF-κB signaling plays a critical role in bile acid-induced inflammatory response and liver injury in cholestasis.