清解化攻方调控NLRP3/TLR4/NF-κB信号通路对重症急性胰腺炎小鼠模型胰腺组织的保护作用

Protective effect of Qingjie Huagong decoction on pancreatic tissue of mice with severe acute pancreatitis by regulating the NOD-like receptor protein 3/Toll-like receptor 4/nuclear factor-kappa B signaling pathway

目的观察清解化攻方对重症急性胰腺炎(SAP)小鼠模型的治疗作用,探索清解化攻方抗炎症反应的作用机制。方法将36只C57BL/6J雄性小鼠随机分成空白组,模型组,清解化攻方低、中、高剂量组,西药组(乌司他丁),每组6只,除空白组小鼠,余各组小鼠采用逆行胰胆管注射5%牛黄胆酸钠建立SAP模型,清解化攻方低、中、高剂量组在造模后分别予以清解化攻方1、2、4 g/kg灌胃,西药组在造模后予以腹腔注射乌司他丁(5×10^(4) U/kg),共干预7 d。采用苏木素-伊红染色观察胰腺组织病理改变;酶联免疫吸附测定法(ELISA)检测小鼠α-淀粉酶、脂肪酶、IL-1β、IL-6、IL-8、IL-18和TNF-α水平;RTqPCR检测胰腺组织NOD样受体蛋白3(NLRP3)、Toll样受体4(TLR4)、核因子-κB(NF-κB)mRNA表达水平;免疫组化检测胰腺组织NLRP3、TLR4、NF-κB的阳性表达率;Western Blot技术检测NLRP3、TLR4、NF-κB、IL-1β、IL-6蛋白的表达水平。计量资料多组间比较采用方差分析,进一步两两比较采用LSD-t检验。结果与空白组相比,模型组小鼠胰腺组织结构弥漫性破坏、胰腺小叶间隔局灶性扩张、腺泡萎缩和大量炎症细胞浸润,α-淀粉酶、脂肪酶、IL-1β、IL-6、IL-8、IL-18和TNF-α含量明显升高(P值均<0.05),NLRP3、TLR4、NF-κB mRNA表达水平及阳性表达率均明显上升(P值均<0.05),NLRP3、TLR4、NF-κB、IL-1β、IL-6蛋白表达均明显上调(P值均<0.05)。与模型组相比,清解化攻方各剂量组和西药组可见小鼠胰腺组织结构稍紧密、完整,胰腺腺泡细胞排列有序,伴少量炎症细胞浸润和胰腺小叶出血灶,α-淀粉酶、脂肪酶、IL-1β、IL-6、IL-8、IL-18和TNF-α含量明显下降(P值均<0.05),NLRP3、TLR4、NF-κB mRNA表达水平及阳性表达率均明显降低(P值均<0.05),NLRP3、TLR4、NF-κB、IL-1β、IL-6蛋白表达水平均明显减弱(P值均<0.05)。结论清解化攻方可能通过抑制NLRP3/TLR4/NF-κB信号通路相关蛋白的激活,减少炎症介质的释放,防止炎症级联反应增强,进而对SAP小鼠胰腺组织发挥保护作用。

Objective To investigate the therapeutic effect of Qingjie Huagong decoction(QJHGD)on a mouse model of severe acute pancreatitis(SAP)and the mechanism of action of QJHGD against inflammatory response.Methods A total of 36 male C57BL/6J mice were randomly divided into blank group,model group,Western medicine group(ulinastatin),and low-,middle-,and high-dose QJHGD groups,with 6 mice in each group.All mice except those in the blank group were given 5%sodium taurocholate by retrograde pancreaticobiliary injection to establish a model of SAP.After modeling,the mice in the low-,middle-,and high-dose groups were given QJHGD(1,2,and 4 g/kg,respectively)by gavage,and those in the Western medicine group were given intraperitoneal injection of ulinastatin(5×10^(4) U/kg),for 7 days in total.HE staining was used to observe the histopathological changes of the pancreas;ELISA was used to measure the levels ofα-amylase,lipase,interleukin-1β(IL-1β),interleukin-6(IL-6),interleukin-8(IL-8),interleukin-18(IL-18),and tumor necrosis factor-α(TNF-α)in mice;RT-qPCR was used to measure the mRNA expression levels of NOD-like receptor protein3(NLRP3),Toll-like receptor 4(TLR4),and nuclear factor-kappa B(NF-κB)in pancreatic tissue;immunohistochemistry was used to measure the positive expression rates of NLRP3,TLR4,and NF-κB in pancreatic tissue;Western blot was used to measure the protein expression levels of NLRP3,TLR4,NF-κB,IL-1β,and IL-6.An analysis of variance was used for comparison of continuous data between multiple groups,and the least significant difference t-test was used for further comparison between two groups.Results Compared with the blank group,the model group had diffuse destruction of pancreatic tissue structure,focal dilatation of pancreatic lobular septum,pancreatic acinar atrophy,and massive inflammatory cell infiltration,as well as significant increases in the content ofα-amylase,lipase,IL-1β,IL-6,IL-8,IL-18,and TNF-α(all P<0.05),the mRNA expression levels and positive expression rates of NLRP3,TLR4,and NF-κB(all P<0.05),and the protein expression levels of NLRP3,TLR4,NF-κB,IL-1β,and IL-6(all P<0.05).Compared with the model group,the low-,middle-,and high-dose QJHGD groups and the Western medicine group had slightly tighter and more intact structure of pancreatic tissue,ordered arrangement of pancreatic acinar cells,a small amount of inflammatory cell infiltration,and hemorrhagic foci of pancreatic lobules,as well as significant reductions in the content ofα-amylase,lipase,IL-1β,IL-6,IL-8,IL-18,and TNF-α(all P<0.05),the mRNA expression levels and positive expression rates of NLRP3,TLR4,and NF-κB(all P<0.05),and the protein expression levels of NLRP3,TLR4,NF-κB,IL-1β,and IL-6(all P<0.05).Conclusion QJHGD may exert a protective effect on the pancreatic tissue of SAP mice by inhibiting the activation of NLRP3/TLR4/NF-κB signaling pathway-related proteins,reducing the release of inflammatory mediators,and preventing the enhancement of inflammatory cascade response.