过表达TRIM27通过抑制NFκB/MAPK信号通路减轻小鼠重症急性胰腺炎

Overexpression of TRIM27 alleviates severe acute pancreatitis in mice by inhibiting NFκB/MAPK signaling pathway

目的探讨过表达三基序蛋白27(tripartite motif-containing,TRIM27)对小鼠重症急性胰腺炎(severe acute pancreatitis,SAP)的保护作用及其可能机制。方法24只小鼠按随机数字表法分为假手术+对照病毒组(AAV-GFP组)、假手术+过表达TRIM27组(AAV-TRIM27组)、SAP+对照病毒组(SAP+AAV-GFP组)、急性+过表达TRIM27组(SAP+AAV-TRIM27组),每组各6只。腹腔注射L-精氨酸(4mg/kg)制作小鼠SAP模型,假手术组注射等体积生理盐水,病毒组通过腹腔注射对照或者TRIM27过表达腺相关病毒(2×10^(11)μg/只)。各组小鼠建模后72h处死收集血清及胰腺组织标本,通过酶联免疫吸附试验检测小鼠血清淀粉酶、脂肪酶、肿瘤坏死因子-α(tumor necrosis factorα,TNF-α)、白介素1b(interleukin-1b,IL-1b)、IL-6、人巨噬细胞趋化蛋白-1(macrophage chemoatractant protein-1,MCP-1)以及胰腺组织中丙二醛(malondialdehyde,MDA)、超氧化物歧化酶(superoxide dismutase,SOD)、谷胱甘肽(glutathione,GSH)等表达,通过苏木精-伊红染色观察胰腺组织病理损伤,通过免疫组化观察小鼠胰腺组织中髓过氧化物酶(myeloperoxidase,MPO)、Ly6g阳性炎症细胞表达,通过蛋白免疫印迹试验测定胰腺组织中p-p65、p65、p-ASK1、ASK1、p-JNK、JNK、p-p38、p38等蛋白表达。结果小鼠SAP后胰腺组织中TRIM27表达显著下调(P<0.05);通过腺相关病毒过表达TRIM27后,小鼠胰腺组织中TRIM27表达显著上调(P<0.05)。AAV-GFP组与AAV-TRIM27组小鼠各指标比较差异无统计学意义(P>0.05)。与SAP+AAV-GFP组相比,SAP+AAV-TRIM27组小鼠血清淀粉酶、脂肪酶以及TNF-α、IL-1b、IL-6、MCP-1水平显著降低,胰腺组织中MDA降低、SOD和GSH升高,MPO、Ly6g等炎症细胞表达显著减少,同时p-p65、p-ASK1、p-JNK、p-p38等蛋白表达显著下调(P<0.05)。结论TRIM27过表达能够通过抑制炎症反应和氧化应激减轻小鼠急性,其机制可能是通过抑制NFκB/MAPK信号通路。

Objective To investigate the protective effect of overexpressed tripartite motif containing(TRIM27)on severe acute pancreatitis(SAP)in mice and its possible mechanism.Methods Twenty-four mice were randomly divided into the sham operation+control virus group(AAVGFP group),sham operation+overexpression of TRIM27 group(AAV-TRIM27 group),SAP+control virus group(SAP+AAV-GFP group),SAP+overexpression of TRIM27 group(SAP+AAV-TRIM27 group),with 6 mice in each group.SAP model of mice was established by intraperitoneal injection of L-arginine(4 mg/kg).The sham operation group was injected with equal volume of normal saline,and the virus group was injected with control or TRIM27 overexpression adeno-associated virus(2×10^(11)μg/per mice),The serum and pancreatic tissue samples were collected 72 h afer modeling.The levels of serum amylase,lipase,tumor necrosis factorα(TNF-α),interleukin-1b(IL-1b),IL-6,macrophage chemoattractant protein-1(MCP-1)and the expression of malondialdehyde(MDA),superoxide dismutase(SOD)and glutathione(GSH)in pancreatic tissue were detected by enzyme-linked immunosorbent assay.Hematoxylin eosin staining was used to observe the pathological damage of pancreatic tissue.The expressions of myeloperoxidase(MPO)and Ly6g positive inflammatory cells in mouse pancreas were observed by immunohistochemistry.The expression of p-p65,p65,p-ASK1,ASK1,p-JNK,JNK,p-p38 and p38 in pancreatic tissue were detected by Western blot.ResultsThe expression of TRIM27 in pancreatic of mice was significantly down regulated after SAP(P<0.05);after overexpression of TRIM27 by adeno-associated virus,the expression of TRIM27 in mouse pancreas was significantly up-regulated(P<0.05).There was no significant difference in the indexes of mice between the AAV-GFP group and AAV-TRIM27 group(P>0.05).Compared with the SAP+AAV-GFP group,the levels of serum amylase,lipase,TNF-α,IL-1b,IL-6 and MCP-1 in mice of the SAP+AAV-TRIM27 group were significantly decreased,MDA in pancreatic tissue was decreased,SOD and GSH were increased,MPO and Ly6g inflammatory cells were significantly decreased,and p-p65,p-ASK1,p-JNK,and p-p38 protein expression were down regulated.Conclusions Overexpression of TRIM27 alleviates SAP in mice by inhibiting inflammatory response and oxidative stress,and its mechanism may be through inhibiting NFκB/MAPK signaling pathway.