Toll样受体4与NOD样受体3炎性小体在肥胖合并急性坏死性胰腺炎大鼠肝损伤中的作用

Role of Toll-like receptor 4 and NOD-like receptor 3 inflammasome in liver injury of acute necrotizing pancreatitis rats with obesity

目的探讨Toll样受体4(TLR4)与NOD样受体3(NLRP3)炎性小体在肥胖合并急性坏死性胰腺炎(ANP)大鼠肝损伤中的作用。方法将24只SD大鼠随机分为非肥胖正常组、非肥胖ANP组、肥胖正常组、肥胖ANP组。采用高脂饲料连续喂养8周建立肥胖大鼠模型,然后经胆胰管逆行注射5%牛磺胆酸钠制备ANP模型。造模成功后12 h处死大鼠,用全自动生化仪测定血清淀粉酶(AMY)、脂肪酶(LIP)、丙氨酸转氨酶(ALT)、天冬氨酸转氨酶(AST)、三酰甘油(TG)及总胆固醇(TC)的水平;光镜下观察大鼠胰腺、肝脏病理变化并进行病理评分;免疫荧光法检测肝脏髓过氧化物酶(MPO)、白细胞分化抗原68(CD68)、TLR4、NLRP3、白细胞介素1β(IL-1β)水平;免疫组织化学法检测核因子κB(NF-κB)、及半胱氨酸蛋白酶3(caspase-3)的水平。结果肥胖ANP组大鼠血清ALT、AST水平较非肥胖ANP组明显升高[(233.00±34.44)U/L比(102.83±8.90)U/L,(388.00±41.60)U/L比(282.00±21.06)U/L],肥胖ANP组肝脏病理评分较非肥胖ANP组明显升高(6.66±1.21比3.33±1.03),肥胖ANP组肝脏CD68+/TLR4+、CD68+/NLRP3+、TLR4+/NLRP3+、MPO、NF-κB、IL-1β及caspase-3的水平较非肥胖ANP组明显增加(24.16±1.47比6.66±1.21,25.00±2.60比7.00±1.41,14.16±1.47比5.50±1.04,35.33±6.88比20.83±2.48,58.80±6.75比37.63±2.96,50.00±2.36比35.00±2.82,66.00±4.04比55.00±2.60),以上差异均具有统计学意义(P值均<0.05)。结论肥胖大鼠ANP时肝损伤更加严重,可能与肥胖增加肝组织中TLR4/NLRP3信号通路活化程度、导致更多的炎性因子释放有关。

Objective To investigate the role of Toll-like receptor 4(TLR4)and NOD-like receptor 3(NLRP3)inflammasome in the liver injury of acute necrotizing pancreatitis (ANP) rat with obesity. Methods Twenty-four SD rats were randomly divided into normal group, ANP group, obesity group and obesity ANP group. The obesity rat model was established by continuously feeding high fat diet and the ANP model was induced by retrograde infusion of 5% sodium taurocholate into the biliopancreatic duct. Rats were killed at 12 h after model establishment, and automatic biochemical immune analyzer were used for detecting serum AMY, LIP, ALT, AST, TG and TC. Pathological changes of pancreas and liver tissue samples were observed by miscroscopy and pathological score was recorded. The levels of MPO, CD68, TLR4, NLRP3 and IL-1β in liver tissue were detected by immunofluorescence, and NF-κB and caspase-3 in liver tissue were detected by immunohistochemistry. Results The serum ALT and AST in obesity ANP group were significantly increased than those in ANP group (233.00±34.44 U/L vs 102.83±8.90 U/L, 388.00±41.60 U/L vs 282.00±21.06 U/L);and liver pathologic score was also significantly higher than ANP group (6.66 ±1.21 vs 3.33±1.03);and CD68+/TLR4+, CD68+/NLRP3+, TLR4+/NLRP3+, MPO, NF-κB, IL-1β and caspase-3 level were all greatly higher in obesity ANP than those in ANP group, respectively (24.16±1.47 vs 6.66±1.21, 25.00±2.60 vs 7.00±1.41, 14.16±1.47 vs 5.50±1.04, 35.33±6.88 vs 20.83±2.48, 58.80±6.75 vs 37.63±2.96, 50.00±2.36 vs 35.00±2.82, 66.00±4.04 vs 55.00±2.60);and all the differences were statistically significant (all P<0.05). Conclusions Liver injury was more severe in ANP rats with obesity, which may be related to the fact that obesity may enhance the activation of TLR4/NLRP3 signal pathway and result in the release of more inflammatory factors.