脂多糖通过NOD样受体蛋白3炎症小体通路诱导大鼠急性肝损伤实验研究

Experimental study on acute liver injury in rats induced by LPS through NLRP3 inflammasomes pathway

目的:探讨脂多糖(LPS)诱导大鼠急性肝损伤的机制。方法:对SD大鼠腹腔注射10 mg/kg LPS 24 h后,比较对照组(3只)与LPS组(3只)大鼠外周血转氨酶水平;取肝组织制片后HE染色观察两组大鼠肝组织有无损伤;免疫组化检测两组大鼠肝组织中NOD样受体蛋白3(NLRP3)与凋亡相关微粒蛋白(ASC)表达情况。另取健康SD大鼠分离肝细胞进行原代培养,设空白对照组、L-LPS组和H-LPS组,分别在含0、100、1000 ng/ml LPS的培养基中培养24 h,测定各组细胞增殖与凋亡情况,并采用PCR与Western blot测定各组细胞NLRP3、ASC及白介素-1β(IL-1β)mRNA及蛋白表达情况。结果:LPS组大鼠注射LPS 24 h后,血液转氨酶水平较对照组明显升高(均P<0.05);肝组织HE染色显示LPS刺激可导致大鼠急性肝细胞损伤;肝组织免疫组化染色结果显示NLRP3、ASC蛋白表达阳性。各组肝原代细胞受到LPS刺激后,增殖率均有所降低,凋亡率均有所升高,且H-LPS组细胞增殖率低于L-LPS组,细胞凋亡率高于L-LPS组(均P<0.05);各组肝原代细胞NLRP3、ASC、IL-1βmRNA与蛋白表达量均明显增加,且H-LPS组均高于L-LPS组(均P<0.05)。结论:LPS刺激会造成大鼠急性肝损伤,其机制可能与上调肝组织中NLRP3炎症小体通路有关。

Objective:To investigate the mechanism of acute liver injury in rats induced by LPS.Methods:After intraperitoneal injection of 10 mg/kg LPS to SD rats for 24 hours,the transaminase levels in peripheral blood of SD rats in the control group(3 rats)and the LPS group(3 rats)were compared;the liver tissue was taken for production,and then HE staining was used to observe whether there was liver tissue damage;the expression of NLRP3 and ASC protein in liver tissue was detected by immunohistochemistry.The liver cells of healthy SD rats were isolated for primary culture,and three groups were set for experiment:blank control group,L-LPS group and H-LPS group.Cell proliferation and apoptosis were measured,and the expression of NLRP3,ASC and IL-1βmRNA and protein were detected by PCR and Western blot.Results:24 hours after the LPS injection,the levels of hepatic enzymes in the blood of LPS group were significantly higher than those of control group(all P<0.05);HE staining of liver tissue showed that LPS stimulation could cause acute liver cell damage in rats;the results of immunohistochemical staining of liver tissue showed that the expression of NLRP3 and ASC protein was positive.After LPS stimulation,the cell proliferation rate decreased and the apoptosis rate increased,and the cell proliferation rate in H-LPS group was lower than that in L-LPS group,and the apoptosis rate was higher than that in L-LPS group(all P<0.05);the expression of NLRP3,ASC and IL-1βmRNA and protein increased significantly,and H-LPS group was higher than those in L-LPS Group(all P<0.05).Conclusion:LPS can cause acute liver injury in rats,and the mechanism may be related to the up-regulation of the NLRP3 inflammasome pathway in liver tissue.