降脂颗粒调控UCP2和JNK/c-Jun介导的NLRP3炎症小体活化减轻非酒精性脂肪性肝炎小鼠脂毒性肝损伤

Jiangzhi Granule alleviates lipotoxic liver injury in nonalcoholic steatohepatitis mice through regulating UCP2 and JNK/c-Jun-mediated NLRP3 inflammasome activation

目的:基于炎症小体活化的调控,探讨中药复方降脂颗粒改善非酒精性脂肪性肝炎(NASH)小鼠脂毒性肝损伤的机制。方法:雄性C57BL/6J小鼠随机分为对照组、模型组、降脂颗粒组,每组8只。对照组小鼠给予正常饲料;其余小鼠给予蛋氨酸-胆碱缺乏(MCD)饮食诱导NASH,降脂颗粒组小鼠同时灌胃给予0.86 g/kg降脂颗粒溶液。干预6周后,检测小鼠血清丙氨酸氨基转移酶(ALT)和天冬氨酸氨基转移酶(AST)水平;HE染色后光镜下观察肝组织形态学变化;PCR检测肝组织NOD样受体蛋白3(NLRP3)、凋亡相关斑点样蛋白(ASC)、天冬氨酸特异性半胱氨酸蛋白酶1(Caspase-1)、白介素-1β(IL-1β)、白介素-18(IL-18)、解偶联蛋白2(UCP2)的mRNA表达水平;Western blot检测肝组织Caspase-1前体与剪切型(pro-Caspase-1、cleaved-Caspase-1)、JNK与c-Jun及其磷酸化的蛋白表达水平。结果:降脂颗粒能够降低MCD饮食诱导的NASH小鼠血清ALT、AST水平,减轻肝组织脂肪变性和炎症细胞浸润。降脂颗粒作用能够显著下调模型小鼠肝组织NLRP3、Caspase-1、IL-1β、IL-18、UCP2的mRNA表达水平(P<0.05,P<0.01),降低cleaved-Caspase-1与pro-Caspase-1蛋白表达比值(P<0.01),同时p-c-Jun和p-JNK蛋白表达受到明显抑制(P<0.05)。结论:降脂颗粒可减轻NASH小鼠脂毒性肝损伤,其部分作用机制可能是下调UCP2表达及抑制JNK/c-Jun信号通路激活,进而抑制NLRP3炎症小体的活化。

Objective: To explore the therapeutic mechanism of Jiangzhi Granule on the lipotoxic liver injury of nonalcoholic steatohepatitis(NASH) mice based on regulation of inflammasome activation. Methods: Male C57 BL/6 J mice were randomly divided into the control group, model group and Jiangzhi Granule group, 8 mice in each group. The control group was fed with the standard diet, and the other groups were fed with methionine-choline deficient(MCD) diet to induce NASH. Meanwhile, the Jiangzhi Granule group was treated with Jiangzhi Granule solution at a dose of 0.86 g/kg by intragastric administration. After intervention for six weeks, the serum levels of alanine aminotransferase(ALT) and aspartate aminotransferase(AST) were detected. The morphological changes of liver tissue were observed under the light microscope after HE staining. The mRNA expressions of Nod-like receptor protein 3(NLRP3), apoptosis-associated speck-like protein containing a CARD(ASC), cysteinyl aspartate specific proteinase-1(Caspase-1), interleukin-1β(IL-1β),interleukin-18( IL-18) and uncoupling protein 2( UCP2) in the liver tissue were detected by PCR. The protein expression levels of pro-Caspase-1,cleaved Caspase-1 as well as JNK,c-Jun and their phosphorylation in the liver tissue were detected by Western blot.Results: Jiangzhi Granule could reduce the serum ALT and AST levels in NASH mice induced by MCD diet,and alleviate the steatosis and inflammatory cell infiltration. Jiangzhi Granule could significantly down-regulate the mR NA expression levels of NLRP3,Caspase-1,IL-1β,IL-18 and UCP2 in the liver tissue of model mice( P<0.05,P<0.01),reduce the protein expression ratio of cleaved-Caspase-1 to pro-Caspase-1( P<0.01),and obviously inhibit the protein expressions of p-c-Jun and p-JNK( P<0.05). Conclusion: Jiangzhi Granule can alleviate the lipotoxic liver injury in NASH mice, and its partial mechanism may be suppressing the activation of NLRP3 inflammasome through down-regulating the expression of UCP2 and inhibiting the activation of JNK/c-Jun signaling pathway.