双氢青蒿素对肝纤维化大鼠肠道菌群及NLRP3炎性小体的影响

Effects of dihydroartemisinin on intestinal flora and activation of NLRP3 inflammatory bodies in rats with hepatic fibrosis

目的 观察双氢青蒿素对肝纤维化大鼠肠道菌群及NOD样受体蛋白3(NLRP3)炎性小体相关蛋白表达的影响,探讨双氢青蒿素抗肝纤维化的作用机制。方法 将30只SD大鼠随机分为假手术组、肝纤维化组及双氢青蒿素组,每组10只。肝纤维化组和双氢青蒿素组大鼠通过结扎胆总管的方法制备肝纤维化模型,假手术组剥离胆总管但不结扎。自术后第15天起,双氢青蒿素组大鼠给予20μg/g的双氢青蒿素溶液5 mL灌胃,假手术组和肝纤维化组大鼠给予含0.6%的二甲基亚砜生理盐水5 mL灌胃,均1次/d,连续灌胃2周。HE染色观察3组肝组织的病理形态,实时荧光定量PCR技术分析肠道特定菌群量,Western blot法检测肝组织中Ⅰ型胶原、α-平滑肌肌动蛋白(α-SMA)、NLRP3、半胱氨酸天门冬氨酸酶1(Caspase-1)、白细胞介素-1β(IL-1β)蛋白表达情况。结果 与假手术组比较,肝纤维化组大鼠肝小叶坏死区明显,小叶周围结缔组织和胆总管数目增多;肠道内大肠杆菌相对表达量增加(P<0.05),双歧杆菌和乳酸杆菌相对表达量减少(P均<0.05);肝组织中Ⅰ型胶原、α-SMA和NLRP3、Caspase-1、IL-1β蛋白相对表达量均明显升高(P均<0.05)。与肝纤维化组比较,双氢青蒿素组大鼠肝小叶坏死区缩小,小叶周围结缔组织和胆总管数目减少;肠道内大肠杆菌相对表达量减少(P<0.05),双歧杆菌和乳酸杆菌相对表达量增加(P均<0.05);肝组织中Ⅰ型胶原、α-SMA和NLRP3、Caspase-1、IL-1β蛋白相对表达量均明显降低(P均<0.05)。结论 双氢青蒿素能减轻肝纤维化大鼠肝纤维化程度,机制可能与调节肠道菌群和拮抗NLRP3炎性小体活化有关。

Objective It is to observe the effects of dihydroartemisinin(DHA)on the intestinal flora and the expression of related proteins of NOD-like receptor protein 3(NLRP3)in liver tissue of experimental liver fibrosis rats,and to explore the mechanism of DHA in improving liver fibrosis.Methods Thirty SD rats were randomly divided into sham operation group,liver fibrosis group,and DHA group,with 10 rats in each group.The rats in the liver fibrosis group and DHA group were suffered from common bile duct ligation to establish liver fibrosis models,while the common bile duct of the rats in the sham operation group were separated without ligation.Starting from the 15th day after surgery,the rats in the DHA group were given 20μg/g DHA solution 5 mL by gavage,the rats in the sham operation group and liver fibrosis group were given normal saline containing 0.6%dimethyl sulfoxide 5 mL by gavage,all once a day,continuously gavaged for weeks.The pathological morphology of liver tissue in the three groups were observed by HE staining,the specific gut microbiota was analyzed by real-time fluorescence quantitative PCR technology,and the expression of type I collagen,α-smooth muscle actin(α-SMA),NLRP3,Caspase-1,interleukin-1β(IL-1β)in liver tissue were determined by Western blot method.Results Compared with the sham operation group,there were significant necrosis in the hepatic lobules of the rats in the liver fibrosis group,with an increase of connective tissue and common bile ducts around the lobules;The relative expression of Escherichia coli in intestinal tract were increased(P<0.05),while that of Bifidobacterium and Lactobacillus were decreased(P<0.05);The relative expression of Type I collagen,α-SMA and NLRP3,Caspase-1,IL-1βproteins in liver tissue were significantly increased(all P<0.05).Compared with the liver fibrosis group,the necrosis area of liver lobules in the DHA group was decreased,and the connective tissue and common bile ducts around the lobules were decreased;The relative expression of Escherichia coli in intestinal tract was decreased(P<0.05),while that of Bifidobacterium and Lactobacillus was increased(P<0.05);The relative expression of TypeⅠcollagen,α-SMA and NLRP3,Caspase-1,IL-1βproteins in liver tissue were significantly decreased(all P<0.05).Conclusion Dihydroartemisinin can reduce the degree of liver fibrosis in rats with liver fibrosis,and its mechanism may be related to regulate intestinal flora,and inhibit the activation of NLRP3 inflammatory body.