目的 研究四逆散对Mdr2(Abcb4)基因缺陷(Mdr2^-/-)小鼠胆汁淤积性肝纤维化的缓解作用,并探究其作用机制。方法 C57BL/6J小鼠作为对照组;C57BL/6J背景的Mdr2^-/-小鼠作为模型小鼠,设模型组和四逆散低、高剂量(按生药量计为3.12、6.24 g·kg^-1)组。四逆散组连续3周ig给予四逆散水提物,每天1次,对照组给予纯水。试剂盒法检测血清丙氨酸氨基转移酶(ALT)、天冬氨酸氨基转移酶(AST)、总胆汁酸水平;取肝脏、脾脏称质量,计算肝脏、脾脏系数;结合小鼠肝组织HE染色,Masson染色,纤连蛋白(Fibronectin)、细胞角蛋白19(CK19)的免疫组化染色,明确四逆散对Mdr2^-/-小鼠肝纤维化及胆汁淤积的影响。基于小鼠肝脏转录组学测序技术,挖掘四逆散改善Mdr2^-/-小鼠肝损伤的作用靶点,并通过实时荧光定量PCR(qRT-PCR)法检测肝纤维化[fibronectin(Fn1)、胶原蛋白1(Col1a1)、角蛋白19(krt19)]、炎症[白细胞介素-1β(Il1β)、Il6、肿瘤坏死因子-α(Tnfα)、一氧化氮合成酶(Inos)]、细胞焦亡[凋亡相关斑点样蛋白(Pycard)、Il18]、胆汁酸合成[细胞色素P450家族成员7A1(Cyp7a1)]及转运[胆盐输出泵(Abcb11)、ATP结合盒转运蛋白(Abcc3)、钠离子-牛磺胆酸共转运蛋白(Slc10a1)]相关基因的转录水平。结果 与模型组比较,四逆散高剂量显著降低血清总胆汁酸的水平(P＜0.05);明显缓解了Mdr2^-/-小鼠肝脏中央静脉及胆管周围炎性细胞的浸润和胶原纤维的沉积,并显著抑制胆管反应的发生。转录组学及qRT-PCR结果共同表明,四逆散下调Mdr2^-/-小鼠肝脏纤维化、炎症、细胞焦亡相关基因的转录(P＜0.05);同时,四逆散下调胆汁酸合成关键限速酶调控基因Cyp7a1和调控胆汁酸向肝内转运的基因Slc10a1的转录,并上调调控胆汁酸外排的基因Abcb11、Abcc3的转录。结论 四逆散能缓解Mdr2^-/-小鼠胆汁淤积性肝纤维化,机制可能与其调控炎症反应、细胞焦亡以及胆汁酸的合成和转运有关。

Objective To clarify the ameliorative effects of the aqueous extract of Sini San(SNS) on cholestatic hepatic fibrosis in Mdr2 gene-deficient(Mdr2^-/-) mice and to ulteriorly explore its mechanism of action.Methods C57BL/6J mice were used as the control group, Mdr2^-/-mice with C57BL/6J background were used as model mice, and were divided into a model group and a low and high dose group of SNS(calculated as 3.12 and 6.24 g·kg^-1 according to the dosage of raw materials). The SNS group was given an ig of SNS water extract for three consecutive weeks, once a day, while the control group was given pure water. The reagent kit method was used to detect serum alanine aminotransferase(ALT), aspartate aminotransferase(AST), and total bile acid levels. Take the mass of the liver and spleen, and calculate the liver and spleen coefficients. By combining H&E staining, Masson staining,immunohistochemical staining for Fibronectin, CK19 of liver section and the detection of serum total bile acid level, the effects of SNS on hepatic fibrosis and cholestasis in Mdr2^-/-mice were clarified. Besides, based on the transcriptomic sequencing, the potential targets of SNS to ameliorate liver injury in Mdr2^-/-mice were excavated. The genes expression related to liver fibrosis [Fibronectin(Fn1), Collagen-1(Col1a1), Keratin-19(krt19)], inflammation [Interleukin-1β(Il1β), Il6, tumor necrosis factor-α(Tnfα), nitric oxide synthase(Inos)], pyroptosis[apoptosis associated spot like proteins(Pycard), Il18], bile acid synthesis [cytochrome P450 family member 7A1(Cyp7a1)], and transport [bile salt efflux pump(Abcb11), ATP binding cassette transporter(Abcc3), and sodium taurocholic acid cotransporter(Slc10a1)] were quantified via real time PCR(qRT-PCR).Results Compared with the model group,high doses of SNS significantly reduced the level of serum total bile acids(P＜0.05). SNS significantly alleviated the infiltration of inflammatory cells and deposition of collagen fibers around the central vein and bile ducts, simultaneously inhibited the occurrence of bile duct reactions in the liver of Mdr2^-/-mice.Results of qRT-PCR and transcriptomics showed that SNS greatly down-regulated the transcription of genes related to hepatic inflammation and pyroptosis in Mdr2^-/-mice liver(P＜0.05), and decreased the gene expression of Cyp7a1, the key rate-limiting enzyme regulatory gene for bile acid synthesis. Furthermore, SNS also regulated bile acid transport in Mdr2^-/-mice liver which was characterized by down-regulated Slc10a1, Slcolb2 genes(responsible for the transport of bile acid into liver) expression and up-regulated Abcb11, Abcc3 genes(responsible for the excretion of bile acids from liver)expression, which remarkably reliving the pressure of cholestasis of Mdr2^-/-mice.Conclusion SNS alleviated hepatic injury, hepatic fibrosis and cholestasis in Mdr2^-/-mice which may be related to its modulation on inflammatory response, pyroptosis, bile acid synthesis and transport.

R285.5

青年科学基金项目(82004029)