Effect of Sini San on improving cholestatic liver fibrosis in Mdr2-/- mice
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.
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