目的 研究四逆散對Mdr2(Abcb4)基因缺陷(Mdr2^-/-)小鼠膽汁淤積性肝纖維化的緩解作用,并探究其作用機(jī)制。方法 C57BL/6J小鼠作為對照組;C57BL/6J背景的Mdr2^-/-小鼠作為模型小鼠,設(shè)模型組和四逆散低、高劑量(按生藥量計為3.12、6.24 g·kg^-1)組。四逆散組連續(xù)3周ig給予四逆散水提物,每天1次,對照組給予純水。試劑盒法檢測血清丙氨酸氨基轉(zhuǎn)移酶(ALT)、天冬氨酸氨基轉(zhuǎn)移酶(AST)、總膽汁酸水平;取肝臟、脾臟稱質(zhì)量,計算肝臟、脾臟系數(shù);結(jié)合小鼠肝組織HE染色,Masson染色,纖連蛋白(Fibronectin)、細(xì)胞角蛋白19(CK19)的免疫組化染色,明確四逆散對Mdr2^-/-小鼠肝纖維化及膽汁淤積的影響。基于小鼠肝臟轉(zhuǎn)錄組學(xué)測序技術(shù),挖掘四逆散改善Mdr2^-/-小鼠肝損傷的作用靶點(diǎn),并通過實(shí)時熒光定量PCR(qRT-PCR)法檢測肝纖維化[fibronectin(Fn1)、膠原蛋白1(Col1a1)、角蛋白19(krt19)]、炎癥[白細(xì)胞介素-1β(Il1β)、Il6、腫瘤壞死因子-α(Tnfα)、一氧化氮合成酶(Inos)]、細(xì)胞焦亡[凋亡相關(guān)斑點(diǎn)樣蛋白(Pycard)、Il18]、膽汁酸合成[細(xì)胞色素P450家族成員7A1(Cyp7a1)]及轉(zhuǎn)運(yùn)[膽鹽輸出泵(Abcb11)、ATP結(jié)合盒轉(zhuǎn)運(yùn)蛋白(Abcc3)、鈉離子-?；悄懰峁厕D(zhuǎn)運(yùn)蛋白(Slc10a1)]相關(guān)基因的轉(zhuǎn)錄水平。結(jié)果 與模型組比較,四逆散高劑量顯著降低血清總膽汁酸的水平(P＜0.05);明顯緩解了Mdr2^-/-小鼠肝臟中央靜脈及膽管周圍炎性細(xì)胞的浸潤和膠原纖維的沉積,并顯著抑制膽管反應(yīng)的發(fā)生。轉(zhuǎn)錄組學(xué)及qRT-PCR結(jié)果共同表明,四逆散下調(diào)Mdr2^-/-小鼠肝臟纖維化、炎癥、細(xì)胞焦亡相關(guān)基因的轉(zhuǎn)錄(P＜0.05);同時,四逆散下調(diào)膽汁酸合成關(guān)鍵限速酶調(diào)控基因Cyp7a1和調(diào)控膽汁酸向肝內(nèi)轉(zhuǎn)運(yùn)的基因Slc10a1的轉(zhuǎn)錄,并上調(diào)調(diào)控膽汁酸外排的基因Abcb11、Abcc3的轉(zhuǎn)錄。結(jié)論 四逆散能緩解Mdr2^-/-小鼠膽汁淤積性肝纖維化,機(jī)制可能與其調(diào)控炎癥反應(yīng)、細(xì)胞焦亡以及膽汁酸的合成和轉(zhuǎn)運(yùn)有關(guān)。

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

青年科學(xué)基金項(xiàng)目(82004029)