水飞蓟宾对利福平和异烟肼致肝损伤中胆汁酸转运体Ntcp与Bsep的影响

Effects of silybin on bile acid transporters Ntcp and Bsep in liver injury induced by rifampicin and isoniazid via Nrf2/ARE signal pathway

目的 初步探讨水飞蓟宾能否通过激动Nrf2/ARE信号通路,激活下游胆汁酸相关的转运体牛磺胆酸钠转运蛋白(Ntcp)及胆盐输出泵(Bsep)Bsep的表达,从而改善异烟肼/利福平(INH/RFP)INH/RFP引起的肝损伤。方法:选用SPF级ICR小鼠随机分成5组:对照组(CON组),模型组(IR组),水飞蓟宾干预组(SY50组,SY100组,SY200组),除对照组外,干预组及IR组根据体质量给予75 mg/kgINH+150 mg/kgRFP,对照组给予空白溶剂,干预组分别同时给予水飞蓟宾低剂量50 mg/kg(IR+SY50),中剂量100 mg/kg(IR+SY100)以及高剂量200 mg/kg(IR+SY200),连续灌胃给药14 d。第15天给予异氟烷吸入麻醉后摘眼球取血,离心后用于生化指标测定,取脏组织于10%福尔马林固定用于病理切片、HE染色;4%多聚甲醛固定用于目标蛋白免疫组化测定;Western blot测定目标蛋白Ntcp、Bsep及核Nrf2的表达。结果 与CON组相比,IR组均能够引起生化指标ALT、AST、TBil、DBil、ALP、TBA的显著变化,与IR组相比,低剂量的水飞蓟宾干预组ALT、Tbil、TBA降低明显,中剂量的水飞蓟宾能显著降低ALT、AST、Tbil、TBA,高剂量的水飞蓟宾能显著降低除ALP以外的所有指标,差异有统计学意义(P<0.05)。IR组Ntcp,Bsep蛋白表达均下调,而水飞蓟宾能显著上调这些蛋白的表达,IR组下调Nrf2核蛋白的表达,与水飞蓟宾合用后,Nrf2核蛋白的表达显著上调。结论 水飞蓟宾可能通过激活Nrf2入核,调控下游胆汁酸转运体Ntcp、Bsep的表达,治疗INH/RFP引起的肝损伤。

Objective To explore whether silybin can activate the expression of bile acid related transporters the Sodium-taurocholate Cotransporting Polypeptide(Ntcp)and the Bile Salt Export Pump(Bsep)by activating Nrf2/ARE signal pathway,so as to improve the liver injury caused by isoniazide/rifampicin. Methods SPF ICR mice were randomly divided into 5 groups:control group(CON group),model group(IR group)and silybin intervention group(SY50 group,SY100 group and SY200 group). In addition to the control group,the intervention group and IR group were given 75 mg/kg INH+150 mg/kg RFP according to body weight,the control group was given blank solvent,and the intervention group was given silybin 50 mg(IR+SY 50),100 mg(IR+SY100)and 200 mg(IR+SY200)respectively for 14 days. On the 15th day,after isoflurane inhalation anesthesia,eyeball blood was taken,centrifuged for biochemical index determination,and liver tissue was fixed in 10% formalin for pathological section and HE staining;4% paraformaldehyde was fixed for immunohistochemical determination of target protein;the expressions of target proteins Ntcp,Bsep and Nrf2 were determined by Western blot. Results Compared with the control group,the IR group could cause significant changes in biochemical indexes ALT,AST,Tbil,Dbil,ALP and TBA.Compared with the model group,the ALT,Tbil and TBA in the low-dose silybin intervention group decreased significantly,Medium dose silybin could significantly reduce ALT,AST,TBIL and TBA,and high dose silybin could significantly reduce all indexes except ALP(P<0.05). The expression of Ntcp,Bsep and nuclear Nrf2 protein were downregulated in the IR group,while silybin could significantly up-regulate the expression of these proteins compared with those in the IR group. Conclusion Silybin may regulate the expression of downstream bile acid transporters Ntcp and Bsep by activating Nrf2 into the nucleus to treat liver injury caused by INH/RFP.