葛根素调控miR-7抗小鼠肝细胞缺氧-复氧存活和细胞凋亡的作用机制

Mechanism of puerarin regulating miR⁃7 against hypoxia⁃reoxygenation survival and apoptosis in mouse hepatocytes

目的探讨葛根素对缺氧-复氧(H-R)诱导小鼠肝细胞损伤的影响及其分子机制。方法将小鼠肝细胞采用随机数字表法分为对照组、H-R组、不同浓度(70μmol/L、140μmol/L、280μmol/L、560μmol/L)葛根素组、H-R+葛根素组、H-R+miR-7组、H-R+miR-7阴性对照(miR-con)组、H-R+葛根素+anti-miR-7组、H-R+葛根素+anti-miR-7阴性对照(anti-miR-con)组、H-R+葛根素+佛波酯(PMA)组。实时荧光定量PCR(qPCR)检测miR-7水平,四甲基偶氮唑盐微量酶反应比色法(MTT法)检测细胞活性,流式细胞仪检测细胞凋亡,蛋白质印迹法(Western Blot)检测细胞中细胞核相关抗原Ki-67(Ki-67)、B细胞淋巴瘤/白血病-2(Bcl-2)、Bcl-2相关X蛋白(Bax)、核因子-κB(NF-κB)p65蛋白表达。结果葛根素预处理后,H-R小鼠肝细胞活性明显增强(P<0.05),Ki-67蛋白表达量、miR-7表达量显著增加(P<0.05)。与H-R组比较,280μmol/L葛根素预处理后,H-R小鼠肝细胞凋亡率[(14.72±1.20)%比(25.85±1.58)%]、Bax、NF-κB p65表达量降低,Bcl-2蛋白表达量增加(均P<0.05)。与H-R+miR-con组相比,过表达miR-7显著提高H-R小鼠肝细胞miR-7水平、细胞活性[(81.29±7.56)比(0.28±0.04)]、Ki-67、Bcl-2蛋白水平,降低细胞凋亡率[(15.65±1.56)%比(26.65±1.25)%]、Bax蛋白水平(均P<0.05)。干扰miR-7表达部分逆转葛根素对小鼠肝细胞H-R后细胞凋亡、Bax和NF-κB p65表达的抑制作用,以及部分逆转葛根素对小鼠肝细胞H-R后细胞活性、miR-7、Ki-67、Bcl-2表达的促进作用。结论葛根素显著促进H-R诱导的小鼠肝细胞增殖并抑制细胞凋亡,可能是通过调控miR-7表达,抑制NF-κB信号通路活性发挥作用。

Objective To investigate the effect and molecular mechanism of puerarin on hepatocytes injury induced by hypoxia and reoxygenation(H⁃R)inmice.Methods mouse hepatocytes were randomly divided into control group H⁃R group,puerarin group with different concentrations(70μmol/L,140μmol/L,280μmol/L,560μmol/L),H⁃R+puerarin group,H⁃R+microRNA⁃7(miR⁃7)group,H⁃R+miR⁃7 negative control miR⁃con group,H⁃R+puerarin+anti⁃miR⁃7 group,H⁃R+puerarin+anti⁃miR⁃7 negative control anti⁃miR⁃con group,H⁃R+puerarin+phorbol ester(PMA)group by random number table method.miR⁃7 level was determined by real⁃time quantitative PCR(qPCR),cell viability was detected by methyl thiazolyl tetrazolium(MTT)assay,cellapoptosis was de⁃tected by flow cytometry,and expressions of Nuclear associated antigen Ki67(Ki⁃67)、B cell lymphoma/lewkmia⁃2(Bcl⁃2),Bcl⁃2 associated X protein(Bax)and nuclear factor⁃κB(NF⁃κB)p65 were analyzed by Western Blot.Results After pretreatment with puerarin,the cell viability of mouse hepatocytes in H⁃R was obviously increased(P<0.05),and the expression of Ki⁃67 protein、miR⁃7 notably increased(P<0.05).Compared with H⁃R group,after pretreatment of 280μmol/L puerarin,the apoptosis rate[(14.72±1.20)%vs.(25.85±1.58)%],expressions of Bax and NF⁃κB p65 decreased and Ki⁃67、Bcl⁃2 protein expressions in⁃creased in H⁃R mouse hepatocytes,and all the differences reached significant(all P<0.05).Compared with H⁃R+miR⁃con group,overexpression of miR⁃7 greatly increased the level of miR⁃7,cell viability[(81.29±7.56)vs.(0.28±0.04)]and Bcl⁃2 protein in H⁃R mouse hepatocytes,and decreased the apoptosis rate[(15.65±1.56)%vs.(26.65±1.25)%]and Bax protein level,and all the dif⁃ferences reached significant(all P<0.05).Interfering with miR⁃7 expression partially reversed the inhibitory effect of puerarin on apoptosis,Bax and NF⁃κB p65 expression after H⁃R of mouse hepatocytes,and partially reversed the promoting effect of puerarin on cell activity,miR⁃7,Ki⁃67 and Bcl⁃2 expression after H⁃R of mouse hepatocytes.Conclusion Puerarin significantly promotes the proliferation and inhibits the apoptosis of mouse hepatocytes induced by H⁃R,possibly by regulating the expression of miR⁃7 and inhibiting the the activity ofNF⁃κB signaling pathway.