黄连素通过下调miR-1224-3p缓解过氧化氢诱导的胰岛β细胞损伤研究

Study on berberine relieves hydrogen peroxide-induced injury of isletβcells by down-regulating miR-1224-3p

目的:探讨黄连素对过氧化氢(H_(2)O_(2))诱导的胰岛β细胞损伤的影响及可能机制。方法:体外培养小鼠胰岛β细胞MIN6,经不同剂量(1.0、2.5、5.0μmol·L^(-1))黄连素预先干预24 h后用H_(2)O_(2)诱导、或H_(2)O_(2)处理转染miR-1224-3p抑制剂的MIN6细胞,或使用5.0μmol·L^(-1)黄连素预先干预转染miR-1224-3p模拟物的MIN6细胞24 h后用H_(2)O_(2)诱导,采用CCK-8法检测细胞增殖抑制率,流式细胞术检测细胞凋亡,蛋白印迹法检测细胞中Bax和Bcl-2蛋白表达,ELISA法检测细胞培养上清液中IL-6、IL-1β和TNF-α水平,RT-qPCR检测细胞中miR-1224-3p表达。结果:黄连素预先处理可降低H_(2)O_(2)诱导的MIN6细胞抑制率、凋亡率及Bax蛋白表达(P<0.05),而促进Bcl-2蛋白表达(P<0.05),降低IL-6、IL-1β和TNF-α的分泌水平及miR-1224-3p表达(P<0.05)。下调miR-1224-3p可降低H_(2)O_(2)诱导的MIN6细胞抑制率、凋亡率及Bax蛋白表达,而促进Bcl-2蛋白表达(P<0.05),降低IL-6、IL-1β和TNF-α的分泌水平(P<0.05)。结论:黄连素可能通过下调miR-1224-3p表达抑制H_(2)O_(2)诱导的胰岛β细胞MIN6凋亡和炎性因子表达。

Objective:To investigate the effect of berberine on the injury of isletβcells induced by hydrogen peroxide(H_(2)O_(2))and its possible mechanism.Methods:The mouse pancreatic isletβcells MIN6 were cultured in vitro.MIN6 cells were preintervened by different doses of berberine(1.0,2.5,5.0μmol·L^(-1))for 24 hours and then induced with H_(2)O_(2),or H_(2)O_(2)induced MIN6cells transfected with miR-1224-3p inhibitor,or 5.0μmol·L^(-1) berberine pre-intervened MIN6 cells transfected with miR-1224-3p mimic for 24 hours,then induced with H_(2)O_(2).The cell proliferation inhibition rate was detected by CCK-8 method,apoptosis was detected by?ow cytometry,the protein expression of Bax and Bcl-2 in the cells were detected by western blot.The levels of IL-6,IL-1βand TNF-αin the cell culture supernatant were detected by ELISA.And the expression of miR-1224-3p in cells was detected by RT-qPCR.Results:Pre-intervention of berberine in MIN6 cells induced by H_(2)O_(2)reduced the inhibition rate,apoptosis rate of MIN6cells and the protein expression of Bax(P<0.05),and promoted the protein expression of Bcl-2(P<0.05),reduced the levels of IL-6,IL-1β,TNF-αand the expression of miR-1224-3p(P<0.05).Down-regulation of miR-1224-3p reduced the inhibition rate,the apoptosis rate and the protein expression of Bax of MIN6 cells induced by H_(2)O_(2)(P<0.05),promoted the protein expression of Bcl-2(P<0.05),and reduced the levels of IL-6,IL-1βand TNF-α(P<0.05).Conclusion:Berberine may inhibit the apoptosis and the expression of in?ammatory factors of H_(2)O_(2)-induced isletβcells by down-regulating the expression of miR-1224-3p.