小檗碱对果糖诱导人肾小管细胞内质网应激PERK凋亡通路的影响

Effect of berberine on endoplasmic reticulum stress PERK apoptosis pathway in HK-2 cells by high fructose

目的探讨小檗碱对果糖诱导下肾小管上皮细胞凋亡的影响。方法人肾小管上皮细胞株(HK-2细胞)于含10%胎牛血清的DMEM培养基中进行体外培养,然后随机分为正常组(C组)、果糖组(F组,含25mmol/L果糖培养液)、小檗碱组(B组,25mmol/L果糖+10μmol/L小檗碱)和TUDCA组(T组,25mmol/L果糖+2μmol/L TUDCA培养液)。培养24h后收集细胞,Western blotting检测葡萄糖调节蛋白78(GRP78)、CHOP蛋白表达水平及蛋白激酶R样内质网激酶(PERK)、真核启始因子2α(e IF2α)的磷酸化水平,流式细胞仪检测各组细胞周期情况,TUNEL法检测各组细胞凋亡率。结果与C组比较,F组GRP78、CHOP蛋白表达水平上调,同时p-PERK、p-e IF2α水平明显上升(P<0.05);与F组比较,B组、T组GRP78、CHOP、p-PERK、p-e IF2α表达水平均明显下降(P<0.05);T组与B组GRP78、CHOP、p-P E R K、p-e I F 2α表达水平比较无明显差异。与C组比较,F组H K-2细胞活力明显降低,细胞凋亡指数明显升高(P<0.05);与F组比较,B组和T组肾小管上皮细胞活力明显增加,细胞凋亡指数明显下降(P<0.05);T组B组细胞活力指数、细胞凋亡率比较无明显差异。结论持续采用果糖培养HK-2细胞可激活细胞内PERK通路,引起内质网应激的发生,小檗碱能够抑制果糖诱导的PERK和e IF2α的磷酸化,下调GRP78、CHOP表达,从而调控PERK通路缓解细胞周期阻滞现象,降低细胞凋亡率。

Objective To investigate the effect of berberine on endoplasmic reticulum stress PERK apoptosis pathway in HK-2 cells by high fructose. Methods HK-2 cells were grown in DMEM/F12, containing 10% fetal bovine serum（FBS） and divided randomly into four groups： normal control group（Group C）; Fructose group（Group F）： it contains 25mmol/L fructose culture; Berberine group（Group B）： 25mmol/L fructose ＋ 10μmol/L berberine treatment group; TUDCA group（Group T）： 25mmol/L fructose ＋2μmol/L TUDCA culture group; Cells were collected after culturing 24 h. The expression of glucose-regulated protein 78（GRP78）, CHOP protein and the phosphorylation levels of PERK, e IF2α were tested by Western blotting. The cell cycles were detected by flow cytometry and the apoptosis of cells were detected by TUNEL staining. Results Western blotting showed that the expression of GRP78 and CHOP protein in group F was significantly higher than that in group C, and the levels of p-PERK and p-e IF2α in group F were significantly higher than those in group F. Compared with group F, GRP78, CHOP, p-PERK and p-e IF2α in group B and T were significantly lower（P〈0.05）. The expression of GRP78, CHOP, p-PERK and p-e IF2α in group B had no significant difference. Flow cytometry and TUNEL staining showed that the cell viability of fructose group was significantly lower than that of C group, and the apoptotic index was significantly higher than that of group C（P〈0.05）. Compared with group F, the activity of HK-2 cells in group B and T significantly increased, otherwise the apoptosis index significantly decreased, and the difference was statistically significant（P〈0.05）. Compared with group T, the viability index and apoptosis rate of group B had no significant difference（P〉0.05）. Conclusion Persistent high fructose can activate the intracellular PERK pathway in HK-2 cells, causing endoplasmic reticulum stress. Berberine can inhibit the fructose-induced PERK and e IF2α phosphorylation, down-regulated the expression of GRP78, CHOP protein, thus by regulating PERK Pathways to alleviate cell cycle arrest and reduce cell apoptosis.