摘要目的 观察7,8-二.羟基黄酮(7,8-DHF)对缺氧诱导的人近端肾小管上皮细胞内质网应激(ERS)损伤的影响,并探讨其可能的分子机制.方法 体外培养人近端肾小管上皮细胞(HK-2),采用缺氧培养箱建立细胞缺氧损伤模型,排除含血清培养基对缺氧的影响后,实验分为5组,分别为缺氧0h、4h、8h、12 h、16h组.实时荧光定量PCR(RT-PCR)法筛选缺氧诱导ERS的最佳缺氧时间.用不同浓度(50、100、150、200、250 μmol/L)7,8-DHF处理HK-2细胞,采用细胞增殖与活性实验筛选7,8-DHF的安全浓度.用7,8-DHF预处理HK-2细胞缺氧模型,按以下处理因素分组:对照组、缺氧+DMSO组、缺氧+7,8-DHF组(包括100 μmol/L组和150 μmol/L组),通过检测细胞增殖与活性筛选最佳给药浓度.后将细胞随机分为缺氧组、缺氧+7,8-DHF组(100 μmol/L),Western印迹法检测细胞蛋白激酶(Akt)、磷酸化蛋白激酶(p-Akt)、富含半胱氨酸蛋白61 (Cyr61)、ERS相关促凋亡蛋白CCAAT增强子结合蛋白(CHOP)的表达.用重组Cyr61慢病毒载体构建稳定表达Cyr61蛋白的Cyr61-HK-2细胞株进行缺氧实验,按如下处理因素分组:缺氧+空质粒转染组,缺氧+Cyr61过表达组,膜连蛋白V和碘化丙啶(Annexin V-FITC/PI)染色后,采用流式细胞仪检测细胞凋亡,Western印迹法检测Cyr61和CHOP蛋白的表达情况.结果 (1)与对照组细胞相比,RT-PCR实验结果显示12h为诱导ERS的最佳缺氧时间(P＜0.01).(2)与对照组相比,细胞增殖与活性实验结果显示100 μmol/L的7,8-DHF组为最佳给药浓度(P＜0.01).(3)与缺氧+DMSO组相比,缺氧+7,8-DHF细胞p-Akt、Cyr61表达量均明显增加,CHOP表达量明显降低(均P＜0.05);LY294002处理可抑制p-Akt的表达,减少Cyr61及增加CHOP的表达(均P＜0.05).(4)与缺氧+空质粒转染组相比,过表达Cyr61组细胞的凋亡率明显降低;Cyr61表达量明显增加,CHOP表达量明显降低(P＜0.01).结论 缺氧可诱导HK-2细胞发生ERS,7,8-DHF可能通过激活Akt通路上调Cyr61,阻止ERS下游凋亡蛋白CHOP的活化,抑制细胞凋亡以及减轻缺氧诱导的HK-2细胞损伤,提不7,8-DHF可能对AKI发挥保护作用.

abstractsObjective To observe the effects of 7,8-dihydroxyflavone (7,8-DHF) on hypoxia induced endoplasmic reticulum stress (ERS) in human proximal tubular epithelial cells (HK-2).Methods The mRNA level of ERS associated biomarkers was evaluated by RT-PCR assay in cell hypoxia damaged model.And HK-2 cells were pretreated with different concentrations of 7,8-DHF through CCK-8 assay;meanwhile CCAAT/enhancer-binding protein homologous protein (CHOP),Cyr61,Akt and p-Akt were determined by western blotting assay.Moreover,HK-2 cells were pretreated by LY294002,a kind of PI3K/Akt inhibitor,to inhibit the PI3K/Akt signaling,and its effects on protein level induced by 7,8-DHF was detected.HK-2 cells was then over-expressed Cyr61 and exposed to hypoxia Apoptosis rate and CHOP expression were determined.Results Compared to hypoxia group (P ＜ 0.01),Hypoxia for 12h was effective in inducing ERS (P ＜ 0.01),while pretreatment with 7,8-DHF (100 μmol/L) increased cell proliferation significantly.The protein expressions of Cyr61 and p-Akt in H+7,8-DHF group were higher,but the level of CHOP was decreased (P ＜ 0.05).With LY294002 pretreated,the expression of Cyr61,p-Akt was down-regulated (all P ＜ 0.05) while the expression of CHOP was up-regulated (P ＜ 0.05).In comparison to empty plasmid group,when cells were transfected with over-expression of Cyr61 plasmid and exposed to hypoxia,the number of apoptotic tubular cells was decreased (P ＜ 0.01).And over-expression of Cyr61 significantly reducedCHOP expression compared with the empty plasmid group (P ＜ 0.01).Conclusion Pretreatment of 7,8-DHF could protect cells from hypoxia injury and inhibit ERS,which may involve the Akt-Cyr61 signaling pathway.