补肾活血方含药血清对AGE-BSA诱导的HK-2细胞凋亡及PERK-ATF4-CHOP信号通路的影响

Effect of Bushen Huoxue Formula Containing Serum on AGE-BSA Induced Apoptosis of HK-2 Cells and the PERK-ATF4-CHOP Signaling Pathway

目的 观察补肾活血方(BSHXF)含药血清对糖基化终产物修饰的牛血清白蛋白(AGE-BSA)诱导的人肾小管上皮细胞(HK-2)凋亡的影响及其机制。方法 采用AGE-BSA诱导HK-2细胞建立凋亡损伤模型,将HK-2细胞分为牛血清白蛋白(BSA)组、AGE-BSA组、不同浓度(2.5%、5%、10%、20%)BSHXF含药血清组,CCK-8法检测各组细胞活力。确定10%、20%BSHXF含药血清可显著提高HK-2细胞活力后再将HK-2细胞分为BSA组、AGE-BSA组、BSHXF低剂量组(BSHXF-L,10%含药血清)和BSHXF高剂量组(BSHXF-H,20%含药血清),流式细胞仪检测各组细胞凋亡情况。以4-苯基丁酸钠盐为阳性对照,设BSA组、AGE-BSA组、BSHXF-L组、BSHXF-H组和4-PBA组,运用Western Blot和RT-PCR法检测补肾活血方对HK-2细胞葡萄糖调节蛋白78 (GRP78蛋白)及mRNA表达的影响。将蛋白激酶样内质网激酶(PERK)-shRNA转染HK-2细胞,采用RT-PCR和Western Blot检测各组PERK mRNA和蛋白的表达。以干扰效果最佳的PERK-shRNA为阳性对照,设BSA组、AGE-BSA组、BSHXF-L组、BSHXF-H组、PERK-shRNA组,Western Blot检测各组HK-2细胞p-PERK/PERK、p-真核起始因子2α(eIF2α)/eIF2α、活化转录因子4(ATF4)、C/EBP同源蛋白(CHOP)、B淋巴细胞瘤2(Bcl-2)、Bcl-2相关X蛋白(Bax)、切割型半胱天冬酶-3(Cleaved-caspase-3)、切割型半胱天冬酶-9(Cleaved-caspase-9)蛋白表达的影响。结果 与BSA组比较,AGE-BSA组HK-2细胞的活力下降,凋亡率增加(P<0.01),GRP78蛋白及mRNA表达升高(P<0.01),Bcl-2蛋白表达下降(P<0.01),p-PERK/PERK、p-eIF2α/eIF2α、ATF4、CHOP、Bax、Cleaved-caspase-3、Cleaved-caspase-9蛋白表达升高(P<0.01)。与AGE-BSA组比较,BSHXF-L和BSHXF-H组可明显提高HK-2细胞的活力(P<0.01),降低HK-2细胞的凋亡率(P<0.01),同时,显著下调GRP78蛋白及mRNA表达(P<0.05,P<0.01),上调Bcl-2蛋白表达(P<0.05,P<0.01),抑制p-PERK/PERK、p-eIF2α/eIF2α、ATF4、CHOP、Bax、Cleaved-caspase-3、Cleaved-caspase-9蛋白表达(P<0.05,P<0.01)。结论 补肾活血方含药血清对于AGE-BSA诱导的HK-2细胞凋亡具有保护作用,其机制可能与抑制PERK-ATF4-CHOP信号通路,减轻内质网应激有关。

Objective To observe the effect of Bushen Huoxue Formula(BSHXF)containing serum on apoptosis of human renal tubular epithelial cells(HK-2)induced by advanced glycosylation end product modified bovine serum albumin(AGE-BSA) and its mechanism.Methods HK-2 cells were divided into the bovine serum albumin(BSA)group,the AGE-BSA group,and different concentrations(2.5%,5%,10%,20%)of BSHXFcontaining serum groups.The cell viability of each group was detected by the CCK-8 method.After it was determined that 10% and 20% of the drug-containing serum of BSHXF could significantly increase the viability of HK-2 cells,HK-2 cells were divided into the BSA group,the AGE-BSA group,the BSHXF low-dose group(BSHXF-L,10% drugcontaining serum),and the BSHXF high-dose group(BSHXF-H,20% drug-containing serum).Flow cytometry was used to detect apoptosis in each group.Sodium 4-phenylbutyrate was used as a positive control.HK-2 cells were divided into the BSA group,the AGE-BSA group,the BSHXF-L group,the BSHXF-H group,and the 4-PBA group.Western Blot and RT-PCR were used to detect glucose-regulated protein 78 protein and mRNA expression in each group of HK-2 cells.The protein kinase R(PKR)-like ER kinase(PERK)-sh RNA was transfected into HK-2 cells,and the expression of each group of PERK mRNA and protein was detected by RT-PCR and Western Blot.The PERKsh RNA with the best interference effect was identified as a positive control.HK-2 cells were divided into the BSA group,the AGE-BSA group,the BSHXF-L group,the BSHXF-H group,and the PERK-shRNA group.Western Blot was performed to detect the expression of p-PERK/PERK,p-eukaryotic initiation factor 2α(e IF2α)/e IF2α,activating transcription factor 4(ATF4),C/EBP homologous protein(CHOP),B cell lymphoma 2(Bcl-2),Bcl-2-associated X(Bax),Cleaved-caspase-3,and Cleaved-caspase-9 proteins in each group of HK-2 cells.Results Compared with the BSA group,HK-2 cells in the AGE-BSA group showed decreased viability and increased apoptosis(P<0.01),GRP78 protein and mRNA expression increased(P<0.01),Bcl-2 protein expression decreased(P<0.01),and p-PERK/PERK,p-eIF2α/eIF2α,ATF4,CHOP,Bax,Cleaved-caspase-3,and Cleaved-caspase-9protein expression increased(P<0.01).Compared with the AGE-BSA group,Both the BSHXF-L group and the BSHXF-H group significantly increased the viability(P<0.01) and the apoptosis rate of HK-2 cells decreased(P<0.01).Meanwhile,it significantly downregulated GRP78 protein and mRNA expression(P<0.05,P<0.01),upregulated Bcl-2 protein expression(P 0.05,P 0.01),and inhibited p-PERK/PERK、p-eIF2α/eIF2α,ATF4,CHOP,Bax,Cleaved-caspase-3,and Cleaved-caspase-9 protein expression(P 0.05,P 0.01).Conclusion BSHXF containing serum has a protective effect on AGE-BSA-induced apoptosis of HK-2 cells.Its mechanism may be related to inhibiting the PERK-ATF4-CHOP signaling pathway and alleviating endoplasmic reticulum stress.