4-PBA通过抑制内质网应激减轻碘普罗胺诱导的HK-2细胞损伤

The protective effect of 4-Phenybutyric acid on iopromide-induced injury in HK-2 cells through inhibiting endoplasmic reticulum stress

目的探讨内质网应激抑制剂4-苯基丁酸(4-Phenylbutyric acid,4-PBA)对碘普罗胺引起的人近端肾小管上皮细胞(HK-2细胞)损伤的保护作用及其可能机制。方法实验分为对照组、碘普罗胺组、碘普罗胺+不同浓度4-PBA处理组(分别加入1、2.5、5 mmol/L 4-PBA处理细胞)。采用CCK-8法检测各组细胞细胞活力,用DCFH-DA染色显微镜下观察并用流式细胞仪检测各组细胞活性氧(Reactive oxygen species,ROS)水平,DAPI染色和罗丹明123(Rhodamine,Rh123)染色分别检测细胞核形态和线粒体膜电位(ΔΨm),Western blot法检测细胞凋亡相关蛋白Cleaved-Caspase-3、Bax、Bcl-2表达以及内质网应激相关蛋白CHOP、GRP78、p-JNK、JNK、p-eIF-2α、eIF-2α、p-IRE1α表达。结果与对照组相比,碘普罗胺组细胞存活率及ΔΨm明显降低,ROS水平,凋亡细胞数,Bax/Bcl-2、p-JNK/JNK及p-eIF-2α/eIF-2α比值,Cleaved-Caspase-3、CHOP、GRP78、p-IRE1α蛋白表达明显增高,而加入不同浓度的4-PBA处理后,可部分逆转上述效应,提高细胞存活率及ΔΨm,下调细胞ROS水平,减少细胞凋亡数,降低凋亡相关蛋白以及内质网应激相关蛋白的表达水平。结论4-PBA可通过抑制内质网应激减轻碘普罗胺引起的肾小管上皮细胞损伤。

Objective To investigate the protective effect and possible mechanism of endoplasmic reticulum stress inhibitor 4-Phenylbutyric acid(4-PBA)on the injury of human proximal tubular epithelial cells(HK-2 cells)induced by iopromide.Methods Cells were subdivided into 5 groups,including control group,iopromide group,iopromide with different concentrations of 4-PBA groups(cells were exposed to 1,2.5,5 mmol/L 4-PBA,respectively).Cell viability of each group was detected by CCK-8.The level of reactive oxygen species(ROS)was measured by DCFH-DA staining method and flow cytometry.The morphology of nucleus and mitochondrial membrane potential(ΔΨm)of cells were observed by DAPI staining and Rhodamine 123 staining,respectively.The expression of apoptosis-related proteins including Cleaved-Caspase-3,Bax,Bcl-2 and endoplasmic reticulum stress-related proteins including CHOP,GRP78,p-JNK,JNK,p-eIF-2α,eIF-2α,p-IRE1αwere measured by Western blot.Results Compared with the control group,cell viability andΔΨm in iopromide group were significantly decreased.The level of ROS,the number of apoptotic cells,the ratio of Bax/Bcl-2,p-JNK/JNK and p-eIF-2α/eIF-2α,the expression of Cleaved-Caspase-3,CHOP,GRP78 and p-IRE1αwere significantly increased in iopromide group.However,after adding different concentrations of 4-PBA,the above effects were partially reversed.Compared with the iopromide group,the cell viability andΔΨm were up-regulated,while the level of ROS,the number of apoptotic cells,the expression of apoptosis-related proteins and endoplasmic reticulum stress-related proteins were down-regulated after treatment with 4-PBA in a concentration-dependent manner.Conclusion These results indicated that 4-PBA appears to suppress iopromide-induced HK-2 cells injury by inhibiting endoplasmic reticulum stress.