摘要
目的:基于B细胞淋巴瘤-2(Bcl-2)相关X蛋白(Bax)/胱天蛋白酶-3(Caspase-3)/消皮素E(GSDME)通路探讨丹酚酸F改善高糖诱导的肾小管上皮细胞(HK-2)损伤的机制。方法:细胞增殖与活性检测(CCK-8)法检测不同浓度(2.5、5、10、20μmol·L^(-1))丹酚酸F对高糖诱导的HK-2细胞相对活力的影响及给予丹酚酸F不同干预时间条件下HK-2细胞的相对活力;乳酸脱氢酶(LDH)试剂盒和酶联免疫吸附测定法(ELISA)试剂盒分别检测细胞培养基上清中LDH和白细胞介素-1β(IL-1β)的含量;流式细胞术结合异硫氰酸荧光素(Annexin V-FITC)/碘化丙啶(PI)和Hoechst 33342/PI染色检测丹酚酸F对高糖诱导的HK-2细胞PI阳性率的影响;蛋白免疫印迹法(Western blot)评价丹酚酸F对高糖诱导的HK-2细胞中Bax、Bcl-2、细胞色素C(Cyt C)、胱天蛋白酶-9(Caspase-9)、Caspase-3、GSDME蛋白表达及激活情况的影响。2,7-二氯二氢荧光素二乙酸酯荧光探针法(DCFH-DA)及线粒体膜电位(JC-1)考察丹酚酸F对高糖诱导的HK-2细胞中活性氧(ROS)产生及线粒体膜电位的影响。结果:与空白组比较,模型组细胞活力显著降低(P<0.01),乳酸脱氢酶、炎症因子IL-1β、PI阳性细胞比例均显著升高(P<0.01);Bax、Cyt C、Caspase-9、Caspase-3、GSDME蛋白表达显著升高(P<0.01),Bcl-2蛋白表达显著降低(P<0.01),HK-2细胞线粒体膜电位的降低,ROS产生过量蓄积。与模型组比较,丹酚酸F组有效改善高糖诱导所致HK-2细胞的损伤(P<0.05),明显降低细胞培养基上清中LDH及IL-1β含量(P<0.05,P<0.01),显著减少PI阳性细胞的比例(P<0.01);丹酚酸F组降低Bax、Cyt C、Caspase-9、Caspase-3、GSDME蛋白表达,明显升高Bcl-2蛋白表达(P<0.05,P<0.01),HK-2细胞线粒体膜电位的升高,ROS的过量蓄积减少。结论:丹酚酸F通过减少ROS的产生,改善线粒体膜电位失衡,抑制Bax/Caspase-3/GSDME信号通路介导的细胞焦亡发挥改善高糖诱导所致HK-2细胞损伤的作用。
Objective:To investigate the mechanism of salvianolic acid F(Sal F)in repairing the high glucose-induced injury in human kidney-2(HK-2)cells via the B-cell lymphoma-2(Bcl-2)-associated X protein(Bax)/cysteinyl aspartate-specific proteinase 3(Caspase-3)/gasdermin-E(GSDME)pathway.M ethod:The cell counting kit-8(CCK-8)was used to measure the relative viability of HK-2 cells exposed to high glucose and different concentrations(2.5,5,10,20μmol·L^(-1))of Sal F and the relative viability of HK-2 cells treated with Sal F for different time periods.The levels of lactate dehydrogenase(LDH)and interleukin-1β(IL-1β)in the supernatant of the cell culture were measured by the LDH assay kit and enzyme-linked immunosorbent assay(ELISA)kit,respectively.Flow cytometry combined with Annexin V-FITC/propidium iodide(PI)and Hoechst 33342/PI staining was employed to reveal the proportion of PI-positive HK-2 cells exposed to high glucose.Western blotting was employed to determine the protein levels of Bax,Bcl-2,cytochrome C,cysteinyl aspartate-specific proteinase(Caspase)-9,Caspase-3,and GSDME in the HK-2 cells exposed to high glucose and treated with Sal F.The 2,7-dichlorodihydrofluorescein diacetate fluorescence probe(DCFH-DA)and mitochondrial membrane potential assay kit(JC-1)were used to determine the production of reactive oxygen species(ROS)and the mitochondrial membrane potential in the HK-2 cells exposed to high glucose and treated with Sal F.Result:Compared with the blank group,the model group showed decreased cell viability(P<0.01),elevated levels LDH and IL-1β,increased proportion of PI-positive cells(P<0.01),up-regulated protein levels of Bax,cytochrome C,Caspase-9,Caspase-3,and GSDME(P<0.01),down-regulated protein level of Bcl-2(P<0.01),decreased mitochondrial membrane potential,and excessive ROS accumulation.Compared with the model group,Sal F repaired the high glucose-induced injury in HK-2 cells(P<0.05),lowered the levels of LDH and IL-1β(P<0.05,P<0.01),and decreased the proportion of PI-positive cells(P<0.01).In addition,Sal F down-regulated the protein levels of Bax,cytochrome C,Caspase-9,Caspase-3,and GSDME and up-regulated the protein level of Bcl-2(P<0.05,P<0.01),increased the mitochondrial membrane potential,and decreased the accumulation of ROS in HK-2 cells.Conclusion:Sal F can reduce the production of ROS,restore the balance of mitochondrial membrane potential,and inhibit pyroptosis via the Bax/Caspase-3/GSDME signaling pathway to repair the high glucose-induced injury in HK-2 cells.
作者
石贤聪
谢治深
赵靓
王佳俊
段亚飞
王潘
张振强
张效威
徐江雁
SHI Xiancong;XIE Zhishen;ZHAO Liang;WANG Jiajun;DUAN Yafei;WANG Pan;ZHANG Zhenqiang;ZHANG Xiaowei;XU Jiangyan(Academy of Chinese Medical Sciences,Henan University of Chinese Medicine,Zhengzhou 450046,China)
出处
《中国实验方剂学杂志》
CAS
CSCD
北大核心
2024年第9期56-64,共9页
Chinese Journal of Experimental Traditional Medical Formulae
基金
国家重点研发计划项目(2020YFE0201800)
国家自然科学基金项目(82104471)
河南省重点研发专项(221111520300)
河南省重点研发与推广专项(科技攻关)(232102310454)
河南省高校科技创新人才支持计划项目(24HASTIT072)
中国博士后科学基金项目(2020M682315)。