罗沙司他通过上调HIF-1α减轻热打击诱导的肾小管上皮细胞凋亡与衰老  

Roxadustat attenuates heat stress-induced apoptosis and senescence in renal tubular epithelial cells by upregulating HIF-1α

作　　者：宋用为 王玲 陈文婷 张明洋 杨学森[2] 戴欢子 SONG Yongwei;WANG Ling;CHEN Wenting;ZHANG Mingyang;YANG Xuesen;DAI Huanzi(Department of Rheumatism Immunology,Army Medical Center of PLA,Chongqing,400042;Department of Tropical Medicine,Faculty of Military Preventive Medicine,Army Medical University(Third Military Medical University),Chongqing,400038,China)

机构地区：[1]陆军特色医学中心风湿免疫科,重庆400042 [2]陆军军医大学(第三军医大学)军事预防医学系热带医学教研室,重庆400038

出　　处：《陆军军医大学学报》2024年第10期1092-1099,共8页Journal of Army Medical University

基　　金：重庆市自然科学基金面上项目(CSTB2023NSCQ-MSX1101)。

摘　　要：目的探讨罗沙司他对热打击诱导的肾小管上皮细胞(HK-2细胞)凋亡与衰老的影响及其机制。方法体外培养HK-2细胞,用不同浓度(10、20、30、40、50μmol/L)的罗沙司他处理24 h,通过CCK-8法确定罗沙司他的最佳干预浓度。将HK-2细胞分为4组(n=3):对照组、罗沙司他组(30μmol/L、24 h)、热打击组(43℃、2 h)、热打击+罗沙司他组(30μmol/L罗沙司他处理24 h后热打击2 h)。通过CCK-8检测细胞活力;Western blot检测缺氧诱导因子-1α(hypoxia-inducible factor-1,HIF-1α)、Cleaved Caspase-3、p16、p21蛋白表达量;免疫荧光检测HIF-1α分布;细胞衰老β-半乳糖苷酶染色试剂盒检测SA-β-Gal活性;TUNEL染色检测细胞凋亡情况。结果用30μmol/L罗沙司他处理的细胞活力最高。与对照组比较,热打击组细胞活力显著降低(P<0.05);HIF-1α、Cleaved Caspase-3、p16、p21蛋白表达量显著增加(P<0.05);SA-β-Gal活性显著增加(P<0.05);TUNEL阳性细胞占比显著增加(P<0.05)。与热打击组比较,热打击+罗沙司他组Cleaved Caspase-3、p16、p21蛋白表达量显著降低(P<0.05);SA-β-Gal活性显著降低[(65.44±5.00)%vs(77.15±2.61)%,P<0.05];TUNEL阳性细胞占比显著降低[(16.73±2.20)%vs(46.40±13.87)%,P<0.05],细胞活力显著增加[(86.33±4.51)%vs(66.33±8.50)%,P<0.05];HIF-1α蛋白表达量进一步增加(P<0.05)。免疫荧光结果显示HIF-1α主要分布在细胞核及细胞核周围。结论罗沙司他通过上调HIF-1α减轻热打击诱导的肾小管上皮细胞凋亡和衰老。Objective To investigate the effect and underlying mechanism of roxadustat on apoptosis and senescence of renal tubular epithelial cell line HK-2 induced by heat stress.Methods After HK-2 cells were treated with different concentrations of roxadustat(10,20,30,40 and 50μmol/L)for 24 h,CCK-8 assay was used to determine the optimal intervention concentration of roxadustat.HK-2 cells were divided into 4 groups(n=3):control group,roxadustat group(30μmol/L,24 h),heat-stress group(43℃,2 h),and heat-stress+roxadustat group(30μmol/L roxadustat treatmnet for 24 h followed by heat-stress 2 h).Cell viability was detected by CCK-8 assay.Expression of hypoxia-inducible factor-1α(HIF-1α),Cleaved Caspase-3,p16 and p21 at protein level was detected by Western blotting.Immunofluorescence assay was employed to observe the distribution of HIF-1α.β-galactosidase staining kit was utilized to detect SA-β-Gal activity.TUNEL staining was used to measure cell apoptosis.Results The highest cell viability was observed in the cells after 30μmol/L roxadustat treatment.Heat stress resulted in a significant decrease in cell viability(P<0.05),elevated protein levels of HIF-1α,Cleaved Caspase-3,p16 and p21(P<0.05),enhanced SA-β-Gal activity(P<0.05)and increased percentage of TUNEL-positive cells(P<0.05)when compared with the cells in the control group.In comparison with the heat-stress group,the heat-stress+roxadustat group showed significant decrease in the protein levels of Cleaved Caspase-3,p16 and p21(P<0.05),reduced activity of SA-β-Gal[(65.44±5.00)%vs(77.15±2.61)%,P<0.05]and decreased percentage of TUNEL-positive cells[(16.73±2.20)%vs(46.40±13.87)%,P<0.05],but increase in cell viability[(86.33±4.51)%vs(66.33±8.50)%,P<0.05]as well as HIF-1αprotein expression(P<0.05).Furthermore,immunofluorescence assay showed that HIF-1αwas mainly distributed in the nucleus and perinucleus.Conclusion Roxadustat attenuates heat stress-induced apoptosis and senescence of renal tubular epithelial cells by upregulating HIF-1α.

关 键 词：热打击 罗沙司他 HIF-1Α 细胞凋亡 细胞衰老 

分 类 号：R594.11[医药卫生—内科学] R692.9[医药卫生—临床医学] R977.6