摘要
目的观察klotho蛋白对硫酸吲哚酚(Is)诱导人脐静脉内皮细胞损伤的作用,并探讨内质网应激是否参与此过程中及可能机制。方法(1)体外培养人脐静脉内皮细胞,分别以5、25、50mg/LIS干预细胞48h,以50mg/LIS干预细胞12、24、48h,CCK-8法测定细胞活性,观察IS对细胞活性的影响。(2)50mg/LIS处理细胞48h,同时给予0、1、10、100肛g/Lklotho蛋白干预,CCK-8测定细胞活性,观察不同浓度klotho蛋白对Is毒性的影响。(3)细胞分为对照组、IS组(50mg/LIS)、klotho组(50mg/LIS+100μg/Lklotho)和磷酸腺苷酸活化蛋白激酶(AMPK)通路抑制剂CompoundC组(50mg/LIS+100μg/Lklotho+10μmol/LCompoundC)干预48h,CCK-8测定细胞活性,流式细胞术检测细胞凋亡,实时荧光定量PCR、Western印迹分别检测糖调节蛋白78(GRP78)、CCAAT/增强子结合蛋白同源蛋白(CHOP)的mRNA和蛋白表达,Western印迹检测磷酸化AMPK表达。结果IS以时间依赖性和浓度依赖性的方式抑制细胞活性。50mg/LIS组细胞活性低于对照组(P〈0.05)。klotho蛋白可部分恢复IS抑制的细胞活性且呈浓度依赖。100μg/Lklotho组细胞活性高于IS组(P〈0.05)。与对照组比较,IS组GRP78、CHOP表达上调,细胞凋亡率增加,磷酸化AMPK降低(均P〈0.05);与IS组比较,klotho组GRP78、CHOP表达降低,细胞凋亡率降低,磷酸化AMPK上调(均P〈0.05)。klotho蛋白的作用可被CompoundC阻断,上述指标CompoundC组与klotho组差异均有统计学意义。结论IS对人脐静脉内皮细胞具有毒性作用,可抑制细胞活性,诱导内质网应激及细胞凋亡;klotbo蛋白可部分拮抗IS对内皮细胞的毒性作用,其机制可能与活化AMPK通路和降低内质网应激相关的细胞凋亡有关。
Objective To investigate the effect of klotho on the human vein umbilical endothelial cells (HUVECs) injury induced by indoxyl sulfate (IS) and to explore its mechanism and the role of endoplasmic reticulum stress (ERS) in this process. Methods (1) The cell vitalities of HUVECs incubated with different concentration of IS (5, 25, 50 mg/L) for 48 h and with 50 mg/L IS for different time points (12, 24, 48 h) were measured by CCK-8 assay. (2) HUVECs were incubated with 50 mg/L IS and different concentration of klotho (0, 1, 10, 100 μg/L) for 48 h and their ceil viabilities were measured by CCK-8 assay. (3) HUVECs were divided into four groups: control group, IS group (50 mg/L IS), klotho group (50 mg/L IS+100 μg/L klotho) and Compound C group (50 mg/L IS+100 μg/L klotho+ 10 μmol/L Compound C). The cell vitality and the apoptosis of HUVECs were evaluated by CCK- 8 assay and flow cytometry, respectively. The mRNA and protein expressions of GRP78 and CHOP were measured by real-time PCR and Western blotting. The phosphorylation level of AMPK was tested by Western blotting. Results IS inhibited cell vitality in the time- dependent and concentration-dependent manner. The cell viability of HUVECs with 50 mg/L IS was lower than normal control (P〈 0.05). The inhibited cell vitality induced by IS was partly restored by klotho in concentration-dependent manner. The cell viability was higher in 100 μg/L klotho+50 mg/L IS group than 50 mg/L IS group (P 〈 0.05). Compared with control group, the expressions of GRP78 and CHOP and cell apoptosis increased, however, the level of phosphorylated AMPK (p-AMPK) decreased in IS group (all P 〈 0.05). Compared with IS group, the expressions of GRP78 and CHOP and cell apoptosis decreased and the level of p- AMPK increased in klotho group (all P 〈 0.05). Furthermore, the above effects of klotho could be partly blocked by Compound C. The above indexes showed statistical differences between Compound C group and klotho group. Conclusions IS can inhibit the HUVECs cell vitality, and induce ERS and cell apoptosis. Klotho protein could antagonize the above effects, probably through activating AMPK pathway and reducing ERS-mediated cell apoptosis.
出处
《中华肾脏病杂志》
CSCD
北大核心
2017年第9期698-703,共6页
Chinese Journal of Nephrology
基金
基金项目:江苏省教育厅六大人才高峰项目(WSN-056)
江苏省临床医学科技专项(BL2014080)
扬州大学附属医院院级课题