隐花色素1基因在肿瘤坏死因子α诱导的肾小管上皮细胞凋亡中的作用研究  

Role of cryptochrome 1 gene in tumor necrosis factor-α-induced apoptosis of renal tubular epithelial cells

作　　者：李锴[1] 朱志辉 Li Kai;Zhu Zhihui(Department of Endocrinology and Nephrology,People’s Hospital of Hanzhong City,Shaanxi Province,Hanzhong 723000,China)

机构地区：[1]陕西省汉中市人民医院内分泌肾脏病科,723000 [2]陕西省汉中市勉县医院肾病内科

出　　处：《临床内科杂志》2021年第4期262-266,共5页Journal of Clinical Internal Medicine

摘　　要：目的探讨隐花色素1(CRY1)对肿瘤坏死因子α(TNF-α)诱导的肾小管上皮细胞凋亡的影响。方法采用10 ng/ml的TNF-α处理肾小管上皮细胞HK-2,分别培养0 h(0 h组)、12 h(12 h组)、24 h(24 h组)、48 h(48 h组),并以正常培养的细胞作为对照(NC)组。将si-con、si-CRY1质粒转染至HK-2中后(si-con组和si-CRY1组)再用10 ng/ml的TNF-α培养24 h,分别记为TNF-α+si-con组和TNF-α+si-CRY1组。采用蛋白质免疫印迹法(Western Blot)检测裂解半胱氨酸天冬氨酸蛋白酶-3(Cleaved caspase-3)、Bcl-2相关X蛋白(Bax)、B细胞淋巴瘤/白血病-2(Bcl-2)、CRY1、p38丝裂原活化蛋白激酶(p38 MAPK)和磷酸化p38 MAPK(p-p38 MAPK)蛋白表达水平;采用流式细胞术检测细胞凋亡率;采用酶联免疫吸附试验(ELISA)检测白细胞介素-1β(IL-1β)、干扰素γ(IFN-γ)、白细胞介素-6(IL-6)水平;采用实时荧光定量聚合酶链反应(RT-qPCR)检测CRY1 mRNA表达水平。结果与0 h组相比,12 h、24 h、48 h组肾小管上皮细胞细胞凋亡率、Cleaved caspase-3、Bax、CRY1表达水平、IL-1β、IFN-γ、IL-6水平显著升高,Bcl-2表达水平显著降低;与si-con组相比,si-CRY1组肾小管上皮细胞凋亡率、CRY1、Cleaved caspase-3、Bax表达水平、IL-1β、IFN-γ、IL-6水平显著降低,Bcl-2表达水平显著升高;与TNF-α+si-con组相比,TNF-α+si-CRY1组肾小管上皮细胞细胞凋亡率、CRY1、Cleaved caspase-3、Bax表达水平、IL-1β、IFN-γ、IL-6水平显著降低,Bcl-2表达水平显著升高;与si-con组相比,si-CRY1组肾小管上皮细胞p-p38 MAPK表达水平显著降低;与NC组相比,TNF-α组肾小管上皮细胞p-p38 MAPK表达水平显著升高;与TNF-α+si-con组相比,TNF-α+si-CRY1组肾小管上皮细胞p-p38 MAPK表达水平显著降低(P<0.05)。结论CRY1可抑制TNF-α诱导的肾小管上皮细胞凋亡和炎症因子分泌,其机制可能与p38 MAPK信号通路有关。Objective To investigate the effect of cryptosene 1(CRY1)on tumor necrosis factor-α(TNF-α)-induced apoptosis of renal tubular epithelial cells.Methods Renal tubular epithelial cells HK-2 were treated with 10 ng/ml TNF-α,and cultured for 0 h(0 h group),12 h(12 h group),24 h(24 h group),48 h(48 h group),and normal cultured cells were used as control(NC)group.After transfecting si-con and si-CRY1 plasmids into HK-2(si-con group and si-CRY1 group),they were cultured with 10 ng/ml TNF-αfor 24 h,denoted as TNF-α+si-con group and TNF-α+si-CRY1 group,respectively.Western blotting was used to detect cleaved caspase-3,Bcl-2 associated X protein(Bax),B cell lymphoma/leukemia-2(Bcl-2),CRY1,p38 mitogen-activated protein kinase(p38 MAPK)and phosphorylated p38 MAPK(p-p38 MAPK)protein expression levels.Flow cytometry was used to detect apoptosis rate.Enzyme-linked immunosorbent assay(ELISA)was used to detect interleukin-1β(IL-1β),interferon-γ(IFN-γ),interleukin-6(IL-6)levels.Real-time quantitative PCR(RT-qPCR)was used to detect CRY1 mRNA expression.Results Compared with the 0 h group,the apoptosis rates of renal tubular epithelial cells,the expression levels of Cleaved caspase-3,Bax,CRY1 and the levels of IL-1β,IFN-γ,IL-6 in the 12 h,24 h,and 48 h group were significantly increased,the expression level of Bcl-2 was significantly reduced;compared with the si-con group,the renal tubular epithelial cell apoptosis rate,CRY1,Cleaved caspase-3,Bax expression level,IL-1β,IFN-γ,IL-6 levels were significantly reduced in si-CRY1 group,and Bcl-2 expression level was significantly increased;compared with the TNF-α+si-con group,the apoptosis rate of renal tubular epithelial cells,CRY1,Cleaved caspase-3,Bax expression levels,IL-1β,IFN-γ,IL-6 levels were decreased significantly in the TNF-α+si-CRY1 group,and the expression level of Bcl-2 was increased significantly;compared with the si-con group,the expression level of p-p38 MAPK in the renal tubular epithelial cells of the si-CRY1 group was significantly decreased;compared w

关 键 词：隐花素1 肿瘤坏死因子-Α 肾小管上皮细胞 凋亡 p38丝裂原活化蛋白激酶信号通路 

分 类 号：R692.5[医药卫生—泌尿科学]