基于NF-κB信号通路探讨低分子量肝素对糖尿病肾病足细胞增殖、凋亡和炎症的影响  

The effects of low molecular heparin on the proliferation,apoptosis and inflammation of podocytes in diabetic nephropathy based on the NF-κB signaling pathway

作　　者：王淮淮 赵学慧 王会芳[1] 魏晓岩[1] 贾军利[1] WANG Huaihuai;ZHAO Xuehui;WANG Huifang;WEI Xiaoyan;JIA Junli(Department of Nephrology,the Second Affiliated Hospital of Hebei North University,Zhangjiakou,Hebei 075000,China)

机构地区：[1]河北北方学院附属第二医院肾内科,河北张家口075000

出　　处：《检验医学与临床》2024年第10期1365-1370,1376,共7页Laboratory Medicine and Clinic

基　　金：2021年度河北省医学科学研究课题计划(20210125)。

摘　　要：目的基于核转录因子-κB(NF-κB)信号通路的变化探讨低分子量肝素(LMH)对糖尿病肾病足细胞增殖、凋亡和炎症的影响。方法体外培养人肾小球足细胞(HGPC),将HGPC分为对照(Con)组(以含5 mmol/L D-葡萄糖的培养基培养)、高D-葡萄糖(Glu-H)组(以含30 mmol/L D-葡萄糖的培养基培养)、LMH组(以含30 mmol/L D-葡萄糖的培养基培养,并分别给予1、5、10、15、20μmol/L的LMH进行干预)、BAY11-7082组(以含30 mmol/L D-葡萄糖的培养基培养,并给予1μmol/L的BAY11-7082干预)、LMH+BAY11-7082组(以含30 mmol/L D-葡萄糖的培养基培养,并给予最适剂量LMH联合1μmol/L的BAY11-7082干预)、LMH+Prostratin组(以含30 mmol/L D-葡萄糖的培养基培养,并给予最适剂量LMH联合20μmol/L的Prostratin干预)。先用细胞计数试剂盒-8(CCK-8)将不同浓度LMH对高糖诱导的HGPC细胞活力进行检测,以筛选出最适的LMH;采用Hoechst 33258染色法检测HGPC的凋亡情况;采用5-乙炔基-2′-脱氧尿苷(EDU)法检测HGPC的增殖情况;采用酶联免疫吸附试验(ELISA)检测HGPC裂解液中肿瘤坏死因子-α(TNF-α)、白细胞介素(IL)-1β水平;采用蛋白免疫印迹法检测HGPC中NF-κBp65、磷酸化(p)-NF-κBp65、半胱氨酸天冬氨酸蛋白水解酶(Caspase-3)、细胞周期蛋白D1(Cyclin D1)的表达量。结果经1、5、10、15、20μmol/L LMH处理的HGPC细胞活力逐渐升高,选用20μmol/L作为LMH最适剂量。与Con组比较,Glu-H组细胞凋亡率升高(P<0.05),TNF-α、IL-1β水平及Caspase-3、p-NF-κBp65蛋白相对表达量升高(P<0.05),细胞增殖率及Cyclin D1蛋白相对表达量则下降(P<0.05);与Glu-H组比较,LMH组和BAY11-7082组细胞凋亡率降低(P<0.05),TNF-α、IL-1β水平及Caspase-3、p-NF-κBp65蛋白相对表达量下降(P<0.05),细胞增殖率及Cyclin D1蛋白相对表达量明显升高(P<0.05);与LMH组比较,LMH+BAY11-7082组细胞凋亡率降低(P<0.05),TNF-α、IL-1β水平及Caspase-3、p-NF-κBp65蛋白相对表达量下降(P<0.05Objective To investigate the effects of low molecular heparin(LMH)on podocyte proliferation,apoptosis and inflammation of podocytes in diabetic nephropathy based on the changes of nuclear factor-κB(NF-κB)signaling pathway.Methods Human glomerular podocytes(HGPC)cells were cultured in vitro,then HGPC cells were divided into control Control(Con)group(culture medium with 5 mmol/LD-glucose),high D-glucose(Glu-H)group(culture medium containing 30 mmol/L D-glucose),LMH group(cultured in medium containing 30 mmol/L D-glucose,and given 1,5,10,15,20μmol/L LMH intervention),BAY11-7082 group(cultured in medium containing 30 mmol/L D-glucose and treated with 1μmol/L BAY11-7082),LMH+BAY11-7082 group(cultured in medium containing 30 mmol/L D-glucose and given the optimal dose of LMH combined with 1μmol/L BAY11-7082 intervention),LMH+Prostratin group(cultured in medium containing 30 mmol/L D-glucose and given the optimal dose of LMH combined with 20μmol/L Prostratin intervention).The viability of HGPC cells induced by high glucose was measured with different concentrations of LMH by cell counting kit-8(CCK-8),so as to screen out the most suitable LMH.Hoechst 33258 staining was used to detect the apoptosis of HGPC.The proliferation of HGPC was detected by 5-acetylidene-2′-deoxyuridine(EDU)method.The levels of tumor necrosis factor(TNF)-αand interleukin(IL)-1βin HGPC lysate were detected by enzyme-linked immunosorbent assay(ELISA).The relative expression levels of NF-κBp65,phosphorylated(p)-NF-κBp65,Caspase-3 and Cyclin D1 in HGPC were detected by Western blot.Results The viability of HGPC cells treated with 1,5,10,15 and 20μmol/L LMH increased gradually,and 20μmol/L was selected as the optimal dose of LMH.Compared with Con group,the apoptosis rate increased in Glu-H group(P<0.05),TNF-αand IL-1βlevel,Caspase-3 and p-NF-κBp65 protein relative expression levels increased in Glu-H group(P<0.05),while the cell proliferation rate and Cyclin D1 protein relative expression levels decreased(P<0.05).Compared with Glu-H gr

关 键 词：糖尿病肾病 人肾小球足细胞 高糖 低分子量肝素 细胞增殖 炎症 NF-ΚB信号通路 

分 类 号：R587.2[医药卫生—内分泌]