摘要
目的探讨低分子量肝素(LMH)对糖尿病肾病(DN)足细胞损伤的影响及机制。方法将人肾小球足细胞(HGPC)分为对照(NC)组、高浓度葡萄糖(HG)组、1、5、10、15、20μmol/L LMH组,NC组细胞以含5 mmol/L葡萄糖的培养基培养;HG组细胞以含30 mmol/L的葡萄糖培养基培养;1、5、10、15、20μmol/L LMH组细胞以含30 mmol/L葡萄糖+不同浓度(1、5、10、15、20μmol/L)LMH的培养基共同培养,筛选出最佳浓度15μmol/L LMH后,又将细胞分为NC组、HG组、LMH组、BAY11-7082组、LMH+BAY11-7082组、LMH+Prostratin组。LMH组细胞以含30 mmol/L葡萄糖+15μmol/L LMH的培养基共同培养;BAY11-7082组细胞以含30 mmol/L的葡萄糖+1μmol/L BAY11-7082的培养基培养;LMH+BAY11-7082组细胞以含30 mmol/L葡萄糖+15μmol/L LMH+1μmol/L BAY11-7082的培养基培养;LMH+Prostratin组细胞以含30 mmol/L葡萄糖+15μmol/L LMH+20μmol/L Prostratin的培养基培养。采用活细胞计数法(CCK-8)检测HGPC细胞的细胞活力;Hoechst 33258染色法检测HGPC细胞的凋亡状况;Transwell小室实验检测HGPC细胞的侵袭能力;酶联免疫吸附实验检测HGPC裂解液中肿瘤坏死因子(TNF)-α、白细胞介素(IL)-6表达;蛋白免疫印迹法检测HGPC细胞中核转录因子kappa-B(NF-κB)p65、磷酸化(p)-NF-κBp65、波形蛋白(Vimentin)、纤连蛋白(FN)、神经型钙黏蛋白(N-cadherin)、上皮型钙黏蛋白(E-cadherin)表达。结果CCK-8结果显示,LMH的最适浓度为15μmol/L。药物处理24 h后,与NC组相比,HG组细胞凋亡率、侵袭细胞数升高(P均<0.05);与HG组相比,LMH组和BAY11-7082组细胞凋亡率、侵袭细胞数下降(P均<0.05);与LMH组相比,LMH+BAY11-7082组细胞凋亡率、侵袭细胞数下降(P均<0.05),LMH+Prostratin组细胞凋亡率、侵袭细胞数升高(P均<0.05)。与NC组相比,HG组TNF-α、IL-6、p-NF-κBp65、Vimentin、FN、N-cadherin蛋白表达升高,E-cadherin蛋白表达降低(P均<0.05);与HG组相比,LMH组和BAY11-7082组TNF-α、IL-6、p-NF-κBp65、Vimentin、FN、N-cadherin蛋白表达下降,而E-cadherin蛋白表达上调(P均<0.05);与LMH组相比,LMH+BAY11-7082组TNF-α、IL-6、p-NF-κBp65、Vimentin、FN、N-cadherin蛋白表达下降(P均<0.05),而E-cadherin蛋白表达上调(P均<0.05);LMH+Prostratin组TNF-α、IL-6、p-NF-κBp65、Vimentin、FN、N-cadherin蛋白表达升高,E-cadherin蛋白表达降低(P均<0.05)。结论LMH可抑制HG诱导的DN足细胞损伤,其机制可能是抑制NF-κB信号通路的活性和EMT进程。
Objective To investigate the effect and mechanism of low-molecular-weight heparin(LMH)on podocyte injury in diabetic nephropathy(DN).Methods Human glomerular podocytes(HGPC)were divided into the control(NC)group,high glucose(HG)group,and 1,5,10,15,20μmol/L LMH group.The cells in the NC group were cultured in medium containing 5 mmol/L glucose.Cells in the HG group were cultured in medium containing 30 mmol/L glucose.Cells in 1,5,10,15,and 20μmol/L LMH groups were co-cultured with medium containing 30 mmol/L glucose+different concentrations(1,5,10,15,and 20μmol/L)of LMH.After the optimal concentration of LMH was screened,the cells were divided into the NC group,HG group,LMH group,BAY11-7082 group,LMH+BAY11-7082 group,and LMH+Prostratin group.Cells in LMH group were co-cultured with medium containing 30 mmol/L glucose and 15μmol/L LMH.Cells in BAY11-7082 group were cultured in medium containing 30 mmol/L glucose and 1μmol/L BAY11-7082.Cells in LMH+BAY11-7082 group were cultured in medium containing 30 mmol/L glucose,15μmol/L LMH and 1μmol/L BAY11-7082.Cells in LMH+Prostratin group were cultured in medium containing 30 mmol/L glucose,15μmol/L LMH,and 20μmol/L Prostratin.The viability of HGPC was detected by CCK-8.Hoechst 33258 staining was used to detect the apoptosis of HGPC.Transwell chamber assay was used to detect the invasion ability of HGPC.The expression levels of tumor necrosis factor(TNF)-αand interleukin(IL)-6 in HGPC solution of lysis were detected by enzyme linked immunosorbent assay(ELISA).Western blotting was used to detect the relative expressionlevels of nuclear transcription factor kappa-B(NF-κB)p65,phosphorylated(p)-NF-κBp65,Vimentin,Fibronectin(FN),N-cadherin and E-cadherin in HGPC.Results CCK-8 results showed that the optimal dose of LMH was 15μmol/L.Compared with the NC group,the apoptosis rate and the number of invasive cells increased in the HG group(both P<0.05).Compared with the HG group,the apoptosis rate and the number of invasive cells in LMH group and BAY11-7082 group decreased(all P<0.05).Compared with the LMH group,the apoptosis rate and the number of invasive cells in the LMH+BAY11-7082 group decreased(both P<0.05),and the apoptosis rate and the number of invasive cells in the LMH+Prostratin group increased(both P<0.05).Compared with the NC group,the protein expression levels of TNF-α,IL-6,p-NF-κBp65,Vimentin,FN and N-cadherin increased,and the protein expression of E-cadherin decreased in the HG group(all P<0.05).Compared with the HG group,the expression levels of TNF-α,IL-6,p-NF-κBp65,Vimentin,FN and N-cadherin decreased,while the expression of E-cadherin was up-regulated in the LMH group and BAY11-7082 group(all P<0.05).Compared with the LMH group,the protein expression levels of TNF-α,IL-6,p-NF-κBp65,Vimentin,FN and N-cadherin in the LMH+BAY11-7082 group decreased(all P<0.05),while the protein expression of E-cadherin increased(P<0.05).The protein expression levels of TNF-α,IL-6,p-NF-κBp65,Vimentin,FN,and N-cadherin increased,and the protein expression of E-cadherin decreased in the LMH+Prostratin group(all P<0.05).Conclusion LMH can inhibit HG-induced podocyte injury in DN,and its mechanism may be achieved by inhibiting the activity of NF-κB signaling pathway and the progression of EMT.
作者
王淮淮
赵学慧
王会芳
魏晓岩
贾军利
WANG Huaihuai;ZHAO Xuehui;WANG Huifang;WEI Xiaoyan;JIA Junli(Department of Nephrology,The Second Affiliated Hospital of Hebei North University,Zhangjiakou 075000,China)
出处
《山东医药》
CAS
2023年第22期41-45,共5页
Shandong Medical Journal
基金
河北省医学科学研究课题(20210125)。
关键词
糖尿病肾病
人肾小球足细胞
高糖
低分子量肝素
细胞凋亡
NF-ΚB信号通路
diabetic nephropathy
human glomerular podocytes cells
high glucose
low-molecular-weight heparin
apoptosis
nuclear transcription factor kappa-B signaling pathway
