摘要
目的 探讨天麻素注射液(GI)对高糖(HG)诱导的糖尿病肾病(DN)人肾小球足细胞(HGPC)增殖、迁移及上皮-间质转化(EMT)进程的影响,以及分析GI是否通过调控蛋白Janus激酶(JAK)2/信号转导和转录激活因子(STAT)3信号通路而发挥作用。方法 通过含30 mmol/L D-葡萄糖的培养基刺激HGPC建立HG损伤模型作为HG组,同时将体外培养的HGPC细胞分为对照组(Con组,不做干预),不同浓度GI组(10、20、30、40μmol/L GI组)进行预实验,通过细胞活力实验筛出最佳作用浓度作为GI组进行后续实验。后续实验将细胞分为6组,分别是Con组(不做干预)、HG组(加入30 mmol/L D-葡萄糖处理)、GI组(加入最佳作用浓度的GI处理)、JAK2/STAT3信号通路抑制剂(AG490)组(加入30 mmol/L D-葡萄糖+40μmol/L AG490处理)、GI+AG490组(加入30 mmol/L D-葡萄糖+最佳作用浓度的GI+40μmol/L AG490处理)、GI+JAK2/STAT3信号通路激活剂(C-A1)组(加入30 mmol/L D-葡萄糖+最佳作用浓度的GI+20μmol/L C-A1),每组干预时间均为24 h。采用细胞计数试剂盒检测HGPC活力;采用5-乙炔基-2′-脱氧尿苷法检测HGPC增殖能力;通过Transwell小室实验检测HGPC迁移能力;采用蛋白免疫印迹法检测HGPC中EMT相关蛋白及JAK2/STAT3信号通路相关蛋白水平。结果 20μmol/L GI组、30μmol/L GI组、40μmol/L GI组细胞活力均高于HG组(P<0.05),且20μmol/L GI组效果最好,因此后续实验选用20μmol/L作为GI的最佳作用浓度。与Con组相比,HG组细胞迁移数和p-JAK2、p-STAT3、波形蛋白(Vimentin)、纤连蛋白(FN)、神经型钙黏蛋白(N-cadherin)蛋白水平均明显升高(P<0.05),细胞增殖率和上皮型钙黏蛋白(E-cadherin)蛋白水平均降低(P<0.05);GI组、AG490组细胞迁移数和p-JAK2、p-STAT3、Vimentin、FN、N-cadherin蛋白水平均低于HG组(P<0.05),细胞增殖率和E-cadherin蛋白水平均高于HG组(P<0.05);与GI组相比,GI+AG490组细胞迁移数和p-JAK2、p-STAT3、Vimentin、FN、N-cadherin蛋白水平均降低(P<0.05),细胞增殖率和E-cadherin蛋白水平均升高(P<0.05);与GI组相比,GI+C-A1组细胞迁移数和p-JAK2、p-STAT3、Vimentin、FN、N-cadherin蛋白水平均升高(P<0.05),细胞增殖率和E-cadherin蛋白水平均降低(P<0.05)。结论 GI可以通过抑制JAK2/STAT3的磷酸化来抑制HGPC的迁移及EMT进程,促进细胞增殖。
Objective To explore the effects of gastrodin injection(GI)on high glucose(HG)induced diabetic nephropathy(DN)human glomerular podocytes(HGPC)proliferation,migration and epithelial-mesenchymal transformation(EMT)process,and to analyze whether or not GI playing the role by regulating the Janus kinase(JAK)2/signal transduction and transcriptional activator(STAT)3 signaling pathway.Methods The HGPC cells were stimulated by the medium containing 30 mmol/L D-glucose to establish the HG injury model as the HG group.At same time,the in vitro cultured HGPC were divided into the control group(Con group without intervention)and different concentrations of GI groups(10,20,30,40μmol/L GI groups)for conducting the pre-experiment.The optimal action concentration was screened by the cell viability experiment to conduct the follow up experiment.The cells were divided into 6 groups by the follow up experiment,which were the Con group(without intervention),HG group(treating by adding 30 mmol/L D-glucose),GI group(treating by adding the optimal action concentration GI),JAK2/STAT3 signaling pathway inhibitor(AG490)group(treating by adding 30 mmol/L D-glucose+40μmol/L AG490),GI+AG490 group(treating by adding 30 mmol/L D-glucose+optimal action concentration GI+40μmol/L AG490),GI+JACK2/STAT3 signaling pathway activator(C-A1)group(adding 30mmol/L D-glucose+optimal action concentration GI+20μmol/L C-A1),and the intervention time in each group was 24 h.The cell count reagent was adopted to detect the HGPC activity;the s-ethynyl-2′-deoxyuridine method was used to detect the HGPC proliferation ability;the HGPC migration ability was detected by the Transwell chamber experiment;the EMT related protein and JAK2/STAT3 signaling pathway related protein levels in HGPC were detected by Western blot.Results The cell viability of the 20μmol/L GI group,30μmol/L GI group and 40μmol/L GI group were all significantly higher than that of the HG group(P<0.05),moreover the effect of the 20μmol/L GI group was the best,so the follow up experiment selected 20μmol/L as the GI optimal action concentration.Compared with the Con group,the cell migration number and protein levels of p-JAK2,p-STAT3,Vimentin,fibronectin(FN),neural cadherin(N-cadherin)in the HG group were significantly increased(P<0.05),while the cellular proliferation rate and the level of epithelial cadherin(E-cadherin)were decreased(P<0.05).The cell migration number and the protein levels of p-JAK2,p-STAT3,Vimentin,FN and N-cadherin in the GI group and AG490 group were lower than those in the HG group(P<0.05),and the cellular proliferation rate and E-cadherin protein level were increased(P<0.05).Compared with the GI group,cell migration number and levels of p-JAK2,p-STAT3,Vimentin,FN,and N-cadherin proteins in the GI+AG490 group were decreased(P<0.05),and the cell proliferation rate and E-cadherin protein level were increased(P<0.05).Compared with the GI group,the cell migration number and protein levels of p-JAK2,p-STAT3,Vimentin,FN and N-cadherin in the GI+C-A1 group were increased(P<0.05),and the cellular proliferation rate and E-cadherin protein level were decreased(P<0.05).Conclusion GI could inhibit the HGPC transformation and EMT process by inhibiting the phosphorylation of JAK2/STAT3,and promote the cellular proliferation.
作者
马医林
李锴
赵芳芳
MA Yilin;LI Kai;ZHAO Fangfang(Department of Endocrine Nephrology,Hanzhong Municipal People′s Hospital,Hanzhong,Shaanxi 723000,China)
出处
《检验医学与临床》
2025年第1期18-23,共6页
Laboratory Medicine and Clinic
基金
国家卫生健康委员会“十四五”规划重点课题(YYWS9499)。
作者简介
马医林,女,主治医师,主要从事内分泌肾病方向的研究。;通信作者:赵芳芳,E-mail:15457515@qq.com。
