低分子量肝素抑制JAK2/STAT3信号通路缓解高糖诱导的足细胞损伤

Low molecular weight heparin attenuates high glucose-induced podocyte injury by inhibiting JAK2/STAT3 signaling pathway

目的 探究低分子量肝素(LMH)对高糖环境诱导的人肾小球足细胞(HGPC)功能的调节作用及Janus激酶2(JAK2)/信号转导与转录激活因子3(STAT3)信号通路变化的机制。方法 体外培养HGPC,以30 mmol/L D-葡萄糖诱导细胞建立体外足细胞损伤模型,并分别以1、5、10、15、20μmol/LLMH进行干预,细胞计数试剂盒-8筛选出最佳的药物浓度进行后续研究。将细胞分为对照组、HG组、20μmol/LLMH组、JAK2/STAT3通路抑制剂(AG490)组、20μmol/LLMH+AG490组、20μmol/LLMH+JAK2/STAT3通路激活剂(C-A1)组。5-乙炔基-2'-脱氧尿苷(EDU)法检测细胞增殖能力;Transwell小室法检测迁移和侵袭能力;蛋白免疫印迹法检测HGPC细胞中JAK2、磷酸化(p)-JAK2、STAT3、p-STAT3、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-cad-herin)的蛋白表达水平。结果 20μmol/LLMH为干预细胞活力的最佳剂量;与对照组比较,HG组细胞增殖率和E-cad-herin蛋白表达水平降低(均P<0.001),迁移、侵袭能力和p-JAK2、p-STAT3、Vimentin、FN、N-cadherin蛋白表达水平升高(均P<0.001);与HG组相比,LMH给药后细胞增殖率和E-cadherin蛋白表达水平升高(均P<0.001),迁移、侵袭能力和p-JAK2、p-STAT3、Vimentin、FN、N-cadherin蛋白表达水平降低(均P<0.001);与20μmol/L LMH组相比,AG490与LMH合用可以显著增加LMH的作用效果(均P<0.05),而C-A1与LMH合用可以显著减弱LMH的作用效果(均P<0.05)。结论 LMH可通过抑制JAK2/STAT3信号通路蛋白的磷酸化抑制HGPC迁移、侵袭及上皮-间充质转化。

Objective To investigate the regulatory effect of low molecular weight heparin(LMH)on podo-cyte function induced by high glucose(HG)environment and the mechanism of Janus kinase 2(JAK2)/signal transducer and activator of transcription 3(STAT3)signaling pathway.Methods Human glomerular podocytes(HGPC)were cultured in vitro and induced by 30 mmol/L D-glucose to establish an in vitro podocyte injury model,the cells were treated with 1,5,10,15,and 20μmol/L LMH,respectively,and the best drug concentra-tion was screened by cell counting kit-8 for subsequent study.The cells were divided into a control group,HG group,20μmol/L LMH group,AG490 group,20μmol/L LMH+AG490 group and 20μmol/L LMH+C-A1 group,cell proliferation was detected by 5-Ethynyl-2'-deoxyuridine(EDU)assay,and transwell chamber meth-od detected cell migration and invasion.Western blotting was used to detect JAK2,phosphorylated(p)-JAK2,STAT3,p-STAT3,Vimentin,Fibronectin(FN),N-cadherin and E-cadherin in HGPC cells.Results Twentyμmol/L LMH was the best dose to interfere with cell viability.Compared with control group,the prolifera-tion rate and E-cadherin protein expression level of the HG group were decreased(all P<0.001),the migra-tion,invasion ability and p-JAK2,p-STAT3,Vimentin,FN,N-cadherin protein expression levels were increased(all P<0.001).Compared with the HG group,the cell proliferation rate and E-cadherin protein expression lev-el were increased after LMH administration(all P<0.001),the migration and invasion ability and p-JAK2,p-STAT3,Vimentin,FN,N-cadherin protein expression levels were decreased(all P<0.001).Compared with the 20μmol/L LMH group,the combination of AG490 and LMH significantly increased the effect of LMH(all P<0.05),while the combination of C-A1 and LMH significantly weakened the effect of LMH(all P<0.05).Conclusion LMH can inhibit high glucose-induced podocyte migration,invasion and epithelial-mes-enchymal transition by inhibiting the phosphorylation of JAK2/STAT3 signaling pathway proteins.