黄芪甲苷对单核细胞与肾小管上皮细胞相互作用的影响及分子机制
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摘要
目的研究单核细胞与肾小管上皮细胞在肾间质纤维化过程中相互作用的分子机制,并探讨黄芪甲苷(AS-IV)对其相互作用的影响机制。方法利用共培养体系在体外共培养U973单核细胞与HK-2人近端肾小管上皮细胞,用黄芪甲苷进行处理,Real-time PCR法检测M1型单核细胞标志基因诱导型一氧化氮合酶(iNOs)和M2型单核细胞标志基因精氨酸酶1(Arg-1)的mRNA表达情况。用流式细胞仪检测U973细胞表面分子Toll样受体4(Toll-like receptors 4,TLR-4,又称CD284)表达,并检测黄芪甲苷对TANK结合蛋白激酶(TBK)/干扰素调节因子3(IRF3)信号通路mRNA和蛋白水平的影响。结果黄芪甲苷处理抑制了HK-2细胞刺激单核细胞发生M1转化引起的iNOs上升和Arg-1下降,同时也抑制了TLR-4的上升,并且阻断了TBK/IRF信号通路。结论黄芪甲苷抑制肾脏纤维化的作用可能是通过阻断TBK/IRF3信号通路,抑制U973细胞TLR-4表达,抑制U973细胞发生M1型转化,最终减轻炎症因子的产生。
Objective To explore the molecular mechanism of the interaction between monocytes and renal tubular epithelial cells in the process of renal interstitial fibrosis and the effects of astragaloside IV on the interaction of the two cells.Methods By using in vitro co-culture system,monocyte cells(U973) and human renal proximal tubular epithelial cells(HK-2)were cultured and treated with astragaloside IV.The mRNA expression of Arg,marker gene of M1 monocytes iNOs and M2 monocytes were tested by real-time PCR.The cell surface marker of U973 TLR-4 was detected by FACS,and the change of TBK/IRF3 signaling expression was explored through mRNA and protein level.Results Astragaloside IV treatment inhibited the increase of iNOs and the decrease of Arg-1 induced by M1 transformation in HK-2 cells stimulated monocytes.Further,the surface marker TLR-4 was also decreased and the TBK/IRF3 signaling pathway was blocked by astragaloside IV.Conclusion Astragaloside IV inhibits renal interstitial fibrosis by blocking the TBK/IRF3 signaling pathway,inhibiting M1 differentiations of U973 cell and the expression of TLR4,and then relieves the production of inflammatory factors.
Objective To explore the molecular mechanism of the interaction between monocytes and renal tubular epithelial cells in the process of renal interstitial fibrosis and the effects of astragaloside IV on the interaction of the two cells.Methods By using in vitro co-culture system,monocyte cells(U973) and human renal proximal tubular epithelial cells(HK-2)were cultured and treated with astragaloside IV.The mRNA expression of Arg,marker gene of M1 monocytes iNOs and M2 monocytes were tested by real-time PCR.The cell surface marker of U973 TLR-4 was detected by FACS,and the change of TBK/IRF3 signaling expression was explored through mRNA and protein level.Results Astragaloside IV treatment inhibited the increase of iNOs and the decrease of Arg-1 induced by M1 transformation in HK-2 cells stimulated monocytes.Further,the surface marker TLR-4 was also decreased and the TBK/IRF3 signaling pathway was blocked by astragaloside IV.Conclusion Astragaloside IV inhibits renal interstitial fibrosis by blocking the TBK/IRF3 signaling pathway,inhibiting M1 differentiations of U973 cell and the expression of TLR4,and then relieves the production of inflammatory factors.
引文
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[12]ZHOU X,SUN X,GOGN X,et al.Astragaloside IV from Astragalus membranaceus ameliorates renal interstitial fibrosis by inhibiting inflammation via TLR4/NF-κB in vivo and in vitro.[J].Intern Immunoph,2017,42:18-24.
[13]ITALIANI P,BORASCHI D.From monocytes to M1/M2macrophages:phenotypical vs.functional differentiation[J].Frontiers in Immunology,2014,5:512-514.
[14]GUITERAS R,FLAQUER M,CRUZADO M,et al.Macrophage in chronic kidney disease.[J].Clinical Kidney J,2016,9(6):765-771.
[2]LIU F,ZHUANG S.Role of receptor tyrosine kinase signaling in renal fibrosis[J].Intern J Mol Sci,2016,17(6):972-981.
[3]MENG X M,NIKOLIC-PATERSON D J,LAN H Y.Inflammatory processes in renal fibrosis.[J].Nat Rev Neph,2014,10(9):493-503.
[4]SUAREZ-ALVAREZ B,LIAPIS H,ANDERS H J.Links between coagulation,inflammation,regeneration,and fibrosis in kidney pathology laboratory investigation[J].J Technic Meth Path,2016,96(4):378-390.
[5]LI Q,LIU B C,LV L L,et al.Monocytes induce proximal tubular epithelial-mesenchymal transition through NFkappa B dependent upregulation of ICAM-1[J].J Cell Bioch,2011,112(6):1585-1592.
[6]JIALAL I,MAJOR A M,DEVARAJ S.Global toll-like receptor 4 knockout results in decreased renal inflammation,fibrosis and podocytopathy[J].J Diab Its Complic,2014,28(6):755-761.
[7]李青,郑敏,张露,等.TLR4、TBK/IRF3信号通路介导肾小管上皮细胞与单核细胞相互作用[J].中国中西医结合肾病杂志,2015,16(11):994-996.
[8]GUI D,HUANG J,LIU W,et al.Astragaloside IV prevents acute kidney injury in two rodent models by inhibiting oxidative stress and apoptosis pathways[J].Apoptosis,2013,18(4):409-422.
[9]WANG Q,SHAO X,XU W,et al.Astragalosides IV inhibits high glucose-induced cell apoptosis through HGF activation in cultured human tubular epithelial cells[J].Renal Failure,2014,36(3):400-406.
[10]GUI D,HUANG J,GUO Y,et al.Astragaloside IV ameliorates renal injury in streptozotocin-induced diabetic rats through inhibiting NF-κB-mediated inflammatory genes expression[J].Cytokine,2013,61(3):970-977.
[11]徐维佳,牟姗,王琴,等.黄芪甲苷对高糖诱导的肾小管上皮细胞损伤的保护作用[J].中国中西医结合肾病杂志,2012,13(9):765-769.
[12]ZHOU X,SUN X,GOGN X,et al.Astragaloside IV from Astragalus membranaceus ameliorates renal interstitial fibrosis by inhibiting inflammation via TLR4/NF-κB in vivo and in vitro.[J].Intern Immunoph,2017,42:18-24.
[13]ITALIANI P,BORASCHI D.From monocytes to M1/M2macrophages:phenotypical vs.functional differentiation[J].Frontiers in Immunology,2014,5:512-514.
[14]GUITERAS R,FLAQUER M,CRUZADO M,et al.Macrophage in chronic kidney disease.[J].Clinical Kidney J,2016,9(6):765-771.