The Role Of TXNIP On Autophagy In Mouse Glomerular Mesangial Cells Induced By High Glucose And Its Related Molecular Mechanism

Objective: Diabetic nephropathy?DN?is one of the most common diabetic microvascular complications.Once obvious proteinuria occurred,the disease is difficult to reverse.Furthermore,the disease can develop to the end-stage renal kidney failure,which is the primary reason for the diabetes patients die.In the past decades,the previous study proposed that the cell autophagy?Autophagy?plays an important role in the DN.Additionally,autophagy is a basic process,which is helpful for degrading cell components by transporting the them into lysosome,facilitating the balance and integrity of cell.In normal state,the function of cell autophagy is under the basic level,beneficial for the normal physiological founction.Moreover,an increasing number of studies have found that there is close relation between the injury of cell autophagy and DN,and the the injury of cell autophagy was responsible for the occurrence and development of the diabetes.In the renal tubular epithelial cells of early diabetic mice induced by STZ,the increase of autophagy marker LC3-II and the autophagy-lysosome degradation products of P62 shows that the activity of cell autophagy may be reduced.Thioredoxin-interacting protein?TXNIP?is widespread in a cell,which can be induced to be over expressed by hyperglycemia,leading to Oxidative Stress Response.As a result,the cells were injured with the accumulation of extracellular matrix proteins to accelerate the apoptosis of cell.What is more,our previous research confirm that the expression of TXNIP in HG-induced MMCs is significantly increased,the ROS?Reactive oxygen species,ROS?generates more and appeareds apoptosis with phenotype transformation in MMCs.Recently,the latest researches suggest that the activity of autophagy in tubular epithelial cell reduced with the autophagosome accumulating,while the expression of autophagy-lysosome degradation products of P62 and autophagy marker LC3-II significantly enhanced.As for the HK-2 renal tubular epithelial cells,the activity of autophagy obviously enhanced with the TXNIP in HK-2 cells being knowdowned,indicating TXNIP is related to the injury of autophagy in diabetic nephropathy tubular cell.Nevertheless,the role of TXNIP in the glomerular mesangial cells induced by high sugar autophagy has not been investigated yet.In our work,not only the relationship between autophagy and TXNIP in the MMC cells was investigated,but also its molecular mechanism was explored in detail by conducting culturing experience of the mesangial cells?Mouse mesangial cells,MMCs?in vitro.Methods: For pBAsim-U6-Neo-TXNIP siRNA interference plasmid,TXNIP si RNA plasmid and blank plasmid are used to transfect MMCs respectively with FuGENER6 transfection reagent.37°C,5% CO₂ in incubator,Cells are cultured in DMEM-F12 containing 10% fetal bovine serum.Experimental cells:?1?normal control group?5.5 mmol/L glucose,NG?,mannitol control group?5.5 mmol/L glucose+24.5 mmol/L mannitol,M?,high glucose?30 mmol/L glucose,HG?,It needs to collect cells with stimulus for 2,6,12,24,48,72 h?2?normal control group?5.5 mmol/L glucose,NG?,mannitol control group?5.5 mmol/L glucose+24.5 mmol/L mannitol,M?,high glucose?30 mmol/L glucose,HG?,blank plasmidcontrol group?30 mmol/L glucose+pBAsim-U6-Neo-blank plasmid,HG+V?,TXNIP siRNA plasmid group?30 mmol/L glucose+pBAsim-U6-Neo-TXNIP siRNA plasmid,HG+T?,bafilomycin A1 group?5.5 mmol/Lglucose+50 nmol/L bafilomycin A1,BA?,nitrotyrosine group?30 mmol/L glucose+p BAsim-U6-Neo-TXNIP siRNA plasmid+0.02 mg/ml Nitrotyrosine,HG+T+N?,rapamycin group?3 mmol/L glucose+1 ng/ml rapamycin,HG+R?.They need to collect cells with stimulus for 24 h to detect the expression of TXNIP,LC3-II,P62,nitrotyrosine,p-mTORC1 by Western blot and mRNA of TXNIP,LC3-II,P62 byReal-time PCR.We detect the morphology of cells by the inverted electron microscope,the autophagosome by the electron microscopy and onfocal microscope,the ROS by the flow cytometry and MitoSOX.Results:1 The effect of hyperglycemia on the mesangial cell autophagy.The expressed value of the TXNIP,LC3-II and P62 after being induced by the hyperglycemia displayed volcanic trend,and reached the maximum after the 24 hour time.Whereas,there is no obvious change of expression occurred in TXNIP,P62 and LC3-II.2 Mesangial cell morphological changes.The results of the inverted microscope suggest that the mesangial cells in normal and mannitol control group present star polygon,irregular,adherent growth.After being induced by high glucose and bafilomycin A1,these mesangial cells are oval with declined bottle adhesion.While the mesangial cell morphology became polygon with the bottle bottom adhesion increasing in the TXNIP knockdown group.3 The effect of siRNA TXNIP on the autophagosome in HG-induced MMCs.In contrast to the normal comparison group,the TEM images show that the autophagosomes increase significantly being induced by the high glucose group and bafilomycin A1 group,but no obvios change occurred in the mannitol controlled group.However,the autophagosome in siRNA TXNIP group was less than that in high glucose induced group.4 The effect of siRNA TXNIP on autophagy related protein expression in HG-induced MMCs.Compared to the normal controlled group,the expression of TXNIP,LC3-II,P62 and p-mTORC1 enchanced significantly,but there is no obvious change in mannitol group on the protein expression.Additionally,the expression of TXNIP,LC3-II,P62 and p-mTORC1 in mesangial cells knocked by TXNIP reduced.5 The effect of siRNA TXNIP on ROS in HG-induced MMCs.Compared to the normal control group,the ROS in the MMCs increased after being induced by high glucose,Bafilomycin A1 and Nitrotyrosine,while the ROS decrease obviously with the siRNA TXNIP.6 The effect of siRNA TXNIP on p-mTORC1 in HG-induced MMCs.Compared to the normal control group,the expression of TXNIP,LC3-II,P62,p-mTORC1 and Nitrotyrosine in the HG-induced MMCs increased,while the expression of TXNIP,LC3-II,P62,p-mTORC1 and Nitrotyrosine decrease obviously in the rapamycin group.Conclusion:1 HG may inhibit the activity of cell autophagy.2 HG is responsible for the overexpresssion of TXNIP and increased autophagosome in cells.3 TXNIP interference achieves regulating function by ROS,and suppress the expression of LC3-II and P62,by the mTOR signaling pathway to regulate the activity of autophagy in HG-induced MMCs.