The Role Of JAKs/STATs Signal Pathway In The Inhibition Of The Apoptosis Of Rat Glomerular Mesangial Cell By TIMP-1

The proliferation,differentiation and apoptosis of renal cells and the balance between the synthesis and degradation of extracellular matrix(ECM) in kidney play important roles in the maintenance of normal renal function and renal diseases.Tissue inhibitor of metalloproteinases-1(TIMP-1) can extensively inhibit matrix metalloproteinases (MMPs), accordingly regulating the balance between the synthesis and degradation of ECM.Recent researches find that TIMP-1 can also stimulate cell proliferation,inhibit cell apoptosis,regulate angiogenesis,affect cell resistance to infection.JAKs (Just another kinases;Janus kinases) /STATs (signal transductors and activator of transcriptions) signal pathway also plays important roles in regulation of cell proliferation and apoptosis.We have already found that TIMP-1 inhibits apoptosis of rat glomerular mesangial cell(RMC) through PI3K/AKT and MEK/ERK signal pathways.Whether the JAKs/STATs signal pathway also participates in this process has not been fully elucidated.RMC was selected in the first part of this study in order to investigate the mechanism by which TIMP-1 inhibited RMC apoptosis and the relationship between the mechanism and JAKs/STATs signal pathway.The human TIMP-1 sense and antisense recombinant plasmids were transferred into RMC through liposome,and non-transfected RMC was used as control.After RMC was stimulated with serum withdrawal and AG490(JAK2 specific inhibitor,50μmol/L) for 24 hours,the apoptosis rate was measured by flow cytometry.The expression of TIMP-1,Cyclin D1, Bcl-x1, P27kipl and JAK2 mRNAs was assayed by RT-PCR.The expressionof JAK2, STAT3, STAT5, P-JAK2, P-STAT3 and P-STAT5 proteins was analysed by Western blot. Results:1,The total apoptosis rates of the non-transfected group,sense group and antisense group in the culture medium without serum and AG490,were (10.59±0.96) %, (7.08±0.43) % and (21.91±0.25) %,respectively.The total apoptosis rate of the sense group was significantly lower than that without transfection(P<0.05),and the total apoptosis rate of the antisense group was higher than that without transfection(P<0.01).AG490 treatment enhanced the apoptosis(P<0.01).2, In the groups without AG490,the expression of Cyclin D1 and Bcl-xl mRNAs was the highest in sense group,while it was the lowest in antisense one.The expression of P27kipl mRNA was the highest in antisense one.The treatment with AG490 decreased the expression of TIMP-1,Cyclin D1 and Bcl-xl mRNAs,however,it enhanced the expression of P27kipl mRNA.3, Before RMC was stimulated with AG490,the expression of P-JAK2, P-STAT3 and P-STAT5 was the highest in sense group,however,it was the lowest in antisense one. AG490 treatment significantly depressed the expression of the phosphorylation proteins in all groups. Conclusion: TIMP-1 could activate JAKs/STATs signal pathway feedbackly in protein level under the condition of serum withdrawal,which helped it inhibit the apoptosis of RMC induced by serum deprivation.In recent years,the morbidity of diabetes mellitus(DM) has been increasing in our country. DM can affect the function of many organs, eventually resulting in many serious complications including diabetic nephropathy(DN).Studies have implicated that high glucose(HG) can activate oxidative stress(OS) in kidneys,and it can advance renal fibrosis through JAKs/STATs signal pathway.Whether OS resulted in DN through TIMP-1 has not been clear,therefore the relationship was investigated in the second part of this study among OS,JAKs/STATs signal pathway and TIMP-1 under the condition of HG.After being stimulated with HG,DPI(specific inhibitor of NADPH oxidase) and AG490(specific inhibitor of JAK2) for 24 hours,the apoptosis rate was measured by flow cytometry.The expression of Bcl-xl, Cyclin D1, P27kip,JAK2 and TIMP-1 mRNAs was assayed by RT-PCR.The expression of JAK2, STAT3> STAT5, P-JAK2,P-STAT3 and P-STAT5 proteins wasanalysed by Western blot.Results:1,The total apoptosis rates of HG(25mmol/L),HG+AG490(HG 25mmol/L+AG490 50μmol/L),HG+DPI (HG 25mmol/L+DPI 0.32μmol/L) and HG+AG490+DPI(HG 25mmol/L+ AG490 50μmol/L+DPI 0.32μmol/L) groups were (10.69±0.26 ) %, (20.52±0.51) %, (24.56±0.36) % and (26.01±0.28) %, respectively; AG490 or (and) DPI treatment enhanced the apoptosis.2,AG490 or (and) DPI treatment enhanced the expression of P27kipl mRNA and significantly upregulated the value of (P27kipl mRNA/actin)/(Cyclin D1 mRNA/actin). But they obviously decreased the expression of Bcl-xl,Cyclin D1, JAK2 and TIMP-1 mRNAs.3, AG490 or (and) DPI treatment significantly decreased the expression of P-JAK2,P-STAT3 and P-STAT5 proteins. Conclusion:HG activated JAKs/STATs signal pathway through reactive oxidative species(ROS);the inhibition of OS or JAKs/STATs signal pathway decreased the expression of TIMP-1 mRNA in RMC.