The Inhibition Of TIMP-1 On The Apoptosis In Rat Mesangial Cells And The Role Of PI3K/AKT Pathway

Objective: To investigate the function and mechanisms of PI3K/AKT pathway in the inhibition of TIMP-1 on the apoptosis in rat mesangial cells (RMC) induced by high glucose. Methods: ⑴ RMCs were transfected with vector of pcDNA3, pcDNA3-TIMP-1 and AS pcDNA3-TIMP-1 respectively by using lipofectin. The expressions of human TIMP-1(hTIMP-1) and endogenous rat TIMP-1(rTIMP-1) were detected by RT-PCR. ⑵The survival rates of RMC were calculated by MTT staining after RMC was treated with high glucose (25mmol/l) in the presence or absence of LY294002 (25umol/l) or not for 24 hours.⑶The percentage of apoptosis was examined by flow cytometry.⑷The activity of Caspase 9 was evaluated by assay kit.⑸RT-PCR was used to detect the expression of Bcl-2 mRNA.⑹Western blot was performed to analyzed the protein level of BAD, phospho- BAD(ser136), AKT and phospho-AKT(ser473). Results: TIMP-1 transfection induced TIMP-1 over-expression accompanied by fewer percentage of apoptosis, while TIMP-1AS transfection induced a inhibition of TIMP-1 expression accompanied by much more percentage of apoptosis. The suppression of TIMP-1 on the apoptosis in RMC was reversed by LY294002. TIMP-1 had little effect on the expression of Bcl-2 mRNA, and TIMP-1 did not affect the activity of Caspase 9 either. Finnally, antisense TIMP-1 weakened the the protein level of phospho-AKT, phospho-Bad, while sense TIMP-1 increased the protein level of these mediators. Conclusions: ⑴PI3K/AKT pathway participates in the suppression of TIMP-1 on the apoptosis in RMC induced by high glucose.⑵BAD, the downstream factor of AKT plays an important role in this regulation.⑶ TIMP-1 can not affect the expression of Bcl-2 mRNA efficiently.⑷Caspase 9 inhibition may not be involved in this cytoprotective process.