Tissue Inhibitor Of Metalloproteinase 1 Protects Rat Mesangial Cells Against High Glucose Induced Apoptosis

Objective:Discuss whether Tissue inhibitor of metalloproteinase 1(TIMP-1) can effect rat mesangial cells against high glucose induced apoptosis and the mechanism of it.Methods:1) Rat mesangial cells were cultrued by fresh serum-free medium 1640 containing 5,10,20,30,40 mM glucose and 30 mM mannitol for 24h,48h,72h and then examined apoptosis ratio by flow cytometry AnnexinV/PI double stains.2) Rat mesangial cells were transfected with the empty pcDNA3.1vector, TIMP-1S-pcDNA3.1(PCT), and TIMP-1AS-pcDNA3.1 (PCA) Using liposomal transfection reagent. Then we established overexpression and suppression systems of human TIMP-1 in vitro .We examined human TIMP-1 expression and endogenous rat TIMP-1 expression by PCR and RT-PCR after stable transfection.3) Describe growth curve.4)Mesangial cells were exposed to high glucose for 24h ,then stained by AO. Cells were observed by fluorescence microscope.5) We examined the expression of Bcl-2 and Bax by RT-PCR after exposed cells in 30mM glucose for 24h; Then we examined Caspse-3 activity by CaspACETM Assay System, Colorimetic after exposed cells in 30mM glucose for 24h. Results: 1) High glucose can induce apoptosis by dose-time dependent manner.the coefficient of determination are separately R2=0.925,R2=0.9867.2) PCR and RT-PCR identified that PCT, PCA and control vector could express neor gene;While only groups PCT and PCA could express human TIMP-1 gene.; PCT did not influence the expression of endogenous rat TIMP-1.However PCA significantly reduced the expression of endogenous rat TIMP-1. 3) Growth curve: PCT promoted mesangial cells growth faster than others,and PCA stayed mesangial cells growth. 4) The results of AO stain showed that : PCT had few apoptosis cell;howerver PCA had a lot of apoptosis cells ; the control vector and normal RMC groups were placed in the middle.5)UsingRT-PCR, PCT could reduced bax expression, however PCA increased bax expression; CaspACETM Assay System,Colorimetic assay examined that PCT reduced Caspse-3 activity ,while PCA increased Caspse-3 activity.P<0.01.Conclusions:1)High glucose can induce apoptosis by dose-time dependent manner. 2)Human TIMP-1 can promote rat mesangial cells growth,inhibit apoptosis by inhibition of Bax,Caspase-3 expression.