Inhibitory Effect Of Puerarin On Cathepsin B Inhibits TGF - β - Induced Apoptosis In HUVEC Cells

Purpose:In this study, we detected the function of Cathespsin B(CTSB) in Human Umbilical Vein Endothelial Cell(HUVEC) proliferation and inducing cell apoptosis. In addition, to detect that Puerarin reduce cell apoptosis induced by TGF-beta through high expression of CTSB.Material and method:Cell culture and Cell transfection,Cell viability assays(MTT method),Real-time PCR assay,Western blot analysis,Flow cytometry.Results:1.HUVEC cells transfection with Lenti Virus We use slow virus as the carrier, the method of using transfection will CTSB genes into HUVEC cells. Transfection effect to inflammation, we recycle the infected cells, respectively, to extract the total RNA in cells, and cells of the total protein. We use real- time PCR method to detect the HUVEC cells before and after transfection CTSB m RNA expression levels found, HU- CTSB CTSB is much higher than the level of gene expression of HUVEC and HU- MOCK, and statistically significant results. Then, we using western blot method, tested the three CTSB protein expression levels in cells, found the HU – CTSB, CTSB protein expression level is higher than in HUVEC and HU- MOCK, and the results have statistical significance.2.CTSB effects on growth of HUVEC We use the method of MTT contrast detection HUVEC, HU- MOCK and HU- CTSB three kinds of cell proliferation. In training after 48 hours, the HU- CTSB survival rate was significantly lower than that of HUVEC, HU- the MOCK(P <0.01), and survival rate between HUVEC, HU- MOCK found no apparent change.3.CTSB effects on HUVEC apoptosis HUVEC, HU- MOCK and HU- CTSB three cells after Annexin V- FITC/PI staining and measured by the flow cytometry in training after 24, HU- first appearedCTSB cells apoptosis phenomenon, prompt the beginning of the early apoptosis,while at the same time of HUVEC, HU- MOCK cells did not change obviously. After72 hours of training, HU- total CTSB cell apoptosis rate were higher than the same period of HUVEC, HU- the MOCK.4.CTSB impact on HUVEC apoptosis related gene and protein Real time quantitative PCR method to detect apoptosis gene Bax found that compared with HUVEC, HU- MOCK cell, in 24 hours later, HU-CTSB Bax gene expression level was significantly higher in cells HUVEC and HU-MOCK cells. And as time changes, Bax gene expression were higher than in the other two kinds of cells. And the other two kinds of quantity does not appear Bax gene expression differences between cells. Immune protein imprinting analysis results showed that in training after 72 hours, HU- CTSB Bcl- 2 protein expression in cells HUVEC and HU- MOCK cells decreased significantly, and pro- caspase3 proteins are apparent shear out 17 kd Cleaved caspase3 stripe. And HUVEC and HU- there is no difference between MOCK the Bcl- 2 protein expression in the cell, the shear Cleaved Caspase3 protein expression and no difference.5.HUVEC apoptosis induced by TGF- beta impact on CTSB expression in cells We use different concentration of TGF- beta(0, 100, 200, 500, 1000, 2000,ng/ml) stimulate HUVEC, observe the TGF- found that when beta cells apoptosis induced HUVEC by 100 ng/ml TGF- beta stimulation after 24 hours, which found that cells apoptosis. With the increase of concentration, number of apoptotic cells increased significantly, and has the concentration dependence. When the concentration increased to 1000 ng/ml, a plateau. We recycle the cells, using western blot method to detect CTSB protein expression in cells found that TGF- beta cell apoptosis induced by elevated CTSB expression in cells.6.Puerarin reduces HUVEC apoptosis induced by TGF- beta We use 100 ng/ml TGF- beta stimulate HUVEC 24 hours, then add respectively0.01, 0.02, 0.05 mg/ml kudzu root element joint training found, 0.02 mg/ml kudzu root element can inhibit the TGF- beta induced HUVEC apoptosis.Conclusions:1. Puerarin can resist TGF-beta induced apoptosis of human vascular endothelial cells.2. CTSB within endothelial cells can regulate the expression of Bax gene activation caspase3 pathways, induction of apoptosis.3. Puerarin can inhibit the upregulation of vascular endothelial cells CTSB inhibit cell apoptosis.