The Mechanism Study Of Gingipains On The Proliferation Of Vascular Smooth Muscle Cells

Objective: The purpose of this study is to study the gingipains from Porphyromonas gingivalis on the proliferation,apoptosis,IL-8 and TNF-? mRNA level of the rat aortic smooth muscle cell,and explore the mechanism of gingipains on the proliferation of rat aortic smooth muscle cell.Methods: Part one Gingipains were isolated and purified from supernatants of Porphyromonas gingivalis W83 which was cultured under standard anaerobic conditions,and primary rat aortic smooth muscle cells were cultured.rat aortic smooth muscle cells were exposed to 100?g/ml,50?g/ml,25?g/ml,12?g/ml,6?g/ml,3?g/ml,1?g/ml,0?g/ml activated gingipains,the rat aortic smooth muscle cells proliferation were evaluated by cck-8 assay at 48 h.In addition,rat aortic smooth muscle cells were stimulated with 5?M,10?M,20?M,40?M Arg-gingipain inhibitor(KYT-1),Lys-gingipain inhibitor(KYT-36)in combination with the gingipain extracts respectively,the rat aortic smooth muscle cells proliferation were evaluated by cck-8 assay,and the cell cycle of rat aortic smooth muscle cells were determined by flow cytometry,the osteopontin and ?-smooth muscle actin expression were examined with immunohistochemical staining.The apoptosis assays were performed by flow cytometry.The expression of IL-8 and TNF-? were determined by ELISA,and the difference of function of Rgp and Kgp were compared.Part two Chemically modified osteopontin siRNA and overexpressive plasmid of osteopontin were designed and constructed,then transfected into the rat aortic smooth muscle cells with different concentration.The cell cycle of rat aortic smooth muscle cells were analyzed with the flow cytometry.The mRNA level of proliferating cell nuclear antigen were analyzed with real-time PCR.And the ?-smooth muscle actin and calponin expression were examined by real-time PCR and western blot analysis.Part three The large scale gene expression profiles were used to screen the differential genes of rat aortic smooth muscle cells treated with gingipains,and the real-time PCR were done to verify.Results: 1)The gingipains were extracted from Porphyromonas gingivalis,of which the enzymetic activity of Rgp is 280U/L and the Kgp is 84U/L;2)Gingipains promote the proliferation of rat aortic smooth muscle cells,in which the 12?g/ml was the most obvious(F=42.048,P<0.001).With the inhibition of KYT-1,KYT-36 and KYT-1+KYT-36 on the gingipains,the proliferation of rat aortic smooth muscle cells were decreased dramatically(F=12.109,P<0.001),and there were no difference among the inhibitors(P>0.05).3)After the gingipain stimulation,the cell cycle of rat aortic smooth muscle cells processed from G0/G1 phase to S phase,in which the number of cells in the G0/G1(25.87±4.58)decreased and increase in the S phase(49.90±3.63).4)The expression of osteopontin increased.5)The apoptosis of rat aortic smooth muscle cells were not exmined,and the difference was not statistically significant;6)The level of IL-8 and TNF-? were increased dramatically after gingipain stimulation(IL-8:F=8.241,P=0.0014;TNF-?:F=44.14,P<0.001).Part two 1)The transfectious efficiency of osteopontin-siRNA F02 was the best at 24 h,and the mRNA level of osteopontin decreased after the transfection;2)The transfectious efficiency of osteopontin-overexpression plasmid was 60 percent,and the mRNA level of osteopontin increased after the transfection;3)The cell cycle of rat aortic smooth muscle cells were blocked in the G0/G1 phase after the osteopontin-siRNA transfection,in which the number of cells in the G0/G1 phase(55.43±1.50)increased and decreased in the S phase(26.87±3.52)companying with the decrease of the mRNA level of proliferating cell nuclear antigen(P<0.001).On the contrary,the cell cycle of rat aortic smooth muscle cells processed from G0/G1 phase to S phase,in which the number of cells in the S phase decreased(38.90 ± 2.67),companying with the decrease of the mRNA level of proliferating cell nuclear antigen(P<0.001);4)The mRNA and protein level of ?-smooth muscle actin and calponin increased after the osteopontin-siRAN trasfection,and decreased after the osteopontin-overexpression plasmid transfection(P < 0.001).Conclusions: 1)Gingipains promote the proliferation of rat aortic smooth muscle cells,and affect its cell cycle;2)Gingipains promote the rat aortic smooth muscle cells transform from the contractile phenotype to the synthetic phenotype;3)The effects of gingipains on the apotosis of rat aortic smooth muscle cells are not obvious;4)osteopontin promote the expression of IL-8 and TNF-?,and there are no difference between Rgp and Kgp;5)Gingipains affect the proliferation of rat aortic smooth muscle cells through the ?-smooth muscle actin and calponin.6)Gingipains may regulate the proliferation of vascular smooth muscle cell through the osteopontin-integrin-focal adhesion kinase-cytoskeletal protein pathway.