The Effects Of Recombinant Fimbrillin Of Porphyromonas Gingivalis With ? FimA Genotype On The Function Of Endothelial Cells Under The Effect Of Smooth Muscle Cells Derived Exosomes

Objective: P.gingivalis is the most important pathogenic bacteria in chronic periodontitis,it not only leads to the local inflammation of the oral cavity but also has the effects on the healthy of the cardiovascular system.A large number of studies have shown that P.gingivalis and its virulence factors are directly related to endothelial dysfunction.And researches have shown that the communication between endothelial cells and smooth muscle cells may play an important role in the occurrence and development of atherosclerosis.However,as an important medium of paracrine secretion,exosomes are widely involved in various messenger secretion and its uptake process.We analysed the effects of the Fimbrillin which is the main virulence factors of Porphyromonas gingivalis with ? fim A Genotype on the function of endothelial cells under the effect of smooth muscle cells derived exosomes,in order to explore the possible function of P.gingivalis in the occurrence and development of the atherosclerosis.Methods:(1)HUVECs were cultured in vitro by the collagenase digestive method.HUASMCs were cultured in vitro by tissue explants adherent method.The cells were identified by fluorescence immunoassay.Exosomes were isolated by PEG method.Western blot was used to detect the marker proteins CD63 and CD9 of exosomes.Electron microscope was used to identify the size and morphology of exosomes.Di I-labeled exosomes were co-cultured with HUVECs for 24 h in order to determine that exosomes were taken by HUVECs.(2)Different concentrations(0.1?1?5?10?g/ml)of rFim A co-cultured with HUVECs for different times(2?8?24?48h),CCK8 method was used to detect the proliferative activity of HUVECs.And the best reasonable time and concentration were selected for the next experiments.Then the experiment was divided into four groups:(1)Normal Group: HUVECs without any treatment.(2)Exo Group: HUASMCs-Exo and HUVECs were co-cultured.(3)r Fim A Group: HUVECs were treated by r Fim A(4)HUASMCs-Exo + r Fim A Group: HUVECs were treated by r Fim A after pretreatment by HUASMCs –Exo.FACS was used to detect the apoptosis rate,survival rate and necrosis rate of HUVECs.RT-PCR was used to detect the expression of IL-18 and Caspase-3 m RNA of HUVECs.Western blot was used to detect the protein expression of Cleaved caspase-3 of HUVECs.ELISA was used to detect the expression of IL-18 in the supernatant of HUVECs.Results:(1)The primary cells grew in good state and were confirmed by the fluorescence immunoassay and morphological observation.The HUASMCs-Exo was successfully extracted and identified by PEG method,which could be internalized by HUVECs.(2)The results of CCK-8 showed that when HUVECs were treated with 10?g/ml r Fim A for48 h,the proliferative activity was the lowest(P<0.05).So we selected 10?g/ml and 48 h as the processing concentration and time for the following experiments.The results of FACS showed that the total cell apoptosis rate of the HUASMCs-Exo group had no significant difference compared with the Normal group(P>0.05),the total cell apoptosis rate of the r Fim A group and HUASMCs-Exo+r Fim A group were increased compared with the Normal group(P<0.05).When compared with the r Fim A group,the total apoptotisis rate of the HUASMCs-Exo+r Fim A group was decreased(P<0.05).The results of RT-PCR,Western blot and ELISA showed that the expression of IL-18 m RNA,Caspase-3 m RNA,the main apoptosis effector protein of Cleaved caspase-3 and IL-18 in the supernatant had no significant difference in the HUVECs of the Normal group compared with HUASMCs-Exo group(P>0.05).The expression of IL-18 m RNA,Caspase-3 m RNA,Cleaved caspase-3 and IL-18 were increased in the HUVECs of the r Fim A group and the HUASMCs-Exo+r Fim A group compared with the Normal group(P<0.05).When compared with the r Fim A group,the expression of these detection indexes of the HUASMCs-Exo+r Fim A group were decreased(P<0.05).Conclusion:The r Fim A of Porphyromonas gingivalis with?fim A Genotype could induce apoptosis and up-regulat the IL-18 expression of HUVECs.And HUASMCs-Exo could regulate apoptosis and inflammatory reaction of HUVECs induced by the r Fim A,but it could not change the results of dysfunction of HUVECs ultimately.The Fim A of Porphyromonas gingivalis may paly a role in promoting the occurrence and development of atherosclerosis.