The Effects Of Porphyromonas Gingivalis Fimbrillin On Inducing Inflammation In Endothelial Cells And The Intracellular Signaling Pathways

Objective:To explore the intracellular signaling pathways involved in inflammatory response in HUVECs under the induction of fimbrillin from different strains of P. gingivalis with different fimA genotypes.Methods:1. P. gingivalis WCSP 115 (typeⅡfimA gene) and W83 (typeⅣfimA gene)were cultured anaerobically in standard condition and identified,then P. gingivalis cells were collected to isolate their DNA.After the fimA gene sequence was cloned, we established fimA prokaryotic expression system pET-30a-FimA BL21DE3 which expressing recombinant fimbrillin under the induction of IPTG..2. HUVECs were cultured in vitro and different concentrations of recombinant fimbrillin from P. gingivalis with different fimA genotypes were added to confluent HUVECs monolayers and co-cultured for 2h,6h and 24h. At different time periods, HUVECs were collected, total RNA was extracted from part of them and used for RT-PCR to investigate the levels of mRNA of cluster of differentiation 14(CD14), Toll-like receptor 2(TLR2), Toll-like receptor 4(TLR4) and myeloid differentiation factor 88(MyD88).The other cells were detected by FCM analysis for CD14,TLR2,TLR4 expression.Results:1. When HUVECs were stimulated with the P. gingivalis-rFimA withⅡorⅣfimA genotype, the level of CD 14 mRNA expressed by HUVECs were higher than that of the negative control group(P<0.05). At 2h,the up-regulating effects on CD 14 mRNA expression by P. gingivalis-rFimA withⅣfimA genotype were stronger than that by P. gingivalis-rFimA withⅡfimA genotype. After HUVECs were exposed to different concentrations(0.5μg/ml,5μg/ml,10μg/ml) of P. gingivalis-rFimA withⅣfimA genotype for 24h, the production of CD 14 protein by HUVECs was higher than that of the negative control group(P<0.05). At 2h, however, when the higher dose (5μg/ml or lOμg/ml) of P. gingivalis-rFimA withⅣfimA genotype was used, the level of CD14 protein producted by HUVECs was lower, and there was a discrepancy between the level of CD14 protein and the level of its mRNA. When HUVECs were stimulated with 0.5μg/ml rFimA from P. gingivalis withⅣfimA genotype at 2h,0.5μg/ml,5μg/ml or 10μg/ml rFimA from P. gingivalis withⅣfimA genotype at 6h, and 0.5μg/ml,5μg/ml or 10μg/ml rFimA from P. gingivalis withⅡfimA genotype at 2h,6h and 24h, the protein expression of CD14 were similar to that of the negative control group (P>0.05).2. The expression of TLR2 mRNA in HUVECs induced by P. gingivalis-rFimA withⅡorⅣfimA genotype were higher than that of the negative control (p<0.05). The up-regulating effects on TLR2 mRNA by P. gingivalis-rFimA with IV fimA genotype were stronger than that by P. gingivalis-rFimA withⅡfimA genotype.The up-regulating effects of TLR2 mRNA induced by P. gingivalis-rFimA with IV fimA genotype were in a dose-dependent manner at 24h. In addition, the level of TLR2 protein expressed by HUVECs stimulated with rFimA was higher than that of the negative control group(P<0.05),which was correspond to the gene level; At 2h, however, when the higher dose (5μg/ml or 10μg/ml) of P. gingivalis-rFimA withⅣfimA genotype was used,the level of TLR2 protein was lower, and there was a discrepancy between the level of TLR2 protein and the level of its mRNA. When HUVECs were stimulated with 0.5μg/ml rFimA from P. gingivalis withⅣfimA genotype at 2h,0.5μg/ml,5μg/ml or 10μg/ml rFimA from P. gingivalis withⅣfimA genotype at 6h,and 0.5μg/ml,5μg/ml or 10μg/ml rFimA from P. gingivalis withⅡfimA genotype at 2h,6h and 24h, the protein expression of TLR2 were similar to that of the negative control group(P>0.05).3. When HUVECs were stimulated with the P. gingivalis-rFimA withⅡorⅣfimA genotype, the level of TLR4 mRNA expressed by HUVECs was higher than that of the negative control(P<0.05). As compared with the stimulation of other concentrations (0.5μg/ml and 10μg/ml) of P. gingivalis-rFimA withⅣfimA genotype, the level of TLR4 mRNA reached its maximum with the stimulation of 5μg/ml P. gingivalis-rFimA withⅣfimA genotype at each time point. The level of TLR4 protein expressed by HUVECs were analyzed at 2h,6h and 24h, we observed that P. gingivalis-rFimA failed to stimulate TLR4 expression in HUVECs in each concentration group(p>0.05).4. When HUVECs were treated with the P. gingivalis-rFimA withⅡorⅣfimA genotype, the level of MyD88 mRNA expressed by HUVECs was higher than that of the negative control group(P<0.05). The up-regulating effects on MyD88 mRNA by P. gingivalis withⅡfimA genotype were in a time-dependent manner.Conclusion:Based on these findings, we concluded that P. gingivalis-FimA withⅡorⅣfimA genotype could active HUVECs via expressing CD14 and TLR2 and up-regulating the level of MyD88 mRNA and TLR4 mRNA. It indicated that the inflammation response in HUVECs induced by P. gingivalis may be mediated through its FimA,A,P. gingivalis-FimA is likely a potential risk factor for atherosclerosis and the biological basis underlying the relationship between periodontitis and atherosclerosis.Our data suggest that singnaling pathways of CD14-TLR2 system and MyD88 may be involved in inflammatory response and cytokine production in HUVECs.The difference of P. gingivalis-FimA with fimA genotypes may lead different effects to HUVECs, and the stimulation of P. gingivalis-FimA withⅣfimA genotype was stronger than that of p. gingivalis-FimA withⅡfimA genotype.