| Objective:To explore the mechanisms and cell signaling pathway involved in the innate immune and inflammatory response of HUVECs elicited by P. gingivalis, and to provide new ideas for prevention of As and C VD, by observing the effect of TLRs blockers on the expression of the downstream molecules involved in the cell signal and the production of inflammatory mediators of HUVECs stimulated by P. gingivalis.Methods:P. gingivalis (W83) withⅣfimA genotype were cultured anaerobically in standard condition and 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. Meanwhile, HUVECs were cultured in vitro and the monoclonal antibody of anti-TLR2 and anti-TLR4 were separately added to confluent HUVECs monolayers and co-cultured for 30 minutes. Then different concentrations of recombinant fimbrillin were added in the culture medium and co-cultured for 2h,6h, and 24h. The levels of mRNA of TLR signaling adapter molecule MyD88, proinflammatory cytokines (IL-1β, IL-6 and TNF-α), chemokines (IL-8 and MCP-1) and adhesion molecule (ICAM-1 and VCAM-1) were detected by RT-PCR. NF-κB nuclear translocation was evaluated by IHC, proinflammatory cytokines and chemokines in the culture supernatant were measured by ELISA and the expression of adhesion molecules of HUVECs were analyzed by FCM.Results:1. The effect of TLR2 blocker on the expression of the downstream molecules involved in the cell signaling and the production of inflammatory mediators of HUVECs stimulated by P. gingivalis-rFimA withⅣfimA genotype:(1) Stimulated by P. gingivalis-rFimA withⅣfimA genotype after pretreated with TLR2 blocker for 30min, the levels of the expression of MyD88 mRNA in HUVECs of the experimental groups at 6h (10ug/ml) and 24h (0.5μg/ml,5μg/ml,10μg/ml) were significantly lower than that of positive and negative control groups. (2) The activation rate of NF-κB in HUVECs of the experimental groups at 6h and 24h were significantly lower than that of control group (P<0.05). (3) The expression levels of mRNA and protein of IL-1βand IL-6 by HUVECs of all the experimental groups showed no statistically significant differences with that of positive and negative control groups (P>0.05); the expression of TNF-a mRNA in HUVECs of the experimental groups at 2h (0.5μg/ml),6h (0.5μg/ml), and 24h (0.5ug/ml,5μg/ml, 10μg/ml) groups were significantly lower than that of positive and negative control groups (p<0.05), the expression of TNF-a protein by HUVECs showed the same statistical result as its mRNA level.(4) The expression of IL-8 mRNA in HUVECs of the experimental groups at 2h (0.5μg/ml,5μg/ml),6h (0.5μg/ml,5μg/ml), and 24h (5μg/ml) were significantly higher than that of positive and negative control groups (P<0.05); the expression of MCP-1 mRNA in HUVECs of the experimental groups at 6h (0.5μg/ml),24h (0.5μg/ml) were significantly lower than that of positive and negative control groups (P<0.05); the expression levels of IL-8 and MCP-1 protein showed the same statistical results as their mRNA levels. (5) The expression of ICAM-1 mRNA in HUVECs of the experimental groups at 24h (0.5μg/ml,5μg/ml,10μg/ml) were significantly lower than that of positive and negative control groups (P<0.05); the expression of VCAM-1 mRNA in HUVECs of the experimental groups at 6h (0.5μg/ml,5μg/ml) and 24h (0.5μg/ml,5μg/ml, 10μg/ml) were significantly lower than that of positive and negative control groups (P<0.05); the expression levels of ICAM-1 and VCAM-1 protein showed the same statistical results as their mRNA levels.2. The effect of TLR4 blocker on the expression of the downstream molecules involved in the cell signaling and the production of inflammatory mediators of HUVECs stimulated by P. gingivalis-rFimA withⅣfimA genotype:(1) Stimulated by P. gingivalis-rFimA withⅣfimA genotype after pretreated with TLR4 blocker for 30min, the levels of the expression of MyD88 mRNA in HUVECs of the experimental groups at 2h (0.5μg/ml,5μg/ml) and 24h (0.5μg/ml,5μg/ml, 10μg/ml) were significantly lower than that of positive and negative control groups (P<0.05). (2) The activation rate of NF-κB in HUVECs of the experimental groups at 6h and 24h were significantly lower than that of control group (P<0.05). (3) The expression levels of mRNA and protein of IL-1βand IL-6 in HUVECs of the experimental groups at 2h (0.5μg/ml,5μg/ml),6h (0.5μg/ml,5μg/ml,10μg/ml) and 24h (0.5μg/ml,5μg/ml, 10μg/ml) were significantly lower than that of positive and negative control groups (P<0.05); the expression of TNF-αmRNA in HUVECs of the experimental groups at 6h (0.5μg/ml,5μg/ml, 10μg/ml) and 24h (0.5μg/ml,5μg/ml, 10μg/ml) were significantly higher than that of positive and negative control groups (P<0.05), the expression of TNF-a protein showed the same statistical result as its mRNA level. (4)The expression of IL-8 mRNA in HUVECs of the experimental groups at 6h (0.5μg/ml,5μg/ml, 10μg/ml),24h (0.5μg/ml,5μg/ml, 10μg/ml) were significantly higher than that of positive and negative control groups (P<0.05), the expression of IL-8 protein showed the same statistical result as its mRNA level. The expression of MCP-1 mRNA in HUVECs of the experimental groups at 6h (0.5μg/ml),24h (0.5μg/ml) were significantly lower than that of positive and negative control groups (P<0.05); the expression levels of IL-8 and MCP-1 protein showed the same statistical result as their mRNA level. (5) The expression of ICAM-1 mRNA in HUVECs of the experimental groups at 24h (0.5μg/ml, 5μg/ml, 10μg/ml) were significantly higher than that of positive and negative control groups (P<0.05); the expression of VCAM-1 mRNA in HUVECs of the experimental groups at 2h (0.5μg/ml),6h (5μg/ml) and 24h (0.5μg/ml,5μg/ml,10μg/ml) were significantly higher than that of positive and negative control groups (P<0.05); the expression levels of ICAM-1 and VCAM-1 protein showed the same statistical results as their mRNA level.Conclusion:1. Either TLR2 or TLR4 blocker can inhibit the innate immune signaling of HUVECs via down-regulating or blocking the expression of MyD88 induced by recombinant fimbrillin of P. gingivalis withⅣfimA genotype. As a result, the downstream molecule NF-κB cannot be activated, the transcription and translation of genes which expressed cytokine cannot be initiated, and the production of proinflammatory cytokine by HUVECs is inhibited. The TLR2 blocker can inhibit the secretion of TNF-α,IL-8, MCP-1, ICAM-1 and VCAM-1 of HUVECs, while TLR4 blocker can inhibit the production of IL-1βand IL-6.2.The innate immune response of HUVECs induced by Recombinant Fimbrillin of P. gingivalis withⅣfimA genotype may correlated with the TLR2 and TLR4, TLR2 and TLR4 blockers can inhibit the inflamatory response in HUVECs elicited by recombinant Fimbrillin of P. gingivalis withⅣfimA genotype... |
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