Porphyromonas Gingivalis Peptidyl-arginine Deaminase Regulates HNRNPA2B1 To Destroy The Intestinal Barrier In Caco-2 Cells

Objectives: Periodontitis is a chronic infectious oral inflammatory disease.Inflammatory bowel disease(IBD)is a disease characterized by non-infectious chronic inflammation of the gastrointestinal tract.A large amount of evidence shows that periodontitis is a risk factor for IBD.Porphyromonas gingivalis(P.gingivalis),as a major periodontal pathogen,is believed to be closely related to the progress of IBD.Porphyromonas gingivalis peptidyl-arginine deaminase(PPAD),a unique virulence factor of P.gingivalis,is involved in the development of IBD.However,the mechanism remains unclear.Occludin and zonula occluden-1(ZO-1)constitute the intestinal physical barrier,and the Toll-like receptor 4(TLR4)in intestinal epithelial cells constitute the intestinal immune barrier.The destruction of the intestinal barrier is involved in the occurrence of IBD.The potential of heterologous nuclear ribonucleoprotein A2B1(HNRNP2B1)in the pathogenesis of IBD has been revealed.Caco-2 cells are often used as a model to simulate IBD in vitro.This study aims to explore the potential mechanism of P.gingivalis destroying the intestinal barrier in Caco-2 cells via PPAD and then aggravating IBD.Methods: Caco-2 intestinal epithelial cell model was established and a PPAD mutant was constructed.The experiment was divided into P.gingivalis W83 group,P.gingivalis△ PPAD group,P.gingivalis-LPS group.The m RNA and protein expression levels of HNRNPA2B1,TLR4 and tight junction associated proteins Occludin and ZO-1 were detected by q RT-PCR and Western blot.The expression levels of inflammatory factor tumor necrosis factor-α(TNF-α)and interleukin-1 beta(IL-1β)were detected by q RT-PCR and ELISA.After using si-HNRNPA2B1 and pc DNA-HNRNPA2B1 to transfect Caco-2 cell to silence and over-express HNRNPA2B1,q RT-PCR and actinomycin D assay were utilized to detect the m RNA expression level and degradation rate of TLR4,tight junction associated proteins Occludin and ZO-1,and q RT-PCR and ELISA were used to detect the expression levels of inflammatory factor TNF-α and IL-1β.All data were expressed with mean ± standard deviation,and P<0.05 was considered statistically significant.The software SPSS20.0 was applied to statistical analysis.Results:1.P.gingivalis W83 group,P.gingivalis △PPAD group and P.gingivalis-LPS group activated TLR4 and produced inflammatory factor TNF-α and IL-1 β in Caco-2cell.The expression level of TLR4 and inflammatory factors was the highest in P.gingivalis W83 group.2.P.gingivalis W83 stimulation down-regulated the m RNA and protein expression of HNRNPA2B1 and Occludin,and the protein expression of ZO-1 in Caco-2 cell.However,the above indexes exhibited no change in P.gingivalis △ PPAD group and P.gingivalis-LPS group.3.After silencing HNRNPA2B1,the stability of Occludin m RNA diminished,and the expression of its m RNA and protein was inhibited.After overexpression of HNRNPA2B1,the stability of Occludin m RNA elevated,and the expression of its m RNA and protein was promoted.4.The stability and expression of ZO-1 m RNA had no change after silencing HNRNPA2B1,while its protein expression was suppressed.The stability and expression of ZO-1 m RNA showed no change after overexpression of HNRNPA2B1,while its protein expression was enhanced.5.After silencing HNRNPA2B1,the stability of TLR4 m RNA,its m RNA and protein expression were improved.Instead,after overexpression of HNRNPA2B1,the stability of TLR4 m RNA,its m RNA and protein expression were hindered.6.The expression levels of TNF-α and IL-1 β were increased in the HNRNPA2B1 knock-down Caco-2 cells while decreased in the HNRNPA2B1 overexpressed Caco-2 cells.Conclusion: 1.P.gingivalis might destroy the intestinal epithelial barrier in Caco-2 cells through PPAD.2.PPAD reduced the stability of Occludin m RNA by down-regulation of HNRNPA2B1 to restrain the expression of Occludin m RNA and protein,and down-regulated the protein expression of ZO-1 via down-regulation of HNRNPA2B1 in Caco-2 cells.The reduced level of Occludin and ZO-1 destroyed the intestinal physical barrier in Caco-2 cells.3、PPAD enhanced the stability of TLR4 m RNA and promoted expression of TLR4,TNF-α and IL-1β via down-regulation of HNRNPA2 B,destroying the intestinal immune barrier and aggravating inflammation in Caco-2 cells.