P. Gingivalis Promotes Inflammatory Response And EGFR Expression In Lung Tissue

Purpose:Periodontal disease is a chronic,infectious,inflammatory,destructive disease of the periodontium that develops as a result of pro-inflammatory cytokine production induced by infection with periodontal pathogenic bacteria.Chronic obstructive pulmonary disease(COPD)is a non-communicable disease resulting from proinflammatory cytokine production induced by infection with respiratory microorganisms.Although a positive association between periodontal disease and COPD has recently been reported,the role of periodontal disease in the pathogenesis of COPD remains unclear.Therefore,we hypothesised that some periodontopathogenic bacteria,such as Porphyromonas gingivalis(P.gingivalis),increase susceptibility to COPD by inducing the production of pro-inflammatory cytokines in airway epithelial cells.The aim of this study was to observe the effect of P.gingivalis on the inflammatory response of lung tissue and to find key differential proteins to verify that P.gingivalis promotes the inflammatory response of lung tissue and to investigate its role in the development of the inflammatory response in COPD.Methods:1.Determination of the effect of P.gingivalis on the inflammatory response of rat lung tissue(1)Grouping of animals: 18 SD rats were divided into 2 groups: normal control group and P.gingivalis group,9 rats in each group;the rats in the P.gingivalis group were injected with P.gingivalis bacterial suspension by bronchial drip.(2)Hematoxylin-eosin(HE)was used to observe the morphological changes of lung tissue.(3)ELISA was detected IL-8 and TNF-α in rat serum and alveolar lavage fluid(BALF).(4)RT-q PCR was used to detect IL-8 and TNF-α mRNA expression in lung tissue.2.Proteomic bioinformatics analysis and validation of key inflammatory proteins(1)Proteomic bioinformatics analysis was to screen for differential proteins in P.gingivalis that promote exacerbation of the inflammatory response in COPD.(2)Western blot was detected differential protein expression of EGFR,ERK1/2,TLR4 in rat lung tissue.3.Detection of the effect of P.gingivalis on the inflammatory response of human alveolar epithelial cells(1)Morphological changes of human alveolar epithelial cells(A549)were observed by inverted fluorescence microscopy.(2)RT-q PCR was performed to detect the expression levels of IL-6,IL-8 and TNF-α mRNA in A549 cells.(3)ELISA detected the secretion levels of IL-6,IL-8 and TNF-α in A549 cells.(4)RT-q PCR detected the expression levels of EGFR,ERK,TLR4 mRNA in A549 cells.(3)Western blot was to detect EGFR,PEGFR,ERK1/2,p-ERK1/2,TLR4 protein levels in A549 cells.Results:1.P.gingivalis promoted the inflammatory response of rat lung tissue(1)HE:We observed,in the P.gingivalis group,thickening of the bronchial wall,increased mucus secretion,inflammatory cell infiltration in the bronchial wall and surrounding lung tissues,enlargement of the alveolar cavity,thinning and even rupture of the alveolar wall in many places,and fusion of the alveoli.(2)Compared with the normal control group,the BALF and serum levels of IL-8 and TNF-α in the P.gingivalis group increased,and the expression of IL-6,IL-8 and TNF-α mRNA in lung tissue increased;the difference was statistically significant(P < 0.05).2.Bioinformatic analysis of the proteome identified EGFR,a key protein of P.gingivalis that promotes inflammation in COPD rats.(1)Bioinformatics analysis revealed that EGFR was upregulated and activated the RasERK pathway after P.gingivalis stimulation.(2)P.gingivalis stimulation increased the activity of EGFR and ERK1/2 in rat lung tissue,the difference was statistically significant(P < 0.05).(3)P.gingivalis stimulation increased the expression of TLR4 in rat lung tissue,the difference was statistically significant(P < 0.05).3.P.gingivalis promoted inflammatory responses in human alveolar epithelial cells(1)The morphology of A549 cells became more elongated compared to the control group,and granules of different sizes could appear in the cytoplasm.(2)IL-6,IL-8 and TNF-αmRNA levels of A549 cells were upregulated after P.ginigivalis stimulation,and the content of secreted IL-6,IL-8 and TNF-α increased,with statistically significant differences(P < 0.05).(3)EGFR,ERK and TLR4 mRNA levels were increased in A549 cells after P.ginigivalis stimulation,and EGFR,P-EGFR and TLR4 protein expression and ERK1/2 activity were increased,the difference was statistically significant(P <0.05).Conclusion: P.gingivalis stimulation increased the release of pro-inflammatory cytokines IL-6,IL-8 and TNF-α in rat lung tissue and human alveolar epithelial cells,increasing susceptibility to COPD.And EGFR and ERK1/2 activity and TLR4 expression increased.P.gingivalis enhanced lung inflammation was related to TLR4-ERK1/2 and EGFR-ERK1/2,but the exact mechanism needs further investigation.