Effects On The Expression Of Pro-inflammatory Cytokines In The Liver And Spleen After Oral Administration Of Porphyromonas Gingivalis In Mice

Background and objective: Periodontitis,as a chronic infectious disease,has been demonstrated to be associated with several systemic diseases especially neroinflammation diseases.Porphyromonas gingivalis(P.gingivalis),one of the major pathogens causing periodontal diseases,is thought to play a critical role in possible mechanisms linking periodontitis with other systemic disorders.Studies have proved that P.gingivalis lipopolysaccharide(LPS)could enter and stay in the brain of Alzheimer disease patients.This study is one part of the China science foundation project,previous study of which has observed that P.gingivalis could activate the release of pro-inflammatory cytokines by microglial cells.Therefore,this study hypothesizes that oral administration of P.gingivalis can lead to cognitive dysfunction dependent on the activation of microglial cells via initiating inflammatory reaction of liver and spleen.To verify the hypothesis,thisstudy assessed the changes in production of pro-inflammatory cytokines interleukin-6(IL-6),interleukin-1β(IL-1β)and tumor necrosis factor-α(TNF-α)and histological changes in the liver and spleen as a result of oral administration of P.gingivalis in mice.Method: 1.Thirty female C57BL/6 mice separated into two groups(n = 15 per group): the P.gingivalis group and control group.Oral administration of P.gingivalis was given to the mice of P.gingivalis group to infect the periodontium.2.Quantitative Real-time PCR was performed to evaluate the gene expression of IL-1β、IL-6 and TNF-αin the liver and spleen of the P.gingivalis group and control group.3.ELISA assay was used to measure the protein levels of IL-6、IL-1β and TNF-α in the liver and spleen of the P.gingivalis group and control group.4.Immunohistochemistry staining was used to detect the positive expression of IL-1β、IL-6 and TNF-α from the liver and spleen of the P.gingivalis group and control group.Quantification of positive expression was quantified as the mean optical density.5.Sections of liver and spleen tissue were cutted and stained with haematoxylin and eosin for histological examination.6.The significance of the differences between the P.gingivalis group and control group was analysed using the Student’s t-test in SPSS version 17.0.0.All data are present as mean ± standard deviation(SD).Results: 1.Elevated m RNA levels of the pro-inflammatory cytokines TNF-α、IL-6 and IL-1β were found in the P.gingivalis group.In addition,oral administration of P.gingivalis increased the protein levels of TNF-α 、 IL-6 as well as IL-1β.Immunohistochemical staining of liver tissues also indicated up-regulation of protein expression compared with the control group.There was a significant difference in MOD between the P.gingivalis and the control group(p<0.01).2.Histological analysis of liver tissue showed that scattered microvesicular steatosis were observed in hepatocytes of the P.gingivalis group.A majority of hepatocytes had “foamy” changes where the nuclei were typically centrally located.Furthermore,a few inflammatory cells had infiltrated the tissue.In contrast,liver sections from the control groupshowed no obvious alterations in the hepatic cord structure or hepatic cells.3.Production of pro-inflammatory genes TNF-α、IL-6 and IL-1β were significantly up-regulated in the P.gingivalis group as compared to the control group(p<0.01).Correspondingly,protein levels of TNF-α、IL-6 and IL-1β were also higher.Furthermore,the immunohistochemical staining of spleen tissue from the P.gingivalis group indicated elevated levels of MOD which implied positive expression of TNF-α、IL-6 and IL-1β protein.4.Several visible histological changes were found in the spleen tissue of P.gingivalis administered mice.We observed obvious activation of the white pulp and reduction of the red pulp compared with the control group.In addition,neutrophil infiltration was detected in the spleen tissue of the P.gingivalis group.In contrast,no histological changes of inflammation were detected in spleen tissue obtained from the control group.Conclusion: The production of pro-inflammatory cytokines in the liver and spleen increased as a result of oral administration of P.gingivalis in mice,which could be considered as the direct evidence of the mechanisms linking periodontitis and chronicle liver disorders such as NAFLD.It also provides experimental foundation for our hypothesis that oral administration of P.gingivalis in mice can cause the release of pro-inflammatory cytokines by microglial cells,which leads to cognitive dysfunction in mice.