Role Of PKM2 In The Inflammatory Response To Porphyromonas Gingivalis In Periodontal Tissues

?Objects? In periodontitis,pathogens stimulate the host defense system and activate the secretion of inflammatory cytokines,leading to metabolism reprogramming in the periodontal microenvironment to provide energy and substrates for the activation of immune cells and fibroblasts.Up-regulated expression of pyruvate kinase M2(PKM2),one of the key enzymes in glycolysis,has been reported in inflammatory diseases.The objects of this study include: 1)investigating whether metabolic reprogramming occurs in periodontitis tissues;2)studying the role of PKM2 in the inflammatory response induced by P.gingivalis in periodontal tissues and 3)determining whether the inflammatory response can be regulated by modulating the expression of PKM2.?Methods? 1.Gingival tissues were collected from healthy and chronic periodontitis subjects according to the exclusion and inclusion criteria.Exclusion criteria included the history of systemic diseases,smoking,immune diseases and pregnant or lactating women.The healthy group included gingival tissues harvested from patients undergoing crown extension,third molar extraction or impacted teeth exposure who needed gingivectomy.Healthy control displayed no gingival swelling,negative bleeding on probing,probing depth less than 3 mm and no attachment loss.The chronic periodontitis group included gingival tissues from hopeless teeth with III°mobility.Within six months,the patients received no periodontal treatment and took no drugs like immunosuppressants,hormones and antibiotics.All patients gave informed consent.q PCR,WB and immunohistochemistry were used to analyze the expression of key enzymes in oxidative phosphorylation and glycolysis,as well as the expression of inflammatory cytokines;2.HGFs and THP-1 were stimulated by P.gingivalis.Cell death was measured by LDH assay and CCK-8.The expression of PKM1,PKM2 and inflammatory cytokines were measured by q PCR and WB.Then,PKM2 inhibitors(Shikonin and Compound 3k)and si RNA were used to regulate the expression of PKM2.Using the same method to determine cell death.Finally,after treated by PKM2 inhibitors or si PKM2,HGFs and THP-1 were stimulated by P.gingivalis for 2 h.q PCR and WB were used to examine the expression of PKM1,PKM2 and inflammatory cytokines.?Results? 1.Differences were observed in the expression of key enzymes in oxidative phosphorylation and glycolysis.In periodontitis tissues,the gene transcription of PKM1?PKM2?HK2?PFKFB3?LDHD?Acetyl-Co A?IDH1?IDH2?OGDH?SDHA and SDHB was up-regulated(P <0.05),while the m RNA of FH was down-regulated(P <0.05).Expression of PKM1 and PKM2 in periodontitis tissues was significantly higher than that in normal gingival tissues by immunohistological chemistry;2.Along with the elevation of MOI and infection time of P.gingivalis,the expression of PKM2 increased accordingly.Compared with normal cells,the expression levels of PKM2,ratio of PKM2 to PKM1 and inflammatory cytokines(IL-6,IL-1?,MCP-1 and TNF-?)were significantly increased(P <0.05);3.PKM2 inhibitors and si PKM2 can effectively reduce the expression of PKM2 in normal cells(P <0.05).After treated by PKM2 inhibitors or si PKM2,the expression of IL-6,IL-1?,MCP-1 and TNF-? genes in HGFs and THP-1 stimulated by P.gingivalis was significantly lower than those in untreated P.gingivalis-infected cells(P <0.05).?Conclusions? 1.Metabolic reprogramming occurred in periodontal tissues,HGFs and THP-1 stimulated by P.gingivalis;2.PKM2 is involved in the occurrence of periodontitis,its expression in HGFs and THP-1 increased after stimulated by P.gingivalis;3.By regulating the expression of PKM2,the occurrence and development of periodontal inflammation can be controlled.