Effects Of Succinate-succinate Dehydrogenase-HIF-1? Axis In Porphyromonas Gingivalis-induced Metabolic Reprogramming In Periodontal Tissues

[Objective] Periodontitis is an inflammatory disease that affects supporting periodontal tissues.Host cells may utilize a wide array of defensive strategies to tackle the invasion of anaerobic bacteria and maintain tissue homeostasis,including respiration burst in the lymphocytes to clear the microbial,secretion of chemokines to recruit systemic defense cells,and production of growth factors to enhance defensive capabilities.Such inflammatory responses might be accompanied by fundamental changes in the anabolic and catabolic metabolism in host cells.Succinate is a mature pro-inflammatory metabolite during inflammatory metabolism,and SDH is a key enzyme for its dehydrogenation;in addition,succinate levels affect the activity of HIF-1?,a key transcription factor for pro-inflammatory gene expression.The purpose of this study was to study the role of Succinate-SDH-HIF-1? in the pathogenesis of chronic periodontal disease,and to provide theoretical basis and guidance for understanding and intervening periodontitis.[Methods] Firstly,Porphyromonas gingivalis was used to stimulate periodontal ligament cells,metabolites and metabolic pathways were analyzed by gas chromatography-mass spectrometer,and oxygen consumption rate was detected by Seahorse XF96 in vitro.Healthy and chronic periodontal inflammatory tissues were collected.Patients with histories of systematic diseases were excluded.For the healthy group,gingival tissues were harvested during root lengthening surgery,wisdom tooth extraction or gingivectomy upon exposure of impacted teeth during orthodontic treatment.All the sampling sites displayed no gingival redness and swelling,negative bleeding after probing,probing depth less than 3 mm,no attachment loss.The inflammatory tissues were obtained from patients with severe periodontitis with degree ? mobility who had not received any periodontal treatment and had no medical history of taking immunosuppressive agent and antibiotics in the last 6 months.On the basis of previous studies,key enzymes of glucose metabolism were investigated by real time quantitative polymerase reaction and immunohistology in healthy and periodontal gingival tissue samples.The expression of succinate dehydrogenase(SDH),isocitrate dehydrogenase(IDH),6-phosphofructo-2-kinase/fructose 2,6-biphosphatase 3(PFKFB3)and hexokinase 2(HK2)were analyzed.Through the detection and analysis of periodontal tissue specimens,it is further proved that metabolic enzymes of TCA cycle are involved in the pathogenesis of periodontitis.Furthermore,P.gingivalis was used to stimulate PDLFs,and the expression of enzymes in the glucose metabolism was detected by q PCR and Western Blot,showing that SDH was significantly altered.Then DMM(dimethyl malonate)and 4-OI(4-octyl itaconate)were used to inhibit SDH and SDH sh RNA lentivirus-mediated gene interference was utilized to knock down SDH,q PCR and ELISA were used to detecte the expression of interleukin-1?(IL-1?),interleukin-6(Il-6)and monocyte chemo-attractant protein-1(MCP-1).In order to explore the pathways associated with metabolomics changes,we next analyzed the changes of the classic pro-inflammatory nuclear factor ?B and HIF-1? pathway by Western Blot.2-Methoxyestradiol was used to inhibit the HIF-1? pathway,and q PCR was used to detect levels of IL-1?,IL-6 and MCP-1.[Results] GC-MS analysis showed that PDLFs developed metabolic shifts after 24 h of treatment by P.gingivalis.33 metabolites,such as glutamine,glutathione and 4-aminophenol,were decreased,while 54 metabolites,including citric acid and malic acid were increased.Moreover,bacterial infections affect a variety of ways,including the TCA cycle,arginine and proline metabolism,glyoxylic acid and dicarboxylic acid salt metabolism,proline metabolism,arginine,glyoxylic acid and dicarboxylic acid metabolism.Furthermore,P.gingivalis-treated PDLFs showed decreased basal respiration,maximum respiration,and reserve capacity,suggesting that energy production was rewired from mitochondrial oxidative phosphorylation to cellular glycolysis.Increased SDH,IDH,PFKFB3 and HK2 were observed in the gingival tissues of periodontitis group than healthy group by q-PCR and immunohistology.In P.gingivalis-stimulated PDLFs,SDH expression was significantly increased while its activity was decreased after long time P.gingivalis infection.Intracellular succinate level at 4 h after infection was increased in a multiplicity of infection(MOI)-dependence of infection-dependent manner.Reactive oxygen species(ROS)were increased in bacteria-infected cells by confocal microscopy and flow cytometry analysis.Inflammatory cytokines,IL-1?,IL-6 and MCP,in bacteria-stimulated PDLFs were inhibited after DMM and 4-OI treatment,as well as SDH knock-down.HIF-1? levels were elevated in PDLFs after P.gingivalis treatment,while prolylhydroxylase domain 2(PHD2)and nuclear factor erythroid 2-related factor 2(Nrf2)was significantly reduced.However,the classical pro-inflammatory NF-?B(the main pathway for TLR signal transduction in monocytes and macrophages)was not activated,suggesting that HIF-1? is the main pathway mediating metabolic reprogramming of PDLFs.The HIF-1? pathway inhibitor 2-methoxyestradiol(2-Me OE2)significantly reduced the transcription of pro-inflammatory cytokines.[Conclusions] PDLFs,the principal resident cells with characteristics of MSCs,can dynamically reprogram its metabolic features to adapt to bacterial infection;in addition,accumulation of succinate,altered SDH activity and activation of HIF-1? pathway may drive the pro-inflammatory responses in PDLFs;therefore,regulating succinate-SDHHIF-1? may be useful in host modulation therapy of chronic periodontitis.