Effects Of Ca²⁺/EdnrA/HIF-1α On Glycolytic Metabolism And Expression Of Inflammatory Cytokines In Human Periodontal Ligament Fibroblasts Affected By LPS

Research background and objectiveMetabolic reprogramming is an important pathogenesis of inflammation.Periodontitis,as an inflammatory disease affecting the supporting tissues of the teeth,causes destruction of periodontal tissues and triggers host immune and inflammatory responses accompanied by fundamental changes in the metabolic state of host cells.Glycolysis can rapidly provide energy for inflammatory response and produce a large number of intermediate metabolites for immune and inflammatory protein biosynthesis,so targeting glycolytic metabolism can regulate the level of inflammatory response.Hypoxia-inducible factor-1α HIF-1α)is a key transcription factor of glycolysis-related gene expression,elucidating the factors affecting HIF-1αexpression and its regulatory mechanism can open up new directions for the regulation of inflammatory response from a metabolic perspective.In this study,we observed the effects of HIF-1α on glycolysis and inflammatory cytokines expression in human periodontal ligament fibroblasts(hPDLFs)by constructing an in vitro model of periodontitis through gene knockdown,qPCR,WB,ELISA and bioinformatics analysis.The study also explore the factors affecting the expression of HIF-1α and its regulatory mechanism,so as to provide a new theoretical basis for the selection of therapeutic targets for periodontitis and periodontitis complicated with other diseases.Materials and methods1.HIF-1α-shRNA lentivirus was constructed and set up as NC group,HIF-1αsiRNA group,LPS group and LPS+HIF-1α siRNA group.qPCR and WB were performed to detect the expression of HIF-1α and key enzymes of glycolysis after LPS acted on hPDLFs for 6 hours;ROS levels in hPDLFs were detected with ROS detection kit;The expression of IL-6,IL-1β in the supernatant of each group was measured by ELISA.2.Immunofluorescence was used to detect hypoxia and intracellular Ca2+level in hPDLFs under LPS stimulation;Setting up control group,LPS group,LPS+calcium-free medium group,LPS+calcium chelator group.qPCR and WB were used to detect the expression of HIF-1α and key enzymes of glycolysis;ELISA was used to detect the expression of IL-6 and IL-1β in the supernatant of each group.3.Bioinformatic analysis of the healthy and periodontitis groups and differential genes associated with calcium pathway;Setting up control group,LPS group,LPS+calcium-free medium group,qPCR,WB to detect the expression of NF-κB,STAT3,EdnrA,EdnrB;ELISA to detect the expression of ET-1 in the supernatant of each group;Using STAT3 inhibitor,EdnrA antagonist for reverse validation,qPCR,WB to detect the expression of HEF-1α.Results1.The expression of HIF-1α as well as glycolytic enzymes,ROS levels was increased in hPDLFs under LPS stimulation,and the expression of glycolytic enzymes as well as IL-6 and IL-1β was down-regulated after inhibition of HEF-1α.2.hPDLFs was in a normoxic state after short time and low concentration of LPS stimulation,and the increase of intracellular Ca2+level was a factor that promoted the upregulation of HIF-1α gene expression and stabilized HIF-1α.Depletion of intracellular and extracellular sources of Ca2+ by calcium-free medium or calcium chelator significantly downregulated the mRNA and protein levels of HIF-1α in hPDLFs under LPS stimulation,and also decreased the expression of glycolytic enzymes as well as IL-6 and IL-1β.3.NF-κB and STAT3 were significantly increased at the mRNA and protein levels in hPDLFs by LPS,while the expression of EdnrA and ET-1 was also significantly upregulated,while EdnrB was not significantly changed.The use of calcium-free medium significantly downregulated the expression of STAT3,EdnrA,and ET-1 under LPS stimulation,but had no significant effect on NF-κB and EdnrB.STAT3 inhibitor decreased the mRNA and protein levels of HIF-1α under LPS stimulation,and the use of EdnrA antagonist inhibited the protein expression of HIF-1α by LPS.Conclusions1.HIF-1α regulates glycolysis and the release of inflammatory cytokines in hPDLFs under LPS stimulation.2.Short time and low concentration of LPS stimulation can stabilize HIF-1αexpression in hPDLFs under normoxia,Ca2+ was a factor that promoted upregulation of HIF-1α gene expression and stabilized HIF-1α,and both extracellular and intracellular sources of Ca2+ could affect HIF-1α expression.3.Modulation of Ca2+ levels can control the glycolysis of hPDLFs and the release of inflammatory cytokines by affecting the expression of HIF-1α.4.Under LPS stimulation,EdnrA is involved in the regulation of HIF-1αstabilized by Ca2+ in hPDLFs.STAT3 signal pathway may be an important pathway for Ca2+ to regulate HIF-1α gene expression in hPDLFs.