The Effect Of 4-Octyl Itaconate In The Inflammatory Response Of Macrophages Stimulated By P.Gingivalis LPS

[Objective] In the progression of periodontitis,immune cells produce a large number of proinflammatory cytokines,including tumor necrosis factor(TNF-?),interleukin 1?(IL-1?)and interleukin 6(IL-6),which plays a crucial role in the inflammation process,and Porphyromonas gingivalis(P.gingivalis)is one of the main pathogens involved in the pathogenesis of periodontitis.Recent studies have found that the endogenous metabolite itaconate can regulate macrophage function,but the exact mechanism is not yet clear.We used a model of P.gingivalis LPS-stimulated macrophages to observe how 4-octyl itaconate(OI)affect macrophages on inflammation.[Methods] Phorbol myristate acetate(PMA)was empolyed to induce THP-1 cells into macrophages.First,we used different concentrations of P.gingivalis LPS to stimulate macrophages,using q-PCR or Western Blot to analyze the expression of inflammatory factor related genes,glycolytic key enzymes and TCA related genes,and inflammasome related genes.Then macrophages were stimulated with different concentrations of OI,and the effects of OI on the viability of human-derived macrophages were tested by CCK-8 analysis.The subsequent experiments were conducted on the basis that it was clear that 62.5 ?M OI had no effect on macrophage proliferation.Macrophages were pretreated with OI(62.5 ?M)for 2 h,and then macrophages were stimulated with P.gingivalis LPS(500 ng / m L).We used ELISA and q PCR to detect the expression of inflammatory factors.And we extracted proteins from the macrophages and analyzed the expression of key glycolytic enzymes,TCA related genes and proteins related to inflammasome by Western Blot.Reactive oxygen species(ROS)was also analyzed the in cells by laser confocal microscopy and microplate reader.[Results] Macrophages were stimulated with P.gingivalis LPS,and it was found that they could upregulate transcription of inflammatory genes in macrophages.The m RNA expression levels of TNF-?,IL-1?,and IL-6 was increased in a concentration-dependent manner.P.gingivalis LPS can also increase the expression of metabolism related genes,including 6-phosphofructo-2-kinase-fructose-2,6 biphosphatase 3(PFKFB3),Hexokinase II(HK2)in glocolysis.The succinate dehydrogenase(SDH)in the tricarboxylic acid cycle(TCA)was increased in m RNA levels in early time and decreased in protein levels in late time.The expression of immunoresponsive gene 1(IRG1)is enhanced.In addition,P.gingivalis LPS can increase expression of macrophage inflammasome related genes,including NLRP3,Caspase-1,and pro-IL-1?.CCK-8 analysis showed that the appropriate concentration of OI had no effect on the cell viability of macrophages.The q PCR and ELISA suggested that OI significantly down-regulated the expression levels of inflammatory cytokines TNF-? and IL-1? in macrophages stimulated by P.gingivalis LPS.OI can also down-regulate the expression of P.gingivalis LPS-stimulated macrophages glycolytic key enzymes PFKFB3,HK2 or IRG1.In addition,OI can also reduce the levels of inflammasome-associated proteins in macrophages induced by P.gingivalis LPS,including NLRP3,Caspase-1,and pro-IL-1?.We also observed that OI can also reduce the expression level of ROS in macrophages induced by P.gingivalis LPS.[Conclusions] P.gingivalis LPS can up-regulate the transcription level of inflammatory genes,change the expression of metabolic related genes,and enhance the expression of inflammasome related genes.OI can reduce the level of P.gingivalis LPS-induced inflammation in macrophages.OI can down-regulate the expressions of the key enzymes of glycolysis PFKFB3,HK2 and IRG1 in macrophages caused by P.gingivalis LPS.OI can also down-regulate P.gingivalis LPS-induced macrophage inflammasome-associated protein expression levels including NLRP3,Caspase-1,pro-IL-1? and intracellular ROS expression levels.