| Background and ObjectivesLipopolysaccharide(LPS)can stimulate macrophages to secrete a variety of pro-inflammatory mediators and trigger a series of immune reactions.The metabolites of arachidonic acid(AA)and eicosanoid are disordered when the liver is inflamed.Some eicosanoid metabolized by CYP ?-hydroxylase are key pro-inflammatory mediators.Oridonin is the main active component of Rabdosia rubescens,and its role in treating and preventing inflammatory diseases has been widely studied.The RNA-seq data form cells showed that oridonin can induce the expression of CYP4F11 and liver X receptor ?(LXR?).LXR?,function as a critical signaling node linking inflammation and lipid metabolism,has anti-inflammatory effect on LPS induced inflammation.Many researchers have explored the anti-inflammatory mechanism of oridonin,but the effect of oridonin on AA metabolism and inflammatory regulation mechanisms is still unclear.The aim of this study is to clarify that oridonin through actives LXR?-CYP4F 11 pathway to regulate the oxidative lipid metabolites of AA and alleviate LPS-induced inflammation.This will provide new evidence for the potential therapeutic mechanism for clinical treatment of liver inflammation.Methods1.LPS was used to induce inflammation in THP-1 cells,and the anti-inflammatory effect of oridonin in different concentrations was investigated in vitro.2.A model of acute liver injury in mice was made by injecting LPS/D-GalN,and the protective effect of oridonin on acute liver injury was investigated.3.Western blot and RT-qPCR was used to investigate the effect of oridonin on the expression of LXR? in cells and liver tissues.THP-1 cells were treated with oridonin combined with LXR agonist GW3965 to investigate the effect of LXR on the anti-inflammatory effect of oridonin.4.Luciferase reporter assays were performed to investigate the regulatory effect of LXR? on CYP4F11.The effect of oridonin on the LXR?-CYP4F11 pathway was also evaluated.5.To investigate the effect of anti-inflammatory activity of CYP4F11,we made a stable cell line overexpressing CYP4F11 in THP-1 cells and detect the cytokine after induced by LPS.Besides,the oxidized lipids metabolites of cells were detected by HPLC-MS/MS.Results and Conclusions1.5 ?M and 10 ?M oridonin could significantly reduce the levels of TNF?,IL-6 and IL-1? in the supernatant of THP-1 cells,and the inhibition efficiency of TNF?and IL-6 were higher than IL-1?.Oridonin could inhibit LPS-induced THP-1 inflammation in vitro.2.5 mg/kg and 10 mg/kg oridonin could reduce the plasma ALT,AST and the cytokines mRNA level in acute liver injury mice.Oridonin showed protective effect on LPS/D-GalN-induced acute liver injury.3.Oridonin could induce the expression of LXR? in HepG2 cells and reverse the decrease of LXR? in the liver of mice with acute liver injury.The combination of GW3965 and oridonin induced LXR?,and significantly reduced TNF? and IL-6 of THP-1 cells.The results indicated that oridonin may exert anti-inflammatory effects by inducing LXR?.4.LXRa induced the promoter activity of CYP4F11 through LXRE.The induction effect on CYP4F11 by oridonin was significantly reduced by siRNA.The results showed that oridonin could increase the expression of CYP4F11 by activating the LXR?-CYP4F11 pathway.5.Compared with the lenti-control group,overexpression of CYP4F11 inhibited the TNF? in THP-1 cell.47 oxidized lipid metabolites in THP-1 cells were detected by HPLC-MS/MS and six metabolites were significantly changed.In lenti-CYP4F11 THP-1 cells,the pro-inflammatory mediator LTB4 was decreased significantly while the anti-inflammatory mediator 17-HDHA was increased significantly compare to lenti-control THP-1 cells.CYP4F11 may inhibit LPS-induced inflammation by removing LTB4 and inducing 17-HDHA. |
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