The Protective Mechanism Of Cannabinoid Receptor 2 On Acute Liver Failure By Inhibiting Macrophage Glycolysis

Backgoroud:Acute liver failure（ALF）is a serious liver dysfunction caused by multiple etiologies,and may lead to gastrointestinal bleeding,hepatic encephalopathy etc.with a high fatality rate.Currently,the specific medical treatment for ALF is extremely limited.Liver transplantation is the most effective treatment for ALF at the end stage,but its application is greatly limited due to the tight liver source,high cost and postoperative rejection reaction.Therefore,finding a new therapeutic target for ALF has become one of the urgent scientific problems to be solved in the world.A massive number of hepatic macrophages are activated during hepatic injury to release inflammatory cytokines to magnify the inflammatory response,induce further apoptosis of hepatocytes,and accelerate liver injury.Therefore,targeting hepatic macrophages to control the inflammatory effects would be an effective therapeutic strategy for ALF.Studies have shown that hepatic macrophages are substantially polarized toward M1during liver injury,with the enhanced glycolysis as the metabolic characteristics of M1 type polarization.Cannabinoid receptor 2（CB2R）,mainly distributed in macrophages and other immune cells,can regulate immunity and inhibit inflammation,and has a protective effect on liver injury,but the specific mechanism of the action is still unclear.We hypothesized that inhibition of the inflammatory response caused by macrophages reprogrammed by glycolysis could reduce apoptosis for the improvement of ALF.In this research,we used an LPS+D-galactosamine（D-Gal N）-induced ALF mice model to verify the hepatoprotective effects of CB2R agonists and to investigate the specific mechanisms by which CB2R exert hepatoprotective effects through modulating macrophage glycolysis pathways.Methods:1.We constructed LPS+D-Gal N-induced acute liver failure wild type/CB2R^-/-mouse model,and investigated the phenotypic effects of cannabinoid receptor 2 agonist GW405833（GW）and antagonist SR144528（SR）on ALF mice through survival analysis,biochemical examination of blood and liver histopathology;TUNEL staining to detect the severity of hepatocyte apoptosis;immunohistochemistry to detect M1/M2differentiation of liver macrophages.2.We constructed an in vitro inflammation model with LPS-stimulated RAW264.7 cell line and CB2R knock down（sh CB2R）RAW264.7 cell line,and analyzed the effects of cannabinoid receptor 2agonist GW and antagonist SR on macrophage proliferation by CCK8,inflammatory factor release by enzyme-linked immunoassay,and alteration of glucose metabolism by glycolysis stress test and mitochondrial stress test.3.We constructed HIF-1αknock dowm（sh HIF-1α）RAW264.7 cell line with LPS-stimulated,and examined the effect of cannabinoid receptor2 agonist GW and antagonist SR on proliferation,glucose metabolism alteration and inflammatory factor release of RAW264.7 cells by CCK8,enzyme-linked immunoassay,and glycolysis stress test respectively.LPS+D-GALN-induced ALF model wase constructed in CB2R^-/-mice,and the immunofluorescence double-staining method of F4/80⁺and HIF-1αwas used to explore the effect of CB2R on the expression of HIF-1αin macrophages of ALF mice.4.CB2R^-/-mice were used to construct LPS+D-Gal N-induced acute liver failure mice,verify the action of GW on cannabinoid receptor receptor2 by survival analysis,peripheral blood liver biochemical examination and liver histopathology,TUNEL staining and immunohistochemistry,and detect the specific mechanism of action of activating CB2R to alleviate the inflammatory response in ALF.5.To detect the effect of CB2R on lipid metabolism in RAW264.7cell line by high resolution non-targeted metabolomics analysis using ultra performance liquid chromatography-tandem time of flight mass spectrometry（UHPLC-Q-TOF MS）;Mass spectrometry and 4D-label-free quantitative proteomics were used to analyze the differently expressed proteins of in LPS-induced sh CB2R RAW264.7.Results1.With the pretreatment of CB2R agonist GW405833,the expression of macrophage marker F4/80⁺and M1 polarization marker CD86 were decreased in LPS+D-GALN induced ALF,macrophage M2 polarization marker CD206 were increased,proinflammatory cytokines TNF-αand IL-1βrelease were decreased,and hepatocyte apoptosis were alleviated.And the pathological damage and liver biochemical indexes（ALT,AST and TBIL）induced by LPS+D-Gal N were also improved,and the survival rate of ALF mice was improved with GW405833 pretreatment.SR144528antagonized the effect of GW405833.CB2R agonist GW405833 could not reduce liver pathological damage and liver biochemical indices in LPS+D-GALN-induced ALF of CB2R^-/-mice.2.CB2R agonist GW can inhibit glycolysis of LPS-induced macrophages,reduce the production of lactic acid,inhibit the activities of lactate dehydrogenase and pyruvate kinase,inhibit the proliferation of macrophages and the release of proinflammatory cytokines as TNF-αand IL-1β.SR144528 antagonized the inhibitory effect of GW405833 on macrophage glycolysis,proliferation and release of inflammatory factors.CB2R knockdown can increase the cell proliferation,glycolysis and TNF-αand IL-1βrelease of macrophages induced by LPS.3.CB2R agonist GW405833 reduced the expression of HIF-1αand PKM2 in LPS-induced macrophages.With HIF-1αknockdown,GW could not inhibit the glycolysis and the release of proinflammatory cytokines TNF-αand IL-1βin LPS-induced macrophages.CB2R agonist GW405833reduced the expression of HIF-1αof liver macrophages in wild type ALF mice.CB2R agonist did not reduce the expression of macrophage marker F4/80⁺and HIF-1αexpression in LPS+D-GALN-induced CB2R^-/-mice.4.By multiple omics analysis,differently expressed proteins and metabolites between CB2R knock-down macrophages and nomal contral（NC）macrophages with LPS stimulation,were enriched in glycerophospholipid metabolism pathway and arachidonic acid metabolism pathway.Phosphatidylcholine metabolism was increased with upregulated metabolite,including phosphatidylcholine,glycerol triphosphocholine,glycerol triphosphocholine,choline and acetylcholine.The expression of LPCAT family was upregulated,which activated PKC and mediated MEKK phosphorylation leading to upregulation of p38MAPK.The metabolite 2-AG was transformed into arachidonic acid that can induce HIF-1αexpression by up-regulating ABHD6.Conclusions1.The activation of CB2R can play a protective role in ALF by inhibiting macrophage infiltration and M1 polarization,reducing the release of pro-inflammatory cytokines,alleviating hepatocyte apoptosis mediated by the inflammatory responses of macrophages,subsequently improving liver function and pathological damage.2.The activation of CB2R inhibits the inflammatory response of macrophages by reducing glycolysis,cell proliferation and proinflammatory cytokine release.3.The activation of CB2R inhibits macrophage proliferation,reduces secretion of proinflammatory cytokines,and ultimately reduces the proinflammatory activity of macrophages by decreasing the expression of HIF-1αin macrophages and its mediated glycolysis to alleviate the liver injury.4.The activation of CB2R can inhibit phosphatidylcholine metabolism in macrophages and decrease the arachidonic acid synthetic,which would reduce HIF-1αexpression.