Carnosic Acid Ameliorates Lipid Accumulation And Inflammatory Response In Alcoholic Fatty Liver Disease Via Regulating NLRP3 Inflammasome

Objective: Alcoholic fatty liver disease is the first response of the liver to alcohol abuse,which is induced by the complex interaction between toxic metabolic and immune responses.Currently,no recognized effective drug for alcoholic fatty liver disease has been developed.The current experiment was aimed to reveal that leucodin acid,which is extracted from rosemary,could ameliorate lipid accumulation and inflammatory response in alcoholic fatty liver disease via regulating NLRP3 inflammasome.Moreover,investigating the mechanism via the in vivo experiment of NIAAA model,the in vitro experiment of AML12 stimulated with alcohol and mouse peritoneal macrophages stimulated with the combination of LPS and ATP,could provide new options for clinical drug thearpies of alcoholic fatty liver disease.Methods: 1.In vivo experiment of NIAAA model: C57BL/6 male mice were randomly divided into six groups: Pair-fed,chronic ethanol feeding plus a single binge group(ETOH-fed),carnosic acid low-dose group(ETOH-fed+Car10 mg/kg),carnosic acid high-dose group(ETOH-fed+Car20 mg/kg),single dose and administration group(Car20 mg/kg),silibinin group(ETOH-fed+Silibinin 100 mg/kg).Pair-fed and the single administration group were given Lieber-De Carli control liquid diet for 15 days,chronic ethanol feeding plus a single binge group,the carnosic acid administration group and the silibinin group group were given the control liquid diet for 5 days and then started to be fed with 5% Ethanol liquid diet for ten days.Then are finally given 5 g/kg alcohol of their body weight as the single binge.The level of serum IL-1β,TG,ALT and AST were detected by enzyme-linked immunosorbent assay or with the corresponding kits;the histopathological changes of liver were observed by H&E,Oil Red O staining and immunohistochemical staining;The expression of P2X7 R,SREBP-1,NLRP3,ASC,IL-1β and Caspase-1were detcted by Western blot.The expression of SREBP-1 and FASN genes were further analyzed by RT-PCR experiments;Apoptosis and the expression of NE,MPO,F4/80 and P2X7 R were further analyzed by immunofluorescence staining.2.In vitro experiment of AML12 stimulated with alcohol: AML12 cells were pretated with different concentrations of carnosic acid(8,1.6,0.32 μM)respectively,and then stimulated with alcohol for 24 hours after an interval of 1 h.The cells were harvsted and stained with Oil Red O to observe the morphological changes of the cells;The total protein was extracted and then the expression of SREBP-1,P2X7 R NLRP3,ASC,IL-1β and Caspase-1 were measured by Western blot.The expression of P2X7 R and Caspase-1 were further measured by immunofluorescence experiments.3.In vitro experiment of mouse peritoneal macrophages stimulated with the combination of LPS and ATP: The mouse peritoneal macrophages were incubated with carnosic acid(0-100 μM)for 24 h,the effect of carnosic acid on cell viability was measured by MTT assay.Pretreat mouse peritoneal macrophages with different concentrations of carnosic acid(8,1.6,0.32 μM)for one hour.Then stimulate these mouse peritoneal macrophages with LPS for 4 h and adenosine triphosphate for 30 min.Harvest the cells and supernatant to extract the total protein,the expression of IL-1β,HMGB1 and Caspase-1 in both cells and the supernatant were measured by Western blot.The cleavage and release of IL-1β(Interleukin-1β,IL-1β)were measured by enzyme linked immunosorbent assay.Results: 1.In vivo experiment: Carnosic acid significantly reduced the serum level of IL-1β,AST,ALT and TG.HE and Oil Red O staining results showed that carnosic acid significantly ameliorated lipid accumulation and liver injury induced by alcohol abuse.Carnosic acid effectively inhibited the expression of P2X7 R,NLRP3,SREBP-1,Caspase-1,ASC,IL-1β and FASN genes,Besides,Carnosic acid attenuated apoptosis and the expression of NE,F4/80,and MPO.2.In vitro experiment: Carnosic acid did not show significant toxicity in the 100 μM dose range.Carnosic acid significantly reduced the level of IL-1β in the supernatant of mouse peritoneal macrophages.Carnosic acid can effectively inhibit the cleavage of pro-IL-1β and the release of mature IL-1β in macrophages.Besides,Carnosic acid inhibited the expression of SREBP-1 P2X7 R,NLRP3,Caspase-1,ASC and IL-1βboth in AML12,mouse peritoneal macrophages and the supernatants of mouse peritoneal macrophages。Conclusion: Carnosic acid significantly inhibited the expression of SREBP-1and the activation of P2X7R-Caspase-1-NLRP3 inflammasome.Carnosic acid ameliorated alcoholic fatty liver disease via down regulating lipid accumulation and inflammatory response.Bisease,Carnosic acid showed an inhibitory effect on the expression of F4/80 and NE,which is the marker of macrophages and neutrophils respectly.Carnosic acid ameliorated apoptosis,the activation of macrophages and the recrument of neutrophils,providing new options for clinical drug thearpies of alcoholic fatty liver disease...