The Anti-inflammatory Effect Of Inulin On The Alcoholic Liver Disease By Short-chain Fatty Acids Regulating Macrophage Polarization

Objective Based on the previous evidence that inulin inhibited the inflammation of alcoholic liver disease(ALD),this study further explored the anti-inflammatory mechanism of inulin effected SCFAs and hepatic macrophage(Mψ)subtype polarization.Methods In vivo,mice were divided into 4 groups: pair-fed(PF)group(PF/CON),alcohol-fed(AF)group(AF/CON),PF with inulin(INU)group(PF/INU)and AF with INU group(AF/INU).Each group was fed modified Lieber-De Carli liquid diet with or without alcohol for modeling.After 6 weeks of intervention,feces were collected;mice were anesthetized with chloral hydrate and liver tissues were obtained.We determined gut microbiota SCFAs concentrations by GC-MS,the levels of hepatic iNOS and Arg-1 in hepatic were measured by IHC assay.Hepatic macrophages,TNF-α in hepatic M1 Mψ,IL-10 in hepatic M2 Mψ were detected by flow cytometry.The expression of TNF-α,iNOS,Arg-1,IL-10,TLR-4,MyD88,NF-κB protein in hepatic of each group were examined by western blot.In vitro,RAW264.7 cell lines were polarized to M1 macrophage(Mψ)or M2 Mψsubsets with lipopolysaccharide(LPS)or interleukin-4(IL-4)stimulation,respectively.Then,co-cultured with SCFAs(including sodium propionate,sodium butyrate and sodium valerate),M1 Mψ、M2 Mψ、TNF-α in hepatic M1 Mψ、IL-10 in hepatic M2 Mψ and were detected by flow cytometry.Arg-1 and iNOS were observed by immunofluorescence staining.ELISA was used to examine the expression of TNF-α and IL-10 in the supernatants of medium after the culture experiments.We further evaluated the expression of iNOS and TNF-α in hepatic M1Mψ,Arg-1 and IL-10 in hepatic M2 Mψ upon SCFAs treatment in vitro by western blot.Results The amounts of propionic acid,butyric acid and valeric acid in AF/CON group were dramatically decreased compared to PF/CON group.After inulin intervention,the content of three SCFAs was significantly increased in ALD.Images from IHC showed that,the levels of iNOS were decreased and Arg-1 were significantly increased in liver after dietary inulin treatment in ALD.The results were detected by flow cytometry showed that the proportion of hepatic M1 Mψ and TNF-α in hepatic M1 Mψ were increased in the AF/CON group compared to PF/CON group,but the proportion was significantly reduced in AF/INU group compared with that in AF/CON group.After inulin intervention,the proportion of M2Mψ and IL-10 in hepatic M2 Mψ were elevated in AF/INU group compared with that in AF/CON group.Western blot analysis showed that the levels of Arg-1 and IL-10 were notably increased in the supplementation with dietary inulin in ALD,the level of iNOS and TNF-α levels were reduced,and TLR4,MyD88 and NF-κB protein expressions were also dramatically reduced.In vitro,the results of flow cytometry demonstrated that after treatmented with sodium propionate,sodium butyrate and sodium valerate,the proportions of M1 Mψ and TNF-α decreased,respectively.In contrast,the ratios of M2 Mψ and IL-10 increased significantly.The immunofluorescence assay showed that sodium propionate,sodium butyrate and sodium valerate were suppressed the vitality of iNOS an increase the vitality of Arg-1.The result of ELISA showed that sodium propionate,sodium butyrate and sodium valerate reduced the level of TNF-α levels increased the expression of IL-10.The data of western blot demonstrated that sodium propionate,sodium butyrate and sodium valerate reduced the level of iNOS,TNF-α levels and increased the expression of IL-10,Arg-1.Conclusion In ALD,inulin ameliorates the inflammation via SCFAs-inducing suppression of M1 and facilitation of M2 Mψ,which may potentially contribute to the control of the disease.