Allicin Improves Acrylamide-induced Intestinal Damage By Regulating Microbiota-short Chain Fatty Acids-TLR4/MyD88/NF-?B Cascade Response

This work was supported by the fund from the National Natural Science Foundation of China(No.31571939).Acrylamide(Acrylamide,AA),as an endogenous pollutant formed in the thermal processing of food,can cause neurotoxicity,genetic toxicity,reproductive toxicity,liver toxicity and potential carcinogenicity.Studies have shown that AA is quickly absorbed into the blood by the intestine via the passive process,which has negative effects on the structure and function of the small intestinal mucosa,causing intestinal barrier leakage.However,the impaired intestinal barrier is mostly associated with the microbiota translocation.The gut microbiota provides various benefits to the host by regulating the intestinal permeability,immune regulation,and metabolism.Also,it can produce short chain fatty acids(Short chain fatty acids,SCFAs)to reduce inflammation and improve intestinal barrier function.Allicin has pharmacological effects such as anti-oxidation,anti-bacterial and anti-inflammatory,which can alleviate intestinal microbial disorders and play a prominent role in protecting the intestine.In this study,we established the models of AA infection and allicin intervention to explore the relationship among intestinal microbiota,SCFAs and inflammatory pathways,and further clarified the protection effect and mechanism by which allicin regulated AA-induced intestinal barrier damage and inflammation response.The main research contents and results were as follows:(1)SD(Sprague Dawley,SD)rats were used to establish AA infection and allicin intervention models.The protective effect on AA-induced intestinal injury in rats was investigated via the observation of changes in intestinal pathological sections and related indicators.Compared with the control group,the levels of myeloperoxidase(Myeloperoxidase,MPO),lipopolysaccharide(Lipopolysaccharide,LPS)and D-lactic acid(D-lactic acid,D-LA)were significantly increased,accompanied by the colon tissue damage and inflammatory cells infiltration in the AA-treated group.After the administration of allicin,the indicators were returned to near the control group levels.The measurement of colonic tight junction protein expressions and mucin transcription levels showed that allicin could restore the decrease protein expressions of occludin,claudin-1 and ZO-1(Zonula occludens-1,ZO-1)and the down-regulation transcription levels of mucoprotein 2(Mucoprotein 2,MUC2)and mucoprotein 3(Mucoprotein 3,MUC3)caused by AA.These results indicated that allicin could alleviate the intestinal barrier damage induced by AA.(2)16S rDNA sequencing was used to explore the effects of allicin on the structural composition of the gut microbiota in the AA-induced intestinal barrier damage rats including Alpha/Beta diversity,phylum level,genus level and LEf Se analysis of the rat cecum contents.GC-MS/MS was applied to investigate the changes of SCFAs content and Spearman's correlation analysis was used to determine the microbiota genus related to the synthesis of SCFAs.The significant improvement of the microbial structure was accompanied by a notable increase of the beneficial bacteria and a notable decrease harmful bacteria after being treated with allicin.The microbiota related to the synthesis of SCFAs were negatively affected by AA treatment,whereas allicin regulated the microbiota-SCFAs signaling response to reverse the AA-induced decrease of SCFAs content.Studies have found that Allicin could further regulate the synthesis of SCFAs though adjusting the composition and contents of microbiota to alleviate the AA-induced intestinal environment disorder.(3)The levels of TLR4/MyD88/NF-?B pathway-related proteins and downstream inflammatory factors were detected to deeply investigate the influence and regulatory mechanism of allicin on AA-induced intestinal barrier damage.Pretreatment with Allicin all markedly down-regulated the protein levels of toll-like receptor 4(Toll-like receptor 4,TLR4),myeloid differentiation primary response 88(Myeloid differentiation primary response 88,MyD88)and nuclear factor-kappa B pathway proteins(Nuclear factor-kappa B,NF-?B),accompanied by the remission of p65(p65 nuclear factor-kappa B,p65)nuclear translocation caused via AA and the regulation of inflammatory factors secretion.Then,the correlations of SCFAs,inflammation pathway and inflammatory factors were explored by Spearman's correlation analysis to investigate the relationship between the changes of SCFAs and the intestinal inflammatory response.It has found that total acid,acetic acid,and propionic acid were negatively associated with TLR4,pp65(phospho-p65 nuclear factor-kappa B,pp65),p-IKK(phospho-I-kappa-B-alpha kinase,p-IKK)and all pro-inflammatory factors,suggesting that SCFAs could alleviate AA-induced intestinal inflammation through the TLR4/MyD88/NF-?B pathway.In summary,allicin regulated the microbiota-SCFAs-TLR4/MyD88/NF-?B cascade response,enriched beneficial bacteria and inhibited pathogenic bacteria to increase the synthesis of SCFAs.And then allicin further inhibited the activation of the downstream NF-?B pathway by inhibiting TLR4/MyD88-dependent signaling transduction.Finally,allicin reduced the recruitment of pro-inflammatory factors and the transcription of cytokines required for adaptive immune responses,thereby preserving the intestinal barrier leakage and ameliorating the intestinal inflammation caused by AA.