Study On The Mechanism Of Baijiu-induced Liver Injury And Regulation Of Intestinal Flora Based On Omic Approaches

BackgroundBaijiu is traditional fermented Chinese alcoholic beverage with important cultural and economic value.During the past millennium,Baijiu has formed many theoretical systems related to health.However,due to the insufficient understanding of the composition of Baijiu,the underlying mechanisms remain to be explained.Chemically,Baijiu comprises thousands of chemical compounds in addition to ethanol due to its unique mixed-culture and solid-state fermentation process.However,the chemical components of Baijiu are still insufficiently understood because the previous studies mainly focused on the volatile compounds(VOCs),while the non-VOCs that often influence the perception of Baijiu were rarely studied.In addition,the compositional differences and similarities between Baijiu and Western liquors have never been compared.It has been reported that Baijiu are abundant with various compounds with potential bioactivity.However,evidences of in vivo experiments were insufficient.Recent advances of ALD suggested that gut microbiota can impact intestinal barrier function and play an important role in the development and progression of ALD.Besides,gut microbiota performs a diverse range of metabolic functions.We hypothesized that the potential bioactive compounds in Chinese soy sauce aroma type Baijiu(CB)can attenuate EtOH induced liver injury by regulate gut microbiota.ObjectiveThis thesis aimed to(1)comprehensively analyze the chemical composition of Baijiu,including VOCs and non-VOCs,and systematically compare the similarities and differences between Baijiu and Western liquor;(2)validate whether the chemical compounds in CB can attenuate EtOH induced liver injury and clarify the underlying mechanisms from the perspective of gut microbiota and its metabolome.MethodsHS-SPME and BSTFA were applied for the pretreatment of VOCs and non-VOCs,respectively,in 24 distilled liquors including 6 typical CB and 18 typical Western liquors.GCTOFMS based untargeted metabolomics were then used to detect the VOCs and non-VOCs.Co-occurrence network were used to identify the characteristic components of different distilled liquors.The compositional differences and similarities between Baijiu and Western liquors were than compared by multivariate statistical analysis.By constructing the mice model of ALD,we compared the degree of liver injury between CB-and EtOH-fed mice by phenotypic,biochemical and pathological analysis.The gut microbiota and its metabolome were also analyzed by high-throughput sequencing and metabolomics,respectively.In addition,immunofluorescence and real-time PCR were used to quantify the intestinal barrier function.Finally,mice model of ALD with lower dosage of EtOH and of blending Baijiu(BB)were constructed to validate the hepatoprotective effect of the volatiles in CB Results(1)A total of 879 volatile peaks were detected from 24 distilled liquors with 661 identified compounds.The VOCs comprised with 166 benzene derivatives,159 esters,105 aldoketones and 92 alcohols etc.Chinese soy sauce aroma type liquor had the most abundant VOCs.A total of 268 non-volatile peaks were detected from 24 distilled liquors with 160 identified compounds.The non-VOCs comprised with 46 acids,43 sugars,30 benzene derivatives and 27 alcohols etc.Whisky had the most abundant non-VOCs.By comparing the differences and similarities between Baijiu and Western liquors,we found ethyl esters and some benzene derivatives and alcohols were shared by most distilled liquors.Sugars and esters formed by fatty alcohol differ significantly between the assessed Baijiu and Western liquors.In addition,many of volatile compounds were found to by bioactive with potential antibacterial and antioxidant activity.(2)The food consumption and body weight decreased and liver to body weight ratio increased in both CB-and EtOH-fed mice.However,LIV:BW ratio of CB-fed mice were lower than EtOH-fed mice.Further biochemical analysis found that,although both CB and EtOH feeding lead to significant increase of plasma ALT,AST and hepatic TG,TBARS,the biochemical index of CB-fed mice were lower than EtOH-fed mice during the entire experiment.Consistent with biochemical analysis,both CB and EtOH feeding lead to steatosis and accumulation of lipid droplets and EtOH feeding was worse than CB feeding,as assessed by pathological analysis.(3)High-throughput sequencing targeting at bacteria 16 S rRNA genes suggested that both CB and EtOH feeding lead the alteration of caecal community structure.In addition,clear differences in the bacterial composition were also observed between mice fed CB and those fed EtOH and these differences tended to be expanded.Further RDA found 28 key OTUs in response to CB and EtOH feeding.Amongst these OTUs,9 OTUs belonging to the genera Akkermansia,Ruminococcus,Dorea,Pseudoflavonifractor and partial unclassified S24-7 were decreased by EtOH and 19 OTUs belonging to the genera Prevotella,Bacteroides,Helicobacter and partial unclassified S24-7 etc.were increased by EtOH.(4)Immunofluorescence coupled with real-time PCR analysis suggested that the expression of tight junction protein Occludin and its mRNA in the ileum tissue of CB fed mice were higher than that of EtOH fed mice.In contrast,the expression of Claudin-2 and its mRNA in CB fed mice were lower than EtOH fed mice.Further ELISA analysis found that CB fed mice had significantly lower levels of translocated LPS,a gram-negative bacteria cell wall component,in serum and liver than EtOH fed mice.These results suggested that intestinal barrier functions of CB-treated mice were more complete relative to EtOH-treated mice.(5)Metabolic profiling of the caecal contents indicated that CB and EtOH treatment resulted in two distinct caecal metabolomes.Further differential metabolites analysis found that CB fed mice had higher contents of many functional saturated long chain fatty acids(LCFAs)(arachidic acid,behenic acid,heptadecanoic acid and stearic acid)and fermentable sugars(myoinositol,D-galactose,L-sorbose,rhamnose and sucrose)relative to EtOH fed mice.As fermentable sugars can be metabolized by intestinal bacteria into short-chain fatty acids(SCFAs),we hypothesized that the lower contents of fermentable sugars are accompanied by higher contents of SCFAs.Thus,we quantitatively analyzed the SCFAs in the colon by targeted metabolomic approach.As expected,CB-fed mice had significantly higher colonic concentrations of most SCFAs than were found for EtOH-fed mice,especially for acetic and butyric acids.(6)In the validation mice model of low dosages of ethanol,we observed EtOH feeding lead to obvious liver injury after 12 weeks of intervention,while CB feeding had no obvious effect on liver,as assessed by phenotypic,biochemical and pathological analysis.In the validation mice model of BB,we observed CB feeding and BB feeding lead to same levels of liver injury and lower than EtOH feeding.ConclusionsDerivatization-based GC-TOFMS is highly complementary to the detection range of HSSPME-GC-TOFMS and both methods are indispensable for studying the complete composition of distilled liquors.Both VOCs and non-VOCs are notably different between Chinese Baijius and Western liquors.These differences may be influenced by many factors including raw materials,production processes,microorganisms,and aging vessels.In vivo analysis of mice model suggested that VOCs in Baijiu could protect against ethanol-induced liver injury.The potential mechanism could involve the antioxidant and antimicrobial effect of many VOCs in CB.On the one hand,the functional VOCs could protect against the ethanol-induced depletion of A.muciniphila by regulating the gut microbiota.A.muciniphila could decrease translocation of proinflammatory LPS from the gut to the liver through the portal vein by protecting gut barrier function,which then ameliorates ethanolinduced liver injury.On the other hand,the altered gut microbiota of CB fed mice resulted in the metabolic changes and induced the increased production of functional saturated LCFAs SCFAs.