Effects And Its Mechanism Of Flaxseed Powder In Improving Nonalcoholic Fatty Liver Disease By Gut-liver Axis Of Bile Acid Metabolic Pathway

1.BackgroundNonalcoholic fatty liver disease（NAFLD）is the main cause of chronic liver disease in the world.The occurrence and development of NAFLD involves the complex interaction between abnormal lipid metabolism,inflammation and changes of intestinal flora.The occurrence and development of NAFLD from nonalcoholic simple fatty liver（NAFL）to nonalcoholic steatohepatitis（NASH）is accompanied by lipid metabolism disorder and changes in the level of inflammatory factors.Therefore,a profound study of the regulatory mechanism of lipid metabolism and inflammation is the key to the prevention and treatment of NAFLD.Flaxseed powder is a rich and economical food source ofα-linolenic acid,but its potential role in the prevention and treatment of lipid disorder and inflammation related diseases has been ignored.Bile acids play important roles in NAFLD lipid metabolism homeostasis and regulation of NAFLD inflammation.Gut-liver-bile acid metabolic pathway is one of the common pathways regulating liver lipid metabolism and inflammation.Farnesoid X receptor（FXR）and G-protein-coupled receptor 5（TGR5）are two important targets of this pathway.At present,the effects of FLA on lipid metabolism,inflammation and gut-liver-bile acid metabolism in the occurrence and development of NAFLD have not been reported.2.Method2.1 Study on the interventional effect of flaxseed powder on nonalcoholic simple fatty liver mice and the possible mechanism of gut-liver-bile acid metabolism pathwayC57BL/6J mice were used to carry out the interventional study of FLA on NAFL at different doses.The experiment was set up with normal control group（CO）,NAFL model group（HFD）and three dose groups,a total of 5 groups with 12 mice in each group.The three dose groups were added with FLA based on high-fat formula feed（D12492,with fat energy accounting for60%）,with the addition amounts of 10g/100g,20g/100g and 30g/100g respectively,which were defined as low-dose group（Low）,middle-dose group（Middle）and high-dose group（High）.The three energetic nutrients were adjusted so that their energy proportion and total energy in the interventional groups were the same as those in the NAFL model group.The intervention time was 12 weeks with the establishment of NAFL model in HFD group as the node.After the experiment,the blood,liver,fat and other tissues and organs as well as the feces of mice in each group were collected.The serum levels of total cholesterol（TC）,low density lipoprotein cholesterol（LDL-C）,high density lipoprotein cholesterol（HDL-C）,triglycerides（TG）,insulin,tumor necrosis factorα（TNF-α）and interleukin-6（IL-6）were measured;The liver pathological sections of mice in each group were stained with HE and evaluated by NAFLD activity score（NAS）;Ultra high performance liquid chromatography tandem mass spectrometry（UPLC-MS/MS）was used to quantitatively detect bile acids in feces;The V3+V4 region was amplified by 16S r DNA sequencing,and the diversity of gut flora was analyzed by bioinformatics method;Real time PCR（RT-PCR）was used to detect the specific bile acid receptor G protein coupled receptor 5（TGR5）,farneside X receptor（FXR）and cholesterol 7α-hydroxylase（CYP7A1）and toll like receptor 4（TLR4）;The protein expressions of nuclear factor-κB（NF-κB）,CYP7A1,fibroblast growth factor receptor（FGFR4）,TLR4,TGR5 and FXR in liver and/or colon were detected by Western Blot.2.2 Study on the interventional effect of flaxseed powder on nonalcoholic steatohepatitis mice and the possible mechanism of gut-liver-bile acid metabolism pathwayTo study the effect of FLA intervention on NASH mice at different doses,and mainly focus on the effect and significance of FLA intervention in the process of NAFL developing into NASH.Therefore,based on the successful establishment of NAFL model,this study conducted three doses of FLA intervention（for 28 weeks）.The experiment set up normal control group（CO）,NASH model group（HFD）and three dose groups（low-,middle-and high-dose groups）,a total of 5 groups,with 8 mice in each group.The observation and measurement indexes of the study are the same as those in the first chapter,and focus on the impact of FLA intervention on the occurrence and progression of NASH.This study also detected the expression of key proteins on the bile acid signaling pathway mediated by bile acid specific receptors FXR and TGR5 in the intestine and liver to determine whether FLA intervention has the biological function of activating FXR and TGR5 to mediate lipid-lowering and anti-inflammatory by affecting the bile acid metabolism of gut flora.2.3 Based on TGR5-knocked mice to explore the key role of flaxseed powder on NAFLD through gut-liver-bile acid metabolism pathway and its mechanism verificationBased on the first two experiments,confirmatory tests were carried out to construct TGR5^-/-model mice,further reverse verify the accuracy of action pathway,and indirectly distinguish whether FXR and TGR5 play a synergistic or separate role under the intervention of FLA.In this experiment,the average value of the effective dose in the three groups was selected as the interventional dose of this experiment,that is 25g/100g FLA added,to intervene wild-type（WT）and TGR5^-/-mice respectively.The experiment set up six groups with 8 mice in each group:WT mice normal control group（WT＿CO）,WT mice NAFLD model group（WT＿HFD）,WT mice flaxseed powder intervention group（WT＿FLA）,TGR5^-/-mice normal control group（KO＿CO）,KO mice NAFLD model group（KO＿HFD）and KO mice flaxseed powder intervention group（KO＿FLA）.The levels of serum lipids（TC,LDL-C,HDL-C and TG）,inflammatory factors（TNF-αand IL-6）levels,pathological changes,and the expression of key proteins regulated by liver（CYP7A1,TLR4 and NF-κB）in type TGR5^-/-and WT mice were examined and verified.In addition,the intestinal flora and BA profiles of wild-type（WT）and TGR5^-/-mice after intervention were detected and compared to observe whether TGR5gene knockout affected the effect of FLA on them.3.Results3.1 Study on the interventional effect of flaxseed powder on nonalcoholic simple fatty liver mice and the possible mechanism of intestinal liver bile acid metabolism pathway（1）Compared with NAFL model group,middle-dose and high-dose FLA intervention could significantly reduce the body weight,body fat and serum TG and LDL-C levels of mice;In addition,high-dose FLA intervention can significantly reduce the serum TNF-αlevel in mice,suggesting that FLA intervention has the health benefits of lipid-lowering and anti-inflammatory.The results of NAS score showed that the intervention of high-dose FLA could significantly improve hepatic steatosis.（2）The results of gut flora test show that,at the phylum level,compared with NAFL model group,FLA intervention significantly reduced the relative abundance of Firmicutes and significantly increased the relative abundance of Bacteroides（P<0.05）.At the genus level,FLA significantly increased the species at the genus level;Compared with HFD group,FLA intervention significantly reduced the relative abundance of Romboutsia and increased the relative abundance of Bacteroides（P<0.05）.In addition,FLA intervention significantly increased the relative abundance of Lachnospiraceae＿NK4A136＿group,Parasutterella and Eubacterium＿xylanophilum＿group,which was closely related to the improvement of NAFL;At the same time,reduce the relative abundance of two harmful genera of bacteria that Coriobacteriaeae＿UCG-002 genus and[Eubacterium]＿fissicatena＿group.（3）FLA intervention affects the metabolism of bile acids in the intestine,which is characterized by the decrease of the main components of conjugated bile acids in feces,including glycoursodeoxycholic acid（GUDCA）,glycocholic acid（GCA）,tauroursodeoxycholic acid（TUDCA）,taurohydroxycholic acid（THDCA）,taurodeoxycholic acid（TDCA）,taurine-α-mouse cholic acid（T-α-MCA）and taurine-β-mouse cholic acid（T-β-MCA）and the increase of the main components of unconjugated bile acids,dehydrolithocholic acid（DHLCA）,allolithic acid（allo LCA）,isolithic acid（iso LCA）,including ursodeoxycholic acid（UDCA）and lithocholic acid（LCA）.In addition,Spearman correlation analysis between fecal bile acid components with significant differences among the five groups and gut flora with significant differences showed that the difference of intestinal microflora was almost related to the fecal content of conjugated bile acids.The results suggest that FLA intervention can play an important role by affecting the uncoupling of gut flora to bile acids and regulating the composition of bile acid spectrum.（4）FLA intervention can exert lipid-lowering and anti-inflammatory effects by activating bile acid specific receptors FXR and TGR5 in the intestine,which is specifically manifested in the activation of intestinal FXR and the inhibition of the expression of CYP7A1 in the liver mediated by FGFR4;In addition,intestinal TGR5 activation inhibits the expression of liver NF-κB p65 mediated by TLR4;The two pathways play the role of lipid-lowering and anti-inflammatory respectively.The above results were significant in the three dose FLA groups,and the effect of middle-and high-dose FLA intervention on the protein in the above signal pathway was more obvious.3.2 Study on the interventional effect of flaxseed powder on nonalcoholic steatohepatitis mice and the possible mechanism of gut-liver-bile acid metabolism pathway（1）Compared with NASH model group,middle-dose and high-dose FLA intervention could significantly reduce the body weight and the levels of serum TG,LDL-C,ALT and TNF-α;In addition,the intervention of middle-dose FLA could increase the level of HDL-C in mice,suggesting that FLA intervention can significantly reduce hyperlipidemia and inflammation.The results of NAS score showed that middle-and high-dose FLA intervention could significantly improve hepatic steatosis,inflammation and balloon degeneration.（2）At the phylum level,compared with the NASH model group,FLA intervention significantly reduced the relative abundance of Firmicutes and significantly increased the relative abundance of Bacteroidetes,Actinobacteria,TM7 and Deferribacters phylum（P<0.05）.At the genus level,the relative abundance of Bacteroides and unidentified＿S24-7 in FLA intervention group was significantly higher than that in HFD group,and the relative abundance of unidentified＿Clostridiales,Mucispirillum and Desulfovibrio in FLA intervention group was significantly lower than that in HFD group（P<0.05）.The results showed that the intervention of FLA could improve the microbiota structure of NASH mice and regulate the relative abundance of bacteria with bile acid metabolizing enzyme activity.（3）FLA intervention increased non-12Αoh bile acids level in NASH mice;with the increase of intervention dose,the interventional effect is more significant.High-dose FLA increases the concentration of eight bile acids,including UDCA,hyodeoxycholic acid（HDCA）,LCA,α-muricholic acid（α-MCA）,β-muricholic acid（β-MCA）,glycohyodeoxycholic acid（GHDCA）,THDCA and TUDCA.Spearman correlation analysis showed that the phylum of Bacteroidetes,especially S24-7 family,had a significant positive correlation with HDCA,LCA,UDCA,GHDCA,α-MCA andβ-MCA.The phylum of Firmicutes was significantly negatively correlated with HDCA,LCA,UDCA,GHDCA,α-MCA andβ-MCA,especially the genus of unidentified＿Clostridiales was significantly negatively correlated with HDCA,LCA and UDCA,and the genus of Lactobacillus was significantly negatively correlated withα-MCA andβ-MCA.（4）FLA intervention could regulate bile acids metabolism and reduce NASH through FXR-FGF15 and TGR5-NF-κB signaling pathways;Among them,the effect of neutralization and high-dose FLA group on the protein of the above signal pathway is more obvious.3.3 Based on TGR5-knocked mice to explore the key role of flaxseed powder on NAFLD through gut-liver-bile acid metabolism pathway and its mechanism verification（1）TGR5-knockout aggravated the disease degree of NAFLD.Specifically,TGR5^-/-mice have higher body weight gain,body fat,blood lipid,inflammation level and pathological score than wild-type（WT）mice.The results of Western Blot showed that TGR5-knockout blocked the inhibition of FLA intervention on key proteins NF-κB and TLR4 in inflammatory regulation pathway,and may also affect the inhibition of CYP7A1 by FXR activation.It shows that the regulation of liver inflammation by FLA through bile acids metabolic pathway is mainly related to the activation of TGR5.The regulation of liver lipid by FLA intervention may be related to the co-synergistic effect of FXR and TGR5,but it still needs to be verified by further research.（2）At the phylum level,FLA intervention in WT mice mainly changed the relative abundance of Proteobacteria,Bacteroidetes and Verrucomicrobia.Among them,compared with WT＿HFD group,Proteobacteria decreased and Bacteroides and Verrucomicrobia increased significantly in WT＿FLA group（P<0.05）.However,in TGR5^-/-mice,compared with KO＿HFD,FLA intervention had no significant improvement on other bacteria except that it significantly reduced Verrucomicrobia in KO＿FLA group.These results suggest that there may be a relationship between the changes of gut flora and the expression of TGR5 receptor.Further comparing the differences between WT mice and TGR5^-/-mice under the same formula feed,the results showed that the role of FLA in regulating key flora in TGR5-/-mice and WT mice is consistent.The results showed that FLA intervention significantly increased Bifidobacterium,Akkermansia and Bacteroides in the feces of WT and TGR5^-/-mice,and significantly decreased Desulfovibrio and Dehalobacterium（P<0.05）.The results showed that the beneficial bacteria Akkermansia,Sutterella and Clostridiaceae＿Clostridium were significantly lower in the feces of TGR5^-/-mice than those of WT mice,while the relative abundance of harmful bacteria Desulfovibrio was significantly higher（P<0.05）.（3）FLA intervention affects bile acid metabolism in the intestine.FLA intervention makes fecal MDCA,LCA-3S,HDCA,TUDCA,7-KLCA,HCA,THCA,UCA,3β-HDCA,GUDCA,TLCA,UDCA,3β-UDCA and LCA had the same change trend in WT mice and TGR5^-/-mice.The results further showed that the main factor affecting the change of bile acids came from dietary factors.TGR5 knockout may have no obvious effect on bile acids.However,Spearman correlation analysis showed that the correlation between intestinal flora and the changes of BAs in TGR5^-/-mice was much lower than that in WT mice.This result is consistent with the previous results that the change of intestinal flora of TGR5^-/-mice by FLA intervention is weaker than that of WT mice.The results show that TGR5 gene knockout weakens the relationship between intestinal flora and bile acids.4.ConclusionFLA intervention has good health benefits for preventing and delaying NAFL and NASH in the two stages of NAFLD,can significantly improve the disorder of lipid metabolism and inflammation in the two stages of NAFLD,can significantly reduce the levels of serum TG,LDL-C,TC and TNF-α,and has significant effects on delaying weight gain.The mechanism is that FLA intervention can regulate the composition of bile acid spectrum by regulating the relative abundance of gut flora with bile acid metabolic enzymes,especially the increase of LCA,TUDCA and UDCA and the decrease of T-α-MCA and T-β-MCA,and activate two specific signal pathways FXR-CYP7A1 and TGR5-NF-κB mediated by intestinal FXR and TGR5,to play the role of lipid-lowering and anti-inflammatory.