The Regulatory Mechanism Of Albumin On "Gut-Liver Axis" In Liver Failure

Objective:Liver failure is a syndrome of severe liver disease,accompanied by a fairly high level of mortality rate.Plasma albumin level is vital in diagnosing and predicting outcome for patients with liver failure.The supplementation of albumin is one of the main treatments for liver failure.Besides,albumin supplementation can also effectively decrease the rate of hepatic complications and improve survival of liver failure patients.In addition to maintaining oncotic pressure,albumin also holds many other functions,such as transportation,immune regulation,anti-oxidation and nutrition.Microbiome can produce a variety of active substances including metabolites,hormones and peptides,and participate in the growth,development and pathogenic process of the human body."Gut-liver axis"is defined as the connection between the liver and the gut（including the intestinal microbiome）.Numerous studies have shown that the axis is vital in the etiology and progression of various liver diseases including liver failure.As the core part of the"gut-liver axis",evidences suggest that changes in microbiome and intestinal barrier function are involved in this process.However,the mechanisms of albumin in liver failure and the inter-relationship between albumin and the"gut-liver axis"are still unclear.In this study,chronic and acute on chronic liver failure models were constructed in analbuminic rats and SD rats to explore the regulation mechanism of albumin on the"gut-liver axis"in liver failure.Materials and Methods:This study was divided into three parts.The first part was further divided into clinical and animal studies.In clinical research,patients diagnosed with PBC-AIH OS from the Department of Gastroenterology&Hepaptology of West China Hospital were included based on the including and excluding criteria.We divided the selected patients into liver failure and non-liver failure groups,compared the baseline information of the two groups and performed a 1:4 Propensity Score Matching（PSM）between patients with liver failure and non-liver failure according to gender and age.The baseline information and liver fibrosis scores of the two groups before and after PSM were analyzed,and the results of 16s r DNA analysis of fecal samples after PSM were compared.The 16s r DNA sequencing data were analyzed in 3 levels:microbiome composition,αdiversity analysis andβdiversity analysis.In addition,we analysed the correlation between micriobiome features and albumin,FIB-4 score.In animal studies,Sprague-Dawley（SD）rats Bile Duct Ligation（BDL）were used to construct a chronic liver failure model（n=7）,and the sham operation（Sham group）was used for control group（n=4）.The biochemistry markers,including albumin,ALT,total bilirubin,creatinine,ammonia,and triglycerides were studied between groups.Serum LPS was measured by LAL assay.The pathological changes of the liver tissue including Hematoxylin and Eosin（HE）staining,Sirius red staining,TUNEL were performed.For intestine,ileum was stained with HE,and tight junction proteins were analyzed by Western Blot of the relative expression of ZO-1,Claudin-4 and Occludin.In the second part of this study,Nagase Analbuminemic Rat（NAR）model was introduced.We divided the experimental animals into three groups:SD group,NAR group and NAR+albumin group.Serum albumin,ALT,total bilirubin,creatinine,ammonia and triglycerides were analysed.Liver tissue samples were stainded by HE,Sirius red staining and TUNEL.The protein level of tight junction markers include ZO-1,Claudin-4 and Occludin were compared in ileum tissues in Western Blot.In the third part,we performed BDL and BDL+LPS in SD and NAR respectively to construct chronic liver failure and acute on chronic liver failure（ACLF）models,and finally divided into control groups,chronic liver failure groups and ACLF groups.The control groups were SD+Sham group（n=4）,NAR+Sham group（n=4）,chronic liver failure groups were SD+BDL group（n=6）,NAR+BDL group（n=8）,SD+BDL+albumin group（n=5）and NAR+BDL+albumin group（n=5）,ACLF groups were SD+BDL+LPS group（n=5）,NAR+BDL+LPS group（n=5）,SD+BDL+LPS+albumin group（n=8）and NAR+BDL+LPS+Albumin group（n=8）.Rats were sacrificed on the 28^th day of modeling,the baseline information and hemodynamics（mean arterial pressure and portal pressure）were analyzed,and plasma biochemistry markers were detected,including albumin,ALT,total bilirubin,creatinine,ammonia and triglycerides.The circulatory LPS level was measured by LAL assay,and circulatory nucleosome was measured by corresponding ELISA kit.Assay of HEK-Blue^TM h TLR4 reporter cells and HEK-Blue^TM IL18/IL1βreporter cells were performed to measure the relative concentration of circulating ligands of TLR4and IL1β/IL18.HE staining,Sirius red staining,TLR4 immunohistochemistry（IHC）and TUNEL were performed in liver tissue.TLR related genes were explored by RT²PCR profiler in liver tissue to detect TLR4 related pathway regulation.Data was further analyzed by GO,KEGG,PSF which further explored the role of albumin in different pathways.For intestinal tissues,HE staining was performed to evalutate ileum injury,IHC and Western Blot detection of tight junction proteins expression were performed to study gut barrier function.Results:In the first part of the clinical study,42 patients of non-liver failure group and 6patients of liver failure group diagnosed with PBC-AIH OS were included.After 1:4PSM,24 patients of non-liver failure group and 6 patients of liver failure group were matched and selected.Comparison of baseline information indicated that regardless of PSM,patients with liver failure had higher bilirubin levels,lower albumin levels,and higher FIB-4（Fibrosis 4）scores.In addition,the mortality of liver failure patients was also significantly higher than that of non-liver failure group.For 16s r DNA analysis,240,4035 valid sequences were obtained from the 30 samples after PSM.Theαdiversity analysis indicated that observed＿species,Chao1,Ace in the liver failure group were all significantly reduced.Correlation analysis indicated that Ace index,observed＿species index,and Chao1 index were positively correlated with albumin levels（P<0.05）;Ace index,observed＿species index,and Chao1 index were negatively correlated with FIB-4 score（P<0.05）.LEf Se（LDA Effect Size）suggested in the non-liver failure group,increased expression of microbiome were as follows:f＿Prevotellaceae,g＿Agathobacter,and g＿Lachnoclostridium etc;in the liver failure group,increased micriobiome were:s＿Lactobaccccus＿bronidentified＿agilis,s＿Ruminococcus＿bronidentified,c＿inino,P＿Actinobacteria etc.Correlation analysis indicated that Lachnoclostridium,Faecaltalea were positively correlated with albumin level（P<0.05）,and Lactobacillus＿agilis was positively correlated with FIB-4 score（P<0.05）.Compared with SD+Sham group,LAL assay indicated that LPS level was upregulated in SD+BDL group,but the difference was not statistically significant.In the first part of the animal experiment,the plasma biochemistry showed in SD+BDL group,albumin level was significantly reduced,and the bilirubin and ammonia levels were significantly increased which was consistent with the definition of chronic liver failure clinically.The HE staining of SD+BDL group indicated that there were inflammatory infiltration and fibrosis in the liver.In the pathology of the ileum,the SD+BDL group had ileum hyperemia,villi were broken and shedding,accompanied by extensive inflammatory cell infiltration,and the number of goblet cells was significantly reduced,but the basement membrane remained intact.Tight junction proteins expression of ileum tissue in the SD+BDL group was significantly down-regulated.In the second part of the study,the albumin level of the NAR group was significantly reduced,and the ALT and Triglyceride levels were significantly increased.After the supplementation of albumin,the albumin level was significantly increased,ALT and Triglyceride levels were significantly reduced in NAR.The levels of bilirubin,creatinine,and ammonia were comparable among groups.In the liver tissues,HE staining of the NAR group showed mild inflammatory infiltration and mild edema of hepatocytes,TUNEL showed scattered focal positive staining.In the ileum tissue,IHC indicated that ZO-1 was less expressed in the NAR group,meanwhile,Western Blot indicated lower expression of ZO-1,Claudin-4 and Occludin in the NAR group.In the third part of the study,the results suggested（1）the mortality of rats in the NAR+BDL+LPS group was significantly increased,accompanied by instability of blood coagulation and hemodynamics.（2）For plasma biochemistry markers,the ACLF group in SD and NAR showed a significantly increase in ALT and creatinine levels which were significantly restored after albumin supplementation.（3）In terms of liver pathology,chronic liver failure and ACLF groups both showed liver inflammatory infiltration,hepatocyte necrosis,and liver fibrosis,but the latter was more severe and accompanied by changes in liver lobular structure which were alleviated in rats supplemented with albumin.（4）In the pathology of the ileum,chronic liver failure and ACLF groups both showed villi loss,breakage,and goblet cell reduction.Chiu’s score indicated that the ACLF group had more serious pathological damage,accompanied by injury of intestinal mucosa basement membrane which were relieved in groups supplemented with albumin.（5）For barrier function,the expression of tight junction proteins was decreased in chronic liver failure and ACLF group,and the decrease was more obvious in ACLF group.（6）The microarray analysis of TLR-related pathway genes in liver tissue suggested compared with SD rats,NAR were activated in both TLR2 and TLR4 pathways,and ultimately led to pro-inflammatory responses and T cell activation.When the NAR+BDL+ALB group was compared with the NAR+BDL group,the TLR2 and TLR4 pathways were inhibited,and the level of pro-inflammatory response was decreased.（7）In the validation of TLR4-related pathways in liver tissue,on the gene level,LPS binding protein in chronic liver failure and ACLF group was down-regulated,accompanied by the up-regulation of TLR4,tumor necrosis factor,and IL1βlevels.（8）In terms of protein level,IHC showed that in NAR group of chronic liver failure and ACLF model,TLR4 expression was increased compared to SD groups and NAR groups with albumin supplementation;Western Blot showed liver tissue cytoplasmic TLR4,Cleaved caspase3 and Nuclear phosphorylation of NF-κB p65 were up-regulated in the chronic liver failure group,and down-regulated after albumin supplementation.（9）Circulatory LPS measured by LAL assay was significantly higher than the remaining groups.The nucleosome level measured by ELISA kit was significantly higher in ACLF model,which was more significant in SD+BDL+LPS group.The HEK-Blue^TM h TLR4 reporter cells and HEK-Blue^TM IL18/IL1βreporter cells assay confirmed that the number of TLR4 and IL18/IL1βligands in plasma samples of chronic liver failure and ACLF groups increased significantly,while the albumin supplementation reduced this trend.Conclusion:（1）The diversity of the microbiome in PBC-AIH OS patients with liver failure decreased significantly,accompanied by a significant increase in the expression of harmful bacteria and a significant decrease in the expression of beneficial bacteria.In the rat liver failure model,it was found that albumin was significantly reduced and the gut barrier function was impaired.（2）Na?ve NAR showed liver and intestinal barrier dysfunction,and supplementation with albumin can alleviate liver injury and protect the intestinal barrier.NAR is a proper model for further investigations focusing on the role of albumin in liver failure.（3）Chronic liver failure and ACLF models in NAR showed increased mortality and significant multi-organ/system dysfunction.Albumin supplementation can protect the gut barrier,reduce bacterial translocation and inhibit TLR4 pathway,thereby protecting the"gut-liver axis"and improving survival rate.