| Chronic liver disease is a progressive disease which liver damage is caused by different pathogenic factors such as viruses,alcohol or drugs.Persistent progression of chronic liver disease can lead to a vicious circle of progressive liver injury,inflammation,and repair.Patients usually experience the processes of hepatitis,liver fibrosis,and cirrhosis,etc.If the disease condition cannot be effectively controlled,it can eventually develop liver cancer.Early hepatitis is mainly characterized by the damage of liver cells caused by local inflammatory reactions in the liver.The persistent presence of inflammation can further cause chronic hepatitis or even cirrhosis.Once the patient enters the stage of cirrhosis,it will cause irreversible damage to the liver and eventually form liver cancer.Hepatocellular Carcinoma?HCC?is the most common type of liver cancer.China has a vast number of liver cancer cases.According to statistics,more than half of the world's liver cancer cases occurred in China in 2019,and 80 percent of new cases were in the advanced stage of cancer.In clinical practice,liver cancer is mainly treated by surgical methods such as surgical resection or liver transplantation.But the five-year survival rate of liver cancer is extremely low,which has brought a heavy burden to the social public health system and patients'health.Therefore,it is particularly important to detect and intervene chronic liver disease in the early stage in order to delay its progression to liver cancer.The gut microbiota plays an important role in the immune regulation,metabolic balance,and nutrient intake of the body,and is known as a forgotten metabolic"organ".The liver is connected to the intestines through the portal vein,a pathway known as the gut-liver axis.The liver transports biological active substances such as bile acids to the intestines to sustain the body's needs.However,the microorganisms and their metabolites in the intestines may cause liver damage through travelling backwards along the portal vein to the liver.In recent years,with the development of high-throughput sequencing technology,more and more clinical studies have shown that intestinal dysbiosis play an important role in the development of many chronic liver diseases such as alcoholic liver disease?ALD?,non-alcoholic fatty liver disease?NAFLD?,liver cirrhosis and liver cancer.At present,most of the researches have been focused on the association between intestinal flora and a particular liver disease,but the relationship between the structural distribution of intestinal flora and the development of chronic liver disease needs to be studied in depth.The imbalance of gut microbiota can lead to the release of a large number of bacterial lipopolysaccharide?LPS?produced by the harmful bacteria.LPS binding to its receptor TLR4,activates the MyD88-NF-?B pathway in immune cells.Finally the imbalance promotes the gene transcription and synthesis secretion of pro-inflammatory cytokines such as IL-6?IL-1?and TNF-?,thereby exacerbating the inflammatory response.Probiotics can improve the gut microbiota imbalance and delay the onset and development of chronic liver disease,thus reducing the progression and mortality of liver disease.However,probiotics cannot be colonized in the intestine for a long time,and the functional differences between different strains are huge,which cause the dispute of its therapeutic effect on liver disease.Fecal microbiota transplantation?FMT?is a method of transplanting healthy human intestinal flora into the patient's gastrointestinal tract to remodel the intestinal flora,which has been widely used in diseases such as Clostridium difficile infection,inflammatory bowel disease,intractable constipation,and intestinal immunodeficiency.At present,there are few studies on the application of FMT in the treatment of chronic liver disease,and the cause of which is related to the structural characteristics of the gut microbiota in chronic liver disease that have not been thoroughly revealed.Furthermore,the mechanism of FMT treatment of chronic liver disease is not clear.To understand the composition of gut microbiota in patients with various chronic liver diseases,and then to explore the mechanism of FMT treatment of chronic liver diseases,this study intends to use high-throughput sequencing technology?16S rRNA?to detect the gut microbiota of patients with hepatitis,cirrhosis,HCC and healthy individuals,thereby analyzing the structure and composition of gut microbiota of different etiologies and stages of chronic liver disease,and searching for the relation between gut microbiota and the progression of chronic liver disease.In order to open up a new field for the diagnosis and treatment of chronic liver disease in clinical practice,we use FMT to intervene on cirrhotic rats by constructing a rat model of cirrhosis,and then we explore the treatment mechanism of FMT on cirrhotic rats by combining microbiome and metabolomics techniques.I.Structural Diversity of the Intestinal Flora of Chronic Liver Disease Methods:?1?High-throughput 16S rRNA was used for hepatitis,cirrhosis,HCC,and healthy individuals to detect the structure and composition of the gut microbiota and analyze the changes in the gut microbiota of chronic liver disease at different stages;?2?Patients with HCC were divided into liver cirrhotic-induced HCC?LC-HCC:52 cases?and nonliver cirrhotic-induced HCC?NLC-HCC:23 cases?.These groups were used to analyze the effect of cirrhosis on the gut microbiota of HCC patients by comparison with cirrhosis group;?3?Patients with HCC were divided into HBV-induced HCC group?HBV-HCC:35 cases?,HCV-induced HCC group?HCV-HCC:25 cases?and Alcohol-induced HCC group?ALD-HCC:15 cases?.The effects of different etiologies on the structure and composition of the gut microbiota of HCC patients were analyzed in three groups.Results:?1?Compared with the healthy group,hepatitis group and HCC group,the diversity of gut microbiota was significantly reduced in the liver cirrhosis group.In the phylum level,the abundances of Verrucomicrobia and Proteobacteria were significantly increased?p<0.05?,whereas the Tenericutes was significantly less abundant?p<0.001?.At the genus level,the abundances of Phyllobacterium,Sphingomonas,Enterococcus and Erysipelatoclostridium were significantly increased?p<0.05?,while the abundances of Ralstonia,Catenibacterium,and Lachnospira were decreased?p<0.05?.Compared to the healthy and hepatitis groups,there was no significant change in the diversity of gut flora in the HCC group?p>0.05?,while the abundance of the Fusobacteria was significantly higher?p<0.05?,and the abundances of the Ralstonia,Clostridiates,and Sarcina were significantly higher?p<0.05?.These results indicated among the four groups,the gut microbiota imbalance in the cirrhosis group was the most serious.Although the gut microbiota diversity in the HCC group did not change significantly,the proportion of gut microbiota may be related to the occurrence of HCC.?2?Compared with the cirrhosis group,there was no significant change in the diversity of the gut microbiota in the LC-HCC group,while the diversity of the gut microbiota in the NLC-HCC group increased significantly?p<0.05?.The results of the genus level analysis showed that the abundance of short-chain fatty acid-producing bacteria such as Phascolarctobacterium and Odoribacter,and beneficial genera such as Bifidobacterium and Akkermansia decreased significantly?p<0.05?,while the abundance of harmful LPS-producing genera,such as Bacteroides,Erysipelotrichaceae and Desulfovibrio increased significantly?p<0.05?in the LC-HCC group compared with cirrhosis group.The results suggested that the decrease of beneficial bacteria and increase of harmful bacteria in the gut microbiota might be one of the reasons for the progression of liver cirrhosis to HCC.?3?Among the HBV-HCC,HCV-HCC,and ALD-HCC groups,alpha diversity and beta diversity analysis showed no significant differences?p>0.05?.Although 18differential genera such as Enterococcus were found in the three groups,there was no statistical difference?p>0.05?at the phylum level.So the different etiologies of HCC are not significantly associated with HCC gut microbiota disorders?.Therapeutic effects and Mechanism of FMT on cirrhotic ratsMethods:Two rat models of liver cirrhosis were constructed using CCL4 and CCL4 in combination with alcohol,respectively,and healthy rats were treated with fecal microbiota solution daily.?1?After 12 weeks of continuous modeling,the living status,body weight,ascites formation time,survival time and mortality rate of each group of rats were compared and analyzed;the liver function indexes and the degree of liver fibrosis in each group of rats were detected by automatic biochemical analyzer and HE staining technique,and the protective effect of FMT on cirrhotic rats was investigated.?2?ELISA was performed to determine the content of inflammatory factors such as IL-6,TNF-?and IL-1?and endotoxin in the plasma samples from each group of rats;The bacterial content of blood,mesenteric lymph nodes and liver tissue and the integrity of the intestinal mucosal barrier in each group of rats were measured;RT-PCR and Western-blot were used to determine the expression levels of genes and proteins related to TLR4-MyD88-NF-?B pathway in liver tissues.The above steps preliminarily explore the therapeutic mechanism of FMT in cirrhotic rats.Results:?1?Compared with the liver cirrhosis group,the life status of rats in the FMT intervention group was effectively improved,body weight increased,ascites formation time and survival time were significantly prolonged,and the mortality rate was significantly reduced.Compared with the cirrhosis group,the living status of rats in the FMT intervention group was effectively improved,including the increase of weight,ascites formation time and survival time and significantly lower mortality?p<0.01?;Liver function parameters such as ALT,AST and GGT in serum were significantly reduced?p<0.05,p<0.01?,while albumin parameters were significantly increased?p<0.05?;The number of nodules on the surface of the liver tissue,the pseudobullet structure in the liver tissue and the degree of connective tissue hyperplasia was reduced.The above results indicate that FMT can effectively improve the living status of cirrhotic rats,restore liver function and delay the development of cirrhosis.?2?ELISA results showed that the levels of inflammatory factors such as IL-6,TNF-?and IL-1?and endotoxin in the serum samples from the FMT intervention group rats were significantly reduced?p<0.05?;The number of bacterial colonies in blood,mesenteric lymph nodes and liver tissue was significantly reduced?p<0.05?,and the structure and barrier function of the intestinal mucosa were effectively improved;The expression levels of genes and proteins related to TLR4-MyD88-NF-?B pathway were significantly reduced in rat liver tissue.The above results indicate that FMT can effectively improve the intestinal mucosal structure and barrier function and reduce bacterial migration in cirrhotic rats.FMT improves the status of inflammation in cirrhotic rats by down-regulating the expression levels of genes and proteins related to the TLR4-MyD88-NF-?B pathway.?.Effects of FMT on gut microbiota composition and metabolism in cirrhotic ratsMethods:?1?16S rRNA high-throughput sequencing was applied to detect the gut microbiota of each group of rats,thereby analyzing and comparing the structure and composition of the gut microbiota of each group of rats,and explore whether FMT can effectively improve the gut microbiota disorder of cirrhotic rats.?2?UPLC-Q/TOF-MS technology was used to identify and analyze the differential metabolites in the plasma of each group of rats,and to investigate whether FMT could play a protective role in modulating the metabolic pattern of cirrhotic rats.Results:?1?The abundance of beneficial genera,such as Lactobacteriaceae,in the gut microbiota of cirrhotic rats decreased significantly,while the abundance of harmful genera,such as Clostridiaceae and Lachnospiraceae,increased significantly,and the structure and diversity of the flora were disturbed;After FMT,the abundance of beneficial genera,such as Lactobacteriaceae,increased,while the abundance of harmful genera,such as Clostridiaceae and Lachnospiraceae,decreased significantly.So FMT can improve the flora structure and maintain the balance of gut microecology,and then delay the process of liver cirrhosis through the gut-liver axis.?2?The plasma content of sphingosine and LysoPC?18:1?9Z??in cirrhotic rats was significantly reduced,while retinol,8,9-epoxyeicosatrienoic acid,9-cis-retinaldehyde,and arachidonic acid metabolites were significantly increased;FMT regulates metabolic pathways such as arachidonic acid metabolism and retinol metabolism.The above results suggest that the therapeutic mechanism of FMT may be to slow down the progression of cirrhosis by ameliorating the disturbance of gut microbiota in cirrhotic rats and modulating the pathways of arachidonic acid metabolism and retinol metabolism.Conclusion:1.The occurrence of chronic liver disease is closely related to the disorder of gut microbiota.The disorder of gut microbioa of HCC patients is closely related to liver cirrhosis.There is no significant correlation with different etiologies of HCC?such as HBV infection,HCV infection or excessive drinking?.The progression of liver cirrhosis to HCC may be associated with a decrease in the abundance of beneficial genus such as Bifidobacterium and Akkermansia and short-chain fatty acid-producing bacteria such as Phascolarctobacterium and Odoribacter,while increasing with the abundance of LPS-producing bacteria such as Bacteroides,Erysipelotrichaceae and Desulfovibrio.2.FMT can effectively improve the intestinal mucosal structure and barrier function in cirrhotic rats and reduce bacterial migration,thus delaying the development of cirrhosis.FMT can down-regulate the expression of genes and proteins related to the TLR4-MyD88-NF-?B pathway levels and reduce inflammation levels in cirrhotic rats;FMT also modulates arachidonic acid and retinol metabolic pathways to alter metabolic patterns,thereby slowing the disease progression in cirrhotic rats.Innovation:1.In this study,we characterized for the first time the structure of gut microbiota in patients with chronic liver diseases such as hepatitis,cirrhosis and HCC in northeastern China.It was found that the disturbance of the gut microbiota of HCC was closely related to liver cirrhosis,which was not significantly related to the different etiologies of HCC.2.This study verified the role of TLR4-MyD88-NF-?B signaling pathway in the development of HCC by constructing a rat model of liver cirrhosis.FMT was found to improve symptoms associated with cirrhotic rats by inhibiting the activation of TLR4-MyD88-NF-?B signaling pathway.3.This study combined microbiomics and metabolomics techniques to elucidate the therapeutic mechanism of FMT in cirrhotic rats,and found that FMT could slow down the progression of cirrhosis by modulating the gut microbiota structure and the metabolic disorders of arachidonic acid and retinol in cirrhotic rats. |
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