The Role Of Group 3 Innate Lymphoid Cells In Washed Microbiota Transplantation Against Metabolic Associated Fatty Liver Disease And Its Relationship With Traditional Chinese Medicine Syndrome Type

Objective:1.To evaluate the clinical efficacy of washed microbiota transplantation(WMT)on metabolic-associated fatty liver disease(MAFLD).2.To evaluate the changes in the number and migration ability of group3 innate lymphoid cells(ILC3s),which are closely related to the intestinal flora,before and after WMT.3.To evaluate the characteristic of ILC3s in peripheral blood from MAFLD patients and assess its association with MAFLD severity and traditional Chinese medicine(TCM)syndrome type.4.To explore the source of ILC3s in the liver and clarify its role in MAFLD progression.Method:1.Medical records of MAFLD patients who underwent WMT treatment were retrospectively collected.The changes in liver fat attenuation index,liver stiffness,liver enzymes,insulin resistance index,and serum lipid profiles of MAFLD patients before and after WMT were analyzed.2.The proportion change of ILC3s and their subsets(CXCR3~+ILC3s and CXCR4~+ILC3s)in peripheral blood mononuclear cells(PBMCs)from MAFLD patients before and after WMT treatment was examined by flow cytometry(FCM);MAFLD model was constructed by feeding with methionine and moline deficient L-amino acid diet,and that were transplanted with fecal bacterial fluids from healthy mice and fecal bacterial fluids from diseased mice,respectively,and the expression of ILC3s in the liver and peripheral blood of MAFLD mice after fecal bacterial transplantation were observed by FCM.3.The proportions of ILC3s in PBMCs from MAFLD patients and healthy control(HC)were examined by FCM.The association between ILC3s and clinical indices was analyzed.4.The clinical data on TCM syndrome were collected to analyze the distributions of TCM syndrome types.The differences in clinical indices and the proportion change of ILC3s among TCM syndromes types in MAFLD patients were analyzed.5.The altered diversity and composition of gut microbiota of MAFLD mice at different stages were analyzed by 16Sr RNA Gene Sequencing.The numbers of ILC3s in PBMCs,liver,small intestine,and spleen of MAFLD mice were examined by FCM.The number of ILC3s in PBMCs,intestine,liver,and spleen of MAFLD mice were examined by FCM.The proliferation ability of ILC3s in the liver of MAFLD mice was examined by FCM.ILC3s were isolated from the small intestines of CD45.2~+mice and adoptively transferred to CD45.1~+MAFLD mice through the lateral tail vein.The number of CD45.2~+ILC3s in the liver,intestinal tissues,and the other tissue of MAFLD mice was examined by FCM.The chemokine receptor expression of ILC3s was examined by FCM.Differential gene expression of chemokine in the liver of MAFLD mice was evaluated by RNA sequencing.6.The level of liver triglyceride(TG),alanine aminotransferase(ALT),aspartate aminotransferase(AST),Serum total cholesterol(TC),and TG in ILC3s-injection MAFLD mice were assessed by the automatic biochemical analyzer.The severity of liver tissue injury was detected by hematoxylin and eosin(HE)staining in ILC3s-injection MAFLD mice and evaluated by NAFLD activity score(NAS).Results:1.After the first and second courses of WMT,the liver fat attenuation index was significantly lower than that before WMT.2.After WMT treatment,the proportion of ILC3s in PBMCs from MAFLD patients was significantly decreased,but the proportion of CXCR4~+ILC3s was increased;Besides,the proportion of liver and peripheral blood ILC3s was increased when the MAFLD mice was transplanted with fecal bacterial fluids from healthy mice.3.The proportion of ILC3s in PBMCs of MAFLD patients was higher than that in HC and was positively correlated with AST,TC,low-density lipoprotein cholesterol,and endotoxin levels.The proportion of ILC3s in PBMCs from MAFLD patients was decreased after metabolic drug treatment.4.The predominant TCM syndrome type in MAFLD patients was liver stagnation and spleen deficiency,followed by phlegm-damp retention,damp and heat Stagnating,intermingled phlegm and blood stasis,and liver-kidney yin deficiency.There is no significant difference in the distributions of ALT,AST,liver fat attenuation index,TC,TG,high-density lipoprotein cholesterol,low-density lipoprotein cholesterol,fasting blood glucose,Hemoglobin A1c among TCM syndrome types in MAFLD patients.The proportion of ILC3s in PBMCs from MAFLD patients with liver depression and spleen deficiency was lower than that from MAFLD patients without liver depression and spleen deficiency.5.With the development of MAFLD,the composition of intestinal flora and the abundance of bacteria was changed.Meanwhile,the number of ILC3s in the small intestine,PBMCs,and liver of MAFLD mice was significantly increased.In the liver of MAFLD mice,most of the ILC3s were Ki-67 negative.After the intestinal-derived CD45.2~+ILC3 were adoptively transferred to CD45.1~+MAFLD mice,most of the intestinal-derived CD45.2~+ILC3s were resident in the liver tissue of MAFLD mice.Most of the ILC3s in the liver of MAFLD mice were CXCR3 and CXCR4 positive.Besides,the RNA levels of CCL12 and CXCL10 were significantly increased in the liver tissues of MAFLD mice.6.The levels of AST,ALT,liver TG and the NAS were significantly decreased in ILC3s-injection MAFLD mice.Conclusion:1.The proportion of ILC3s in peripheral blood from MAFLD patients was increased,which was positively correlated with the severity of liver function,lipid level,and intestinal barrier function.2.The predominant TCM syndrome type in MAFLD patients was liver stagnation and spleen deficiency,followed by phlegm-damp retention,damp and heat stagnating,intermingled phlegm and blood stasis,and liver-kidney yin deficiency.The proportion of ILC3s in PBMCs was significantly decreased in MAFLD patients with liver depression and spleen deficiency,which may have the potential to be a diagnostic marker for liver depression and spleen deficiency.3.With the development of MAFLD,the intestinal-derived ILC3s may migrate to the liver in response to chemokines/chemokine receptors axes and play a therapeutic role.4.WMT treatment can significantly reduce the liver fat deposition of MAFLD patients,which may promote the homing of ILC3s to the liver by regulating the expression of chemokines of ILC3s.