Study On The Effect And Mechanism Of Kaili Sour Soup On Non-alcoholic Fatty Liver Disease

Objective:Non-alcoholic fatty liver disease(NAFLD)refers to a clinicopathological syndrome in which diffuse hepatocyte fat becomes the main feature except for alcohol and other clear liver damage factors,including simple fat Studies have shown that the The occurrence of steatohepatitis and cirrhosis is closely related to various metabolic syndromes.With the general improvement of people's quality of life,the incidence of NAFLD is increasing all over the world,which has become a public health problem closely concerned by countries all over the world,which has seriously affected the quality of human life.Due to the long-term and comprehensive effects of multiple factors,its pathogenesis has not yet been clear.Previous studies have confirmed that NAFLD is inseparable from unhealthy lifestyles such as genetics,living environment,excessive dietary energy intake,and insufficient exercise.At present,in addition to basic drug treatment,improving people's life and eating habits to achieve the purpose of preventing and treating NAFLD has become a new research hotspot.Since the establishment of the "enterohepatic axis" theory,a large number of studies have shown that the changes in the species and abundance of intestinal flora,the resulting changes in intestinal barrier function and intestinal endotoxemia play an important role in the occurrence and development of NAFLD.When the intestinal mucosal barrier is broken,the intestinal flora and its metabolites can enter the liver through the "gut-liver axis" circulation and trigger a series of immune and inflammatory reactions,thereby inducing NAFLD and even liver cirrhosis or liver cancer.Sour soup is a characteristic food originated in Qiandongnan region of Guizhou Province,its production technology is the representative of the intangible cultural heritage of Guizhou Province.This study is based on the epidemiological investigation of NAFLD among healthy people in Kaili Sour Soup of southeastern Guizhou Province.Because of the preference for sour soup,the incidence of NAFLD in Kaili area is significantly lower than the average level of the whole China..Combined with hot medical research theory "intestine-liver axis" and diet intervention NAFLD related research,on the basis of animal experimental research,from the research direction of diet-intestinal microecology-metabolic inflammation,the effect and mechanism of Kaili Sour soup decoction on NAFLD were discussed.Methods:(1)The epidemiological investigation of NAFLD in Kaili area: the healthy persons in the second affiliated Hospital of Guizhou Medical University were randomly selected and filled in the "Fatty liver Epidemiology questionnaire".The basic conditions,living habits and general health status of the subjects were obtained,and the serum alanine aminotransferase(ALT),aspartate aminotransferase(AST),triglycerides(TG),total cholesterol(TC),total bile acids(TBA)and fasting blood-glucose(FBG)were determined by biochemical analysis.Abdominal ultrasound confirmed whether suffering from fatty liver disease.According to the guidelines for the prevention and treatment of NAFLD(updated in 2018),1314 healthy people were included in this NAFLD epidemiological survey,and were divided into two groups: healthy group and NAFLD group.Through statistical analysis,we found the epidemic trend and risk factors of NAFLD in Kaili area,and made clear the role of Kaili Sour soup in the epidemiology of NAFLD.(2)The second part,the effect and mechanism of Kaili acid decoction on NAFLD induced by high-fat diet in rats: 34 6-week-old male SD rats without specific pathogen grade(Specific pathogen free,SPF)were randomly divided into 4 groups:normal control group(ND,n=8),normal diet + Kaili Sour soup group(NS,n=8),model control group(HF,n=10)and high-fat diet + Kaili Sour soup group(HS,n=8).Rats in ND and NS groups were fed with standard diet,HF and HS groups were fed with high-fat diet to replicate NAFLD rat model.At the time,NS group and HS group were given intragastric administration of Kaili sour soup,the same volume of double distilled water give rats in ND group and HF group.The Sour soup was boiled on the same day,cooled naturally and then given intragastrically at a dose of 1ml / 100 g /week for 6 days / week,1 day / time.After feeding for 12 weeks,2 rats in the HF group were killed to determine the success of the model,and the fecal samples of rats in each group were taken and killed in each group.Collect blood,liver tissue and ileal tissue samples.The weight of body and liver of rats in each group were weighed,and calculate the liver index and Lee's index,the levels of ALT,AST,TG,TC,high density lipid-cholesterol(HDL-C),low density lipid-cholesterol(LDL-C),TBA and FBG in serum were determined by biochemical analysis.The changes of liver and ileum were observed by HE staining.The histopathological changes of liver and the deposition of steatosis were observed by oil red O staining,and the ultrastructure of liver cells was observed by electron microscope.The changes of intestinal flora in rat feces were detected by 16 s r RNA high-throughput sequencing..The levels of fasting serum insulin(INS),interleukin-6(IL-6),interleukin-18(IL-18),tumor necrosis factor-?(TNF-?)and lipopolysaccharide(LPS)were detected by ELISA.The expression of tight junction protein ZO-1,Claudin-1,Occludin,IL-6,TNF-?,Toll-like receptor 4(TLR-4),myeloid differentiation factor 88(My D88),nuclear factor kappa B p65(NF-kappa B)and NF-? B p-p65 in ileal mucosa was detected by WB,and the expression of ZO-1 in ileal mucosa,TNF-? junction and My D88 in liver tissue was detected by IHC.The expression of TLR-4 and NF-? B p65 was detected by IF,and the relative expression of insulin receptor farnesoid X receptor(FXR),substrate-1(IRS-1),NF-? B p-p65 and TLR-4 in liver tissue was detected by QPCR.(3)The effect of Kaili Sour Soup decoction on liver proteomics of NAFLD rats:the proteomic changes of rat liver tissue were analyzed by tandem mass spectrometry relative quantitative proteomics TMT.The differential proteins of liver tissue protein changes induced by acid decoction were found and verified by WB test.Results:(1)The first part: The NAFLD epidemiological survey in Kaili area found that among the 1,314 participants,239 were NAFLD patients,and the prevalence rate was18.19%.Among them,there were 161 NAFLD patients in the 721 male survey subjects,with a prevalence rate of 22.33%,and 78 of the 593 female survey subjects,with a prevalence rate of 13.15%.With age,the prevalence of NAFLD is on the rise.20 to 49 years old: males are higher than females(P<0.05),over 50 years old: the difference between males and females is not statistically significant(P>0.05).Multivariate logistic regression analysis found that ALT,AST,TBA,FBG,TG,waist circumference,body mass index(BMI),age,and gender were independent risk factors for the onset of NAFLD in the region,while exercise and sour soup diet became factors Protective factors,and eating sour soup 3 times or more a week,the prevalence of NAFLD has been reduced.(2)The rat model of NAFLD was successfully established by feeding with high-fat diet for 12 weeks.At the same time,rats in HS group were given sour soup intragastric intervention.After 12 weeks of feeding and intervention,the body weight,liver weight,liver index,Lee's index,serum levels of ALT,AST,TC,TG,LDL-C,HDL-C,TBA,FBG,INS and IR in HS group were improved as compared with those in HF group.The expression of tight junction proteins ZO-1,Claudin-1 and occludin increased in the ileum of rats.The species and abundance of intestinal microflora increased in HS group.At the "phylum" level,the change of flora is mainly reflected in the decrease in the number of thick-walled bacteria,the increase in the number of Bacteroides and actinomycetes,the decrease in the number of Bacteroides and actinomycetes,and the increase in the number of Brevibacterium methanogenes and Lactobacillus at the genus level.In HS group,the contents of inflammatory cytokines IL-6,IL-18,TNF-?and LPS in serum decreased,the levels of TNF-?,TLR-4,NF-? BP-p65 and NF-?Bp65 in liver tissue decreased,and the expression level of FXR and IRS1 increased.(3)A total of 6158 proteins were identified by TMT quantitative proteomics.A total of 294 differentially expressed proteins were screened according to the criteria(FC > 1.2fold and P < 0.05).Results: 1.Among them,Compared with ND group,53 proteins were up-regulated and 49 proteins were down-regulated in HF group,while 6proteins were up-regulated and 21 proteins were down-regulated in HS and HF group.According to Venn diagram analysis,We can get up-regulated in HF group compared with ND group,and down-regulated in HS group and HF group,there are 12 kinds of differentially expressed proteins in both cases;down-regulated in HF group and ND group,and up-regulated in HS group and HF group,there are 4 kinds of differentially expressed proteins in both cases..These 16 different proteins may be the key proteins of the improving effect of sour soup on the liver of NAFLD rats.The differential proteins were verified by WB test,and four kinds of differential proteins,Slc12a2,Pcif1,CYP3A1 and Sult1e1,which were of clinical significance for the improvement of NAFLD,were obtained.Conclusion:(1)The epidemiological survey in Kaili area shows that the prevalence of NAFLD in this area is 18.19%,which is lower than the national average of 27.37%.The prevalence of NAFLD increases with age,before the age of 49: males are significantly higher than females.ALT,AST,TBA,FBG,TG,waist circumference,BMI,age,and gender are independent risk factors for the onset of NAFLD in this area,while exercise and sour soup diet are protective factors,and sour soup is consumed 3 times or more a week.Conducive to the prevention and control of NAFLD.(2)Kaili Sour Soup decoction can effectively improve the indexes of NAFLD rats induced by high-fat diet,and its mechanism is to improve the intestinal microecology,regulate bile acid metabolism and reduce the inflammation of TLR-4/NF-? B signal pathway.(3)The dietary habit intervention of Kaili Sour Soup decoction affected the changes of liver proteomics in NAFLD rat model.After experimental verification,the differentially expressed proteins Slc12a2,Pcif1,CYP3a1 and Sult1e1 in this study may become a new direction in the diagnosis,prevention and treatment of non-alcoholic fatty liver disease.