The Relationship Between TCM Constitution And NAFLD Based On Cohort And The Role Of Osteopontin Neutralizing Antibody In The Regression Of Fatty Liver Fibrosis In Rats

background: Fatty liver disease(NAFLD)refers to a group of clinical pathological syndromes characterized by hepatic steatosis caused by metabolic disorders of the body.NAFLD is reversible at an early stage.The changes in risk factors of NAFLD during the progression of the disease are important factors influencing the development of fatty liver status from NAFL to NASH or even cirrhosis or gradual regression.The constitution of TCM can determine to a large extent the susceptibility of the individual to the pathogenic factors inside and outside the NAFLD and the syndromes that may be manifested after the disease,and more importantly,the individual's different effects on the treatment effect.Therefore,the study of constitutional identification and the impact on NAFLD disease has become an important part of the research on personalized treatment of traditional Chinese medicine.Studying the relationship between TCM constitutional factors and indicators of fatty liver and risk factors,and its impact on the occurrence and regression of fatty liver,will provide a scientific basis for further prevention and treatment of NAFLD.Ostepontin(OPN)is a secreted phosphorylated glycoprotein with cytokines.In recent years,a series of studies have shown that OPN has a profibrotic effect during the development of liver fibrosis.It can not only induce the activation,proliferation and migration of hepatic stellate cells(HSC),but also cause intrahepatic type I.Collagen,type III collagen,transforming growth factor beta receptor m RNA(TGF-? m RNA)and protein production,and also significant chemotaxis to hepatic macrophages.This study is to use the physical examination cohort of colleges and universities to study the characteristics of TCM constitution on the occurrence and development of fatty liver,the distribution characteristics and clinical characteristics of different TCM constitutions in the population,and its impact on the occurrence and regression of fatty liver.To provide a scientific basis for further prevention and treatment of cardiovascular disease risk factors such as NAFLD.The study also used the data of the population of the medical examination cohort to evaluate the predictive value of baseline OPN on the occurrence and regression of NAFLD and the influence of TCM constitution on it.Finally,this study used the rat model of fatty liver fibrosis to investigate the specific binding of OPN neutralizing antibodies to OPN protein and antagonize the biological function of OPN,and to observe the presence or absence of intervention and changes in liver pathological changes and related fibroblasts under different interventions.In this case,it is preliminary to evaluate whether the OPN immunoneutralization regimen is feasible and effective in treating liver fibrosis.An in-depth study of the mechanisms of liver fibrosis associated with OPN can help explore new strategies or new drug targets for the prevention and treatment of fatty liver fibrosis.research content:1.Study on the relationship between TCM constitution and non-alcoholic fatty liver based on cohort:2.Predictive evaluation of serum Osteopontin on the occurrence and regression of nonalcoholic fatty liver disease;3.The role of Osteopontin neutralizing antibody in the regression of fatty liver fibrosis in rats;Part 1 Relationship between TCM constitution and NAFLD based on cohort Objective :This study was based on a combination of cross-sectional studies and prospective studies in a population of physical examination cohorts to study the relationship between TCM constitutional factors and the distribution and changes of NAFLD risk factors,and the influence of TCM constitution types on the occurrence and regression of fatty liver..Methods: A cross-sectional survey and a cohort study were used to investigate a group of people over the age of 40 who participated in physical examination for five consecutive years.The physical examination indicators and the TCM constitution questionnaire were tested,and the survey data were statistically described and analyzed.Result:Cross-sectional survey:1.The composition of fatty liver in the crowd was mainly phlegm-dampness,qi-deficiency,and damp-heat.The number of phlegm-dampness patients was the highest in both moderate fatty liver and mild fatty liver.The damp-heat people were mainly moderate fatty liver,and the qi-deficiency people are mainly mild fatty liver.2.From the tendency to susceptible to fatty liver compared gender differences,in the bloody sputum,qi stagnation,damp heat quality males were more likely to be detected fatty liver,in Yin deficiency,yang deficiency women were more likely to be detected Fatty liver.3.With the increase of age,the total detection rate of fatty liver was increasing.Qi deficiency and peace are gradually increasing with the increase of age,and the detection rate of moist heat was gradually decreasing with age.The 50-55 age group was the intersection of the detection rate of fatty liver in men and women.The detection rate of fatty liver in women before the intersection is lower than that in men.The detection rate of fatty liver in women is higher than that in men.Queue crowd survey:1.The annual incidence of fatty liver is significantly higher than the annual regression rate of fatty liver.The general trend is that the number of patients is increasing.2.The new fatty liver population is mainly composed of qi deficiency,damp heat and dampness.Fatty liver retreat population is dominated by phlegm-dampness,qi-deficiency,and damp-heat quality.The conversion rate of new fatty liver from high to low are: qi deficiency,phlegm dampness,damp heat,yang deficiency.The conversion rate of regression in people from high to low are: yang deficiency,peace and quality,and qi deficiency.Part II Predictive evaluation of the occurrence and regression of osteopontin in nonalcoholic fatty liver diseaseObjective:The predictive value of OPN for the occurrence and regression of NAFLD was evaluated by comparing the changes of serum OPN levels before and after non-alcoholic fatty liver diseaseMethod:The research subjects came from a college to participate in the physical examination population.A total of 360 subjects who met the baseline and follow-up study data from the population who completed the 3-year follow-up were included in the study.The following four groups were classified according to the presence and absence of fatty liver at the end of follow-up: no occurrence occurred.Group,new group,maintenance group,and elimination group.All subjects completed liver ultrasound diagnosis,physical examination,TCM constitution identification,serum OPN concentration,anthropometric parameters,various glucose and lipid metabolism indicators and laboratory tests of liver enzymes.Logistic regression was used to analyze the predictive role of baseline serum OPN in the development and regression of NAFLD.Result:Baseline serum OPN levels were significantly higher in the newly-occurring NAFLD population than in those without NAFLD(48.47 ng/m L(40.1,62.9)vs 38.18 ng/m L(34.2,51.2),(P<0.001).Baseline serum OPN levels were significantly higher in the maintenance group than in the remission group(54.16 ng/m L(33.2,71.3)vs 50.71 ng/m L(36.7,75.9),(P <0.05).Serum OPN levels in the maintenance group were significantly higher than those in the non-prone fatty liver,59.11 ng/m L(55.2,73.5)vs 50.96 ng/m L(47.8,70.8),(P<0.05).There was no significant difference between the non-occurring group,the new-issued group and the extinction group.Multiple stepwise regression showed that baseline serum OPN levels and BMI were independent predictors of risk factors for predicting NAFLD.ROC curve analysis showed that baseline serum OPN+BMI predictive models had good predictive value for NAFLD after 3 years(area under the curve was 0.821(95% CI 0.773–0.839).Part III The Role of Osteopontin Neutralizing Antibody in the Process of Fatty Liver Fibrosis Regression in RatsObjective:To investigate the effect of Osteopontin(OPN)neutralizing antibody on fatty liver fibrosis in rats and to explore its potential therapeutic value.To study the expression changes of liver fibrosis after intervention of OPN neutralizing antibody in rats with fatty liver fibrosis Its relationship with glycolipid metabolism.Method:1)60 SD rats,including the control group(N=8)and the NAFLD model(N=52),the classic CCl4 induced high-fat diet feeding method for fatty liver fibrosis modeling,and the successful modeling in the 6th week Eight rats were randomly selected as model group to be sacrificed,and 24 randomly selected were divided into natural regression group,OPN neutralizing antibody intervention group and colchicine group.The OPN neutralizing antibody intervention group was injected with OPN antibody(titer: 1:15)through the tail vein every other day from 7 weeks for 4 weeks(0.15 m L each time);the colchicine group started colchicine at the 7th week.Intervention treatment,rats were administered with 0.02mg/100 g body weight for 4 weeks;NAFAD natural regression group stopped high-fat diet from the 7th week and returned to normal diet for 4 weeks.At the 0th,4th,6th,8th,and 10 th weekends,5,5,8,5,and 5 rats were randomly selected for biochemical,pathological,and western blot analysis to evaluate modeling and related indicators Changes.2)Real-time PCR and immunoblotting were used to detect the expression of OPN and ?-SMA in rat liver tissue;enzyme-linked immunosorbent assay was used to detect liver fibrosis related indicators;pathological histology and HE and Masson staining were used to detect rats.Pathological changes of liver fibrosis;liver function indicators,TC,TG,FBG,FINS,HOMA-IR,ISI were calculated using rat serum.Result:1)The m RNA levels and protein expression levels of OPN and ?-SMA in the OPN antibody intervention group were significantly lower than those in the natural regression group and the model group(P<0.01).2)After intervention with OPN antibody,the pathological area of rat liver tissue decreased,fibrous tissue hypertrophy,and inflammatory cell infiltration decreased.3)Col I,PCIII,MMP-3,TGF-?1,?-SMA and TNF-? in the antibody intervention group and the colchicine group were significantly lower than those in the natural regression group(P<0.05 or P<0.01).4)FPG,FINS,HAMO-IR and TG in the antibody intervention group were significantly lower than those in the natural regression group(P<0.05 or P<0.01);FPG,FINS,HAMO-IR,TG,ISI and TG in the antibody intervention group were significantly lower than those in the antibody intervention group.There were no significant statistical differences between the two groups in the TC index.Conclusion:1 The distribution characteristics of TCM constitution and non-alcoholic fatty liver are as follows:1)Composition ratio: non-alcoholic fatty liver is mainly composed of phlegm-dampness,qi-deficiency,damp-heat physique;susceptibility: phlegm-dampness,qi-deficiency,damp-heat physique is more susceptible to fatty liver;in male blood sputum,Qi stagnation,damp heat physique is more likely to detect fatty liver,and it is easier to detect fatty liver in female yin deficiency and yang dysfunction.2)Non-alcoholic fatty liver occurrence and regression changes:The new fatty liver population is mainly composed of qi deficiency,damp heat and phlegm dampness.The conversion rate of new fatty liver from high to low is: qi deficiency,phlegm and d Nampness,dampness and heat;Qi deficiency and damp heat are the main factors;the conversion rate of fatty liver regression is from high to low: qi deficiency,qi stagnation and yang deficiency.2.Human serum OPN level is an independent risk factor for the occurrence of NAFLD,and can be a new serum marker for predicting NAFLD.The type of TCM constitution that is susceptible to fatty liver is also an independent risk factor for NAFLD.3.In the process of fatty liver fibrosis modeling,OPN high expression or activation of ?-SMA,up-regulation of downstream HSC-related mechanisms lead to liver fibrosis,and targeted inhibition of OPN can significantly improve liver fibrosis caused by non-alcoholic fatty liver At the same time,it can also significantly improve insulin resistance and reduce serum TG levels.