Study On Long-term Cohort And Expression Of 90K/Mac-2BP In Patients With PBC Treated By Tongdan Decoction

Background:Primary biliary cholangitis(PBC)is a chronic cholestatic disease induced by immune dysfunction.With the unceasing progress of diagnostic technology of PBC,the diagnostic accuracy of this disease has increased year by year in China.There is still no specific remedy for PBC.Ursodeoxycholic acid(UDCA)is the only drug used for PBC treatment approved by the FDA.However,only about 60% of patients receive biochemical remission.Therefore,seeking new treatment methods attracted increasing attention in the field of current research.Since 2002,Prof.Tong's research group has explored the traditional Chinese medicine treatment.Through years of clinical experience,it has been confirmed that Tongdan Decoction combined with UDCA can evaluate clinical efficacy.In our present study,we attempted to observe the effect of Tongdan Decoction with UDCA on improving the prognosis and further explore the underlying mechanism of PBC through this long-term cohort study.Based on Macrophage-2-binding protein(M2BP),the potent mechanism of Tongdan Decoction treating PBC was investigated by molecular assessments.Objective:To observe the effect of Tongdan Decoction on improving long-term PBC prognosis,we administrated Tongdan Decoction combined UDCA in 124 cases of patients diagnosed as early and mid-term PBC,controlled by UDCA monotherapy.Meanwhile,C57BL/6 wild type and M2 BP knockouted mice were used to established PBC model to investigate whether Tongdan Decoction could improve the situation of cholestasis of PBC model mice by down-regulating the expression of M2 BP.Methods:Clinical trialequally randomized into treatment and control groups.2 patients in the control group were excluded as receiving less than 4 weeks treatment.According to the relevant definitions,9 and 10 patients in the treatment group and the control group were diagnosed as early-term PBC respectively.54 and 51 were mid-term patients.2.Methods2.1 Group: treatment group: Tongdan Decoction + basic treatment + UDCA;control group: basic treatment + UDCA.Basic treatment includes Compound Glycyrrhizin and polyolacylcholine,which was generally not carried out more than 3 months.2.2 Regimen and course of treatment: 1)treatment group: UDCA,13 to 15 mg/kg/day,3 times/day.The maximum daily dose is 1000 mg.Tongdan Decoction,1 dosage/day,100 ml in the morning and 100 ml in the afternoon,more than 5 dosages per week.Control group: treated with UDCA plus the basic treatment.The dosage and method were the same as the treatment group.2)The course of treatment was 48 weeks,as a randomized controlled trial.After 48 weeks,it turned to an open trial.Moreover,the total time of taking Chinese medicine in the treatment group was not less than 12 weeks every year.2.3 Clinical definition: stage I: without clinical symptoms,normal liver biochemical laboratory examination,AMA positive,with PBC pathological manifestation;stage II: without obvious clinical symptoms,abnormal liver biochemical laboratory examination(such as ?-GT,ALP,etc.);Stage III: abnormal liver biochemical laboratory examination,clinical symptoms such as fatigue,pruritus and the like;Stage IV: occurrence of liver decompensation such as elevated serum bilirubin,low serum ALB and portal hypertension and so on.Stage I and II were defined as the early clinical stage.However,stage III was defined as the clinical mid-term stage and stage IV as the end stage of liver disease2.4 Definition of disease progression: Supposing that in the event there existed liver biopsy data,progression could be confirmed through the evidence of histopathology.If noninvasive liver fibrosis or cirrhosis examination was carried out previously,by satisfying at least one basic noninvasive imaging,including ultrasound semi quantitative score and transient elastography assessment and at least one of the non-invasive serum fibrosis arrays,such as APRI,FIB-4 index,Forns index and WFA +-M2 BP,disease progression could be confirmed.2.5 Curative effect observation: blood routine examination,liver biochemical laboratory examination,liver histopathology,noninvasive liver fibrosis or cirrhosis examination.Disease progression was sequentially accessed after treatment.Animal experiment:1.Modeling: 30 M2 BP gene knockouted mice and 30 C57BL/6 wild type mice were purchased,established PBC model by administrated 2OA-BSA for 8 weeks,then evaluated successfully or not.2.Group and intervention methods2.1 PBC established mice were divided into group A(M2BP gene knockouted mice)and group B(C57BL/6 wild type mice).Group A and B were sequentially randomly divided into group A1,A2,B1,B2.Each group contained 10 mice.2.2 Group A1 and B1 were treated with PBS as control;Group A2 and B2 were administrated Tongdan Decoction.2.3 The mice were sacrificed after 12-week treatment.blood sample were collected through orbital plexus and liver tissues were isolated.2.4 Test methods: liver biochemical laboratory examination(ALT,AST,GGT,ALP,TB and ALB),liver histology(H&E staining),M2 BP detection(immunohistochemistry staining,Western Blot and q-PCR).3.Control group: 12 6-week C57BL/6 wild type mice(2 litters)were used as control group.3 mice from one of the litters were first sacrificed to exam the M2 BP mRNA level of liver tissues and the rest were sacrificed after administrated 2OA-BSA for 8 weeks to test the liver M2 BP mRNA level.6 mice from the other litter were intragastric administrated PBS for 8 weeks.5 of those were sacrificed to carry out the liver biochemical laboratory and liver histopathology examination,compared with Group A and B respectively.Results:1.A 48-week randomized controlled study showed that liver biochemical laboratory examination reflecting liver function of the two groups were significantly improved.Compare with the control group,the improvement of treatment group was more significant in the treatment group(P<0.05).2.A total of 19(15.3%)patients suffered endpoint events after treatment,including 6 in the treatment group(9.5%)and 13 in the control group(21.3%),based on the Kaplan-Meier method of survival analysis defined previously.It revealed that a notably lower incidence of adverse events of the treatment group than of the control group(P = 0.045,Log-rank(Mantel-Cox)test).The Hazard Ratio(logrank)was 0.3941(95% CI 0.1599 to 0.9714).3.We conducted a Cox regression analysis of the subjects' gender,age,group(treatment difference),serum TB,ALT,AST,ALP,TBA,ALB,?-GT,CHOL,IgM,IgG,and IgA.The results showed gender,treatment difference,and high TB of the enrolled baseline was an independent risk factor for the progression of disease(HR: 6.60,15.87,1.35;P<0.05.)4.Logistic regression was performed on the group,baseline TB and ALP.The regression equation was calculated as: Logit(P)=-21.366 + 4.015 × group + 0.013 × ALP + 0.263 × TB.Predicted probability of each patient was P = e Logit(P)/ 1 + e Logit(P).Long-term prognosis for each patient was predicted according to the above equation.The area under the curve was 0.970(sensitivity: 94.7%,specificity: 93.3%).5.In the treatment group,the Paris II standard showed higher predictive value(P <0.05)than the Barcelona and Paris I standards in terms of disease prognosis.However,in the control group,there was no significant difference in predicting the disease progression according to these three standards(P> 0.05).6.Serum WFA(+)-M2 BP level was measured in part of the patients.We found that the WFA(+)-M2 BP levels in these two groups were both higher after treatment.Additionally,no significant difference existed in the treatment group(P= 0.32),but one in the control group(P = 0.01).The increase in the control group is more obvious than the treatment group(P = 0.004).Thus,the WFA(+)-M2 BP level could promisingly reflect the presence of disease progression.Compared with the other judgment methods described in the article,the Kappa value was 0.848,showing better agreement.In addition,when comparing the baseline WFA(+)-M2 BP levels between patients with disease progression and those without progression,the former WFA(+)-M2 BP level was significantly higher than that of the latter(1.34±0.27 vs.0.77±0.10,P=0.000).7.M2 BP gene in C57BL/6 mice was knockouted successfully by Gene Sequencing.8.Establishment of PBC model mice: Compared with the control group,lymphocyte infiltration was observed around the portal area of the liver tissue of the PBC model mice induced by 2OA-BAS.Furthermore,some of bile duct disappeared while epithelioid granuloma appeared.The ALP and ?-GT reflecting the severity of cholestasis were markedly higher than those of the control group.The level of M2 BP mRNA in the liver tissues was significantly increased after modeling in the wild type mice(P<0.01).9.After the intervention of Tongdan Decoction,the liver function and cholestasis indices of mice in group A2 and B2 were significantly improved respectively.The biochemical improvement extent of group B2 was more obviously higher than that of group A2(P<0.05).The M2 BP level in group B2 was decreased significantly after treatment.However,M2 BP in group A1 did not show increased expression,neither the biochemical improvement.No significant changes of biochemistry or M2 BP level of group B1 were found.Conclusion:1.The 48-week randomized controlled clinical trial of Tongdan Decoction combined with UDCA for patients with PBC during the early and mid-term showed that the combination treatment could significantly improve the biochemical indicators and release the liver inflammation.Through Cox regression analysis and logistic regression model,it was shown that combination with Tongdan Decoction can achieve better curative effect than UDCA monotherapy.Moreover,high TB level of baseline and male are independent risk factors for disease progression.Retrospective WFA(+)-M2 BP detection showed that there existed a positive correlation with the progression of PBC.2.A further 12-year long-term cohort study and Kaplan-Meier survival analysis showed that the incidence of adverse events in the treatment group was significantly lower than that in the control group.3.Among patients with early and mid-term PBC treated with UDCA,higher predictive values could be obtained by accessing biochemical responses according to Paris II standard.4.CRISPR/Cas9 technology could successfully knockout M2 BP gene.After establishing a model of PBC induced by 2OA-BSA in C57BL/6 mice for 8 weeks,pathological and biochemical changes similar to those of PBC patients can be obtained.5.The underlying mechanism of Tongdan Decoction improving cholestasis is likely to decrease the expression of M2 BP in C57BL/6 PBC model mice.However,the detailed pathway remains to be further explored.