Gandou Fumu Decoction In The Treatment Of Wilson’s Disease Liver Fibrosis And Its Mechanism On Liver Fibrosis In TX-J Mice

1 Objective:In the clinical part,Fibro Touch was used to evaluate the efficacy of Gan Dou Fu Mu Decoction(GDFMD)combined with Western medicine in the treatment of hepatolenticular degeneration(Wilson disease,WD)liver fibrosis.It is a non-invasive diagnosis and traditional Chinese medicine for WD liver fibrosis.Provide basis for intervention efficacy.Combined with animal experimental studies,observe the effects of GDFMD on the JNK signaling pathway and downstream factors of Toxic Milk(TX-J)by regulating the WD model,further clarify the molecular mechanism of GDFMD against liver fibrosis,and provide a theory for the clinical application of GDFMD Basic and scientific basis.2 Methods2.1 Clinical researchA total of 60 WD patients who were admitted to the outpatient and inpatient department of the Brain Disease Center of The First Affiliated Hospital of Anhui University of Traditional Chinese Medicine from September 2019 to September 2020 were selected.According to the principle of randomized control,they were divided into treatment group and control group,with 30 in each group.example.The patients were instructed to have a low-copper diet,the control group was treated with liver-protecting and copper-eliminating western medicine,and the treatment group was given oral administration of GDFMD based on the control group.The curative effects were evaluated 24 weeks after treatment,and the liver stiffness measurements(LSM),liver function(ALT,AST),liver fiber(HA,LN,PC-Ⅲ,C-IV),liver stiffness measurements(LSM),liver function(ALT,AST),and liver fiber(HA,LN,PC-Ⅲ,C-IV)were compared before and after treatment.TCM syndrome score,Uniform Wilson Disease Rating Scale(UWDRS)liver function score.2.2 Experimental researchAfter genetic identification,50 homozygous TX-J mice were selected,and 10wild-type TX-J mice were selected as the control group,all of which were 6 months old.Divided 50 TX-J mice were randomly divided into model group,penicillamine group and GDFMD high,medium and low dose groups,each with 10 mice.Different drugs and doses were given by gavage according to different groups.The control group and model group were given normal saline gavage for 4 weeks.2.2.1 Observe the effects of GDFMD on TX-J mice serology and liver histopathology:ELISA method was used to detect serum ALT,AST,HA,LN,MDA,SOD indicators;HE and Masson staining to observe histopathological changes of liver fibrosis;2.2.2 Observe the effect of GDFMD on the mitochondrial function of TX-J mice:A kit was used to detect the mitochondrial ROS content,ATP content and MMP changes in liver tissue.2.2.3 Observe the influence of GDFMD on the JNK pathway and downstream factors of TX-J mice:Western Blot was used to observe the protein expression of JNK,P-JNK,caspase-3,Bax and Bcl-2.3 Results3.1 Clinical part:3.1.1 Clinical curative effect: GDFMD combined with western medicine has better curative effect than western medicine alone.3.1.2 Comparison of LSM values: Before treatment,there was no statistically significant difference in LSM between the two groups(P>0.05);compared with before treatment,the LSM values of the two groups were significantly decreased(P<0.01);after treatment,the LSM of the treatment group was compared The value decreased more significantly than the control group(P<0.05).3.1.3 Comparison of serum indexes: Before treatment,there was no statistically significant difference in ALT,AST,HA,LN,PC-Ⅲ,and C-IV between the two groups(P>0.05);compared with before treatment,the two groups of ALT,AST,HA,LN,PC-Ⅲ,and C-IV all decreased significantly(P<0.01);after treatment,the values ??of ALT,AST,HA,LN,and C-IV in the treatment group decreased more significantly than those in the control group(P<0.01).3.1.4 Comparison of scale scores: There was no significant difference in UWDRS liver function scores and TCM syndrome scores between the two groups before treatment(P>0.05);compared with before treatment,the scores of the two scales decreased significantly(P<0.01);Comparison between groups after treatment,the scores of the two scales decreased more significantly than those of the control group(P<0.01,P<0.05).3.1.5 Comparison of adverse reactions between the two groups: the total incidence of adverse reactions in the control group was 3.33%,and the incidence of adverse reactions in the treatment group was 7.14%,but the difference between the two groups was not statistically significant(P>0.05).3.2 Experimental part3.2.1 Comparison of serum ALT,AST,HA and LN of TX-J mice in each group:(1)Compared with the normal group,the contents of ALT,AST,HA and LN in the model group were all increased(P<0.01).(2)Compared with the model group,the contents of ALT,AST,HA and LN in the PCA group and GDFMD low,medium and high dose groups were all decreased(P<0.01).(3)Compared with the PCA group,ALT and AST in the GDFMD low-dose group increased(P<0.05),and the content of HA and LN increased(P<0.01).(4)Compared with the high-dose GDFMD group,the ALT activity of the low-dose GDFMD group was increased(P<0.05),and the content of AST,HA and LN increased(P<0.01);the AST of the middle-dose GDFMD group increased(P<0.01).3.2.2 Comparison of serum SOD and MDA of TX-J mice in each group:(1)Compared with the normal group,the SOD content of the model group decreased,and the MDA content increased(P<0.01).(2)Compared with the model group,the SOD content of PCA group and GDFMD low,medium and high dose group increased(P < 0.01),GDFMD low dose group(P<0.05),PCA group and GDFMD medium and high dose group MDA content decreased(P<0.01).(3)Compared with the PCA group,the SOD content of the GDFMD low-dose group decreased,and the MDA content increased(P<0.01).(4)Compared with the GDFMD high-dose group,the GDFMD low-dose group had lower SOD content and higher MDA content(P<0.01).3.1.3 Comparison of mitochondrial ATP,ROS and MMP in liver tissue of TX-J mice in each group:(1)Compared with the normal group,the ROS content of the model group increased,while the ATP content and MMP decreased(P<0.01);(2)Compared with the model group,GDFMD low-dose group decreased ROS content(P < 0.05);GDFMD medium,high-dose group and PCA group decreased ROS content,while ATP content and MMP changed increased(P<0.01);(3)Compared with GDFMD high-dose group,GDFMD low-dose group ROS content increased,ATP content and MMP decreased(P<0.01);ROS content increased in GDFMD middle-dose group(P<0.05);ROS content increased in PCA group,MMP decreased(P<0.01),ATP content decreased(P<0.01)0.05);(4)Compared with the PCA group,the ROS content in the GDFMD low-dose group increased,while the ATP content and MMP decreased(P<0.01).3.1.4 Comparison of the pathological morphology of the liver tissue of TX-J mice in each group: Observed under a microscope,the cell structure of the normal group was normal,and no obvious pathological changes were seen.Compared with the normal group,the model group showed liver cell degeneration and necrosis,inflammatory cell infiltration,unclear liver lobule structure,and obvious fibrous tissue deposition.Compared with the model group,the histopathological recovery of the GDFMD low-dose group was not obvious,while the PCA group,and the GDFMD high and medium-dose groups reduced fibrous tissue deposition to varying degrees.Among them,the GDFMD high-dose group had the most obvious effect.3.1.5 The expression levels of JNK,p-JNK,p-JNK/JNK in each group of TX-J mice:(1) Compared with the normal group,the expression levels of p-JNK,p-JNK/JNK in the liver tissue of the model group were up-regulated(P<0.01).(2)Compared with the model group,the expression levels of p-JNK and p-JNK/JNK in the PCA group and GDFMD low,medium and high dose groups were down-regulated(P<0.01).(3)Compared with the PCA group,the expression levels of p-JNK and p-JNK/JNK in the GDFMD low-dose group were up-regulated(P<0.01).(4)Compared with the high-dose GDFMD group,the expression levels of p-JNK and p-JNK/JNK were up-regulated in the low-dose GDFMD group(P<0.01);the expression levels of JNK and p-JNK/JNK were up-regulated in the medium-dose GDFMD group(P<0.05);The expression levels of JNK and p-JNK/JNK were up-regulated in the PCA group(P<0.05,P<0.01)3.1.6 The expression levels of Caspase-3,Bcl-2,and Bax in TX-J mice in each group:(1)Compared with the normal group,the expression of Caspase-3 and Bax in the liver tissue of the model group increased,and the expression of Bcl-2 decreased(P<0.01).(2)Compared with the model group,the expression of Caspase-3 and Bax in the PCA group decreased(P<0.05),and the expression of Bcl-2 decreased(P<0.01,P<0.05);the expression of Caspase-3 and Bax decreased in the GDFMD medium-dose group(P<0.05).0.05,P<0.01),the expression of Bcl-2 decreased(P<0.01);the expression of Caspase-3 and Bax decreased in the GDFMD high-dose group(P < 0.01),and the expression of Bcl-2 decreased(P < 0.01).(3)Compared with PCA group,Caspase-3expression in GDFMD low-dose group GDFMD low-dose group was up-regulated,Bcl-2 expression was down-regulated(P<0.05),and Bax expression was up-regulated(P<0.01).(4)Compared with the GDFMD high-dose group,the GDFMD low-dose group Caspase-3 expression was up-regulated,Bcl-2 expression was down-regulated(P<0.05),Bax expression was up-regulated(P<0.01);Bax expression in the penicillamine group was up-regulated(P<0.01).4 Conclusion(1)After a long course of treatment,GDFMD combined with western medicine treatment can improve clinical efficacy,reduce LSM,reduce liver function and liver fibrosis,and improve the clinical symptoms and signs of patients,and there are fewer clinical adverse reactions.((2)GDFMD can effectively improve the pathology of liver fibrosis in TX mice,reduce ALT,AST,HA,LN,MDA content,increase SOD activity,and reverse WD liver fibrosis;(3)GDFMD can reduce ROS content,increase ATP content,increase mitochondrial membrane potential,restore damaged mitochondrial function,and reduce oxidative damage;(4)GDFMD can down-regulate the level of key phosphorylated proteins in the JNK signaling pathway and the expression of Caspase-3 and Bax,up-regulate the expression of Bcl-2,and inhibit hepatocyte apoptosis.