| Objective:1.To investigate and analyze the clinical efficacy of Gandouling(GDL)tablets in improving neurological function impairment by the regulation of autophagy in Wilson’s disease(WD)patients,and provide a further effective clinical evidence for GDL on Wilson disease.2.To explore the pharmacodynamic mechanism and target of GDL in the treatment of Wilson’s disease,by observing the behavior,detecting the mitochondrion structure and the levels of related mitophagy genes and proteins in mitophagy signaling pathway in TX model mice of WD.Methods:1.Sixty WD Patients exhibiting neurological impairment were randomized into two groups for a 8-coures trial period:a GDL group(n=30)and a Control group(n=30).Additionality,thirty healthy subjects were selected as a reference.Individuals in the GDL group were given GDL+DMPS.The internal dose of GDL was 0.09g/kg/day.For the intravenous DMPS,the internal dose was 5 mg/kg/day,and then gradually increased to 20mg/kg/day during the period of 2 week.Each treatment course was treated with DMPS for 6 consecutive days,then stopped 2 days to supply zinc.In the case of Control group,all patients were given intravenous DMPS and oral zinc.As for the usage and dosage of the drugs,there were the same as those in Control group.During the treatment,the levels of p62,and LC3/LC3Ⅱ was measured by ELISA and RT-PCR.The scales of GAS and BI were used to assesses the neurological impairment of WD.Moreover,24-h urine copper excretion,hematological and biochemical parameters such as aminotransferase,bilirubin,albumin,creatinine,and blood urea nitrogen levels,and instances of adverse effects were also recorded and analyzed.2.Thirty-nine TX mice(age=4 months)were randomized into 3 groups:Wilson group,penicillamine(DPA)group and Gandouling(GDL)group,with 13 mice in each group.12 DL mice were divided into Control group.Mice in GDL group were given GDL at a dose of 0.486 g/kg/d.For the mice in DPA group,penicillamine was given by at a dose of 0.09g/kg/d.8 weeks were performed for the administrations.Morris water maze,traction test,and pole test were used for the evaluation of animal behaviors.SOD,GSH-Px,and MDA were detected by ELISA.DCFH-DA probe was conducted for the detection of ROS and JC-1.Mitophagy was detected by transmission electron microscopy.Pink1、parkin、NIX、FUNDC1,immunofluorescence(IF).RT-qPCR and WB were used for the detection of p62,and LC3,respectively.Results:1.clinical research①Traditional Chinese medicine syndromes(TCMS):Relative to those at baseline,the TCMS scores in all WD patients declined obviously(P<0.01);Obvious differences in two groups were observed at 8-course follow-up time points(P<0.05).②GAS and BI scores:Compared with baseline,the GAS scores in GDL groups decreased in all the courses of treatment(P<0.05 and P<0.01).As for those in Control group,the GAS scores slightly elevated in the first 2 courses.After 4 courses of treatment,it began to gradually declined(P<0.01).Obvious differences in GAS were found between them(P<0.01).Correspondingly,the BI scores in almost all WD patients increased slightly after 4 courses of treatment(P>0.05).Then they further elevated after 8 courses of treatment.Clear differences were found between them(P<0.01).③Serum p62 and LC3:Relative to those at baseline,the levels of serum p62 in all groups were slightly declined in the first 2-course,and then in tune increased at 8 follow-up time(P<0.01).As for the indicator of LC3,the level in most WD patients were slightly increased in the first 2 courses,and then decreased at 8 follow-up time(P<0.01),relative to those at baseline.Additionality,relative significance in the two indictors were observed in GDL group at 8-course treatment(P<0.01).④The p62 and LC3Ⅱ mRNA:Relative to those normal people,the p62 mRNA in most of the WD patients were all significantly decreased.Relatively,the LC3Ⅱ mRNA was significantly increased(P<0.01).After 8 courses treatment,they were all changed significantly(P<0.01).Clear differences were found between them(P<0.05,P<0.01).⑤The correlation of LC3Ⅱ,GAS,TCMS and BI:The correlation in LC3Ⅱ and GAS,TCMS and BI was analyzed.It revealed that there were positive correlations between GAS,TCMS and LC3II.Other more,negative correlation between BI and LC3Ⅱ in TCMS was also observed.⑥Ceruloplasmin and urinary copper levels:Serum ceruloplasmin levels did not significantly differ between patients on either regimen at the 8-coures follow-up time point.After 2 courses of treatment,24-hour urinary copper excretion sharply elevated for those patients in the GDL group(P<0.01).After 4-course of treatment,it then turned to decreased.Significance in 24-hour urinary copper excretion were observed in either regimen at each follow-up time point(P<0.05,P<0.01).⑦Liver function and kidney function:The ALT and AST levels were slightly increased at first 2 courses(P<0.05),After 4-course therapy,they began to declined(P>0.05).No clear differences were found between them(P>0.05).The levels of TBIL and ALB all showed obvious improvement in most of the WD patients.Significances in TBIL were found at the 8-course time(P<0.05).Renal function remained stable throughout copper removal therapy in all patients.⑧Blood cells:The WBC counts at 2 and 4 courses therapy in GDL group were decreased,compared to the baseline.However,it still failed to recover to baseline at 8 courses.Clear differences were found at 4,8-course follow-up(P<0.05,or P<0.05).2.Animal experiment(1)Behaviors:compared with control group,the TX model mice in Wilson group sharply improved in the time of escape latencies,the frequency across the platform,the traction test,and the times of the pole test(P<0.01).After GDL interruption,these behaviors indicators were all improved significantly(P<0.01).(2)The effect of autophagy①HE:Regular hippocampal cells in Control group were observed.As for those in Wilson model group,amounts of apoptotic cells were found.Additionality,few apoptotic cells and abnormal form of cells morphology in GDL group were observed.②Transmission electron microscopy:In Wilson group,amounts of mitochondria displayed cristae decrease swelling,or mitochondrial vacuolation.After GDL treatment,few mitochondrial swellings,vacuolization,or autophagosomes were observed,and no obvious mitochondria ultrastructure abnormalities were found.③Immunofluorescence:the LC3 level in Wilson group was sharply increased(P<0.01).correspondingly,the p62 level declined significantly.After GDL interruption,these results can be changed(P<0.05).④RT-qPCR:In the Wilson group,the LC3Ⅱ mRNA levels were significantly elevated.Additionality,the p62 was remarkably declined(P<0.01).After GDL intervention,these indicators were all significantly reversed(P<0.05 or P<0.01).⑤WB:The rate of LC3II/I was elevated significantly in the Wilson group.Relatively,the p62 was remarkably declined(P<0.05 or P<0.01).GDL intervention could improve these indicators significantly(P<0.05).(3)The effect of mitophagy①The level of ROS and JC-1:The release of ROS in Wilson group was sharply elevated(P<0.01).After GDL interruption,these indicators were all decreased significantly(P<0.01).Correspondingly,the JC-1 levels in Wilson group increased significantly(P<0.01).After GDL intervention,this indicator was significantly reversed(P<0.01).②The level of SOD,GSH-Px,MDA:The SOD,GSH-Px levels dropped sharply in Wilson group(P<0.01).Relatively,the level of MDA elevated remarkably.After GDL interruption,these indicators were all inproved significantly(P<0.01).③RT-qPCR:The expression of pink1、parkin、NIX、FUNDC1 mRNA were significantly elevated in the Wilson group(P<0.01).After GDL intervention,these indicators were all significantly reversed(P<0.05 or P<0.01).④WB:The proteins level of pink1、parkin、NIX、FUNDC1 were significantly elevated in the Wilson group(P<0.01).GDL intervention could improve these indicators significantly(P<0.01).Conclusion1.In this study,we found that autophagy was involved in the development of Wilson disease,and GDL intervention can significantly inhibit the excessive autophagy in neurological Wilson disease.2.The significant correlations between autophagy and the neurological impairment of WD patients was found,and GDL intervention can significantly reverse and improve the impairment in neurological Wilson disease.3.Amounts of neuron cells damage with obvious mitochondrial damage were observed in TX model mice.LC3 and p62 levels changed significantly,and GDL could reverse the expression of these indictors,and improve the neuron damage.4.Significantly abnormalities in the levels of ROS,SOD,GSH-PX,MDA and related mitophagy proteins(pink1,parkin,FUNDC1,and NIX)were detected in the TX model mice of Wilson disease.These abnormalities indicate that excessive mitophagy is involved into the pathogenesis of WD.5.Significantly abnormalities in the indictors of ROS,JC-1,SOD,GSH-PX,MDA and related mitophagy proteins were all reversed by GDL intervention.These results indicates that GDL could inhibit excessive mitophagy through the regulation of the mitophagy pathway in the TX mouse brain of WD. |
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