The Protective Effect Of Penicillamine Combined With Lipoic Acid On Mitochondrial Damage Of Neurons In Wilson Disease

ObjectiveThe aggravation of neurological symptoms,the high mortality and disability of patients with Wilson disease have become urgent issues among clinicians.Taking mitochondrial damage of WD brain neurons and therapy of copper removal combined with mitochondrial protection as key points,the present study explores the mechanism of mitochondria damage in WD cranial nerve cells and uses copper removal in combination with mitochondrial protection treatment to assess the changes of mitochondrial damage-related proteins and the regulatory mechanisms through the application of clinical cases,animal and cell experiments,aiming to regulate cell damage at organelle levels and repair mitochondrial function,in order to provide new ideas for clinical treatment of brain type WD.Methods1 Brain-type WD imaging analysis and clinical preliminary study1.1 Clinical and imaging methods of WD brain mitochondrial injuryFirstly,the clinical data of the cranial MRI scans of 364 patients diagnosed with brain type WD from January 2008 to December 2017 were retrospectively analyzed to reveal the injury characteristics.Then the clinical data,cranial MRI,MRS,blood lactic acid and other biochemical indicators of 58 patients with brain type WD clinically confirmed but with poor treatment outcome from January 2018 to December 2018 were selected for the study.Meanwhile,another 63 age-matched non-WD patients were selected as the control group to assess their brain MRI,MRS and blood lactic acid,hoping to detect the clinical evidence of mitochondrial injury in brain type WD.1.2 Retrospective analysis of PCA combined with LA in treatment of brain type WD patientsClinical PCA combined with LA or PCA alone was randomly selected to treat brain type WD.The number of cases with deterioration of neurological symptoms in two groups was compared one month after treatment.2 Effects of penicillamine combined with lipoic acid on mitochondrial damage of brain neurons in copper-loaded SD Rats / TX mice2.1 Animal experiments,study on mitochondrial injury of brain neurons in SD rats with copper loading and mitochondrial protective treatment with copper removalWD animal model was successfully established using copper-loaded rats,which were divided into five groups,namely,group A(SD control group),group B(copper-loaded SD model group),group C(PCA treatment group),group D(LA treatment group)and group E(combined treatment of LA and PCA).Then the biochemical indexes of serum copper were detected,the copper content in brain tissues was determined by AAS,and the mitochondrial electron microscopic structures of brain neurons were observed.LC3,Cyt C,XIAP and ERK1/2 protein expressions were assessed by immunohistochemistry and Western blot,searching for the evidence of copper loading on mitochondrial damage of brain neurons in animal experiments and confirming the application of copper removal combined with mitochondrial protection as an optimal treatment.2.2 Cell experiments,study on mitochondrial injury of brain neurons in TX suckling rats and mitochondrial protective treatment of copper removalThe cells were successfully cultured using primary TX model mouse nerve cell culture technique,and the rates were divided into five groups,namely,group A(DL control group),group B(TX suckling rat model group),group C(PCA treatment group),group D(LA treatment group)and group E(combined treatment of LA and PCA).The mathematical differential centrifugation method was used to obtain WD genetic model--TX suckling mouse mitochondria in brain tissues,flow cytometry was employed to detect ROS and mitochondrial membrane potential,spectroscopy and enzyme labeling were applied to assess mitochondrial complex and key enzyme activities,and Western blot was used to detect LC-3,Cyt C,XIAP,ERK1/2 protein expressions,determining mitochondrial damage in brain neurons and confirming the application of copper removal combined with mitochondrial protection as an optimal treatment.Results1 Clinical outcome1.1 Craniocerebral MRI findings of 364 cases of brain type WDRetrospective analysis of the clinical data of 364 WD patients showed a definite diagnosis of WD neurological symptoms.Craniocerebral MRI examination found abnormalities in at least one brain region,with the most common abnormalities in the putamina,followed by those in the pons and thalamus.The putamina was the most frequently damaged region in patients with brain type WD regardless of the clinical neurological symptoms.Brain damage in WD patients under the age of 10 was shown only in putamina;most brain injuries in WD patients with short course of disease were abnormal putamina,and the injuries gradually extended with the extension of the course of disease.1.2 Findings of MRI,MRS and blood lactic acid in 58 patients with brain type WDThe abnormal rate of brain injury in 58 cases of brain type WD craniocerebral injury patients was higher than that of healthy controls(P<0.05),except for that in cerebellum(P=0.228).MRS detection of temporal lobe of brain showed that the occurrence of abnormal Las peak and the abnormal NAA decline rate were significantly higher than those of healthy controls(P<0.001).Meanwhile,blood lactic acid and the ratio of lactic acid/pyruvic acid in WD patients under stationary state were markedly higher than those of healthy controls(P<0.001).1.3 Preliminary results of PCA combined with LA in treatment of brain type WD patientsThe aggravation of neurological symptoms occurred in only one case among the 23 cases of brain-type WD patients treated with PCA combined with LA,which,however,was detected in 6 cases among the 21 cases of brain-type WD patients treated with PCA alone.2 Experimental results2.1 Results of animal experiments2.1.1 Results of blood copper biochemistry and AAS detection of copper content in cerebral tissues of copper-loaded SD ratsDamage of liver function was detected in copper-loaded SD rats,and the serum copper and CP content decreased,which were in line with the characteristics of liver function and copper metabolism in WD patients.Copper-loaded SD rats were proved to be an accurate and reliable animal model for WD study.Besides,increased copper content in brain tissues of copper-loaded rats and unstable walking and other symptoms were traced during the experiments,which were similar to those of brain type WD patients.2.1.2 Results of electron microscopic structures of brain neurons of copperloaded SD ratsResults of electron microscope structures of brain neurons of copper-loaded SD rats showed that the mitochondrial damage in brain neurons in group B was more obvious than that in group A,and the mitochondrial structures of brain neurons in group E were ameliorated compared with those in group B,C and D.2.1.3 Immunohistochemistry and Western blot analysis of brain neurons in copper-loaded SD ratsAs shown by immunohistochemistry and Western blot,LC3 and Cyt C protein expression of group B were significantly up-regulated compared with those of group A(P<0.01).After treatment,LC3 and Cyt C in brain neurons in group E(n=7)were down-regulated compared with those in group B(n=7),group C and D(n=7)(P<0.01).ERK1/2 and XIAP protein expressions were markedly down-regulated compared with group A(P<0.01),and ERK1/2 and XIAP protein expressions in brain neurons of group E(n=7)increased compared with those of group B(n=7)and group C and D(n=7)after treatment(P<0.01).2.2 Results of cell experiments2.2.1 ROS and JC-1 of neuron cells of TX/DL suckling mice in each group detected by flow cytometryAs detected by flow cytometry,ROS release in TX mouse neurons increased and oxidative stress injury could be observed,while ROS production in neurons of Group E could be significantly reduced and oxidative stress injury was improved.The mitochondrial membrane potential level markedly decreased,which,however,could be raised with the addition of LA and PCA,and JC1 was mainly in a polymer state;the difference was statistically significant compared with group B(P<0.01).Besides,the fluorescence intensity of JC-1 in group E was apparently higher than that in group C or D.2.2.2 Results of mitochondrial complex and key enzyme activities of TX/DL suckling mouse neurons in each group detected by spectroscopy and enzyme markerThe ?-? activities of mitochondrial complex in TX rat neurons were found to be reduced,in which the decreased activities of complex ? were the most obvious.The activities of important dehydrogenases in mitochondria of TX rat neurons,such as malate dehydrogenase(NAD-MDH),pyruvate dehydrogenase complex(PDH)and cisaconitase(Aco)were all found to be reduced.The activities of NAD-MDH,PDH and Aco in TX rat neurons in group E were boosted compared with those in group B,and the difference was statistically significant(P<0.01).2.2.3 Mitochondrial proteins in neurons of TX/DL suckling mice in each group detected by Western blotWestern blot analysis showed that LC3 and Cyt C protein contents in neurons of group B significantly increased,while XIAP and erk1/2 proteins evidently decreased,and the differences were statistically significant(P<0.01).After treatment,LC3 and Cyt C protein expressions in group E were markedly down-regulated,while XIAP and ERK1/2 protein expressions were significantly up-regulated compared with those in group B(P<0.01).Conclusions1.The most common and the first involved site of brain injury in WD patients is the putamen,and the neuronal mitochondrial damage can be found in the detection of craniocranial MRS and blood lactic acid.2.Aggravation of neurological symptoms rarely appears in brain type WD patients treated for the first time with the selection of combined treatment of PCA and LA.3.The mitochondrial damage of brain neurons in copper-loaded SD rats is reflected in not only the abnormal mitochondrial structure,but also the abnormal expression of mitochondria-related proteins,such as LC3/Cyt C/XIAP/ ERK1/2.After the treatment of copper removal combined with mitochondrial protection,the indicators of mitochondrial damage can be significantly improved compared with those of the control and model groups.4.After cortical neuron culture of TX suckling mice,abnormalities can be detected in mitochondrial indicators,such as ROS,JC-1,mitochondrial complex,mitochondrial key enzymes and LC3/Cyt C/XIAP/ ERK1/2.After the treatment of copper removal combined with mitochondrial protection,mitochondrial damage indicators are all significantly improved compared with those of the control and model groups.5.A new approach to the treatment of brain type WD is copper removal combined with mitochondrial protection,which may be effective in preventing the aggravation of neurological symptoms and reducing the rate of death and disability.