The Research Of GDT Decoction On Mitophagy Effect And Mechanism Of HT-22 Cell Induced By High Copper

Wilson’s（Wilson’s Disease,WD）is an autosomal recessive hereditary dis ease with copper metabolism disorder due to mutation of ATP7B gene on chrom osome 13.The disease is complicated and changeable,so it can be divided into l atent type（presymptomatic type）,brain type,visceral type,intracerebral visceral t ype and so on.Brain type is the most common type,accounting for about 60%of the total number of patients.The clinical manifestations of brain-type WD pati ents are mainly motor disorders,mental disorders and Parkinson’s syndrome.Thei r clinical manifestations are closely related to the deposition of copper ions in th e brain,resulting in the involvement of related cognitive areas such as hippocam pus and basal ganglia.Previous studies have shown that the impairment of learni ng and memory ability of copper-loaded rats may be related to the activation of mitogen-activated protein kinase（MAPK）signaling pathway induced by high co pper and the injury of hippocampus-associated neurons.Therefore,hippocampal cel ls（HT-22）were selected for related study in this experiment.Therefore,it is an important scientific problem to study the mechanism of hippocampal injury indu ced by high copper and its possible intervention pathway in patients with brain-t ype WD.The accumulation of copper in cells can increase the release of reactive oxygen species（ROS）,induce oxidative stress and induce autophagy.The traditi onal western medicine,such as penicillamine,sodium dimercaptopropionate and s o on,has a good effect on removing copper from WD,but the adverse reaction is large.It may even lead to further deterioration of neuropsychiatric symptoms,whichhugely affects the status and prognosis of the patients with this disease.Ga ndou decoction of clearing heat and detoxification and clearing Fu and promoting dampness was used in the treatment of WD patients.Previous studies have foun d that Gandou decoction can down-regulate the expression of ASM,Cer,NK and other proteins in the brain of TX mice,thus improving the symptoms of brain i njury in TX mice.However,these studies can not fully clarify the specific therap euticmechanism of Gandou decoction with high copper-induced neuronal injury.Hi gh copper can induce autophagy death of neurons,so we can assume that Gando u decoction protects neurons by regulating mitochondrial autophagy-associated pro tein to block the injury of neurons induced by high copper autophagy.The symp tomsof brain injury in patients with WD were improved.Objective:to study the effect of Gandou decoction on mitochondrial autophagy induced by high copper in HT-22 cells and its mechanism so as to provide new therapeutic means and research ideas for those diagnosis and treatment of cerebral WD patients by traditional Chinese medicine（TCM）.Methods:（1）MTT method was used to detect the effect of different doses of CuSO₄ on t he survival rate of HT-22 cells at different time points.The concentration and ti me of CuSO₄ model were screened to simulate the WD cell model.（2）LDH release of LDH was used to test the leakage of LDH.（3）the content of ROS in cells was detected by dichlorodihydrofluorescein aceto acetate（DCFH-DA）probe.（4）fluorescent dye JC-1 was used to detect the mitochondrial membrane potentia l.（5）the morphological changes of the cells were observed by electromicroscopy.（6）the expression of mitochondrial autophagy-associated protein（PINK1,Parkin,L C3 A/B,BNIP3L）was detected by Western Blot.Results:（1）the results of MTT showed that the harm of HT-22 cells induced by CuSO₄ showed a certain dose-time relationship.With the increase of dose or time of CuSO₄,the survival rate of HT-22 cells decreased.（2）the results of MTT showed that the leakage rate of LDH in HT-22 cells treated with CuSO₄ increased significantly（P<0.01）.Gandou decoction decreased the leakage rate of LDH in HT-22 cells damaged by CuSO4（P<0.01,P<0.05）.（3）Flow cytometry showed that CuSO₄ significantly increased the production of reactive oxygen species in HT-22 cells after DCFH-DA fluorescence staining,an d Gandou decoction serum significantly inhibited CuSO4-induced intracellular RO S production（P<0.01,P<0.05）,while Gandou decoction serum significantl y inhibited CuSO₄-induced intracellular ROS production（P<0.01,P<0.05）.（4）JC-1 staining showed that the mitochondrial membrane potential of HT-22 ce lls induced by CuSO₄ was significantly decreased（P<0.01）.10%and 15%G andou decoction serum inhibited the decrease of mitochondrial membrane potentia l induced by CuSO₄（P<0.01）.（5）the results of transmission electron microscopy showed that the morphology of mitochondria in normal group was intact,the size of mitochondria was norma l,and the number of autophages was less.In the model group,the number of m itochondria decreased,and the structure was disordered and vacuolated,the conte nts of some vacuoles could be seen in some vacuoles,and some mitochondria w ere wrapped in vesicles,and the autophagy was larger and more number of auto phages was also increased obviously.In Gandou decoction group,a small numbe r of vacuoles were found in the cells,and the morphology and structure of mito chondria were close to the normal state,and autophagy decreased significantly co mpared with the model group.（6）the results of Western blot showed that the expression of PINK1,Parkin,LC3 A/B and BNP3L protein in the model group was significantly higher than that in the normal group（P<0 01）.Compared with the model group,the expression of Parkin,LC3 A/B and BNP3L protein in Gandou decoction serum group were significantly lower than those in the model group（P<0 01）.Compared with the model group,the expression of PINK1 protein in 10%and 15%Gandou decoction serum group was significantly lower than that in the model group（P<0.01）.Conclusion:High copper can up-regulate the expression of autophagy-relatedprote ins and genes PINK1,Parkin,LC3 A/B and BNIP3L by inducing mitochondrial oxidative stress in HT-22 cells,and inducing autophagic death in HT-22 cells.Ga ndou decoction could inhibit the oxidative stress of mitochondria of HT-22 cells induced by high copper,down-regulate the expression of autophagy-associatedprot ein PINK1,Parkin,LC3 A/B,BNIP3L,inhibit the occurrence of autophagy,and block the injury of neurons induced by high-copper.So as to play a neuroprotect ive role.