The Research Of Tcm Tongfuyangsui Decoction On Autophagy Effect And Mechanism Of SH-SY5Y Cell Induced By High Copper

Wilson's disease is an autosomal recessive disorder caused by mutations in the ATP7B gene.These mutations can lead to disorders of copper metabolism,resulting in abnormal deposition of copper in the liver,brain and other other organs and tissues,with main clinical manifestations of liver cirrhosis,neurological/psychiatric symptoms and movement disorders.Imaging studies suggest that lesions in brain-type patients were mainly located in the basal ganglia.Experimental studies had also found that high copper induced degeneration of dopaminergic neurons in the substantia nigra in mice.These demonstrations suggest that the damage of dopaminergic neurons in the basal ganglia induced by high copper is a significant mechanism of brain damage among WD patients.Therefore,to study the mechanism of copper-induced dopaminergic neuron injury and possible ways of intervention is an essential scientific issue in the therapeutic study of brain-type WD patients.The excessive accumulation of copper in cells can cause oxidative stress reaction and the promotion of reactive oxygen species?ROS?releasing.ROS accumulation in cells can induce autophagy through multiple pathways.Copper-dopamine complexes can also induce mitochondrial autophagy in dopaminergic neurons of rats.The treatment of WD with metal complexing agents cannot be tolerated by many patients because of its toxicity and adverse reactions,which seriously affects the condition and prognosis of the patients.This paper unit has achieved significant efficacy in the treatment of WD patients with heat-clearing,detoxifying,and phlegm-inducing with dampness-prevention methods.On the basis of this,using drugs for promoting blood circulation and nourishing marrow and treating brain-type WD patients with the method of“tongfuyangsui”have markedly lightend clinical symptoms.Experimental study found that TCM TongFuYangSui decoction could restrain the expression of ASM,Cer,P38,JNK and other proteins in brain tissue of WD animal model of TX mice,and it could relieve copper damage to brain tissue.However,these studies have not been able to elucidate the specific therapeutic mechanism of TCM TongFuYangSui decoction against high-copper-induced dopaminergic neuron damage.Since the hypercopper environment induces autophagic apoptosis of dopaminergic neurons,we hypothesize that TCM TongFuYangSui can protect neuronal by regulating autophagy-related pathways of dopaminergic neurons to block high copper-induced dopaminergic neuronal damage.So as to play a protective role in neurons to alleviate the brain injury symptoms of WD patients.Objective:To explore TCM TongFuYangSui decoction on autophagy effect and mechanism of SH-SY5Y cells induced by high copper environment,to provide new therapeutic targets and research ideas for the prevention and treatment of brain-type WD by Chinese medicine.Methods:?1?The cell viability of SH-SY5Y cells at different time points was measured by MTT at different doses of CuSO₄ and the concentration and time of CuSO₄ modeling were screened out to simulate the cell model of WD.?2?The effect of the rabbit's serum containing TCM TongFuYangSui decoction and Butylphthalide on the survival rate of SH-SY5Y cells in model group was studied at different concentrations and different time points by atomic absorption spectrometry and the concentration of serum and the time of action were screened out.?3?Detection of cell LDH leakage rate using lactate dehydrogenase LDH release assay.?4?The content of reactive oxygen species?ROS?in the cells was measured by flow cytometry?FCM?using dichloro-fluorescein-acetoacetate?DCFH-DA?.?5?Detection of cell mitochondrial membrane potential using fluorescent dye JC-1.?6?Observing the formation of autophagic vacuoles in cells using AO/EB staining.?7?The formation of autophagosomes was observed by transmission electron microscopy.?8?Quantitative analysis of autophagy by flow cytometry with MDC staining.?9?The expression of Cyt-c was detected by Western Blot method.?10?Western blot was used to detect the expression of autophagy-related proteins LKB1,AMPK,p-AMPK,TSC2,LC3A/B,mTOR,P27,ULK1 and p-ULK.?11?The expression of autophagy-related genes LKB1,AMPK,TSC2,mTOR,P27,ULK1 were detected by Real-time PCR method.Results:?1?The results of MTT showed that the damage of CuSO₄ to SH-SY5Y cells showed certain dose-dependent and time-dependent manner?P<0.01?.The survival rate of SH-SY5Y cells showed a decreasing trend with the increase of CuSO₄ dose and the prolongation of the action time?P<0.01?.?2?The results of MTT showed that 10%of the rabbit's serum containing TCM TongFuYangSui decoction and 10%of the rabbit's serum containing Butylphthalide could significantly inhibit CuSO₄-induced cell death,which was significantly different from the model group?P<0.01?.?3?The leakage rate of LDH increased significantly after induce by CuSO₄?P<0.01?,the rabbit's serum containing TCM TongFuYangSui decoction could significantly reduce the LDH leakage?P<0.05?and the rabbit's serum containing Butylphthalide could significantly reduce the LDH leakage in SH-SY5Y cells damaged by CuSO₄?P<0.01?.?4?DCFH-DA fluorescence staining showed that CuSO₄ significantly increased ROS production in SH-SY5Y cells?P<0.01?.Rabbit serum containing TCM TongFuYangSui decoction and Butylphthalide could significantly inhibit intracellular ROS production induced by CuSO₄?P<0.01?.?5?JC-1 staining showed that CuSO₄ induced the decrease of mitochondrial membrane potential of SH-SY5Y cells?P<0.01?,while rabbit serum containing TCM TongFuYangSui decoction and Butylphthalide inhibited the mitochondrial membrane potential induced by CuSO₄ in a dose-dependent manner?P<0.05?.?6?The results of AO/EB staining showed that the control group SH-SY5Y cells had regular morphology,and most of the nuclei were uniformly green after AO staining.In the model group,most of the nuclei chromatin showed shrinkage and shrinkage,the cell morphology changed,and some cells showed strong orange/red fluorescence.After the protection of the rabbit's serum containing TCM TongFuYangSui decoction and the rabbit's serum containing Butylphthalide the few cells were stained with orange-red fluorescent,which indicated that the degree of cell injury was significantly decrease,it is the same as normal cells.?7?Transmission electron microscopy results showed that the mitochondria in the control group had normal mitochondria,complete morphology,normal distribution in the cells,and less number of autophagosomes.Compared with the control group,the chromatin condensation and mitochondrial number were found in the model group.Reduction,mitochondrial structure disorder and more vacuolization,some vacuoles visible content,and some mitochondria were wrapped into vesicles,autophagosome larger,and the number increased significantly.Compared with the model group,no clear vacuoles were found in the TCM TongFuYangSui decoction and Butylphthalide cells.The morphological structure of the cells was close to the control group.?8?The results of MDC staining showed that the fluorescence intensity of the model group was significantly increased compared with the control group?P<0.01?,and the fluorescence intensity of the rabbits with the serum of the group containing TCM TongFuYangSui decoction and Butylphthalide rabbit was significantly reduced compared with the model group?P<0.01?.?9?Western blot results showed that CuSO₄ induced the release of Cyt-c,while the TongFuYangSui decoction and the rabbit's serum containing Butylphthalide dose-dependently inhibited the release of Cyt-c induced by CuSO₄?P<0.01?.?10?Western blot analysis indicated that the expression of LKB1,AMPK,p-AMPK,TSC2,LC3A/B,P27,ULK1 protein was significantly increased and the expression of mTOR and p-ULK protein was significantly decreased compare with the control group induce by high copper?P<0.01?.The expression of LKB1,AMPK,p-AMPK,TSC2,LC3A/B,P27,ULK1 protein were significantly decreased and the expression of mTOR,p-ULK protein was significantly increased in the rabbit's serum containing TCM TongFuYangSui decoction and the rabbit's serum containing Butylphthalide compare with the model group?P<0.01?.?11?Real-time PCR results showed that the expression of LKB1,AMPK,TSC2,P27,ULK1 genes was significantly increased and the expression of mTOR gene was significantly decreased compare with the control group induce by high copper?P<0.01?.The expression of LKB1,AMPK,TSC2,P27,ULK1?P<0.05?genes was significantly decreased and the expression of mTOR and gene was significantly increased in the rabbit's serum containing TCM TongFuYangSui decoction and the rabbit's serum containing Butylphthalide compare with the model group?P<0.01?.Conclusion:High copper can induce autophagic apoptosis in SH-SY5Y cells by inducing intracellular mitochondrial oxidative stress and upregulating the expression of autophagy-related proteins and genes LKB1,AMPK,p-AMPK,TSC2,LC3A/B,P27,ULK1 and down-regulation of autophagy-related proteins and expression of mTOR,p-ULK.But TCM TongFuYangSui decoction can inhibit the occurrence of autophagy and inhibit the expression of autophagy-related proteins and LKB1,AMPK,p-AMPK,TSC2,LC3A/B,P27,ULK1and up-regulation of autophagy-related proteins and expression of mTOR,p-ULK by regulating the LKB1-AMPK signaling pathway.High copper-induced neuronal damage is cut off,thereby exerting a neuroprotective effect.