| Wilson's disease is an autosomal recessive copper metabolic disorder most happened during teenager period.It is also one of the few neurodegenerative diseases that can be treated.Clinical research showed that patients with brain-type WD who had major clinical manifestations of limb torsion,disorder of walking and swallowing,had benefited less from routine copper-chelating therapy,which had bring severe limitations to their learning,work and life,and even become life-threatening.Therefore,the treatment of the with refractory brain-type WD patients is a difficult problem in the current clinical issue.In the previous study,we created Tong Fu Yangsui decoction to treat brain-type WD patients based on the research of Chinese medicine dialectical,and had obtained satisfied effect during the clinical utilization.A series of studies on its therapeutic mechanism from the perspective of neuronal apoptosis and autophagy had been achieved during the past decades.Recent studies had found that intracellular iron homeostasis imbalance caused by disorder of ceruloplasmin(CP)synthesis and ferrous oxidase activity was a significant mechanism of refractory brain-type WD neuronal damage addition to directly copper accumulation.This was also the main reason for the limited efficacy of routine copperchelating therapy towards these patients.Recent studies had also found that iron-dependent non-programmed cell death,ferroptosis,was closely related to the pathogenesis of varieties of neurodegenerative diseases,and was highly correspond with the neuronal injury mechanism of brain-type WD.Further studies had found that p62-Keap1-NRF2 was an important regulatory pathway of ferroptosis,which could inhibit ferroptosis induced by ferroptosis activators such as erastin to perform its cytoprotective effects.Thus,it is a potential treatment target for refractory brain-type WD..Based on this,this study hypothesized that Tong Fu Yang Sui decoction has neuroprotective effects by inhibiting ferroptosis by regulating the p62-Keap1-NRF2 pathway.We try to study the protective mechanism of Tong Fu Yang Sui decoction from the perspective of neuronal injury in WD model TX mice,to provide new treatment methods and ideas for the therapeutic mechanism for refractory brain-type WD patients.[Objective] 1.Part I: To observe the level of ferroptosis oxidative stress and the expression of p62-Keap1-NRF2 signaling pathway in cortical neurons of WD model TX suckling mice.2.Part II: To observe the regulatory effect of TFYSD on the level of ferroptosis oxidative stress and the expression of p62-Keap1-NRF2 signaling pathway in the cortical neurons of WD model TX suckling mice.[Methodology] 1.(1)Grouping conditions of Part I: A.Normal control group: The cortical neurons cultured from the cortex of DL suckling mice born within 24 hours.B.Model group: The cortical neurons cultured from WD model TX suckling mice born within 24 hours.C.Fer-1 group: Ferrostatin-1,a ferroptosis inhibitor with a final concentration of 60 n M,was added to the model group.D.HCQ group: the model group was treated with Hydroxychloroquine,an autophagy inhibitor with a final concentration of 20?M.E.FMK group: Apoptosis inhibitor Z-VAD-FMK with a final concentration of 50?M was added to the model group.F.Nec-1 group: Necrostatin-1,a necrosis inhibitor with a final concentration of 490 n M,was added to the model group.Cells in each group were collected after 24 hours of administration,and relevant indicators were detected.(2)Grouping conditions of Part II: A.Normal control group: The cortical neurons cultured from the cortex of DL suckling mice born within 24 hours.B.Model group: The cortical neurons cultured from TX suckling mice born within 24 hours.C.TCM group: Rabbit serum containing TFYSD with a final concentration of 10% was added into the model group;D.DFO group: Deferoxamine,an iron chelating agent with a final concentration of 300?M to the model group;E.ERA group: Ferroptosis activator erastin with a final concentration of 30?M was added to the TCM group.2.MTT assay was used to detect the survival rate of TX suckling mice cortical neuron and the cell survival rate of each group after 24 h of drug administration.3.ICP-MS was used to detect the level of Fe and Cu element level of each group.4.Elisa was used to detect the expression level of iron-related proteins.5.Lactate dehydrogenase release assay was used to detect the LDH level in the supernatant of cells in each group.6.The levels of SOD,MDA,GSH and GSSG in cells of each group were detected by biochemistry-microplate assay.7.Cell ROS levels in each group were determined by flow cytometry.8.Western Blotting was used to detect the expression of p62,keap1,NRF2,HO1,NQO1 and FTH1 in each group.[Results] 1.MTT: Compared with the normal control group,the survival rate of cells in the model group was significantly reduced,which was about 50% at 7 days of isolation and culture.The cell survival rate of 10% TFYSD group was higher than 5%,15% and 20%.2.Cell survival rate: Compared with the model group,the survival rate of the Fer-1 group,the HCQ group and the FMK group was significantly improved(P<0.01),and that of the Nec-1 group was improved(P<0.05).The survival rate of cells in TCM group,DFO group and ERA group was significantly increased(P<0.01).3.Iron and copper element level: Compared with the normal control group,the element level of iron and copper were significantly increased in the model group(P<0.01).Compared with the model group,the element level of iron and copper of Fer-1 group had no statistical differences(P>0.05).Compared with the model group,the element level of iron in TCM group was significantly decreased(P<0.01),and the element level of copper in TCM group was decreased,while the difference had no statistical significance(P>0.05).4.Iron metabolism-related proteins: Compared with the normal control group,the expressions of DST,TRF,TFR and FER proteins in the model group were significantly down-regulated(P<0.01).Compared with the model group,DST and FER protein expressions were significantly up-regulated in the Fer-1 group,DST,TRF and FER protein expressions were significantly up-regulated in the TCM group(P<0.01),and TFR protein was up-regulated(P<0.05).5.ROS level: Compared with the normal control group,the ROS level in the model group was significantly increased(P<0.01).Compared with the model group,the ROS of Fer-1 group,HCQ group,FMK group and Nec1 group decreased significantly(P<0.01).The cell ROS of TCM group,DFO group and ERA group decreased significantly(P<0.01).6.LDH leakage: Compared with the normal control group,LDH leakage was significantly increased in the model group(P<0.01).Compared with the model group,LDH leakage was significantly decreased in the Fer-1 group,HCQ group,FMK group and Nec-1 group(P<0.01).The LDH leakage rate was significantly decreased in TCM group,DFO group and ERA group(P<0.01).7.SOD level: SOD in the model group was significantly lower than that in the normal control group(P<0.01).Compared with the model group,SOD level of Fer-1 group,HCQ group and FMK group increased significantly(P<0.01),and SOD level of Nec-1 group increased significantly(P<0.05).Compared with the model group,SOD level of cells in the TCM group increased significantly(P<0.01).8.MDA level: Compared with the normal control group,MDA level of cells in the model group increased significantly(P<0.01).Compared with the model group,MDA levels in the Fer-1 group,HCQ group,FMK group and Nec-1 group decreased significantly(P<0.01).Compared with the model group,SOD levels of cells in the TCM group,DFO group and ERA group decreased significantly(P<0.01).9.GSH levels: Compared with the normal control group,GSH levels in the model group were significantly decreased(P<0.01).Compared with the model group,the GSH level of Fer-1 group and HCQ group increased(P<0.05).Compared with the model group,GSH level of cells in the TCM group and DFO group increased(P<0.05).10.GSSG level: Compared with the normal control group,GSSG levels of cells in the model group were significantly increased(P<0.01).Compared with the model group,the GSSG level in the Fer-1 group,HCQ group,FMK group and Nec-1 group decreased significantly(P<0.01).Compared with the model group,the GSSG levels of TCM group,DFO group and ERA group were significantly decreased(P<0.01).11.Western Blotting: Compared with the normal control group,Keap1 protein expression was significantly up-regulated in the model group(P<0.01),while p62,NRF2,NQO1,HO1 and FTH1 protein expression were significantly down-regulated(P<0.01).Compared with the model group,p62,NRF2,NQO1,HO1 and FTH1 proteins were significantly up-regulated(P<0.01)in Fer-1 group,and the expression of Keap1 protein was significantly down-regulated(P<0.01).In the TCM group,p62,NRF2,NQO1,HO1 and FTH1 proteins were significantly up-regulated(P<0.01),while keap1 protein was downregulated(P<0.01).[Conclusions] 1.Ferroptosis-induced oxidative stress injury is an essential mechanism of neuron damage of WD model TX mice.The protection of neurons based on the anti-ferroptosis pathway can reduce the level of oxidative stress of neurons and improve the survival rate of cortical neurons in TX suckling mice.2.Based on the p62-Keap1-NRF2 signaling pathway,the traditional Chinese medicine formula Tongfuyangsui decoction can reduce the level of oxidative stress in cells(NRF2 upgraded,keap1 downgraded),improve the survival rate of cells,and exert the protective effect of neurons,and its protective effect is in a concentration dependent relationship with the concentration.At the same time,Tongzhi Yangsuifang drug-containing serum also has the effect of reducing intracellular iron content to a certain extent. |
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