The Mechanism Of Moxibustion Inhibit Ferroptosis In Parkinson’s Disease Through NCOA4-mediated Ferritintophagy Pathway

ObjectiveParkinson’s disease(PD)is a neurodegenerative disease caused by the loss of dopaminergic neurons in the dense part of substantia nigra,resulting in dopamine deficiency and the disorder of substantia nigra striatum pathway.Its main clinical features are resting tremor,limb stiffness,slow movement,abnormal posture and other nommotor symptoms.The population of PD is mainly in the middle-aged and elderly people,while in the group of age over 60 the morbidity reached 1%.As the world enters an aging society.experts predict that in the next 20 years,the population of PD patients may reach 14 million all over the world.As a chronic progressive disease,PD not only seriously affects the patients’ quality of life,but also causes a great economic burden to patients,which brings a very heavy burden to patients’ families and society.Therefore,it is of great clinical and social significance to conduct in-depth research on the mechanism and treatment of PD.There are many mechanism involved in PD,among which ferroptosis is the research hotspot.Ferroptosis is a novel mode of cell death characterized by the over-production of reactive oxygen species(ROS)and depletion of glutathione.At present,there are many ways of regulating ferroptosis.The previous researches of our research group found that the level of ferritinophagy increased in the process of ferroptosis in PD.However,how ferritinophagy affects ferroptosis in PD is still unclear.So this study took the key gene NCOA4 mediating ferritinophagy as the breakthrough point to deeply explore the mechanism of feritinophagy regulating feroptosis.At present,western medicine is mainly one of the clinical treatment of PD,but patients who take western medicine for a long time are prone to drug resistance and large toxic and side effects.As one of the essential parts of traditional Chinese medicine therapy,moxibustion has played a significant role in the clinical treatment of PD.Moxibustion therapy has the advantages of less side efects,no pain,and high,patient acceptance.Therefore,moxibustion in the treatment of PD has long-term therapeutic advantages and development potential.However,most of the current studies on moxibustion for PD still targets at symptoms remission,and few studies targets at the specific molecular mechanism.Therefore,while our research group deeply explored the pathogenesis of PD,we also explored the molecular mechanism of moxibustion in the treatment of PD,so as to provide more theoretical basis for moxibustion in the treatment of PD and the development of new drrugs in the future.Methods1.Cell experimentsIn this study,the MTS experiment was used to screen out the appropriate 6-OHDA concentration to intervene PC 12 cells to establish a PD cell model.Next,the changes in the levels of total iron,Fe2+,MDA,SOD and ROS levels in the cells were detected by iron ion concentration detection,MDA concentration detection,SOD concentration detection,and flow cytometry;the expression change of TH,GPX4,NCOA4 and FTHl proteins in each group were detected by Western Blot method;PCR technology was used to detect the expression levels of TH,GPX4,NCOA4 and FTH1 mRNA in each group of cells to verify whether the PD model was successful and whether there was ferroptosis in the model.Based on the successful model construction,suitable NCOA4 interference fragments were screened out.After the cells were transfected,the expression changes of TH,GPX4,NCOA4 and FTHI proteins in each group of cells were detected by Western Blot;the expression change of Fe2+,MDA and ROS were detected by iron ion concentration detection,MDA concentrtation detection,and flow cytometry to verify the effect of ferritinophagy on PD ferroptosis.2.Animal experiments2.1 PD rat model experimentsThe PD rat model was established by cerebral stereotactic injection of 6-OHDA.and the successful establishment of the PD model was verified by behavioral testing;on the basis of ensuring the successful establishment of the PD rat model,moxibustion was performed on the PD rats.To determine whether moxibustion has an improving effect on dopaminergic neurons in PD rats model by HE and immunohistochemistry;then,the iron ion concentration detection,MDA concentration detection,SOD concentration detection,and flow cytometry were used to detect various indicators of ferroptosis,to verify whether moxibustion has an inhibitory effect on PD ferroptosis;Western Blot and PCR were used to detect the changes of TH,GPX4,NCOA4 and FTH1 protein and mRNA expression levels to verify whether moxibustion has an effect on PD ferritinophagy pathway and ferroptosis mechanism.2.2 PD mouse model experiments2.2.1 Firstly,The PD mouse model was established by cerebral stereotactic injection of 6-OHDA,and the successful establishment of the PD model was verified by behavioral testing;on the basis of ensuring the successful establishment of the PD rat model,moxibustion was performed on the PD mouses.After treatment,iron ion concentration detection,MDA concentration detection,flow cyometry and Western Blot were used to detect various indicators of ferroptosis to verify whether moxibustion has an inhibitory effect on ferroptosis in PD mice.2.2.2 The rAAV-NCOA4 plasmid was constructed,and the most efficient plasmid was screened by PCR and Western Blot,then the plasmid was transfected into cells for rAAV virus packaging,and the PCR was used to verify the successful packaging of rAAV virus,next calculate the titer and the purity of rAAV virus.2.2.3 The NCOA4 knockout model mice were established by cerebral stereotactic injectionof rAAV virus,and the success of the knockout model was verified by PCR and Western Blot:based on the confirmation of the successful establishment of the NCOA4 knockout mouse model,6-OHDA was used to construct PD mouse model,and mouse behavioral experiments were used to verify the successful establishment of PD model On the basis of the previous model,moxibustion was used for treatment.After the treatment,the substantia nigra tissue of the mice in each group were collected,and then iron ion concentration,MDA concentration detection,flow cytometry,and Western Blot were used to detect the expression of Fe2+,MDA,ROS and GPX4 protein,and the changes of various indicators of PD ferroptosis were observed,so as to verify whether moxibustion played a role in ferroptosis in PD model through NCOA4-mediated ferritinophagy pathway.Results1.Results of cell experiments1.1 In this study,the optimal condition for constructing PD cell model was 50μmol 6OHDA intervening PC 12 cells for 24h.The experiment found that in the PD cell model,compared with the normal control group,the ferroptosis indexes such as total iron,Fe2+,MDA,ROS were all increased,and the expression levels of SOD and GPX4 were decreased,and the difference was statistically significant(P<0.05),indicating that ferroptosis exists in the PD cell model.At the same time,it was found that in the PD cell model group,compared with the normal control group,the protein and mRNA levels of NCOA4 were increased.and the protein and mRNA levels of FTH1 were decreased,indicating that the PD cell model ferritinophagy was activated.1.2 The expression of NCOA4 in PC12 cells was silenced by siRNA,the silencing effect of siRNA was detected by PCR,and the most effective siRNA-NCOA4 interference fragment was screened.After silencing the NCOA4 gene in cells,we found that compared with the PD model group,the levels of Fe2+,MDA and ROS in the siRNA-NCOA4 group were decreased,while the levels of TH.GPX4,and FTH1 were increased,and the difference was statistically significant(P<0.05),the experimental results showed that the degree of ferroptosis in the PD cell model was reduced after silencing NCOA4,indicating that NCOA4-mediated ferritinophagy pathway may promote ferroptosis.2.Results of animal experiments2.1 PD rat model experimentsThis study found that moxibustion can significantly improve the motor function of PD rats meanwhile,moxibustion improved the morphology of dopaminergic neurons and increased the number of dopaininergic neurons and TH-positive neurons in the substantia nigra of rats indicating that moxibustion has a therapeutic effect on PD rats;futher detection of the substantia nigra of rats in each group found that compared with the model group,the levels of total iron,Fe2+,MDA,ROS,and NCOA4 in the moxibustion group were higher,while the levels of SOD,GPX4 and FTHl increased,the difference was statistically significant(P<0.05),indicating that moxibustion can inhibit the ferroptosis phenotype in PD.and at the same time can regulate the NCOA4-FTH1 ferritinophagy pathway.We speculate that moxibustion may inhibit ferroptosis in PD through the NCOA4-FTH1 fernitinophagy pathway,but further experimental verification is needed.2.2 PD mouse model experiments2.2.1 Behavioral experiments found that moxibustion can significantly improve the motor function of PD mouse models.Further experiments found that compared with the model group,the levels of Fe2+,MDA and ROS in the substantia nigra of PD mice were increased,while the levels of GPX4 protein were decreased,and the difference was statistically significant(P<0.05),which is consistent witgh the previous rat experiments,indicating that moxibustion can inhibit ferritinophagy and feroptosis in PD mouse model.2.2.2 It was found that the best plasmid for silencing NCOA4 was PT-4188 and PT4188 was transfected into cells for virus packaging through PCR experiments.2.2.3 After silencing the NCOA4 gene in the substantia nigra of mice,we found that compared with the NC group,the levels of Fe2+,MDA,ROS,and NCOA4 in the substantia nigra of PD mice were decreased,and the expression level of GPX4 protein increased;after moxibustion treatment,compared with the shRNA-NC.OA4 group,the feroptosis indexes in the substantia nigra of the mice in the shRNA-NCOA4 group plus moxibustion group did not improve significantly,and the experimental results were not statistically sigificant(P>0.05).which indicated that moxibustion may be through NCOA4-mediated ferritinophagy pathway to regulate ferroptosis in PD.Conclusions1.6-OHDA can increase iron ion concentration and oxidative stress levels in PC 12 cells.In the occurrence and development of PD,ferroptosis is one of the ways of death of dopaminergic neurons,NCOA4-mediated fenitinophagy pathway can promote PD ferroptosis.When we silenced the NCOA4 gene,ferroptosis was inhibited in PD cell model.2.A ctivation of the ferritinophagy pathway and the occurrence of ferroptosis exist in both rat and mouse model of PD.After the interention of moxibustion,both NCOA4mediated feritinophagy and ferroptosis were inhibited,and the results of the two experiments were consistent,so moxibustion may regulate PD ferroptosis through the NCOA4-mediated ferrtinophagy pathway.3.After silencing the NCOA4 gene,the effects of moxibustion on PD ferroptosis were reduced,which further verified that NCOA4 may be one of targets of moxibustion and regulate the feritinophagy pathway to inhibit PD ferroptosis.