The Therapeutic Effect Of RTMS On Parkinson’s Disease And The Related Mechanism Of FBXO22 Regulating Parkinson’s Disease

Part One Therapeutic effect and related mechanism of transcranial magnetic stimulation on Parkinson’s diseaseBackground : PD is a common degenerative disease of the nervous system.The main pathological changes of PD are the degeneration and death of dopaminergic(DA)neurons in the substantia nigra of the midbrain,the significant decrease of DA content in the striatum,and the appearance of Lewy bodies in the cytoplasm of the remaining neurons in the substantia nigra with α-synuclein as the main component.The exact etiology of PD is still unclear.Aging,oxidative stress,genetic factors,environmental factors and other factors may be involved in the development of PD.In recent years,more and more attention has been paid to the role of cell senescence in the pathogenesis of PD.Cell senescence is a state in which proliferating cells leave the cell cycle and show permanent stagnation of growth.One of the salient characteristics of cell senescence is the secretion of a large number of bioactive substances,namely cell senescence related secretion phenotype(SASP)factors.These factors are often abnormally expressed in patients with Parkinson’s disease.Anti-PD drugs have certain curative effects and corresponding adverse reactions.In recent years,transcranial magnetic stimulation(TMS)uses pulsed magnetic field to pass through the brain without attenuation and acts on the brain tissue,generating induced current of a certain intensity,affecting local cerebral blood flow and metabolism,and affecting neurotransmitters and their transmission in the brain.Different brain regions have significant effects on the expression of various receptors and genes regulating neuronal excitability so as to regulate the functional state of brain.Objective:(1)Correlation analysis of the level of SASP factor and the degree of PD in patients with Parkinson’s disease;(2)The effect of high-frequency r TMS treatment on motor evoked potential of PD patients and the effect of motor symptoms and non-motor symptoms;(3)The effect of r TMS treatment on the expression of serum SASP factor in PD patients.Methods:(1)Clinical study: Hoehn & Yahr classification and motor evoked potential detection were performed in 108 PD patients enrolled.Motor symptoms(UPDRS score,timed motor test,reentry motor test),non-motor symptoms score(non-motor symptom screening questionnaire,Hamilton Depression and Anxiety Scale,cognitive function rating,sleep status rating,autonomic nervous dysfunction rating);The patients were randomly divided into r TMS group(54 patients)and false r TMS group(54 patients)by random number table method.5Hz high frequency r TMS and sham r TMS were given for 2courses of 4 weeks.(2)Laboratory study: Detection of peripheral blood SASP factors(TNF,IL1β,IL 6,MMP-3).Results:(1)Before treatment,there was no significant difference in motor symptom scores and non-motor symptom scores between 2 groups(P>0.05);(2)Compared with before treatment,after treatment and 1 month after treatment,the UPDRS I,UPDRSⅡ,UPDRS "score and total score of patients in r TMS group were significantly decreased,the average 1min movement times of right hand and left hand were significantly increased(P<0.01),and the average duration of 10 m return movement was significantly decreased(P<0.01).The difference was statistically significant(P<0.05).HAMA and HAMD scores were significantly lower than those before treatment(P< 0.01),while MMSE scores were significantly higher than those before treatment(P< 0.01).P300 latency PL was shorter than that before treatment,and amplitude AMP was higher than that before treatment(P< 0.01).NMSQUEST frequency and degree scores were significantly decreased,and the difference was statistically significant(P<0.05),while PDSS scores were slightly decreased,but the difference was not statistically significant(P>0.05).There were statistically significant differences in the above indicators compared with the sham r TMS group after treatment and 1 month after treatment(P<0.05).In the sham r TMS group,the scores of motor symptoms and non-motor symptoms did not change significantly after 1 course of treatment and 1 month after treatment,and the difference was not statistically significant(P>0.05).(3)Before treatment,there were no significant differences in serum TNF,IL-6,IL-1βand MMP-3 between the r TMS group and the sham r TMS group(P>0.05),but they were significantly higher than those in the normal control group(P<0.05).After treatment,TNF,IL-6,IL-1β and MMP-3 in serum of r TMS group decreased compared with before treatment,but the differences were not statistically significant(P>0.05).However,TNF,IL-6,IL-1β and MMP-3 in serum of r TMS group decreased significantly 1 month after treatment.The difference was statistically significant(P<0.05).There were statistically significant differences in the above indicators between the r TMS group 1 month after treatment and the sham r TMS group 1 month after treatment(P<0.05).There were no significant changes in serum TNF,IL-6,IL-1β and MMP-3 in the sham r TMS group after treatment and 1 month after treatment,and the difference was not statistically significant(P>0.05).CONCLUSIONS:(1)5Hz high frequency r TMS can improve motor symptoms and non-motor symptoms in patients with Parkinson’s disease.(2)Serum cell senescence factor were significantly abnormal in patients with Parkinson’s disease,and were significantly correlated with the severity of the patient’s disease;(3)5Hz high frequency rTMS therapy can improve the changes of serum SASP factors(TNF,IL1β,IL 6,MMP-3)in patients with Parkinson’s disease.Part Two Study on the mechanism of FBXO22 ubiquitination degradation of PHLPP1 and activation of AKT pathway to regulate Parkinson’s diseaseBackground: Parkinson’s disease(PD)is a neurodegenerative disease caused by a deficiency of dopaminergic neurons.Rotenone is a highly toxic,fat-soluble,broad-spectrum insecticide extracted from legumes,and is one of the most effective members of rotenoids.Rotenone can specifically cross the blood-brain barrier and accumulate in subcellular organelles.It has been reported that rotenone can induce dopaminergic degeneration,neuroinflammation,oxidative stress in the substantia nigra,especially the accumulation of intracellular ubiquitins and synuclein,which can be used in the successful development of PD models in rats and mice.FBXO22 is a poorly understood protein that may be involved in neurological diseases.However,the correlation of FBXO22 in PD has not been reported.Akt(also known as protein kinase B,PKB)is an important signal transduction molecule in the PI3K/Akt/m TOR signaling pathway,which can regulate cell growth and transport and promote cell survival.In the treatment of PD,a number of studies have shown that activation of PI3K/Akt/m TOR pathway has a protective effect,while inhibition of this pathway has the opposite effect.PHLPP1(PH domain leucine-rich repeat protein phosphatase 1)is a serine/threonine phosphatase belonging to the type 2C phosphatase(PP2C)family that forms a complex with Akt and dephosphorylates it.In neurons,down-regulation of PHLPP also increases the activation of Akt pathway,thus reducing the cytotoxicity induced by MPP+(1-methyl-4-phenylpyridine)and protecting the cells.We induced by use of rotenone SH SY5 Y cells and in rats,to simulate the PD,the basic pathological changes and behavior change,and then carries on the intervention,and through the evaluation of intervention after the change of the relevant molecular markers,to explore FBXO22 of AKT pathway regulating mechanism,to further explore the mechanism of its effect on PD to improve,To provide experimental basis for the new treatment strategy of PD.Objective: To study the regulation of FBXO22 in rotenone-induced Parkinson’s disease.Methods:(1)Establishment of PD cell model: human glioma cells SH-SY5 Y were treated with rotenone,overexpressed by retroviral transfection technique,or FBXO22 or ph LPP1 was knocked out,and cell viability was detected by MTT assay.Western blot was used to detect gene protein expression.Cell apoptosis was detected by flow cytometry.The production of reactive oxygen species was measured by IF.The activity of LDH was detected by LDH-cytotoxicity assay kit.The binding ability of FBXO22 and PHLPP1 was tested by Co-IP.(2)Establishment of PD animal model: male SD rats were subcutaneously injected with rotenone at 1.5 mg/kg/d for 4 consecutive weeks.High titer lentivirus particles LV-FBXO22 or LV-GFP were concentrated and the final concentration was adjusted to 1 ×1010 TU/ m L for injection to overexpress FBXO22 in rats.Experiment groups :(I)CON,(II)ROT,(III)ROT+LV-GFP,(IV)ROT+LV-FBXO22.Each group of 10.Give an athletic ability test.IHC detected TH(tyrosine hydroxylase)positive cells.FBXO22,p HLPP1,PAKT/Akt,p MTOR /m TOR,cleaved caspase 3expression were detected by Western Blot.Results:(1)After rotenone treatment of SH-SY5 Y cells,cell viability was decreased,apoptosis was enhanced,and FBXO22 protein expression was down-regulated.Levodopa had no effect on the expression of FBXO22 in PD cell model.(2)Overexpression of FBXO22 reduced rotenone-induced apoptosis,enhanced cell viability,decreased LDH activity and ROS level;The levels of Pakt/Akt,p MTOR/m TOR and Bcl-2 were enhanced,and the levels of Bax and cleaved caspase 3 were decreased.(3)Ubiquitin degradation of ph LPP1 by FBXO22: the expression of ph LPP1 m RNA did not change after up-regulation of FBXO22,and the decreased expression of ph LPP1 protein proved that FBXO22 affected the expression of ph LPP1 protein,but did not affect the expression of ph LPP1 m RNA,suggesting that the regulation may be achieved through ubiquitination.Co-IP detection results showed that FBXO22 was combined with PHLPP1.The upregulation of FBXO22 enhanced the ubiquitination of PHLPP1.(4)PHLPP1 inhibits AKT activity by down-regulating p AKT/AKT and pm TOR/m TOR levels: After treatment with rotenone,PHLPP1 protein expression was increased,while p AKT/AKT and pm TOR/m TOR levels were decreased.Overexpression of PHLPP1 offset the increased levels of p AKT/AKT and pm TOR/m TOR induced by down-regulation of PHLPP1.(5)PHLPP1 overexpression partially reversed the rotenone-induced neurotoxicity reduction caused by FBXO22 overexpression: On the basis of rotenone treatment,PHLPP1 overexpression saved f BXO22-induced increases in p AKT/AKT and pm TOR/m TOR levels,and reduced FBXO22 overexpression induced increases in Bcl-2 levels and decreased Bax and Cleaved caspase 3 levels.(6)Overexpression of FBXO22 alleviated rotenone-induced symptoms in PD rats;In rotenone induced PD rat model,FBXO22 expression was down-regulated,PHLPP1 expression was up-regulated,p AKT/AKT and pm TOR/m TOR levels were decreased,apoptosis related protein cleaved caspase 3 level was increased,TH expression was decreased;Overexpression of FBXO22 enhanced rotenone induced FBXO22 protein expression,inhibited PHLPP1 protein expression,increased p AKT/AKT and pm TOR/m TOR levels,decreased Cleaved Caspase 3 levels,and enhanced TH protein expression in PD rats.Conclusion: Our work proved that FBXO22 ubiquitination degraded PHLPP1 to ameliorate rotenone induced neurotoxicity by activating AKT pathway.This work suggested that FBXO22 may be an effective biological marker for PD treatment.