The Mechanism Study On The Protective Effect Of MiR-22 On Dopaminergic Neurons In PD

Background:Parkinson’s disease(PD)is the second most common neurodegenerative disease and is characterized by the loss of Dopaminergic neurons(DA)and the formation of Louise bodies in the pars compsa substantia nigra.The main clinical manifestations are motor symptoms(kinesia,static tremor,muscle rigidity,etc.)and non-motor symptoms(sleep disorders,depression,anxiety,cognitive disorders,speech disorders,etc.),which bring serious harm and heavy economic burden to patients and families.Epidemiological survey results show that the incidence of PD in people over 60 years old is about 1%,and the number of deaths is more than 100,000 per year.Its pathogenesis is complex and there is no good clinical treatment.Therefore,the intervention and treatment of PD and the exploration of its mechanism of action have important social and economic significance.Micro RNA(miRNA)are a class of highly conserved non-coding Rnas that play a central role in gene expression regulation by binding to complementary sites of the3’untranslated region(3’UTR)of target m RNA.A large number of studies have confirmed that miRNA is involved in the occurrence and development of a variety of neurodegenerative diseases,including Parkinson’s disease.Some studies have shown that miR-22 alleviates ischemia reperfusion injury through antioxidant and anti-apoptotic effects.In addition,miR-22 has a neuroprotective effect on neurodegenerative diseases such as Huntington’s disease through an anti-apoptotic mechanism,but the role of miR-22 in PD is rarely reported.Therefore,this study mainly explored the protective effect and mechanism of miR-22 on motor function and dopaminergic neurons in PD mice.Purpose:In vitro and in vivo experiments were conducted to determine whether miR-22could improve motor dysfunction in PD mice and its protective effect on dopaminergic neurons.To further explore the mechanism of the protective effect of miR-22 on dopaminergic neurons in PD mice,and provide a new strategy for the treatment and diagnosis of clinical PD.Method:In vitro experiment:1.In vitro,human neuroblastoma cells(SH-SY5Y)were used to induce PD cell model with 1-Methyl-4-phenyl Ridinium Ion(MPP~+);miR-22 mimic was used to perform cell intervention experiments.2.The effect of miR-22 on the activity of SH-SY5Y cells induced by MPP~+was detected by CCK-8 technology.3.The effect of miR-22 on SH-SY5Y cell apoptosis induced by MPP~+was detected by immunofluorescence technique.In vivo experiment:1.Healthy male C57BL/6J mice with a weight of 20-25g were used.1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine(MPTP)was used to induce the PD mouse model in vivo.Adenovirus with overexpression of miR-22 was injected into the striatum region for intervention.2.The influence of miR-22 on the motor function of MPTP-induced PD model mice was detected by rotating rod experiment.3.The effects of miR-22 on the autonomic behavior and inquiry behavior of MPTP-induced PD model mice were detected by open field experiment.4.Cat Walk gait analysis system was used to detect the effects of miR-22 on the gait parameters of MPTP-induced PD model mice.5.Western blot was used to detect the influence of miR-22 on the protein expression levels in striatum and substantia nigra of MPTP-induced PD model mice.6.The effect of miR-22 on the morphology of MPTP-induced PD model mice was detected by immunofluorescence technique.7.Western blot and immunofluorescence were used to verify the possible mechanism of the protective effect of miR-22 on dopaminergic neurons of MPTP-induced PD model mice(apoptosis,oxidative stress,neuroinflammation).Results:In vitro experiment:1)miR-22 had a protective effect on SH-SY5Y cells induced by MPP~+.CCK-8 test results showed that the cell activity of MPP~+model group was significantly decreased compared with the control group,and the cell activity was significantly increased after treatment with miR-22 mimic.2)miR-22 can inhibit cell apoptosis.In order to investigate whether miR-22 could inhibit the apoptosis induced by MPP~+,immunofluorescence was used to detect the apoptosis of cells.The results showed that compared with the control group,the apoptosis of cells was significant in the model group.After being treated with miR-22 mimic,the apoptosis of cells was significantly improved.In vivo experiment:1)miR-22 can improve the exercise ability of MPTP-induced PD model mice.To explore the effect of miR-22 on the exercise ability of MPTP-induced PD model mice.The results showed that compared with the Control group,the retention time of mice in the MPTP model group was significantly shortened.After the intervention of miR-22,the stick retention time of mice was increased.Then,we used the classic Cat Walk gait analysis system to further observe the effects of miR-22 on the gait of MPTP-induced PD model mice.We found that compared with the control group,MPTP-induced PD model mice showed significant motor retardation and decreased motor coordination ability.Parameters such as the time used to pass a specific distance(run duration),single stance time and step cycle increase significantly.average speed and swing speed decreased significantly;After overexpression of miR-22,these indexes were significantly improved compared with the model group.2)miR-22 can improve the exploratory behavior of MPTP-induced PD model mice.Open field experiment showed that,compared with the Control group,the total movement distance,movement frequency and residence time in the central region of mice in the MPTP model group decreased,and the total movement distance,movement frequency and residence time in the central region of mice in the PD model tended to increase after the intervention of miR-22 virus overexpression.At the same time,we also found that the autonomic activity of mice in the MPTP model group was significantly decreased,most of the activities were limited to the open field,and the exploratory behavior of mice was significantly weakened.miR-22 virus overexpression intervention significantly increased the exploratory behavior.3)miR-22 can alleviate the loss of dopaminergic neurons in MPTP-induced PD model mice.Western blot and immunofluorescence techniques were used to test the results.The results showed that,compared with the control group,the number of dopaminergic neurons in the compactus nigra and the number of dopaminergic nerve fibers in the striatum were significantly reduced in the PD model group.After the intervention of miR-22 virus overexpression,the loss of neurons was improved.4)miR-22 plays a neuroprotective role by inhibiting oxidative stress response.In order to further explore the neuroprotective mechanism of miR-22 on MPTP-induced PD model mice,we started from three perspectives of apoptosis,neuroinflammation and oxidative stress,and the results showed that MPTP-induced PD model mice had significant oxidative stress response:Two indexes of reduced glutathione(GSH)and peroxidase(CAT)were increased,and the intervention of miR-22 could alleviate this reaction,and the two indexes were decreased.5)The number of astrocytes increased in MPTP-induced Parkinson’s disease mouse models.Western blot and immunofluorescence were used to detect the expression of astrocytes in the dense part of substantia nigra and striatum.The results showed that there were significant differences between the MPTP model group and the control group in the compact part of substantia nigra and the striatum.Compared with MPTP model group,miR-22 virus did not decrease after intervention.6)miR-22 can improve the morphological changes of microglia cells in MPTP-induced pars densiflora nigra mice in PD model.Activation of microglia can cause astrocytes to transform into pathological A1forms,which contribute to the development of various neurodegenerative diseases such as Parkinson’s disease.Therefore,we further explored whether microglia activation existed in MPTP-induced PD model mice,and whether miR-22overexpression interfered with the activation.Immunofluorescence staining results showed that compared with the Control group,the morphology of microglia cells in the substantia compsa nigra in the MPTP model group changed:the cell bodies in the model group became larger and synapses became shorter.Given miR-22overexpression intervention,microglia activation was improved.7)miR-22 plays a protective role by activating the PI3K/Akt/mTOR signaling pathway.To further explore the mechanism by which miR-22 plays a neuroprotective role?We detected the protein levels of PI3K、Akt and mTOR and the expression of their phosphorylated forms in the striatum.Western blot results showed that the expressions of the two proteins in the model group were significantly decreased,while the expression of the proteins was increased after the intervention of miR-22overexpression.This suggests that overexpression of miR-22 activates the Akt/mTOR signaling pathway and exerts its neuroprotective effect.Conclusion:1.miR-22 can promote neuronal survival and inhibit cell apoptosis.2.miR-22 can improve the motor dysfunction of MPTP-induced PD model mice,as well as the loss of dopamine neurons.3.The number of astrocytes increased in PD model mice induced by MPTP.4.miR-22 can improve the morphological changes of microglia in MPTP-induced PD model mice and miR-22 can activate PI3K/Akt/mTOR signaling pathway by inhibiting oxidative stress induced by PD model mice,thus playing a neuroprotective role.