Study Of The Role Of Microglia NOX2 In SVHRSP Against Parkinson’s Disease

Background:Parkinson’s disease(PD)is a common neurodegenerative disease and the second most common neurodegenerative disease after Alzheimer’s disease(AD).The pathological changes are the degenerative death of dopamine(DA)neurons in the substantia nigra compacta(SNpc),which leads to a decrease in dopamine transmission in the striatal region,resulting in a decrease in nigrostriatal dopamine levels and an abnormal accumulation of alpha-synuclein(α-syn).The main pathogenic mechanisms of PD are oxidative stress,abnormal protein aggregation,neuroinflammation and mitochondrial dysfunction.There is increasing evidence that microglia density is increased in the substantia nigra of PD patients,suggesting that neuroinflammatory responses mediated by microglia activation around dopaminergic neurons are involved in the development of PD.NADPH oxidase 2(NOX2)is a multimeric protein complex consisting of cytoplasmic subunits(p47phox,p67 phox,p40phox,Rac1 or Rac2)and cytosolic catalytic subunits p22 phox and Gp-91 phox,which are highly expressed in microglia and whose activation is an important pathway for microglial The activation is an important pathway for microglia activation,and the production of reactive oxygen species(ROS)has a dual role in regulating apoptosis and the central neuroinflammatory response.In PD,NOX2 inhibition or gene deletion significantly reduces microglia-mediated neuroinflammation,reduces dopaminergic neuronal loss and plays a neuroprotective role.Current treatments for PD can improve symptoms but cannot stop the progression of the disease,nor can they cure it.Currently,our group has synthesized Scorpion Venom Heat Resistance Synthesis Peptide(SVHRSP)and obtained a national patent[ZL201610645111.7].Previous studies have shown that SVHRSP has neuroprotective and inhibitory effects on inflammatory responses,and it was found that knockdown of NOX2 expression by siRNA at the cellular level significantly attenuated the inhibitory effects of SVHRSP on pro-inflammatory factor expression and related neurotoxicity in PD models.However,it is unclear whether SVHRSP can protect dopaminergic neurons by inhibiting NOX2-mediated microglia activation at the animal level.Aims:In the first stage,a Rotenone(Rot)induced PD model was established to investigate the effect of SVHRSP on NOX2 activation.In addition,a mouse model of PD induced by rotenone after stereotaxic injection of Adeno-associated virus(AAV)to knock down NOX2 expression in the nigrostriatal region of C57BL/6J mice was developed to elucidate the role of SVHRSP in the NOX2 pathway of microglia and to provide a theoretical basis for seeking treatment of PD with SVHRSP.Methods:1.Rot-induced PD model establishment: control group(Con),rotenone group(Rot),rotenone+SVHRSP low dose group(Rot+SVHRSP-200μg/kg/day),rotenone+SVHRSP high dose group(Rot+SVHRSP-400μg/kg/day),SVHRSP(400μg/kg/day).2.Stereotaxic injection of AAV to knock down NOX2 in the bilateral substantia nigra region of C57BL/6J mice,followed by Rot-induced PD model establishment:C57BL/6J mice were injected with AAV in bilateral substantia nigra localization,divided into control virus group(CTRL-AAV)and target virus group(NOX2-AAV),and then each group was divided into control group(Con),model group(Rot),and SVHRSP group(Rot-SVHRSP),AAV infection for 6 weeks and Rot induction for 21 days.3.Immunofluorescence assay: The nigrostriatal control virus group(CTRL-AAV)and the target virus group(NOX2-AAV)were examined for AAV binding to IBA-1 and NOX2 in the Con group.4.Immunohistochemical assay: detection of TH+ neuronal loss in the nigrostriatal control virus(CTRL-AAV)and target virus(NOX2-AAV)groups.5.Western blot assay: to detect the effect of oxidative stress in the control virus(CTRL-AAV)and target virus(NOX2-AAV)groups,and to detect the expression levels of oxidative stress-related proteins(Nrf2,Keap-1)in the control virus(CTRL-AAV)and target virus(NOX2-AAV)groups.6.RT-qPCR assay: mRNA levels of M1 phenotypic markers(TNF-α,IL-1β,iNOS)and M2 phenotypic markers(CD206,Arg-1,Ym-1)were detected in microglia.7.Western blot assay: to detect the activation level of Jak-STAT,MAPK and NF-κB signalling pathways,to measure the indicators of mitochondrial damage,and to detect the expression level of mitochondrial fusion and division marker proteins(Drp1,Fis1,Mfn1).Results:1.Effect of SVHRSP on NOX2 activation in PD mice: Western blot results showed compared with the Con group,phosphorylated p47 phox protein and Gp-91 phox protein were significantly increased in the Rot-induced PD model group and significantly decreased after treatment with SVHRSP group(200 μg/kg/day)and Rot-SVHRSP group(400 μg/kg/day),with the Rot-SVHRSP(400 μg/kg/day)group decreasing more The difference was statistically significant(P < 0.05).MDA is an indicator of lipid peroxidation damage,and NADP+/NADPH is a marker of oxidative stress,and the results showed that both MDA and NADP+/NADPH values increased in the Rot-induced PD model group,with statistically significant differences(P < 0.05).,and both MDA and NADP+/NADPH values were reduced significantly after treatment with SVHRSP group(200 μg/kg/day)and Rot-SVHRSP group(400 μg/kg/day).These results suggest that SVHRSP inhibits NOX2 activation in PD mice.2.Effect of knocking down the activation of NOX2 in microglia on the anti-PD neuroprotective effect of SVHRSP: The results of behavioral experiments showed that in the Rot-induced PD model group compared to the Con group,there was a statistically significant difference in functional deficits in learning ability and balance(P < 0.05),and in the control virus group(CTRL-AAV),SVHRSP showed a significant improvement in learning ability and balance in the PD model,while the target virus group(NOX2-AAV)showed the opposite result,and IBA-1 immunofluorescence staining detected no binding of AAV to IBA-1 in the CTRL-AAV-Con group and better binding of AAV to IBA-1 in NOX2-AAV-Con.-91 phox immunofluorescence staining detected no binding of AAV to NOX2 in the CTRL-AAV-Con group and better binding of AAV to IBA-1 in NOX2-AAV-Con.In NOX2-AAV-Con,AAV bound well to NOX2.Tyrosine hydroxylase(TH)immunohistochemistry and Western blot detection of dopaminergic neurons,SVHRSP in the control virus group(CTRL-AAV)had a significant protective effect on dopaminergic neurons in the PD model with statistically significant differences(P < 0.05),while the target virus group(NOX2-AAV),in contrast,did not play a significant role compared with the model group,and the difference was not statistically significant(P>0.05).In conclusion,reducing the activation of microglia NOX2 attenuated the improvement of motor function and the protective effect of SVHRSP on dopaminergic neurons in PD model mice.3.The effect of knockdown of microglia NOX2 activation on SVHRSP against oxidative stress in PD:Western blot results showed that the expression of oxidative stressrelated proteins(Nrf2,Keap-1)was significantly lower in the Rot-induced PD model group compared to the Con group,with statistically significant differences(P < 0.05),and in the control virus group(CTRL-AAV),SVHRSP significantly increased Nrf2 and Keap-1 protein expression in the control virus group(CTRL-AAV)against Rot-induced PD model rats,while no significant increase in Nrf2 and Keap-1 protein expression was observed in the target virus group(NOX2-AAV)against Rot-induced PD model rats,and the difference was not statistically significant(P>0.05).m DA and GSH As indicators of lipid peroxidation damage,the kit results showed that SVHRSP in the control virus group(CTRL-AAV)significantly reduced MDA and GSH/GSSG values in the Rot-induced PD model rats with statistically significant differences(P<0.05),whereas in the target virus group(NOX2-AAV),SVHRSP on the Rot-induced PD model rats MDA and GSH/GSSG values were not significantly increased and the differences were not statistically significant(P>0.05).These results suggest that reducing the activation of NOX2 in microglia attenuated the protective effect of SVHRSP against oxidative stress in PD.4.RT-qPCR results showed that M1 phenotype pro-inflammatory markers(TNF-α,IL-1 β,iNOS),SVHRSP significantly decreased the mRNA expression levels of inflammatory factors in the CTRL-AAV group in the Rot-induced PD model rats with significant differences(P < 0.05),and in the NOX2-AAV group,SVHRSP had no significant effect on the mRNA expression of inflammatory factors in the Rot-induced PD The M2 phenotype,inflammatory markers(CD206,Arg-1)were significantly increased by SVHRSP in the CTRL-AAV group and not significantly increased by SVHRSP in the NOX2-AAV group in the Rot-induced PD model.The differences were not statistically significant(P>0.05).no significant changes were seen in the M2 phenotype inflammatory marker YM-1 in the groups,and the differences were not statistically significant(P>0.05).The results suggest that reducing NOX2 activation attenuated the inhibitory effect of SVHRSP on M1 activation of microglia and M2 phenotype activation.5.Effects of Jak-STAT,MAPK,and NF-κB signaling pathways in SVHRSP via NOX2 pathway: Western blot results showed that in the control virus group(CTRL-AAV),compared with the Con group,in the Rot-induced PD model group,the key proteins of Jak-STAT pathway,STAT1,STAT3 protein In the Rot-induced PD model group,the phosphorylation levels of key proteins STAT1 and STAT3 of the Jak-STAT pathway,P-38 and p-ERK of the MAPK pathway,and P-65 and I-kappb-α of the NF-κB pathway were significantly increased,while the phosphorylation levels of related proteins in the Jak-STAT,MAPK and NF-κB pathways were significantly decreased in the Rot-induced PD model group treated with SVHRSP.The levels of phosphorylated proteins in the JakSTAT,MAPK and NF-κB pathways were significantly reduced in the Rot-induced PD model mice treated with SVHRSP.In the target virus group(NOX2-AAV),there was no significant difference in the phosphorylation levels of related proteins in Jak-STAT,MAPK and NF-κB pathways in the Rot-induced PD model rats by SVHRSP(P>0.05).The results suggest that reducing the activation of NOX2 attenuated the inhibition of the activation of inflammation-related pathways by SVHRSP in PD model rats.6.The effect of knocking down the activation of NOX2 in microglia on the effect of SVHRSP on mitochondrial damage: Western blot results showed that in the control virus group(CTRL-AAV),SVHRSP significantly decreased the expression level of Fis1 protein expression and significantly increased the expression level of Mfn1 protein expression in the Rot-induced PD model mice,the difference was significant(P< In the target virus group(NOX2-AAV),SVHRSP did not significantly increase Fis1 and Mfn1 protein expression in the Rot-induced PD model rats,and the difference was not statistically significant(P>0.05).no significant difference was seen in Drp1 protein expression in each group(P>0.05).The results suggest that reduced activation of NOX2 reduced the protective effect of SVHRSP against mitochondrial damage in PD.Conclusions1.SVHRSP inhibited the activation of NOX2 in PD model mice.2.Reduced NOX2 activation attenuated the inhibitory effects of SVHRSP on microglia M1-type activation,inflammatory response and oxidative stress,and mitochondrial damage.3.Jak-STAT,MAPK,and NF-κB may be important signaling pathways in microglia NOX2 regulation of SVHRSP anti-PD.