The Role And Mechanism Of MiR-7 In PD Animal And Cell Models

Objective:Parkinson’s disease(PD)is the second most common neurodegenerative disease after Alzheimer’s disease.The main pathological feature of PD is massive degeneration and loss of Dopamine(DA)neurons in the substantia nigra.More and more studies have shown that mi R-7 plays an important role in the pathogenesis of neurodegenerative diseases,but the exact mechanism has not been clarified.In clinical reports,compared with the healthy control group,the expression of mi R-7 was up-regulated in peripheral blood lymphocytes of PD patients after treatment,suggesting that the effect of treatment may be related to the increase of mi R-7 expression level and the down-regulation of the expression of target genes,suggesting that mi R-7 may play an important role in the pathogenesis of PD.In addition,the expression level of mi R-7 is affected by a variety of factors.This study intends to further explore the role and possible mechanisms of mi R-7 in animal and cell models of PD.Methods:C57BL/6J mice were given 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine(MPTP)(30 mg/kg)for 5 days to construct PD model mice.The expression of mi R-7 was detected by real-time fluorescence quantitative PCR,and the expression of Zinc Finger SWIM type containing 8(ZSWIM8)was detected by western blotting.In the 1-methyl-4-phenylpyridinium ion(MPP~+)-induced N2a cell model,western blotting was used to detect the expression of the voltage-dependent anion channel 1(VDAC1)and Transferrin Receptor1(Tf R1).The content of Superoxide Dismutase(SOD)was detected by the Superoxide Dismutase kit,and the level of reactive oxygen species(ROS)was detected by flow cytometry.In Lipopolysaccharide(LPS)-induced BV2 cells,western blotting was used to detect nod-like receptor protein 3(NLRP3),tumor Necrosis factor-alpha(TNF-α),and interleukin-1β(IL-1β).Results:1.PD animal model was successfully prepared.(1)In MPTP-induced PD model mice,compared with the control group,the results of pole climbing experiment showed that the mice had longer turning time(P<0.01);the experimental results showed that the residence time on the rotating rod was shortened(P<0.05).In conclusion,the motor coordination ability of MPTP-induced PD model mice was reduced.(2)Compared with control group,the expression of tyrosine hydroxylase(TH)protein was decreased in substantia nigra in MPTP mice(P<0.05);Bax protein levels was increased(P<0.05),suggesting that DA neurons in MPTP mice substantia nigra were damaged.2.Compared with control group,real-time quantitative PCR results showed that expression of mi R-7 was decreased in the substantia nigra of MPTP model mice(P<0.05),western blot results showed that ZSWIM8 protein level was increased(P<0.05);N2a cells were treated with MPP~+(100μM)for 24 h,compared with control group,real-time quantitative PCR results showed that mi R-7 expression decreased in MPP~+(100μM)group(P<0.05),CDR1as expression level increased(P<0.05).3.On N2a cells,the expression of VDAC1、Bax and Tf R1 were detected by western blot after treated with MPP~+(100μM)for 24 h.The results showed that compared with the control group,the protein levels of VDAC1 and Bax were increased(P<0.05,P<0.001),Tf R1 protein level was significantly decreased in MPP~+(100μM)group(P<0.05);after pretreatment with high mi R-7 expression for 24 h,the expression levels of VDAC1 and Bax proteins decreased(P<0.01),Tf R1 expression level was still decreased(P<0.05).4.N2a cells were treated with MPP~+(100μM)for 24 h,and the expression of SOD was detected by SOD kit,the ROS expression was detected by flow cytometry.The results showed that SOD expression in MPP~+(100μM)group was lower than that in control group(P<0.001),ROS expression level was increased(P<0.001);after pretreatment for 24 h with high expression of mi R-7,SOD expression level was increased(P<0.05),ROS expression level was decreased(P<0.001).5.On BV2 cells,the expression of NLRP3、TNF-αand IL-1βwere detected by western blot after 24 h of LPS treatment.The results showed that there was no significant change in NLRP3protein level,TNF-αand IL-1βprotein levels were significantly increased in the LPS-treated group compared with the control group(P<0.01);after pretreatment for 24 h with high mi R-7expression,NLRP3 protein level decreased significantly(P<0.05),TNF-αand IL-1βprotein levels were significantly decreased(P<0.01).Conclusions and Significance:The above results showed that mi R-7 was significantly decreased and ZSWIM8 protein expression level was significantly increased in MPTP model mice.In N2a cells,mi R-7 was significantly decreased and CDR1as was significantly increased after MPP~+treatment.After high expression of mi R-7,the protein levels of VDAC1 and Bax were decreased,SOD expression was increased,ROS release was decreased,and Tf R1 protein level continued to decrease.In BV2 cells,significantly decreased levels of NLRP3,TNF-α,and IL-1βproteins were observed after high expression of mi R-7.These experimental results suggest that the protective mechanism of mi R-7 may be related to the reduction of oxidative stress and inhibition of neuroinflammation.This study provided experimental basis for exploring the protective mechanism of mi R-7 in PD..