Effects Of Inhibiting PGC-1? And Enhancing PGC-1? Expression On Microglia In Mice Model Of PD

Parkinson disease(PD),also known as tremor palsy,is a neurodegenerative disease that has increased dramatically with increasing of age all over the world.The pathogenesis is the accumulation of the Lewy bodies and the loss of dopaminergic neurons in the substantia nigra(SN),at the same time with decreasing of dopamine in the striatum.Genetic susceptibility,inflammation,environmental factors,heavy metal factors,head trauma,and infection with bacteria or viruses can cause PD.Studies have confirmed that inflammation plays an important role in the development of PD,and it has appeared in the early stages of the disease,chronic inflammation leading to impair of nigrostriatal pathway.Under physiological conditions,microglia present a resting phenotype and constantly monitor its surrounding environment.When microglia detect a"dangerous"signal that impairs the homeostasis of the CNS,it is rapidly activated.Activated microglia display macrophage-like amoebic appearance,releasing a large number of neurotoxic substances such as pro-inflammatory cytokines and reactive oxygen species.These neurotoxins lead to apoptosis of dopaminergic neurons.Impaired dopaminergic neurons,in turn,as stimulators to further stimulate the activated microglia,forming a pathological process of the vicious circle.Finally,it will lead to the continued progression of neurodegenerative diseases.Coactivator-1?(PGC-1?),a peroxisome proliferator activated receptor gamma(PPAR?)associated with mitochondrial biogenesis,has been found to have significant protective effects.The previous study of our group showed that transfection of SH-SY5Y cells with lentivirus carrying PGC-1?gene could significantly inhibit mitochondrial autophagy and increase the survival of MPP~+injured SH-SY5Y cells.Stereotactic injection overexpression of lentivirus in the substantia nigra of mice,intraperitoneal injection of MPTP replication PD model,found that the number of dopaminergic neurons increased,but no statistical difference.And microglia activation was significantly higher than the MPTP experimental model group.Therefore,we hypothesized that this protective effect of PGC-1?overexpression on dopaminergic neurons may be related to activation of microglia,in addition to its direct effect on neurons itself.This study is to investigate the effect of changes in the expression of PGC-1?on the polarization of microglia in inflammatory response,and the relationship between the polarization of microglia and PD,in order to reveal the mechanism of PD and the neuroprotection of PGC-1?.And the results will provide basic research materials for PD intervention.In this study,C57BL/6 mice were used as experimental subjects.Injecting lentivirus to the mice SN,then the mice PD subacute model was replicated by intraperitoneal injection of MPTP after lentivirus injection 29 days.Behavioral analysis was used to detect the PD motor symptoms.The expression of TH,PGC-1?and IL-6 protein in substantia nigra was detected by western blot.ELISA method was used to monitor the change of IL-6 and IL-1?in blood.Immunofluorescence staining was used to detect the changes of TH?PGC-1?and other positive cells in SN,which including microglial marker Iba1(ionized calcium-binding adapter molecule 1,Iba1)?M1 microglial cell marker iNOS(inducible nitric oxide synthase,iNOS)?M2 microglial cell marker Arg1(Arginase catalyzes 1,Arg1)and so on.This study aimed to investigate the effect of PGC-1?expression on microglia in PD mice,to reveal the relationship between PGC-1?,microglia polarization and PD.The experimental results obtained are as follows:1.The number of Iba1-labeled microglia and the number of Iba1/PGC-1?double-labeled cells in the substantia nigra in MPTP group were significantly higher than those in the control group(P<0.05).In the overexpression and intervention PGC-1?groups in the substantia nigra of the mice,it was confirmed that the microglia cells were transfected with lentivirus.2.In the PGC-1?overexpression experiment,the MPTP+LV-PGC-1?group compared to the MPTP group showed a decrease in the number of drops and there was a significant difference(P<0.05),the latency increased and there was a significant difference(P<0.05).Immunofluorescence results showed that the number of microglia in the substantia nigra was significantly increased in the MPTP+LV-PGC-1?group compared with the MPTP group(P<0.05).The protein expression of PGC-1?in the substantia nigra of the MPTP+LV-PGC-1?group was higher compared with MPTP group and LV-EGFP group(P<0.05).IL-1?content in blood increased significantly(P<0.05).However,the TH expression in the MPTP+LV-PGC-1?group was elevated but there was no statistical difference compared with MPTP group(P>0.05).3.In the PGC-1?interference experiment,the free-standing rear decreased and there was a significant difference(P<0.05)between MPTP+LV-shRNA-PGC-1?group and MPTP+LV-shRNA-EGFP group.Compared with MPTP group and LV-shRNA-EGFP group,the protein level of PGC-1?was significantly decreased in MPTP+LV-shRNA-PGC-1?group(P<0.05).The number of iNOS positive cells was significantly decreased(P<0.05)in MPTP+LV-shRNA-PGC-1?group compared with MPTP group and LV-shRNA-EGFP group.Compared with MPTP group,the level of IL-6 in blood was significantly decreased in MPTP+LV-shRNA-PGC-1?group(P<0.05).The number of Arg1 positive cells reduced significantly in MPTP+LV-shRNA-PGC-1?group compared with MPTP group(P<0.05).4.Application minocycline after PGC-1?overexpression,the number of TH-positive neurons in substantia nigra of mice was significantly increased(P<0.05)in MPTP+LV-PGC-1?+Mino group compared with MPTP+LV-PGC-1?group,and the number of Iba1 cells activated in SN was significantly decreased(P<0.05).The number of iNOS cells in SN was significantly reduced(P<0.05)in MPTP+LV-PGC-1?+Mino group.Through the analysis of the above results,the following conclusions are drawn:1.Increasing the expression of PGC-1?in the substantia nigra of PD mice can activate microglia and increase the inflammatory factors released by M1 microglia;it can partially improve the motor symptoms of PD mice.2.Decreasing the expression of PGC-1?in the substantia nigra of PD mice can significantly reduce the autonomic activity of PD mice,and the number of M1 and M2microglia decreased significantly.The number of inflammatory factors released by M1microglia was significant reduced.3.The anti-inflammatory drug,minocycline,significantly reduced the activation of microglia in substantia nigra due to overexpression of PGC-1?,and the number of dopaminergic neurons increased significantly.From the above results and conclusions,it can be seen that overexpression of PGC-1?in substantia nigra does activate microglia,but there is no increase in anti-inflammatory M2microglia that the authors hope.And proinflammatory release increase by M1,the inflammatory response is strengthened.From the results of behavioral and morphological tests,increasing the expression of PGC-1?in the substantia nigra can significantly improve the partial motor symptoms of PD mice,and has protective effects on dopaminergic neurons.This seems to suggest that the neuroprotective effect of PGC-1?is not achieved by increasing the number and function of M2 microglia,but by increasing the ability of dopaminergic neurons to resist MPP~+damage.However,in view of the complexity of the differentiation process of microglia,the time of the experiment may be earlier or later than the time of differentiation from M1 to M2,so the above conclusions need further experiments to verify.This study provides some basic data for improving the expression of PGC-1?to prevent and treat PD,but the related mechanism remains to be further studied.