| Objective Parkinson’s disease(PD)is a neurodegenerative disorder characterized by a progressive decrease in the number of dopaminergic(DAergic)neurons in the midbrain.Microglial cells play the most important role in the development of Parkinson’s disease(PD).We performed cell and animal experiments to explore the therapeutic effect of artemisinin on PD and the TLR4/Myd88/NF-κB signalling pathway.But our study cannot explain the relationship between microglia and dopaminergic neurons.Methods1.To assess the ability of artemisinin to alleviate the clinical symptoms of PD model mice,mice were subjected to the pole test and wire hanging test.2.To estimate the protective effect of artemisinin on dopaminergic neurons on PD model mice,the midbrain of mice was detected by WB.And the activation of microglial cell in the midbrain of each group was explored by IHC.3.To clarify the effect of artemisinin on microglial cell,CCK-8、FCM、Elisa、IF were used to detect the activation of microglial cell in BV-2 of each group.4.The expression level of TLR4 was assessed by WB after treatment with artemisinin microglial cells.Adding of LPS group based on method 3,CCK-8、FCM、Elisa、IF were used to detect the activation of microglial cell in BV-2 of each group.5.WB and Elisa were used to estimate the effect of artemisinin on TLR4/Myd88/NF-κB singal pathway in BV-2 of each group.6.WB and Elisa were used to estimate the effect of artemisinin on TLR4/Myd88/NF-κB singal pathway in PD model mice.Results1.Through the behavioral experiment analysis of the climbing rod experiment and the suspension experiment,it was found that compared with the blank group,the climbing rod speed of the model group slowed down and the time spent on getting rid of the dilemma increased(P< 0.05);compared with the model group,the climbing speed of the artemisinin group was accelerated and the time taken to get rid of the dilemma was shortened(P< 0.05).2.By IHC and WB analysis,it was found that compared with the blank group C57 mice,the number of dopaminergic neurons and the content of tyrosine hydrogenase in the midbrain of the model group were significantly reduced(P<0.05).Compared with the model group,the number of dopaminergic neurons and the content of tyrosine hydrogenase in the midbrain of the mice in the artemisinin group increased(P<0.05).WB analysis of the content of IBa-1 in the midbrain of three groups of C57 mice showed that the content of IBa-1 in the model group was significantly increased(P<0.05),and the content of IBa-1 in the artemisinin group was decreased(P<0.05).3.MPP+ induced BV-2 cells,and CCK-8 analysis showed that compared with the blank group,the cell activity of BV-2 in the MPP+ group increased(P<0.05).Compared with MPP+ group,the cell activity of BV-2 in artemisinin group was decreased(P<0.05).At the same time,by detecting apoptosis by flow cytometry,Elisa detection of activated cell secretion and IHC detection of IBa-1content representing microglia activation,it was confirmed that the cell activity of BV-2 in the blank group was higher than that in the MPP+ group(P<0.05),and the cell activity of BV-2 in the artemisinin group was lower than that in the MPP+group(P<0.05).4.The protein was extracted from the four groups of cells in the blank group,MPP+ group,artemisinin group and LPS group.WB analysis showed that the expression of TLR4 in the MPP+ group was higher than that in the blank group(P<0.05).Compared with the MPP+ group,the content of TLR4 protein in the artemisinin group decreased(P<0.05)and the expression of TLR4 in the LPS group was higher than that in the artemisinin group(P<0.05).CCK-8,FCM,Elisa and IHC analysis showed that the addition of LPS could reverse the inhibitory effect of artemisinin on microglia activation(P<0.05).5.WB and Elisa analysis of cell protein and cell supernatant showed that compared with the blank group,the expression of TLR4/Myd88/NF-κB pathway-related proteins in the MPP+ group increased(P<0.05),compared with the MPP+ group,the expression of TLR4/Myd88/NF-κB pathway-related proteins in the artemisinin group decreased(P<0.05),and the expression of TLR4/Myd88/NF-κB pathway-related proteins in the LPS group increased compared with the artemisinin group(P<0.05).6.WB analysis of the midbrain protein extracts of three groups of C57 mice showed that the expression of TLR4/Myd88/NF-κB pathway-related proteins in the MPTP group was higher than that in the blank group(P<0.05),and the expression of TLR4/Myd88/NF-κB pathway-related proteins in the artemisinin group was lower than that in the MPTP group(P<0.05).Conclusions1.Artemisinin can improve the behavioral symptoms of Parkinson ’s disease model mice.2.The protective effect of artemisinin on dopaminergic neurons is related to its inhibition of microglia activation.3.Artemisinin can inhibit the activation of microglia through TLR4/Myd88/NF-κB pathway.In short,Artemisinin exerts a protective effect in the MPTP mouse model of Parkinson’s disease by inhibiting microglial activation via the TLR4/Myd88/NF-KB pathway... |
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