The Neuroprotective Effects And Mechnisms Of Sonic Hedgehog/Pi3K/AK Signaling Pathwayson Neurons In Inflammatory Model Of Parkinson’s Disease

Our experimental application of lipopolysaccharide(LPS)to stimulate the mouse microglia glioma cell line(BV2)to establish the model of inflammatory activation and application of the rat adrenal pheochromocytoma(PC12)cells to simulate dopaminergic neurons.We observed the effects of sonic hedgehog signaling pathways activator activate its signaling pathways on the expression of pro-inflammatory factors,anti-inflammatory factors,related Nurr1 gene of Parkinson’s disease in BV2 cells induced by LPS and its culture’s influence on PC12 cell survival rate after PI3K/AKt signaling pathway inhibitor LY294002 inhibit the signaling pathways;We further application of MPTP to induce mouse model of Parkinson’s disease,blockade of PI3K/Akt pathway by intranasal administration of LY294002 and we observed the effects of activation of SHH signaling pathways in mice by intraperitoneal injection of PM on the behavior of mouse,microglia,inflammation factors and the dopaminergic neurons.This can provide new strategies and theoretical basis for the clinical treatment of Parkinson’s disease.The first part: The effects of Sonic hedgehog/PI3K/AKt signaling pathways on the inflammatory cell models of Parkinson’s diseaseObjective: Applylication of LPS to stimulate the BV2 cell to establish the model of inflammatory activation of microglia cells and investigate activation of SHH signaling pathways play a role in suppression inflammation in BV2 induced by LPS,whether if it related with PI3K/AKt signaling pathway.Methods: Applylication of LPS to stimulate the BV2 cell to induced inflammation,Western Bloting experiments was used to detect the expression of P-AKt protein,which the marker of PI3K/AKt signaling pathway in inflammation and the relationship between SHH signaling pathway and PI3K/AKt signaling pathway.Application of LY294002 to inhibit the PI3K/AKt signaling pathway in BV2 cells,Real-time fluorescent quantitative PCR(Q-PCR)was used to detect the expression of proinflammatory factor IL-1β,TNF-a and anti-inflammatory factor TGF-beta and related Nurr1 gene of Parkinson’s disease in BV2 cells induced by LPS after PM activated SHH signaling pathways;CCK-8 tests was used to detect the effects of each cell culture’s on PC12 cell survival rate.Results: 1)Western Bloting experimental results show that:(1)Compared with control group,there was significantly inhibitory effect on the PI3K/AKt signaling pathway in the inflammation induced by LPS(P < 0.01);(2)Compared with LPS group,the activation of SHH signaling pathway increased the expression of P-AKt protein in the inflammation induced by LPS(P < 0.01;(3)Compared with PM + LPS group,the inhibition of the SHH signaling pathway with Cyclopamine reversed the increased the expression of P-AKt protein(P < 0.01);2)Q-PCR experimental results showed:(1)Compared with LPS group,PM activated SHH signaling pathways could significantly inhibit the expression of inflammatory factor IL-1β,TNF-a and Nurr1 m RNA(all P < 0.01),increased the expression of anti-inflammatory factor TGF-beta m RNA(P < 0.01);(2)Compared with PM + LPS group,the inhibition of PI3K/AKt signal pathway with LY294002 pretreatment could significantly increased the expression of IL-1β,TNF-a m RNA(all P < 0.01)and decreased the expression of Nurr1,TGF-beta m RNA(all P < 0.01);(3)Compared with LPS group,there were significantly increased the expression of IL-1β,TNF-a and TGF-beta m RNA(all P < 0.01)and significantly inhibited the expression of Nurr1 m RNA in LY + LPS group(P < 0.01);3)The CCK 8 results showed:(1)Compared with control group,the survival rate of PC12 cells was significantly decreased induced by the supernatant of LPS group’ culture medium(P < 0.01);(2)Compared with LPS group,PM + LPS group’ culture can promote the survival rate of PC12 cells(P < 0.01);(3)Compared with PM + LPS group,LY294002 + PM + LPS group’ culture has certain inhibiting effect on PC12cell survival(P < 0.05).Conclusion: 1.There was significantly inhibitory effect on the PI3K/AKt signaling pathway in the inflammatory response;2.SHH signaling pathways has certain regulation effect on PI3K/AKt signal pathway;3.The activation of SHH signaling pathway can reduce inflammatory response,promote the expression of anti-inflammatory factor TGF-β,inhibit the expression of Nurr1 gene induced by LPS;4.SHH signaling pathways play a role of inhibition of inflammation and protective of PC12 cells and its related with PI3K/AKt signal pathway.The second part: The effects of Sonic hedgehog/PI3K/AKt signaling pathways on the neuroinflammation、neuroprotective and behavior of Parkinson’s disease modelObjective: MPTP was used to induce acute model of Parkinson’s disease and and investigate activation of SHH signaling pathways protect dopaminergic neurons in neuroinflammation,whether if it’s related with PI3K/AKt signaling pathway in the model of Parkinson’s disease.Methods: MPTP was used to induce acute PD model,Western Bloting experiments was used to detect the expression of PI3K/AKt signaling pathway in PD model and observe the effects of activation of SHH signaling pathways on PI3K/AKt signaling pathway.Blockade of PI3K/Akt pathway by intranasal administration of LY294002 and after activation of SHH signaling pathways in the body by intraperitoneal injection of PM,the suspension experiment tests was used to detect the behavior of mouse,immunohistochemical staining and Western Bloting experiments were used todetect the expression of microglia and DA neurons and Q-PCR experiment was used to detect the expression of related inflammatory factors.Results: 1)Western Bloting(WB)results showed that:(1)Compared with control group,there was significantly inhibitory effect on the PI3K/AKt signaling pathway in the model of PD.(P<0.01);(2)Compared with MPTP group,activation of SHH signaling pathway could increased the expression of P-AKt protein(P < 0.01;(3)Compared with PM + MPTP group,LY294002 pretreatment could significantly inhibit the PI3K/AKt signal pathway;2)Iba1 immunohistochemical staining results showed that:(1)Compared with control group,the number of microglia in PD model of the substantia nigra were increased,the cell body become large,stained darker and cell process become shorter and coarsen;(2)Compared with MPTP group,the number of microglia was decreased,microglia with small cell body and slender enation in PM + MPTP group;(3)The number,size and morphology of Iba1-positive microglial cells in LY+PM+MPTP group are similar to LY+MPTP group;3)Iba1 WB results showed:(1)Compared with control group,the expression of Iba1 protein was significantly raised in MPTP group(P < 0.01);(2)Compared with MPTP group,the activation of SHH signaling pathways could significantly decreased the expression of Iba1 protein,the expression of Iba1 protein increases more significantly in LY+ MPTP group(all P < 0.01);(3)Compared with PM+MPTP group,LY294002 pretreatment could reverse the inhibitory effect of SHH signal on the expression of Ibal protein(P < 0.01);4)TH immunohistochemistry staining results showed that:(1)Compared with control group,the number of DA neurons was decreased significantly,the cell body is incomplete,and cytoplasm staining shallow in the substantia nigra of PD model;(2)Compared with MPTP group,the number of DA neuron was increased,the cell body shape is more complete,clear outline and cytoplasm staining deep in PM + MPTP group;the numbe of DA neurons was slightly reduced in LY+MPTP group,the morphology and staining in MPTP group are similar to LY+MPTP group.(3)Compared with PM + MPTP group,the numbe of DA neurons was decrease,the cell body is incomplete,and cytoplasm staining shallow in LY+PM+MPTP group;5)TH WB results showed:(1)Compared with control group,the expression of TH protein decreased significantly in MPTP group;(2)Compared with MPTP group,the expression of TH protein was increased in PM + MPTP group,the expression of TH protein was decreased in LY+MPTP group;(3)Compared with PM+MPTP group,LY294002 significantly inhibited the expression of TH protein induced by SHH signal;6)Q-PCR results showed:(1)Compared with control group,the expression of inflammatory cytokines IL-1β、TNF-a were increased significantly in MPTP group;(2)Compared with MPTP group,the expression of inflammatory cytokines IL-1β、TNF-a were decreased in PM + MPTP group and the expression of IL-1β、TNF-a were increased in LY+MPTP group;(3)Compared with PM+MPTP group,LY294002 significantly changed the expression of IL-1β、TNF-a induced by SHH signal;7)The suspension experiment showed that:(1)Compared with control group,the scores of mouse model of PD suspension time significantly decreased(P < 0.01);(2)Compared with MPTP group,the scores of mouse model of PD suspension time significantly increased in PM + MPTP group(P < 0.01);(3)Compared with PM + MPTP group,inhibition of PI3K/AKt signaling pathway by LY294002 could significantly inhibit the suspension time points induced by SHH signal(P < 0.01);Conclusion: 1.There was significantly inhibitory effect on the PI3K/AKt signaling pathway in the model of PD;2.SHH signaling pathways has certain regulation effect on PI3K/AKt signal pathway in the body;3.Activation of SHH signaling pathways could improve motor coordination ability,protect the DA neurons in mice,inhibit the activation of microglia and the expression of inflammatory factors are associated with PI3K/AKt signal pathway.