| Parkinson's disease(PD)is a chronic neurodegenerative disease among the elderly people,gradually affects the physical and mental health of middle-aged and elderly people,and brings heavy burdens to patients and society.The pathogenesis of Parkinson's disease is complicated,and the neuroinflammation mechanism has attracted widespread attention recently.Proanthocyanidin(PC)possesses efficient biological functions,especially has strong anti-inflammatory and antioxidant properties.Therefore,the use of proanthocyanidin for the prevention and treatment of Parkinson's disease has become a hot topic in society.This study commences with establishing the cell model of Parkinson's disease,by using LPS to activate BV2 microglia for inflammatory damage,and the supernatant of BV2 cells treated with LPS was used as inflammation-conditioned media to stimulate dopaminergic neuronal SH-SY5 Y cells,then observe the protective effect of proanthocyanidin on SH-SY5 Y dopaminergic neurons damage mediated by activation of BV2 cells and the mechanisms of proanthocyanidin inhibiting microglial activation,in order to provide a basis for exploring new targets and new strategies for the treatment of Parkinson's disease.The experimental scheme is made up of two parts as follows,The first part: Protective effects of proanthocyanidin on microglial activation mediated SH-SY5 Y cell damagePurposesTo observe the protective effect of proanthocyanidins on SH-SY5 Y nerve cell damage mediated by BV2 cell activation by establishment of inflammatory injury model of Parkinson's disease in vitro by activation of BV2 cells by LPS.Methods1.BV2 cells were stimulated with different concentrations of LPS(0.01,0.1,1.0,10.0,20.0 ?g/mL)for 24 h,Griess assay was used to measure the nitric oxide(NO)levels in supernatants of cultures,select the appropriate concentration of LPS to construct inflammatory injury model of BV2 cells in subsequent experiments.2.MTT assay was used to evaluate the effects of LPS as well as proanthocyanidin on the viability of BV2 cells.The experimental groups were divided into the zero setting group,normal control group,LPS(1.0 ?g/mL)treated group,proanthocyanidin(0.1,0.5,2.5,10.0 ?g/mL)intervention groups,and LPS(1.0 ?g/mL)+ PC(0.1,0.5,2.5,10.0 ?g/mL)co-treated groups.Cell viability was measured after24 h in each group.3.The effects of BV2 cell supernatant and LPS as well as proanthocyanidin on the viability of SH-SY5 Y cells were detected by MTT assay.The cell cultures of LPS(1.0 ?g/mL)activated BV2 as inflammatory cell cultures of conditioned medium(CM)to incubate dopaminergic neurons SH-SY5 Y cells,and the experimental groups were divided into control group,LPS group,proanthocyanidin treatment groups with different concentration,resting BV2 supernatant(C-CM)group,LPS-activated BV2 cells conditioned medium(LPS-CM)group and LPS-CM +PC(0.1,0.5,2.5,10.0 ?g/mL)groups,respectively.Cell viability was measured after24 h in each group.4.ELISA assays were used to measure the secretion levels of tumor necrosis factor-?(TNF-?),interleukin-1?(IL-1?)and interleukin-6(IL-6).The experimental groups were divided into control group,LPS group and LPS + PC(0.1,0.5,2.5,10.0?g/mL)co-treated groups.The levels of TNF-?,IL-1? and IL-6 in the supernatants of BV2 cells were measured after 24 h in each group.5.Annexin V-FITC/PI double staining method was used to detect the apoptosis of SH-SY5 Y neurons induced by BV2 conditioned medium.The experimental groups were divided into control group,LPS-activated BV2 cells conditioned medium(LPS-CM)group and LPS-CM + PC(0.1,0.5,2.5,10.0 ?g/mL)groups.Results1.Compared with the control group,there was a significant increase in NO release in BV2 cells treated with various concentrations of LPS.To ensure the establishment of an inflammatory cell model and to reduce the adverse effects of LPS on cells,1.0 ?g/mL LPS was used to stimulate BV2 cells.2.MTT results showed that proanthocyanidins of 0.1,0.5,2.5,10.0 ?g/mL alone or with 1.0 ?g/mL LPS treated BV2 cells for 24 h,presented no significant statistically difference in survival rates compared with the control group(P>0.05),indicating that LPS and proanthocyanidin did not produce toxic effects on BV2 cells within the dose range used in the experiments.3.Compared with the control group,proanthocyanidins of 0.1,0.5,2.5,10.0?g/mL alone or 1.0 ?g/mL LPS alone treated SH-SY5 Y cells for 24 h,presented no significant statistically difference in survival rates(P>0.05),indicating that LPS and proanthocyanidin did not produce toxic effects on SH-SY5 Y cells within the dose range used in the experiments.Resting BV2 cell supernatants of SH-SY5 Y cell lines showed no significant difference(P>0.05).While the groups treated with LPS-activated BV2 cell conditioned medium caused a decline in cell viability(P<0.05),and a co-incubation of proanthocyanidin significantly improved the survival rate of SH-SY5 Y cells(P<0.05).4.Compared with the control group,LPS treatment significantly increased the secreting of inflammatory mediators TNF-?,IL-1?,IL-6 in BV2 cell lines(P<0.05).While different concentrations of proanthocyanidin inhibited the release of inflammatory mediators activated BV2 cells in a dose dependent manner(P<0.05).5.Compared with the control group,SH-SY5 Y cells treated with inflammatory medium conditioned medium(LPS-CM)significantly promoted the apoptosis of SH-SY5 Y cells(P<0.05).Treatment of SH-SY5 Y cells with different concentrations of proanthocyanidins(0.1,0.5,2.5,10.0 ?g/mL)inhibited LPS-CM-induced apoptosis,and the protective effect was significantly enhanced with the increase of proanthocyanidin concentration(P<0.05).ConclusionLPS-activated BV2 cells induce massive release of inflammatory mediators of TNF-?,IL-1? and IL-6,which damage SH-SY5 Y cells.Proanthocyanidin can effectively inhibit the activation of BV2 cells,reduce the expression ofinflammatory mediators,protect SH-SY5 Y cells,and inhibit microglia-mediated neuronal apoptosis.The second part: Study on signaling pathways of proanthocyanidin-mediated inhibitory effect on microglia activationPurposesTo explore the role of TLR4-MyD88-ERK/p38/JNK MAPK signaling pathways in proanthocyanidin inhibiting microglia activation by establishment of inflammatory injury model of Parkinson's disease in vitro by LPS-activated BV2 cells.Methods1.The effect of proanthocyanidin on LPS-induced TLR4 and MyD88 protein expression and phosphorylation of ERK,p38 and JNK proteins in BV2 cells was detected by Western Blot.The experimental groups were divided into control group,LPS group and LPS + PC(0.1,0.5,2.5,10.0 ?g/mL)co-treated groups.PC was added1 h earlier than LPS.2.TLR4,ERK,p38 and JNK signaling pathways blockers were used to detect TLR4,MyD88,p-ERK,p-p38 and p-JNK expression levels by Western Blot.The experimental groups were the same as above.Results1.Compared with the control group,the expression of all proteins in LPS stimulated BV2 cells increased significantly(P<0.05).Compared with 1.0 ?g/mL LPS group,pretreatment of BV2 cells with different concentrations of proanthocyanidins(0.1,0.5,2.5,10.0 ?g/mL)could down-regulate the expression of proteins in a dose-dependent manner(P < 0.05).2.The signaling pathways blockers were applied to detect the effects of proanthocyanidin on the expression levels of TLR4,MyD88,and p-ERK,p-p38,and p-JNK protein by Western Blot assay.Compared with the control group,the blocker alone acted on BV2 cells had no significant influence on the protein expression levels(P>0.05),while the protein levels were significantly increased in the 1 ?g/mL LPS exposure group(P<0.05).Compared with LPS group,LPS+blocker group could down-regulate protein expression levels(P < 0.05).Proanthocyanidin intervention also significantly decreased the protein expression levels(P < 0.05).It is confirmed that proanthocyanidin can inhibit the activation of microglia by lipopolysaccharide through the TLR4-MyD88-ERK/p38/JNK MAPK signaling pathways.ConclusionLPS induced the activation of TLR4-MyD88-ERK/p38/JNK MAPK signaling pathways in BV2 cells,and the expression levels of TLR4,MyD88,p-ERK,p-p38,and p-JNK proteins were significantly increased.Proanthocyanidin significantly inhibited protein expressions and inhibited the activation of microglia by LPS through the TLR4-MyD88-ERK/p38/JNK MAPK signaling pathways. |
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