Inhibitory Effect Of Proanthocyanidin On LPS/ATP Induced Activation Of NLRP3 Inflammasome In Microglia And Protective Effect On SH-SY5Y Cell Injury

Parkinson’s disease（PD）is a typical neurodegenerative disease.The incidence rate increased significantly with age.The exact cause of PD is difficult to be explored,but neuroinflammatory response is considered to play a key role in the progression of PD.In recent years,the role of nucleotide-binding oligomerization domain-like receptor protein 3（NLRP3）inflammasome in neurodegenerative diseases has attracted widespread attention.Based on the important role of neuroinflammation and neuron injury in the development of PD,as well as the antioxidant and anti-inflammatory effects of proanthocyanidin,lipopolysaccharide（LPS）and adenosine triphosphate（ATP）induced BV2 cells were used to study the effect of proanthocyanidin on the activation of NLRP3 inflammasome and its related mechanisms.Human neuroblastoma cells（SH-SY5Y）were treated with 1-methyl-4-phenylpyridinium（MPP⁺）to investigate the effect of proanthocyanidin on SH-SY5Y injury and activation of NLRP3 inflammasome.From the two aspects of inflammation and neuron damage,the role of proanthocyanidin in inflammation inhibition and neuron protection was studied for providing a new strategy on the prevention of PD.The main content of this paper is divided into the following three parts:PartⅠ:The effect of proanthocyanidin on activation of NLRP3 inflammasome in LPS/ATP induced BV2 cellsObjective:BV2 cells were used to investigate the effect of proanthocyanin on the secretion of inflammatory cytokines and the activation of NLRP3 inflammasome induced by LPS/ATP.Methods:（1）BV2 cells were treated with LPS（0.1,1.0,10.0,20.0μg/m L）for 24h,and the content of nitric oxide（NO）was determined indirectly by microplate method to confirm the appropriate dose for subsequent experiments.（2）BV2 was treated with LPS（1.0μg/m L）for24 hours following by the treatment of ATP（1.0,2.5,5.0,10.0μmol/L）for 30 minutes.Cell viability was measured by 3-（4,5-dimethylazol-2-yl）-2,5-diphenyltetrazolium bromide（MTT）assay,and the effect of LPS combined with ATP on cell viability at different concentrations was analyzed.（3）The control group,proanthocyanin（0.1,1.0,2.5,5.0μg/m L）,LPS（1.0μg/m L）/ATP（2.5μmol/L）,LPS（1.0μg/m L）/ATP（2.5μmol/L）+proanthocyanin（0.1,1.0,2.5,5.0μg/m L）and the blank group were prepared.The cells were pretreated with proanthocyanin for 1h following by treatment of LPS for 24h and ATP for 30min,respectively.（4）The control group,LPS（1.0μg/m L）/ATP（2.5μmol/L）,LPS（1.0μg/m L）/ATP（2.5μmol/L）+proanthocyanin（0.1,1.0,2.5,5.0μg/m L）group were established.The cells were pretreated with proanthocyanin for 1h following by treatment of LPS for 24h and ATP for 30min,respectively.The content of NO was determined by microplate method.The activity of Lactate dehydrogenase（LDH）was measured by enzymatic labeling.Secretion levels of IL-1βand IL-18 were measured by enzyme-linked immunosorbent assay（ELISA）.（5）The control group,LPS（1.0μg/m L）/ATP（2.5μmol/L）,LPS（1.0μg/m L）/ATP（2.5μmol/L）+proanthocyanidin（0.1,1.0,2.5,5.0μg/m L）group were established.The cells were pretreated with proanthocyanin for 1h following by treatment of LPS for 6h and ATP for 30min,respectively.The expression of NLRP3,ASC,caspase-1,pro-caspase-1 and pro-IL-1βwere measured by Western blot analysis.Results:（1）Compared with the control group,LPS（0.1,1.0,10.0,20.0μg/m L）induced the increase of NO level in BV2cells,the difference was statistically significant（P<0.05）,and the effect was does-dependent（r=0.981,P<0.001）.（2）Compared with the control group,the cell viability of LPS（1.0μg/m L）/ATP（1.0,2.5,5.0,10.0μmol/L）group decreased,the result is significant difference（P<0.05）.（3）Compared with the control group,different concentrations of proanthocyanidin（0.1,1.0,2.5,5.0μg/m L）had no significant effect on cell viability,the difference was not statistically significant（P>0.05）.Compared with the LPS/ATP group,the cell viability of the LPS（1.0μg/m L）/ATP（2.5μmol/L）+proanthocyanidin（2.5,5.0μg/m L）group increased,and a statistically significant differences（P<0.05）.（4）Compared with the control group,NO level of LPS/ATP group was increased,and the difference was statistically significant（P<0.05）.Compared with LPS/ATP group,the NO content in proanthocyanidin（0.1,1.0,2.5,5.0μg/m L）group decreased,showing a statistical meaning（P<0.05）.The inhibitory effect was does-dependent（r=-0.836,P<0.001）.（5）Compared with the control group,the LDH activity of LPS/ATP group was increased,and the difference was statistically significant（P<0.05）.Compared with the LPS/ATP group,proanthocyanidin（0.1,1.0,2.5,5.0μg/m L）pretreatment could reduce the LDH activity,the result is significant difference（P<0.05）,and the effect has dosage dependence（r=-0.982,P<0.001）.（6）Compared with the control group,IL-1βand IL-18 levels in LPS/ATP group increased,and the difference was statistically significant（P<0.05）.Compared with the LPS/ATP group,proanthocyanidin（0.1,1.0,2.5,5.0μg/m L）reduced the release levels of IL-1βand IL-18,the result is significant difference（P<0.05）,and the effect has dosage dependence(r _IL-1β=-0.906,P<0.001;r _IL-18=-0.971,P<0.001).（7）Compared with the control group,the expression of NLRP3,ASC,pro-caspase-1,caspase-1 and pro-IL-1βin the LPS/ATP group increased,the difference was statistically significant（P<0.05）.Compared with the LPS/ATP group,the expression of NLRP3,ASC,caspase-1 decreased in LPS/ATP+proanthocyanidin（1.0,2.5,5.0μg/m L）group,the result is significant difference（P<0.05）.The expression of pro-IL-1βin LPS/ATP+proanthocyanidin（0.1,1.0,2.5,5.0μg/m L）group was decreased,the difference was statistically significant（P<0.05）,and the expression of pro-caspase-1 in LPS/ATP+proanthocyanidin（2.5,5.0μg/m L）group decreased,the difference was statistically significant（P<0.05）.The inhibitory effect was does-dependent(r _NLRP3=-0.818,P<0.001;r _ASC=-0.960,P<0.05;r_{pro-caspase-1}=-0.786,P<0.05;r_caspase-1=-0.742,P<0.05;r_pro-IL-1β=-0.862,P<0.001).PartⅡ:The study on the signaling pathway of proanthocyanidin inhibiting the activation of NLRP3 inflammasome in BV2 cellsObjective:To investigate the related mechanism of proanthocyanidin inhibiting the secretion of inflammatory factors and the activation of NLRP3 inflammasome induced by LPS/ATP in BV2 cells.Methods:（1）The control group,LPS（1.0μg/m L）/ATP（2.5μmol/L）group,LPS（1.0μg/m L）/ATP（2.5μmol/L）+proanthocyanidin（0.1,1.0,2.5,5.0μg/m L）group were prepared.The cells were pretreated with proanthocyanin for 1h following by treatment of LPS for 6h and ATP for 30min,respectively.The expression of TLR4 and MyD88 and phosphorylation of NF-κBp65 were measured by Western blot analysis.（2）The control group,Bay11-7082（5.0μmol/L）group,LPS/ATP group,LPS/ATP+Bay 11-7082（5.0μmol/L）group and LPS/ATP+PC（5.0μg/m L）group were established.The cells were pretreated with proanthocyanin for 1h following by treatment of LPS for 6h and ATP for 30min,respectively.The expression of NLRP3,ASC,caspase-1,pro-caspase-1,pro-IL-1βand phosphorylation of NF-κBp65 were measured by Western blot analysis.（3）The control group,Bay11-7082（5.0μmol/L）,LPS/ATP group,LPS/ATP+Bay 11-7082（5.0μmol/L）and LPS/ATP+PC（5.0μg/m L）groups were established.The cells were pretreated with proanthocyanin for 1h following by treatment of LPS for 24h and ATP for 30min,respectively.The secretion levels of IL-1βand IL-18 were measured by ELISA.Results:（1）Compared with the control group,the expression levels of TLR4,MyD88,and p-NF-κBp65 in the LPS/ATP group increased,the difference was statistically significant（P<0.05）.Compared with LPS/ATP group,the expression of MyD88 and p-NF-κBp65 in LPS/ATP+PC（1.0,2.5,5.0μg/m L）group decreased,the difference was statistically significant（P<0.05）.The expression of TLR4 in LPS/ATP+PC（0.1,1.0,2.5,5.0μg/m L）group decreased,and a statistically significant differences（P<0.05）,and the effect has dosage dependence(r _NF-κB=-0.807,P<0.001;r _TLR4=-0.851,P<0.001;r _MyD88=-0.775,P<0.05).（2）Compared with the control group,the expression of NLRP3,ASC,pro-caspase-1,caspase-1,pro-IL-1β,p-NF-κBp65 in the Bay 11-7082 group did not change significantly,and the difference was not statistically significant（P>0.05）.The expression of NLRP3,ASC,pro-caspase-1,caspase-1,pro-IL-1β,p-NF-κBp65 in LPS/ATP group were all increased,and a statistically significant differences（P<0.05）.Compared with LPS/ATP group,the expression of NLRP3,ASC,pro-caspase-1,caspase-1,pro-IL-1β,p-NF-κBp65 in LPS/ATP+BAY11-7082 group and LPS/ATP+proanthocyanidin group decreased,the difference was statistically significant（P<0.05）.（3）Compared with the control group,the secretion levels of IL-1βand IL-18 in the LPS/ATP group increased,and the difference was statistically significant（P<0.05）.Compared with LPS/ATP group,the levels of IL-1βand IL-18 in LPS/ATP+Bay11-7082（5.0μmol/L）group and LPS/ATP+proanthocyanidin（5.0μg/m L）group decreased,and a statistically significant differences（P<0.05）.PartⅢ:The protective effect of proanthocyanidin on SH-SY5Y cell damage induced by MPP⁺Objective:To study the protective effect of proanthocyanidin on SH-SY5Y injury induced by MPP⁺.Methods:（1）The blank group,control group,MPP⁺（0.1,0.2,0.5,1.0,2.0mmol/L）group were established.The cells were treated with MPP⁺for 24 h.Cell viability was measured by MTT assay.The blank group,control group,PC（0.1,1.0,2.5,5.0μg/m L）group were established.The cells were treated with proanthocyanin for 24h.Cell viability was measured by MTT assay.The blank group,control group,MPP⁺（0.5mmol/L）group,MPP⁺（0.5mmol/L）+proanthocyanidin（0.1,1.0,2.5,5.0μg/m L）group were established.The cells were pretreated with proanthocyanin for 1h following by treatment of MPP⁺for 24h,cell viability was measured by MTT assay.（2）The control group,MPP⁺（0.5mmol/L）group,MPP⁺（0.5mmol/L）+proanthocyanidin（0.1,1.0,2.5,5.0μg/m L）group were established.The cells were pretreated with proanthocyanin for 1h following by treatment of MPP⁺for 24h.LDH activity was measured by microplate method.ROS production was measured by fluorescence probe.IL-1βand IL-18 levels were measured by ELISA.（3）The control group,MPP⁺（0.5mmol/L）,MPP⁺（0.5mmol/L）+proanthocyanidin（0.1,1.0,2.5,5.0μg/m L）group were established.The cells were pretreated with proanthocyanin for 1h following by treatment of MPP⁺for 24h.The expression of NLRP3,caspase-1,pro-caspase-1,pro-IL-1βwere measured by Western blot analysis.Results:（1）Compared with the control group,the cell viability of MPP⁺（0.1,0.2,0.5,1.0,2.0,5.0 mmol/L）decreased,the difference was statistically significant（P<0.05）,the effect was does-dependent（r=-0.926,P<0.001）.（2）Compared with the control group,proanthocyanidin（0.1,1.0,2.5,5.0μg/m L）had no significant effect on cell viability,and the difference was not statistically significant（P>0.05）.The cell viability in the MPP⁺group decreased,showing a statistical meaning（P<0.05）.Compared with the MPP⁺group,the cell viability of the MPP⁺+PC（1.0,2.5,5.0μg/m L）group increased,the difference was statistically significant（P<0.05）.（3）Compared with the control group,the activity of LDH in the MPP⁺group increased,and the difference was statistically significant（P<0.05）.Compared with the MPP⁺group,the MPP⁺+PC（1.0,2.5,5.0μg/m L）group decreased,the difference was statistically significant（P<0.05）,and the effect was does-dependent（r=-0.949,P<0.001）.（4）Compared with the control group,the ROS level of the MPP⁺group increased,the difference was statistically significant（P<0.05）.Compared with the MPP⁺group,the ROS level of the MPP⁺+（1.0,2.5,5.0μg/m L）group decreased,showing a statistical meaning（P<0.05）,and the effect has dosage dependence（r=-0.949,P<0.001）.（5）Compared with the control group,the levels of IL-1βand IL-18 in the MPP⁺group increased,and the difference was statistically significant（P<0.05）.Compared with the MPP⁺group,the level of IL-1βin MPP⁺+PC（1.0,2.5,5.0μg/m L）group decreased,the difference was statistically significant（P<0.05）.the level of IL-18 in MPP⁺+PC（5.0μg/m L）group decreased,the difference was statistically significant（P<0.05）,and the effect has dosage dependence(r _IL-1β=-0.879,P<0.001;r _IL-18=-0.853,P<0.001).（6）Compared with the control group,the expression of NLRP3,pro-caspase-1,caspase-1 and pro-IL-1βin the MPP⁺group increased,the difference was statistically significant（P<0.05）.Compared with the MPP⁺group,the expression of NLRP3 and pro-caspase-1 in MPP⁺+PC（1.0,2.5,5.0μg/m L）group decreased,the result is significant difference（P<0.05）.The expression of pro-IL-1βwas decreased in MPP⁺+PC（0.1,1.0,2.5,5.0μg/m L）group.The expression of caspase-1 in the MPP⁺+PC（2.5,5.0μg/m L）group was decreased,the result is significant difference（P<0.05）.The effect has dosage dependence(r _NLRP3=-0.818,P<0.001;r_{pro-caspase-1}=-0.949,P<0.001;r_caspase-1=-0.796,P<0.001;r_pro-IL-1β=-0.982,P<0.001).Conclusion:（1）LPS/ATP induced increase of inflammatory factor secretion and NLRP3inflammasome activation in BV2 cells,and the expression of NLRP3,ASC,caspase-1,pro-caspase-1 and pro-IL-1βincreased.Proanthocyanidin inhibits LPS/ATP induced secretion of inflammatory factors and activation of NLRP3 inflammasome in BV2 cells.（2）LPS/ATP induced the activation of TLR4,MyD88 and NF-κB signaling pathway in BV2 cells,which plays an important role in the activation of NLRP3 inflammasome and the secretion of inflammatory factors.Proanthocyanidin inhibits LPS/ATP induced inflammatory factor secretion and NLRP3 inflammasome activation through TLR4,MyD88 and NF-κB signaling pathways.（3）MPP⁺induced the increase of ROS,inflammatory factors and the activation of NLRP3 inflammasome in SH-SY5Y cells,the expression of NLRP3,caspase-1,pro-caspase-1and pro-IL-1βincreased.Proanthocyanidin inhibits ROS,inflammatory factor secretion and NLRP3 inflammasome activation of SH-SY5Y cells induced by MPP⁺.