| ObjectiveAccording to the statistics of the United Nations,by 2050,the elderly over 65 years old in China will account for a quarter of the total population.In the elderly,vascular dementia(VaD)is one of the main causes of dementia.Alzheimer’s disease is more common in European and American countries,and vascular dementia is more common in Asian countries.This is similar to cerebrovascular diseases in Asian countries.The incidence rate is higher than that of European and American countries.In recent years,due to the blowout growth of cerebrovascular diseases,people pay more and more attention to the research of VD.Although there is no effective treatment for VD,it is considered to be a kind of dementia syndrome that can be effectively prevented,and this preventability is mainly aimed at the early stage.Therefore,if we can actively intervene in the early stage-vascular cognitive dysfunction,not only can a good treatment effect be achieved,but also the process of becoming an irreversible dementia can be delayed.This topic aims to investigate the effects of the active ingredients of Danggui Shaoyao San,paeoniflorin and butenyl phthalide,on the OGD/R-induced injury of mouse microglia hypoxia and hypoglycemia model,microglia M1/M2 table Type dynamic balance,and the effect of p38/MAPK-PI3K/AKT signaling pathway on vascular dementia and its mechanism are discussed.MethodsThe OGD/R model was established by hypoxia and glucose deprivation injury of microglia,the cell activity of the model group was detected by CCK-8,the modeling conditions were determined,the safe concentration range of paeoniflorin and butenylphthalide was clarified,and the safe concentration range of paeoniflorin and butenylphthalide was evaluated.Cell viability after administration of ester glycosides and butenyl phthalide;ELISA to detect the content of inflammatory factors TNF-α and IL-1β in the cell supernatant,flow cytometry to detect the apoptosis of BV2 cells,and Hoechst33258 staining to observe the fluorescence intensity of cell nuclear damage Change;Western Blot detects the protein expression of PI3K,AKT,p38,ASK,microglia M1 type marker CD16/32,M2 type marker CD206,and detects the expression of LC3B.Caspase3.BAX.BCl-2 and other proteins.Results1.OGD/R construction of in vitro cell hypoxia and hypoglycemia model:Compared with the blank control group.the cell survival rate of microglia BV2 was significantly reduced after 4h.6h,and 8h OGD(P<0.001),hypoxia and hypoglycemia treatment 4h After 12 hours of reoxygenation,the cell survival rate increased slightly;after 6 hours and 8 hours of hypoxia and hypoglycemia treatment,the cell survival rate decreased for 12 hours after reoxygenation.Treating the cells under OGD4h/R12h conditions can make the cell survival rate around 50%,which is the best modeling condition.2.The effect of paeoniflorin and butenylphthalide on the activity of BV2 cells:compared with the blank control group,10μmol·L-1.20μmol L-1.40μmol·L-1,80μmol·L-1 There was no significant difference in the cell survival rate of the paeoniflorin group(P>0.05);5μmol·L1,10μmol·L-1,20μmol·L-1,40μmol·L-1,80μmol·L-1 butene There was no significant difference in the cell survival rate of the phthalide group(P>0.05),indicating that the concentration of paeoniflorin in the concentration range of 10-80 μmol·L-1 and the butenyl phthalide in the concentration range of 5-80μmol·L-1 No obvious toxicity to BV2 cells.3.The effect of paeoniflorin and buteny lphthalide on BV2 cell viability after OGD/R injury:Compared with the blank control group,the cell survival rate of BV2 cells in the model group after OGD/R injury was significantly reduced(P<0.001);Compared with the model group,the cell survival rate of the low,medium and high doses of paeoniflorin and the edaravone group was significantly improved(P<0.01,P<0.001),and it was positively correlated with the dose;butene The cell survival rate of low.medium and high dose groups of phthalide was significantly improved(P<0.05,P<0.001).The results show that paeoniflorin and butenylphthalide can significantly improve the survival rate of BV2 cells after OGD/R injury.4.The effect of paeoniflorin and butenylphthalide on the secretion of inflammatory factor TNF-αand IL-1βin BV2 cells after OGD/R injury:Compared with the blank control group,the inflammatory factor of BV2 cells in the model group after OGD/R injury The concentration of TNF-αand IL-1β increased significantly(P<0.001);compared with the model group,the concentration of TNF-αand IL-1β in the low,medium,and high-dose groups of paeoniflorin and the edaravone group significantly decreased(P<0.001),and compared with the model group There was a positive correlation:the concentration of TNF-αand IL-1βin the low,medium and high dose groups of butenylphthalide was significantly reduced(P<0.001).The results indicate that paeoniflorin and butenylphthalide can significantly reduce the concentration of inflammatory factor TNF-αand IL-1β in BV2 cells after OGD/R injury.5.Flow cytometric analysis of the effect of paeoniflorin and butenylphthalide on the apoptosis of BV2 cells after OGD/R injury:Compared with the blank control group,the BV2 cells of the model group are early after OGD/R injury And the proportion of apoptosis in the middle and late stages increased significantly(P<0.001).and the proportion of total apoptosis increased significantly(P<0.001);compared with the model group.the cells in the low.middle and high doses of paeoniflorin and edaravone groups The proportion of apoptosis in the early and middle and late stages was significantly reduced(P<0.001),and the proportion of total apoptosis was significantly reduced(P<0.001).and the total apoptotic proportion was positively correlated with the dose:butenylphthalide low.medium.and high dose groups and In the edaravone group,the proportion of cell apoptosis in the early and middle and late stages was significantly reduced(P<0.001).and the proportion of total apoptosis was significantly reduced(P<0.001).The results indicate that paeoniflorin and butenylphthalide can significantly inhibit the apoptosis of BV2 cells after OGD/R injury.6.Hoechst33258 staining to detect the effect of paeoniflorin and butenylphthalide on the apoptosis of BV2 cells after OGD/R injury:the blank control group cells are mostly smooth and round,and the nucleus is uniform blue;BV2 cells in the model group Nuclei fragmentation occurs,a large number of cells appear dense and dense,blue and whitish,which are the characteristics of apoptotic cells;nuclei staining in the edaravone group,paeoniflorin and butenylphthalide low,medium,and high dose groups Compared with the model group,the white polarized light is significantly reduced.The results indicate that paeoniflorin and butenylphthalide can inhibit the apoptosis of BV2 cells after OGD/R injury.7.Western Blot detection of LC3B.Caspase3.BAX,BCl-2 protein expression:Compared with the blank control group,the model group LC3B,Caspase3 protein expression increased(P<0.01,P<0.001),BAX/BCl-2 The ratio increased(P<0.001);compared with the model group,the paeoniflorin and butenylphthalide administration group could reduce LC3B,Caspase3(P<0.05,P<0.01,P<0.001),BAX/BCl The ratio of-2 decreased(P<0.01,P<0.001).indicating that the active ingredients of Danggui Shaoyao San and paeoniflorin and butenylphthalide can reduce autophagy induced by OGD/R and inhibit cell apoptosis.8.Western Blot detects the expression of p38,PI3K,AKT,ASK,CD 16/32,CD206 protein:Compared with the blank control group,the expression of p38 and ASK protein in the model group increased(P<0.01,P<0.001),PI3K And AKT protein expression decreased(P<0.01,P<0.001);compared with the model group,the paeoniflorin and butenylphthalide administration group can reduce the expression of p38 and ASK protein(P<0.05,P<0.01),P<0.001),PI3K and AKT protein expression increased(P<0.05,P<0.01,P<0.001),indicating that the active ingredients of Danggui Shaoyao San and paeoniflorin and butenylphthalide can reduce cell OGD/R The induced autophagy reduces inflammation and inhibits cell apoptosis.Compared with the blank control group,the CD16/32 protein expression of BV2 cells in the model group increased significantly after OGD/R injury(P<0.001),and the CD206 protein expression decreased significantly(P<0.001);compared with the model group In comparison,the CD16/32 protein expression of the cells in the low.medium,and high doses of paeoniflorin and the edaravone group were significantly reduced(P<0.001).and the expression of CD206 protein was significantly increased(P<0.001).The dose was positively correlated:the CD16/32 protein expression of cells in the butenylphthalide low,medium,and high dose groups were significantly reduced(P<0.001),and the CD206 protein expression was significantly increased(P<0.05,P<0.01,P<0.001).The results show that OGD/R promotes the expression of CD 16/32 protein in cells and reduces the expression of CD206 protein.Paeoniflorin and butenylphthalide can significantly reduce the protein expression of CD16/32 and increase the protein expression of CD206.ConclusionThe above research results suggest that the active ingredients of Danggui Shaoyao San and paeoniflorin and butenylphthalide can increase the activity of BV2 cells injured by OGD/R,regulate the M1/M2 phenotype homeostasis of microglia,reduce inflammation,and hinder Stagnation of cell autophagy and inhibition of cell apoptosis.The mechanism may be related to the regulation of p38/MAPK and PI3K-AKT signaling pathway. |
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