| ObjectiveThe purpose of this study was to explore the relationship between the anti-inflammatory effects of Tetrahydroxy stilbene glycoside and Osthol and APOE-TREM2 signal transduction pathway and its mechanism in Aβ1-42-induced mouse microglia AD model.MethodsTo establish a method of inducing BV2 cells to replicate AD model in vitro by Aβ1-42,and to determine the concentration of the model combined with the method of CCK-8 to detect cell viability.The safe administration range of Tetrahydrox stilbene glycoside and Osthol was selected.After pretreatment with Tetrahydrox stilbene glycoside and Osthol,the cell viability was detected by CCK-8,the cell morphology was observed by inverted microscope,the secretion of inflammatory cytokines IL-1βand TNF-α was detected by ELISA kit,the expression of M1 microglial marker CD16/32 was detected by immunofluorescence.The effect of BV2 cell culture supernatant on SH-SY5Y morphology was observed by inverted microscope,and the effect of BV2 cell culture supernatant on SH-SY5Y apoptosis was detected by flow cytometry.And the expression of APOE and TREM2 in microglia was detected by qPCR and Western Blot.Using SPSS21.0 statistical software,the results were expressed by mean ±standard deviation(x±s),and the mean values of data among groups were compared by one-way analysis of variance(One-Way ANOVA),pairwise comparison using LSD test,P<0.05 was considered that there was statistical difference.Results1.Selection of modeling concentration of Aβ1-42:CCK-8 assay showed that 30μmol·L-1,20 μmol·L-1 and 10 μmol·L-1 Aβ1-42 decreased the viability of BV2 microglia,but the solvent control group of 30 μmol·L-1 Aβ1-42 and 20 μmol·L-1 Aβ1-42 also decreased the viability of BV2 microglia,while the solvent control group of 10 μmol·L-1 Api-42 had no significant damage to BV2 cells.Therefore,10μmol·L-1 Aβ1-42 was chosen as the modeling concentration of cell AD model.2.Toxicity test of Tetrahydroxy stilbene glycoside and Osthol:after BV2 cells were treated with Tetrahydroxy stilbene glycoside and Osthol at different concentrations for 24 hours,CCK-8 assay showed that Tetrahydroxy stilbene glycoside had no obvious toxic effect on BV2 cells in the concentration range of 0-50μmol·L-1.Osthol had no obvious toxic effect on BV2 cells when the concentration of osthol was 0-25 μmol·L-1,but it had obvious toxic effect on BV2 cells when the concentration of osthol reached 50 μmol·L-1.3.The protective effects of Tetrahydroxy stilbene glycoside and Osthol:the cell viability of each group was calculated by CCK-8 after pretreatment with high,middle and low doses of Tetrahydroxy stilbene glycoside(50 μmol·L-1,10 μmol·L-1,2μmol·L-1)and high,middle and low doses of Osthol(25 μmol·L-1,5 μmol·L-1,1μmol·L-1)for 24 hours.The results showed that compared with the blank group,the cell survival rate of BV2 cells was significantly decreased in the model group(P<0.001).Compared with the model control group,the cell survival rate in the low,middle and high dose groups of Tetrahydroxy stilbene glycoside was significantly increased(P<0.01,P<0.001),and positively correlated with the drug dose.The cell survival rate was significantly increased in the low,middle and high dose groups of osthol(P<0.01,P<0.001),and the effect was the most obvious in the low dose group.4.The results of cell morphology:compared with the blank group,the number of cells in the model group decreased significantly,and the cells changed from resting state to activated state,the antennae elongated,the branches increased,and the cells changed from round to "amoeba-like".Compared with the model group,the activated cells in the high and middle dose groups of Tetrahydroxy stilbene glycoside were significantly decreased,and the cells in the resting state were significantly increased.Although there were more activated cells in the low dose group of Tetrahydroxy stilbene glycoside,they were significantly improved compared with the model group.The number of activated cells in the high,middle and low dose groups of osthol decreased significantly,while the number of cells in resting state increased significantly,and the state of cells in the low dose group of osthole was the best,and the number of activated cells was the least.5.Detection of inflammatory cytokine IL-1β:compared with the blank group,the secretion of inflammatory cytokine IL-1β in the model group was significantly increased(P<0.001).Compared with the model group,the secretion of IL-1βdecreased significantly after pretreatment with Tetrahydroxy stilbene glycoside(P<0.05,P<0.001),and positively correlated with the drug dose.The secretion of IL-1β was significantly decreased in each dose group of osthol(P<0.001),and the effect was the most obvious in the low dose group of osthol.6.Detection of inflammatory cytokine TNF-α:compared with the blank group,the secretion of inflammatory cytokine TNF-α in the model group increased significantly(P<0.001),compared with the model group,Tetrahydroxy stilbene glycoside significantly decreased the secretion of TNF-α(P<0.001),and positively correlated with the drug dose.Osthole groups significantly decreased the secretion of TNF-α(P<0.01,P<0.001),and the effect was the most obvious in the low dose group of Osthol.7.Detection of Ml microglia:in order to evaluate M1 microglia,BV2 cells were stained with CD 16/32 for M1 phenotypic immunostaining.Red showed that the nucleus stained by CD16/32,DAPI,a surface marker of M1 microglia,was blue.Compared with the blank group,the expression of CD 16/3 2 on the surface of BV2 cells in the model group was significantly increased.The expression of CD 16/32 on the cell surface of Tetrahydroxy stilbene glycoside group and osthol group was significantly lower than that of the model group.It is suggested that Aβ1-42 promotes the increase of M1 phenotype of BV2 cells,while Tetrahydroxy stilbene glycoside and Osthol inhibit this change,among which the middle and high dose groups of stilbene glycoside have more obvious effect,while the low dose group of osthol has more obvious effect.8.The effect of BV2 cell culture supernatant on SH-SY5Y:compared with the blank group,the injury of SY5Y cells in the model group was obvious,and the early and late apoptotic cells were significantly increased(P<0.001).Compared with the model group,the cell state was improved and the total apoptotic cells decreased in all stilbene glycosides groups(P<0.01,P<0,001),and the apoptotic cells were the least in the high dose stilbene glycosides group.The cell state was improved and the total apoptotic cells decreased in each dose group of osthol(P<0.05,P<0.01,P<0.001),and the number of apoptotic cells was the least in the low dose group.9.The gene expression levels of APOE and TREM2 were detected by qPCR:compared with the blank group,the relative expression of APOE and TREM2 mRNA in the model group increased significantly(P<0.001),compared with the model group,the gene expression of APOE in high,middle and low dose Tetrahydroxy stilbene glycoside groups decreased significantly(P<0.001,P<0.01),and the gene expression level of APOE decreased with the increase of stilbene glycoside dose.Compared with the model group,the high-dose Tetrahydroxy stilbene glycoside group could reduce the expression of TREM2 at the gene level,but there was no significant difference between the middle and low-dose stilbene glycoside groups and the model group.The gene expression of APOE was significantly decreased in the high,middle and low dose groups of osthol(P<0.001),and the gene level of APOE in the low dose group of osthol was the lowest.The gene expression of TREM2 in the high and low dose groups of osthol could be decreased at the gene level(P<0.05),but there was no significant difference between the middle dose group and the model group.10.The protein expression of APOE and TREM2 was detected by Westren Blot:compared with the blank group,the protein expression of APOE and TREM2 in the model group increased significantly(P<0.001),compared with the model group,the expression of APOE and TREM2 decreased significantly in the high,middle and low dose Tetrahydroxy stilbene glycoside groups(P<0.001,P<0.01),and the protein levels of APOE and TREM2 decreased with the increase of Tetrahydroxy stilbene glycoside dose.The expressions of APOE and TREM2 were significantly decreased in the high,middle and low dose groups of osthol(P<0.001,P<0.01,P<0.05),especially in the low dose group.ConclusionThe above results suggest that Aβ1-42 can damage microglia,induce BV2 cells to change from resting state to M1 active state,and increase the secretion of pro-inflammatory cytokines IL-1β and TNF-α,while Tetrahydroxy stilbene glycoside and Osthol can reduce Aβ1-42-induced apoptosis and inhibit the production of pro-inflammatory cytokines.The mechanism may be related to the decrease of the expression of APOE and TREM2.The high dose of Tetrahydroxy stilbene glycoside and low dose of Osthol had the best anti-inflammatory effect. |
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