| Background: The activation of astrocytes and inflammation aggravate the apoptosis of neurons after cerebral ischemia,leading to the loss of neural function.Resveratrol(Res)can inhibit the activation and inflammation of astrocytes,thus protecting neurons,but its mechanism has not been elucidated.Sonic hedgehog(Shh)signaling pathway plays an important role in neuroprotection after cerebral ischemia,but it is unclear whether it mediates resveratrol to inhibit the activation and inflammation of astrocytes after cerebral ischemia.In addition,resveratrol is a specific agonist of sirtuins 1(Sirt1).The relationship between Shh signal path and Sirt1 needs further discussion.Objective: To explore whether resveratrol can affect the activation and inflammation of astrocytes by activating Shh signaling pathway after oxygen glucose deprivation/reoxygenation(OGD/R)injury,and whether resveratrol can activate Shh signaling pathway through Sirt1.Methods:The primary cultured astrocytes were isolated from the cerebral cortex of newborn SD rats within 24 hours,deprived of oxygen and glucose for 150 minutes,and then reoxygenated for 24 hours.1.The concentration effect of resveratrol on astrocytes after OGD / R injury.The experiment was divided into:(1)Normal group(Normal,Nor),(2)Control group(Control,Ctrl),(3)Vehicle group(Vehicle,Veh),(4)Resveratrol pretreatment group with different concentrations(1,5,20,40,80,100 μ mol / L,Res).2.Effect of resveratrol on activation and inflammation of astrocyte after OGD/R injury.The experiment was divided into:(1)Normal group(2)Control group,(3)Resveratrol pretreatment group with concentration of40μmol/L.3.Effect of resveratrol on Shh signaling pathway of astrocytes after OGD/R injury.The experiment was divided into:(1)Normal group,(2)Control group,(3)Resveratrol pretreatment group with concentration of40μmol/L,(4)Smo inhibitor cyclopamine group(Cyc),(5)40μmol/L Resveratrol+Cyclopamine group(Res+Cyc),(6)40μmol/L Resveratrol+Smo agonist Purmorphamine group(Res+ Pur).4.Effect of Shh signaling pathway on astrocytes activation and inflammation after OGD/R injury.The experiment was divided into:(1)Control group,(2)Resveratrol pretreatment group with concentration of40μmol/L,(3)40μmol/L resveratrol+ Cyclopamine group(Res+Cyc).5.Effect of Sirt1 on Shh signaling pathway.The experiment was divided into:(1)Normal group,(2)Control group,(3)Resveratrol pretreatment group with concentration of 40μmol/L,(4)Sirt1 inhibitor group(Sirtinol),and(5)Shh group(Shh).CCK-8 method was used to detect cell viability,immunofluorescence and Western blotting were used to detect the expression and nucleartranslocation of related proteins,and ELISA was used to detect the levels of related proteins.Result:1.The concentration effect of resveratrol on astrocytes after OGD/R injury in vitro.CCK-8 assay showed that with the increase of resveratrol concentration(5,20,40 μmol/L),the cell viability increased gradually,which was significantly higher than that in the control group(P < 0.01).The cell viability of 40μmol/L group was the highest.After that,with the increase of resveratrol concentration(80 and 100 μmol/L),the cell viability decreased significantly,and the cell viability of 100 μmol/L group was weakest(P<0.05).2.Effect of resveratrol on astrocytes activation and inflammation after OGD/R injury.Edu detection showed that after OGD/R injury,the percentage of edu positive cells in the control group and resveratrol group was significantly higher than that in the normal group(P<0.01),while that in resveratrol group was significantly lower than that in the control group(P<0.01).Western blotting and ELISA showed that the expression or levels of GFAP,S100β,vimentin,IL-10,IFN-β,TNF-α,IL-1β in the control group and resveratrol group were significantly higher than that in the normal group(P<0.05),but the expression or levels of IL-10 and IFN-β in resveratrol group were lower than that in the control group(P < 0.05).3.Effect of resveratrol on Shh signaling pathway of astrocytes after OGD/R injury.Immunofluorescence staining showed that Gli-1 was expressed in the cytoplasm of the normal group.After OGD/R injury,part of Gli-1 was transferred to the nucleus;in the Res group and the Res+Pur group,theexpression of Gli-1 protein in the nucleus was significantly enhanced;in the Cyc group and the Res+Cyc group,the nuclear translocation of Gli-1protein was significantly inhibited,especially in the Cyc group.WB showed that the expression of Shh,Smo,Ptc-1 and Gli-1 proteins in control group,Res group and Res+pur group were significantly higher than that in Normal group,that in Res group and Res+Pur group were significantly higher than that in control group,and that in Res+Pur group was significantly higher than that in Res group(P<0.01);The expression of those proteins in Cyc group was significantly lower than that in control group,Res group and Res+Cyc group;That in Res+Cyc group was significantly lower than that in Res group(P < 0.01).4.Effect of Shh signaling pathway on astrocytes activation and inflammation after OGD/R injury.Edu test showed that after OGD/R injury,the percentage of edu positive cells in Res group and Res+Cyc group was significantly lower than that in Control group(P<0.01),and that in Res+Cyc group was significantly higher than that in the Res group(P<0.01).Western blotting and ELISA showed that the expression or content of GFAP,TNF-α and IL-1β in Res group and Res+Cyc group were significantly lower than that in Ctrl group,and the expression or content in Res+Cyc group was significantly higher than that in Res group.The proteins expression or levels of IL-10 and IFN-β in Res group and Res+Cyc group was significantly higher than that in Ctrl group,and the proteins expression or levels in Res+Cyc group was significantly lower than that in Res group(P < 0.05).5.Effect of Sirt1 on Shh signaling pathway.Immunofluorescence staining showed that Gli-1 accumulated in the cytoplasm of Nor group.After OGD/R injury(Ctrl),part of Gli-1 wastransferred to the nucleus.Most of Gli-1 was transferred to the nucleus in Res group and Shh group.However,Sirt1 inhibitors(sirtinol)group inhibited the nuclear translocation of Gli-1.Western blotting showed that the proteins expression of Shh,Smo,Ptc-1 and Gli-1 in the Control group,Res group and Shh group was significantly higher than that in the Normal group,that in Res group and Shh group was significantly higher than that in the Control group(P<0.01),and that in sirtinol group was significantly lower than that in the Control group(P<0.01).Conclusion: Resveratrol pretreatment can inhibit the activation and inflammation of astrocytes after OGD/R,The mechanism may be that Sirt1 influences Shh signaling pathway. |
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