| Background: Oxidative stress injury is one of the core pathological processes after cerebral ischemia,and the abnormal activation of microglia is closely related to oxidative stress.The activation process of microglia is the polarization process of M1(pro-inflammatory)and M2(anti-inflammatory)microglia.Resveratrol has been proved to have the effects of antioxidant stress and promote the transformation of microglia to M2 type,but the mechanism has not been clarified.Previous studies have shown that resveratrol has neuroprotective or repairing effects via activating Nrf2 and Shh signaling pathways.However,it is unclear whether Nrf2 and Shh signals participate in the microglial M2 polarization process and whether its interact with each other.Therefore,this study investigated the interaction of Nrf2 and Shh signaling and the role of resveratrol in regulating microglial polarization and oxidative stress injury after OGD /R injury in vitro,so as to provide a solid experimental basis for the early useof resveratrol in clinic and the development and design of new drugs.Methods: N9 microglia were cultured in vitro with oxygen glucose deprivation/reoxygenation(OGD/R)model;N9 microglia-HT22 neurons were co-cultured model.1.To explore the role of Nrf2 signaling in resveratrol-regulated microglial polarization and oxidative stress injury after OGD/R injury and its effect on Shh signaling pathway.The experiment was divided into 6groups:(1)normal group(Nor),(2)control group(Ctrl),(3)20umol/L resveratrol group(Res),(4)Nrf2 inhibitor 5umol/L ML385 group,(5)20umol/L resveratrol combined 5umol/L ML385 group(R+M),(6)Nrf2agonist 100nmol/L RTA-408 group.2.To investigate the role of Shh signal in resveratrol regulating microglial polarization and oxidative stress injury after OGD/R injury and its effect on Nrf2 signaling pathway.The experiment was divided into five groups:(1)normal group(Nor),(2)control group(Ctrl),(3)20umol/L resveratrol group(Res),(4)Shh signaling pathway Smo receptor inhibitor5umol/L cyclopramine group(Cyc),(5)20umol/L resveratrol combined5umol/L cyclopramine group(R+C).3.To explore the effect of conditional co-culture of neurons and microglia on HT22 neurons.3.1 To explore the effect of resveratrol mediated by Nrf2 signal on neurons-microglia condition co-culture on neurons.The experiment wasdivided into 6 groups:(1)normal group(Nor),(2)control group(Ctrl),(3)20umol/L resveratrol group(Res),(4)Nrf2 inhibitor 5umol/L ML385 group,(5)resveratrol combined ML385 group(R+M),(6)Nrf2 agonist100nmol/L RTA-408 group.3.2 To explore the effect of Shh signal-mediated resveratrol on neuron-microglia co-culture on neurons.The experiment was divided into five groups:(1)normal group(Nor),2)control group(Ctrl),3)20umol/L resveratrol group(Res),(4)Shh signaling pathway Smo receptor inhibitor 5umol/L cyclopramine group(Cyc),5)resveratrol combined cyclopramine group(R+C).Cell viability,MDA content and SOD activity were measured by Cell Counting Kit-8,TBA and WST-1 assays.Related proteins or mRNA of the polarization of N9 microglia and the expression of Shh and Nrf2 signaling pathway were detected by Immunofluorescence,Western blotting and RT-qPCR.Apoptosis of HT22 neurons was detected by flow cytometry.Results:1.The role of Nrf2 signaling in resveratrol regulating microglial polarization and oxidative stress injury after OGD / R injury and its effect on Shh signaling pathway.After OGD/R injury,compared with the control group,the cell viability,SOD activity,CD206,Arg1,Shh,Ptc-1,SMO,Gli1 related proteins or mRNA expressions in 20umol/L resveratrol group and RTA-408 group were significantly increased,while MDA contents and iNOS,TNF-αproteins or mRNA expressions were significantly reduced(P < 0.05),and Shh protein in resveratrol group was significantly shifted to the nucleus compared with the control group.In ML385 group,the cell activity,SOD activity,CD206,Arg1,Shh,Ptc-1,SMO,Gli1 related proteins or mRNA expressions were significantly decreased,while MDA contents and iNOS,TNF-α proteins or mRNA expressions were significantly increased(P <0.05).Shh protein was mainly expressed in the cytoplasm of the cell.2.The role of Shh signal in resveratrol regulating microglial polarization and oxidative stress injury after OGD / R injury and its effect on Nrf2 signaling pathway.After OGD/R injury,compared with the control group,the cell viability,SOD activity,CD206,Arg1,Nrf2,HO-1,NQO1 related proteins or mRNA expressions in 20umol/L resveratrol group were significantly increased,while MDA contents and iNOS,TNF-α proteins or mRNA expressions were significantly reduced(P<0.05),Nrf2 protein of resveratrol group was significantly shifted to the nucleus compared with the control group.The cell viability,SOD activity,CD206,Arg1,Nrf2,HO-1,NQO1 proteins or mRNA expressions were significantly decreased,while the MDA contents and iNOS,TNF-α proteins or mRNA expressions were significantly increased(P<0.05),Nrf2 protein was mainly expressed in the cytoplasm.3.Effects of conditional co-culture of neuron-microglia on HT22 neurons.After neuron-microglia condition co-culture,the percentage of apoptosis in the normal group of HT22 neurons was the least and the cell viability was the strongest.Compared with the control group,the resveratrol group and RTA-408 group had increased cell viability.However,the percentage of neuronal apoptosis in the ML385 group and the cyclopramine group decreased significantly and the cell viability decreased significantly.Conclusion: Nrf2 may interact with Shh to regulate resveratrol,promote microglial polarization to M2 and inhibit oxidative stress injury after OGD/R injury,so as to improve neuron damage and reduce brain damage.At the same time,the medium released by microglia regulates the apoptosis and viability of peripheral neurons. |
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