Oxygen Glucose Deprivation Induces IL-10 Secretion Of Astrocytes To Suppress Neuronal Apoptosis

Part I Astrocytes injured by OGD release IL-10 to repress neuronal apoptosisObjective: To investigate the expression level of IL-10 and its biological function among the process of oxygen glucose deprivation(OGD)injured to the primary astrocytes and neurons.Methods: The astrocytes and neurons were primary cultivated and injured by OGD.The levels of IL-10 mRNA and protein expressions were detected by Real-time PCR and ELISA techniques,and the cells without OGD injury were served as its control.The cell proliferation and apoptosis in the astrocytes injured by OGD were tested following exogenous IL-10 intervention through the kits of CCK-8 and Annexin V-FITC/PI.Meanwhile,the levels of Ca2+ in the cytoplasm and cell apoptosis in the OGD injured neuron after the IL-10 treatment were tested with calcium imaging and Annexin V-FITC/PI kit,respectively.Results:(1)Primary astrocytes and neurons were respectively identified by GFAP and NSE using immunofluorescence method,and injured by OGD successfully.(2)The mRNA(P=0.026)and protein(P=0.032)expression levels of IL-10 were both significantly increased in the OGD astrocytes compared with the control group;while the levels of IL-10 were not differed in the neuron with or without OGD treatment.(3)Exogenous IL-10 treatment suppressed the early apoptosis(P=0.009)and late apoptosis(P=0.002),and also activated the release of intracellular calcium ions(P<0.0001)in the OGD injured neurons,but didn't significantly affect on the proliferation and apoptosis of the OGD injured astrocytes.Conclusions:(1)OGD injury models are successfully established on the primary astrocytes and neurons.(2)The OGD injury induces astrocytes paracrine IL-10 to repress the apoptosis of OGD injured neurons and activate the release of intracellular calcium ions in the OGD neurons.Part II Molecular mechanism of IL-10 secretion in the OGD induced astrocytesObjective: To explore the molecular mechanism of IL-10 release in the OGD induced astrocytes.Methods: The levels of TLR2 and NF?B p-p65 protein expressions were detected in the OGD injured astrocytes using immunofluorescence and western blotting techniques.The levels of TLR2,p-p65 and IL-10 expressions were deteced in the OGD injured astrocytes following si TLR2 adenovirus infection or PDTC treatment via Western blotting and ELISA techniques.Lastly,CHIP-q PCR was used to examine the interaction between p-p65 and the promoter of IL-10 gene in the primary astrocytes injured by OGD.Results:(1)The levels of TLR2(P=0.003)and p-p65(P=0.0001)protein expressions were significantly increased in the OGD astrocytes compared with those of the control group.(2)With the si TLR2 adenovirus infection,the levels of TLR2,p-p65 and IL-10 potein expressions were statistically lower than those of the OGD + RFP group(P<0.0001).(3)After PDTC treatment,NFkB specific blocker,to the OGD damaged astrocytes,the levels of p-p65(P<0.0001)and IL-10(P=0.012)protein expressions were significantly decreased compared with those of the OGD group.But the expression levels of TLR2 protein were no statistically different in theOGD astrocytes in the present or absent of PDTC treatment.(4)The p-p65 protein was enriched on the promoter of IL-10 gene in the primary astrocytes injured by OGD(P=0.0008).Conclusions: OGD injury induces high level of TLR2 expression in the primary astrocytes and actives NF?B signaling pathway to enhance the p-p65 interaction on the promoter of IL-10,which promots the transcriptional activy of IL-10.