Effects And Mechanisms Of IL-10 On Apoptosis,Neurite Outgrowth And Synapse Formation In Rat Cortical Neurons After OGD Injury

Background and Aim: As an important anti-inflammatory and immunoregulatory cytokine,interleukin-10(IL-10)provides effective neuroprotection after cerebral ischemia.This effect can be confirmed either in middle cerebral artery occlusion(MCAO)-induced injury in vivo or oxygen-glucose deprivation(OGD)-induced injury in vitro.However,it remains uncertain whether IL-10 facilitates neurite outgrowth and synapse formation in cortical neurons followed OGD injury.To verify it,the cultured cortical neurons were taken to OGD injury to establish cerebral ischemia model in vitro.And then we evaluated the effects of IL-10 on apoptosis,neurite outgrowth and synapse formation in OGD-injured cortical neurons.Our study extends the understanding of neuroprotective effect and mechanisms of IL-10 on cerebral ischemia.At the same time,the study provides more evidences to suggest its therapeutic potential against cerebral ischemia.Methods: 1.Primary cortical neurons from rat were separated,cultured and identified in vitro.2.Seven days after plating,the rat cortical neurons were taken to OGD injury for 90 min to establish cerebral ischemia model in vitro.Then the light microscope and laser scanning confocal microscope were used to observe the morphology of OGD-injured cortical neurons.And the expression of IL-10R1 in cortical neurons was detected after OGD injury.3.IL-10,IL-10 NA and/or JAK1 inhibitor GLPG0634 were administered into the cultured rat primary cortical neurons right before and after OGD injury.The experimental groups were designed casually as follows: Control group,OGD group,OGD+IL-10 group,OGD+IL-10+GLPG0634 group and OGD+ IL-10 NA group.4.Then we carried out following experiments: The expression of IL-10R1、Bax、Bcl-2、Netrin-1、synaptophysin and GAPDH were analyzed by q PCR.The expression of IL-10R1、JAK1、p JAK1、STAT3、p STAT3、Bax、Bcl-2、Netrin-1、synaptophysin and GAPDH were detected by western blot and the apoptosis of neurons were assessed by flow cytometry using annexin V/PI staining.After imunofluorescence staining,the neurons were viewed and photographed by a ZEISS LSM 780 confocal microscope followed by LSM Image Browser(V4.2.0.121)to quantify the length of the longest neurites and the number of primary neurites,and Image J software was used to analysis the density of dendritic spines,excitatory synapses and inhibitory synapses.5.Five days after planting,to make sure the role of Netrin-1 in IL-10-induced neurite outgrowth,Netrin-1 sh RNA was used to knockdown Netrin-1 expression.Then scrambled sh RNA was used as control.Forty-eight hours later,the rat cortical neurons were taken to OGD injury for 90 min.IL-10 or IL-10 NA was added into the cultured rat primary cortical neurons right before and after OGD injury.The experimental groups were designed casually as follows: Control group,OGD group,OGD+scrambled sh RNA group,OGD+Netrin-1 sh RNA group,OGD+Netrin-1 sh RNA +IL-10 group and OGD+scrambled sh RNA+IL-10 group.Then the neurons were taken to imunofluorescence staining,take photo and measure the length of the longest neurites and the number of primary neurites.Results: 1.The neurite of cortical neurons are ruptured shortened,and even loss.Some nucleus are karyopyknosis,karyorrhexis and karyolysis after OGD.2.The m RNA level and protein level of IL-10R1 in the OGD group are both markedly increased when compared with the control group.3.IL-10 can activate the JAK1/STAT3 pathway in primary cortical neurons after OGD injury.4.IL-10 suppresses neuronal apoptosis of cultured cortical neurons by down-regulating the Bax expression and up-regulating the Bcl-2 expression after OGD injury.The anti-apoptotic effect can be partly abolished by JAK1 inhibitor GLPG0634.Contrarily,IL-10 NA increases OGD-injured apoptosis by increasing Bax expression and decreasing Bcl-2 expression.5.IL-10 increases the average length of the longest neurites,the average number of primary neurites and the expression of Netrin-1 in cultured cortical neurons after OGD injury.These effects are partly blocked by GLPG0634.Similar to the effect of GLPG0634,IL-10 NA decreases the average length of the longest neurites,the average number of primary neurites and the expression of Netrin-1 in OGD-injured cultured cortical neurons.The knockdown of Netrin-1 not only decreases the average length of the longest neurites and the average number of primary neurites in OGD-injured neurons,but also abolishes the effect of IL-10 on increasing the average length of the longest neurites and the average number of primary neurites after OGD injury.6.IL-10 can increase synaptophysin expression and the density of dendritic spines,excitatory synapses and inhibitory synapses in cultured cortical neurons.These effects can be partly restrained by GLPG0634.In contrast to IL-10,IL-10 NA decreases the expression of synaptophysin,the density of dendritic spines,excitatory synapses and inhibitory synapses in OGD-injured cortical neurons.Conclusions: 1.OGD increases m RNA level and protein level of IL-10R1 in cultured cortical neurons.2.IL-10 attenuates apoptosis in OGD-injured cortical neurons by activating JAK1/STAT3 signaling pathway to down-regulate Bax expression and up-regulate Bcl-2 expression.3.IL-10 facilitates neurite outgrowth in primary cortical neurons after OGD injury via JAK1/STAT3 pathway.4.IL-10 promotes synapse formation in OGD-injured cortical neurons by activating JAK1/STAT3 pathway to increase the expression of synaptophysin.