Mechanism Study Of Endogenous IL-6 Secreted By Bone Marrow Mesenchymal Stem Cells Regulating The Ogd Injured

Part One:The effect of bone marrow mesenchymal stem cells separated coculture on the OGD injured astrocytesObjective To investigate the effect of bone marrow mesenchymal stem cells (MSCs) on the injured astrocytes following oxygen glucose deprivation (OGD) through the model in vitro of MSCs separated coculture with OGD injured astrocytes.Methods Primary astrocytes were cultured and identified by GFAP immunofluorescence staining.The primary astrocytes was damaged by OGD, and then separately cocultured with MSCs. Meanwhile, the astrocytes with or without OGD treatment were set up as its control groups (named as OGD group and control group).The levels of IL-6 secretion were tested in the coculture medium by ELISA kit. The levels of IL-6R, STATS, Bcl-2, Bax mRNA and protein expressions in the injured astrocytes were detected by real-time PCR and western blotting, respectively. Calcium imaging system determined the changes of cytoplasmic Ca2+ concentration in the astrocytes following ATP activation. And the proliferation of the injured astrocytes were detected by CCK-8 kit.Results (1) With identification of GFAP immunofluorescence staining, the purity of primary astrocytes was more than 95%. (2) After cocultured with MSCs, the level of IL-6 secretion in the coculture medium was statistically increased compared with that of OGD group (P<0.05). (3) After separated coculture with MSCs, the level of Bcl-2 expression was significantly increased in the OGD injured astrocytes (P<0.01), and the level of Bax expression slightly decreased, while the ratio of the Bcl-2/Bax expression level was statistically higher than those of other two groups (P<0.05). (4) MSCs coculture significantly induced to upregulate the levels of IL-6R and STAT3 mRNA and protein expressions compared with those of the OGD group.(5) Compared with the control group, ATP activation enhanced Ca2+ release in the OGD injured astrocytes (P<0.001), however, MSCs coculture significantly reduced the level of intracellular Ca2+ in the OGD astrocytes (P<0.05). (6) The proliferation activity of the injured astrocyte was induced by OGD, but the MSCs separated coculture obviously impaired the proliferation capability of injured astrocytes.Conclusion OGD damage leads to decrease of Bcl-2/Bax expression ratio in the astrocytes, increase of intercellular Ca2+ concentration and promotes proliferation activity in the astroxytes. However, MSCs separated coculture upregulates IL-6 secretion level in the cocutlure medium to activate the IL-6R/STAT3 signaling pathway, promotes the ratio of the Bcl-2/Bax expression levels to reduce intercellular Ca2 +release in the OGD injured astrocytes, and suppresses excessive proliferation of injured astrocytes so as to maintain the normal physiological function of the astrocytes.Part two:Endogenous IL-6 of bone marrow mesenchymal stem cells regulates restoration of the damaged astrocytesObjective To evaluate the effects of endogenous IL-6 secreted by MSCs on the OGD damaged astrocytes through siIL-6 MSCs separated coculture with the injured astrocytes.Methods The siIL-6 MSCs were separately cocultured with the OGD injured astrocytes, and the GFP MSCs served as control group. The levels of IL-6 secretion in the cultural supernatant was determined by ELISA kit. The levels of IL-6R, STAT3 expression in the IL-6 pathway and apoptotic factors Bcl-2, Bax mRNA and protein expressions was detected by real-time PCR and western blotting, respectively. The changes of intracellular Ca2+concentration was determined in the injured astrocytes by calcium imaging system, and its proliferation ability was tested by CCK-8 kit.Results (1) Following separately coculture with siIL-6 MSCs, the level of IL-6 secretion in the cultural supernatant was significantly decreased compared with GFP MSCs group (P<0.01). (2) The levels of IL-6R and p-STAT3 expressions were obviously reduced in the OGD injured astrocytes after cocultured with siIL-6 MSCs (P<0.01, P<0.01). (3) Compared with the GFP MSCs group, the siIL-6 MSCs coculture significantly decreased the levels of Bcl-2 (P<0.01), and slightly increased the levels of Bax expression in the injured astrocytes, but the ratio of the Bcl-2/Bax expression level was decreased significantly (P<0.01). (4) ATP induced the increase of Ca2+ release in the OGD injured astrocytes following cocultured with siIL-6 MSCs (P<0.05). (5) The siIL-6 MSCs separated coculture statistically enhanced the proliferation of the damaged astrocytes compared with the GFP MSCs group.Conclusion With the decrease of the IL-6 secretion levels in MSCs, the activation of the IL-6R/STAT3 signaling pathway was supressed in OGD injured astrocytes, the ratio of Bcl-2/Bax expression level was decreased, intracellular Ca2+ release was increased in the OGD injured astrocytes, and the proliferation of injured astrocytes accelerated.Part three:The effect of exogenous cytokines IL-6 on the astrocytes following OGD injuryObjective To compare the effects on the injured astrocytes between the recombinant cytokine IL-6 and endogenous IL-6 from MSCs through OGD astrocytes treated by exogenous IL-6.Methods The OGD damaged astrocytes were treated by recombinant IL-6 for 48h, and the OGD injured astrocytes without IL-6 treatment were served as control. The concentration of IL-6 in the culture medium was determined by ELISA kit. The levels of IL-6R, STAT3 anf Bcl-2, Bax protein expressions were detected by western blotting. The changes of Ca2+ levels in the damaged astrocytes were measured by calcium imaging system, and the proliferation capability of the injured astrocytes was tested by CCK-8 kit.Results (1)After IL-6 treatment for 48h, the concentration of IL-6 in the culture supernatant was increased compared with that of the OGD injured astrocytes, but there were no statistical difference between the two groups (P>0.05). (2) The exogenous IL-6 could upregulate the levels of IL-6R and STAT3 protein expression in the injured astrocytes. (3) The level of Bcl-2 protein expression was significantly increased and the Bax expression was obviously decreased in the injured astrocytes with IL-6 treatment. The ratio of Bcl-2/Bax expression levels was markedly induced in the IL-6 treated group compared with that of IL-6 untreatment (P< 0.01). (4) ATP induced the increase of Ca2+levels in the OGD astrocytes following IL-6 treatment, but there was no significant difference on the increase range between the two groups. (5) The proliferation abilities were not significantly different between the OGD injured astrocytes with, or without IL-6 treatment.Conclusion Exogenous IL-6 can activate the IL-6R/STAT3 signaling pathway, increase the ratio Bcl-2/Bax expression levels to enhance the anti-apoptotic ability of the injured astrocytes. However, the recombinant cytokine IL-6 don’t affect the intracellular Ca2+ concentration and the proliferation of the OGD astrocyte.