| ObjectiveTo explore the underlying mechanisms about the effects of IL-27 on protecting the brain from the damage caused by ischemic-reperfusion,through investigating the experimental models in vivo and vitro,basing on the gp130/STAT3 cell signaling pathway.Methods1.The healthy and clean mice were randomly divided into three groups:sham operation group(SHAM),model group(MCAO),IL-27 treated group(IL-27).The cerebral ischemic reperfusion models were established by 1 hour meddle cerebral artery occlusion followed by reperfusion.IL-27 was injected daily to the mice of IL-27 treated group on consecutive 7 days with the dosage0.2?g/d.The SHAM group only isolated the middle cerebral artery and theinner carotid artery,without ligating or blocking the arteries.Neurological behaviors of the mice were evaluated by applying the Bederson scale and the Beam walking test.The cerebral infarction volumes were calculated by the TTC staining method.All animals were sacrificed after the procedure and the brain tissues were collected and stored in the-80? freezer.Cytokines and chemokines relative to inflammation were detective in brain tissue by applying ELISA method.RT-PCR was utilized to investigate the folds changing of STAT3 mRNA in three groups of mice.The expressional levels of pSTAT3 protein were measured by employing the Western-blot method in three groups of mice.2.In vitro experiment,the primary culture of fetal rat cortical neurons were treated with no glucose and less oxygen condition to establish the OGD cell model.To mimick the ischemic reperfusion state,the neurons were placed back to regular condition,after the OGD procedure.The concentration of LDH(lactate dehydrogenase,LDH)and apoptosis ratios were measured in OGD neurons with and without IL-27 treated.In order to investigate the relationship of gp130/STAT3 cell signaling pathway and IL-27,the gp130 siRNA and the STAT3 phosphate inhibitor stattic were applied to the OGD neurons and control group,and the western blot and colorimetric method were utilized to value the pSTAT3,Bcl2 protein and LDH respectively.Results1.Ischemia reperfusion 7 days later,the Bederson score in IL27 group(1.68±0.55)were lower than that(2.40±0.55)in MCAO group(P<0.05),and the Beam walking test score in IL-27group(2.20±0.45)was lower than that in MCAO group(3.00±0.61)(P<0.05);the percentage of cerebral infarction volume in IL-27 group(12.45±6.18%)was less than that(38.89±5.99%)ofMCAO group(P<0.05).The ELISA detecting results of cytokines and chemokines in cerebral infarction brain showed that the concentrations of TNF-?,IL-1?,MCP-1 in IL-27 treated group(232.24±26.59,36.16±6.63,28.70±2.47)were lower than that(473.70±36.34,51.85±10.19,68.16±7.69)of MCAO group(P<0.05)and the concentration of IL-10,TGF?(23.69±7.60,49.17±8.17)were higher than that(12.56 ±3.78,25.45±5.49)in MCAO group(P<0.05).2.The results of RT-PCR showed that the expression of STAT3mRNA(3.15±0.60folds)in IL-27 treated group was higher than that(2.52±0.430folds)in MCAO group(P<0.05).Western blot results showed that the p STAT3(5728.80±368.11)in group treated with IL-27 was higher than that(3025.20±647.74)of MCAO group(P<0.05).3.The LDH concentration in the IL-27 group(239.19±10.52)was significantly lower than that(371.65±43.34)in the OGD group(P<0.05).The cell apoptosis ratio of IL-27 treated group(55.14±10.76%)was lower than that(81.87±2.81%)of OGD group(P<0.05).4.The expression of the pSTAT3(1023.33±173.08)and Bcl2(1320.0±244.06)of OGD+gp130 siRNA +IL-27 group were lower than that(1426.0±196.55,1790±280.26)of OGD+ CON+IL-27 group(P<0.05),and the expression of pSTAT3(650.0±239.74)and Bcl2(879±204.59)of OGD+gp130siRNA group were lower than that(1035.67±74.01,1035.67±74.01)of OGD+CON group(P<0.05).5.There was no significant difference in LDH concentration between OGD+IL-27+Stattic-10?M group and OGD group(231.05±18.09vs234.71±16.41,P>0.05),but the LDH concentrations in OGD+IL-group and OGD+IL-27+stattic-5 ?M group were higher than that in OGD group(98.90±6.11,130.94±12.03 vs 243.71±16.41,P<0.05).Western blotshowed that the expression level of pSTAT3 in OGD+IL-27+stattic-10 ?M group was slightly higher than that in OGD group(674.33±173.32 vs622.33±137.55,P>0.05),and so did the expression level of Bcl2(531±133.72 vs 466±120.80,P>0.05);and in the OGD+IL-27 group the expression level of pSTAT3 was higher than the OGD group(1373.67±277.29 vs 622.33±137.55,P<0.05)and the expression level of Bcl2 was the same as the pSTAT3(1050.33±272.26 vs 466±120.80,P<0.05);in the OGD+IL-27+stattic-5?M group the expression of p STAT3 was higher than that of the OGD group(1414.67±315.69 vs 622.33±137.55,P<0.05),and the expression of Bcl2 was similar with pSTAT3(901±234.04 vs 466±120.80,P<0.05).ConclusionOur study showed that IL-27 could improve neurological defects and decrease the infarction volume of the cerebral ischemic-reperfusion model mice and gp130 siRNA and Stattic could inhibit the protective effect of IL-27 on OGD neurons;the possible mechanism could be associated with the IL-27 mediating the cell signaling pathway gp130/STAT3 and enhancing the expression of the anti-apoptosis protein Bcl2. |
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