Study Of The Inflammatory Inhibition Effect Of SOCS3 On Pharmacological Preconditioning Of Cerebral Ischemia Induced By Lipopolysaccharides(LPS)

First part: Study of the Inflammatory Inhibition effect of SOCS3 on Pharmacological Preconditioning induced by Lipopolysaccharides(LPS)in mice suffering from cerebral ischemia reperfusion injuryObjective:To investigate the neuro-protective effects of LPS on pharmacol ogical preconditioning in mice suffering from cerebral ischemia-Reperfusion(I/R)injury.To explore whether there are mutual regulations between Janus Kinase 2/Signal transducers and activators of transcription 3/Suppressors of cytokine signaling 3(JAK2-STAT3-SOCS3)signaling pathway and Toll-like receptor 4/myeloid differentiation protein 88(TLR4-My D88)inflammatory pathway or not.Methods:The animals were administered by intraperitoneal injection of low dose of LPS 48 h before Middle Cerebral Artery occlusion(MCAO).The cerebral ischemia-reperfusion injury model was established by MCAO.The left middle cerebral artery was embolised for 2 hours and then restored blood reperfusion after 24 hours.The model of cerebral ischemia-reperfusion in jury in mice was evaluated by Longa’s scoring method.The cerebral infarction volume was determined by TTC staining.The inflammatory cytokines such as IL-6,IL-1β and TNF-ɑ in serum and ischemic brain tissue were detected by ELISA kits.Protein expression of TLR4,My D88,nuclear NF-κB,cytosolic NF-κB,p-JAK2,JAK2,p-STAT3,STAT3 and SOCS3 in ischemic brain tissue were measured by Western-blot method.Results:(1)Compared with the Sham group,The neuroscores was increased significantly in ishemia/reperfusion(I/R)group compared with Sham group.(p<0.05).(2)In comparison with Sham group,LPS pharmacological preconditioning markedly improved the neurological deficit and the neuroscores were significantlydecreased(p<0.05)in the cerebral ischemia-reperfusion group(2)Compared with the Sham group,Infarct volume in I/R group was significantly increased and infarct volume in LLPS+I/R group and HLPS+I/R group were significantly decreased(p<0.05).(3)Compared with the Sham group,the expression of IL-6,IL-1? and TNF-ɑ in the cortical tissue and serum of the LPS+Sham group increased,but there was no statistically significance(p>0.05).Compared with Sham group,the levels of IL-6,IL-1 β and TNF-ɑ were significantly increased in the ischemia-reperfusion(I/R)group(p<0.05).Compared with the I/R group,the level of TNF-ɑ,IL-6 and IL-1βwere significantly decreased(p<0.05)in serum and cortex of mice suffering from ischemia-reperfusion injury after LPS preconditioning.(3)Compared with the Sham group,the expression of TLR4,My D88 and p NF-κB in the cortical tissue of the LPS+Sham group was increased,but there was no statistically significance.Compared with the Sham group,the expressions of TLR4,My D88 and nuclear NF-κ B were significantly increased in the I/R group(p<0.05).Compared with the ischemia-reperfusion(I/R)group,the expression of TLR4,My D88 and p NF-κB in the cortex of LLPS+I/R group and HLPS+I/R group mice decreased,there were statistically significance(p<0.05).(4)Compared with the Sham group,the expression of p-JAK2/JAK2,p-STAT3 /STAT3 and SOCS3 in the cortical tissue of LPS+Sham group were increased,but there were no statistically significance(p>0.05).Compared with the Sham group,the expression of p-JAK2/JAK2,p-STAT3 /STAT3 and SOCS3 were significantly increased in the ischemia-reperfusion(I/R)group(p<0.05).Compared with the ischemia-reperfusion(I/R)group,the expression of p-JAK2 and p-STAT3 in the cortex of LLPS+I/R group and HLPS+I/R group mice were decreased(p<0.05),but SOCS3 was significantly increased(p<0.05).Conclusion:(1)LPS preconditioning is able to improve the symptoms of nerve injury and reduce the volume of cerebral infarction in mice suffering from cerebral ischemia reperfusion injury.(2)LPS preconditioning has the neuroprotective effect by inhibiting TLR4-My D88 inflammatory signaling pathway in the cortical tissue.(3)LPS preconditioning may up-regulate the expression of SOCS3 protein,alleviating cerebral ischemia-reperfusion injury.Second part: The Anti-Inflammatory effect of SOCS3 in Preconditioning induced by LPS in Cortical Cells suffering from Oxygen-Glucose Deprivation injuryObjective:To investigate whether LPS-induced Preconditioning has protective effects on cortical cells suffering from oxygen-glucose deprivation(OGD)injury,and to explore whether the mechanism is related to TLR4-My D88 inflammatory pathway and JAK2-STAT3-SOCS3 pathway.Methods:Primary cortical cultures subjected to oxygen-glucose deprivation(OGD)were used as an in vitro simulated ischemic model.LPS was co-incubated with cortical cells 48 h before OGD.The toxicity of OGD was measured by using 3-(4,5-dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium(MTT)assay and lactate dehydrogenase(LDH)leakage assay.The levels of inflammatory cytokines such as IL-6,IL-1β and TNF-ɑ in cells’ culture medium were detected by ELISA kits.Western-blot methods were used to detect the expression of proteins such as TLR4,My D88,SOCS3,p-JAK2,JAK2,p-STAT3 and STAT3 in cortical cells.Results:(1)Compared with the Sham group,the cells in ―LPS + Sham‖ group had no obvious damage,and the expressions of proteins such as TLR4,p-JAK2/JAK2,p-STAT3 /STAT3 and SOCS3 were increased with no statistically significance(p>0.05).(2)Compared with the Sham group,the viability of the cortical cells in the OGD group decreased and the leakage of LDH increased after 2 hours’ OGD and 24 hours’ reperfusion.Meanwhile,the contents of IL-6,IL-1βand TNF-ɑ in the culture medium were increased in the OGD group.Furthermore,The expression of TLR4,My D88,p-JAK2,p-STAT3,p-STAT3,SOCS3 in the OGD group were increased significantly(p<0.05).(3)Compared with the OGD group,cell injury is alleviated in the ―LPS + OGD‖ group via inhibiting the secretion of inflammatory cytokines such as TNF-ɑ,IL-6 and IL-1β and promoting the expression of SOCS3 in cell culture medium.Meanwhile,the expression of TLR4,p-JAK2/JAK2 and p-STAT3 /STAT3 in the ―LPS + OGD‖ group were significantly decreased(p<0.05).(4)Conclusions:(1)LPS-induced preconditioning is able to reduce the oxygen glucose deprivation injury and exert protective effects in cortical cells.(2)The mechanism of protective effects of LPS-induced preconditioning are complex,they are concerned with up-regulating the expression of SOCS3 protein in cortical cells and inhibiting the TLR4 inflammatory pathway.Conclusion:(1)LPS preconditioning can improve the reduction of oxygen glucose deprivation injury in nerve cells.(2)LPS preconditioning may up-regulate the expression of SOCS3 protein in cells,inhibit the TLR4 inflammatory pathway of oxygen-glucose deprivation,then alleviate OGD injury.