The Role Of HMGB1-TLR4-mediated NF-κB Signaling Pathway In Mullein Isoflavones In Protecting Cerebral Ischemia-reperfusion Injury

Objective:By preparing a middle cerebral artery occlusion model（MCAO）and an oxygen and glucose deprivation/reoxygenation（OGD/R）model of PC12 cells,the cerebral ischemia-reperfusion injury was simulated in vivo and in vitro,respectively.In this research,we sought to investigate the effect of phytoestrogens calycosin on HMGB1-TLR4-mediated NF-κB signaling pathway,and to explore the neuroprotective mechanism of calycosin on cerebral ischemia reperfusion injury.Methods:（1）SD rats weighing 230-280 g were randomly divided into a sham operation group,MCAO group,and calycosin administration group.in which the concentration administrated was 5 mg/kg（low concentration group）,10 mg/kg（medium concentration group）,and 20 mg/kg（high concentration group）with 10 rats in each group.Two weeks after the intraperitoneal injection,the modeling treatment was performed.The middle cerebral artery were blocked with thread plugs in the MCAO group and the calycosin administration group.The blood flow was blocked for 2 hours and recanalized for 24 hours.In the sham operation group,the corresponding neck operation was performed without inserting the thread plug.After the modeling was completed,neurological scores and brain tissue TTC（2,3,5-triphenyltetrazolium chloride）staining were performed to observe cerebral infarction.Brain tissue proteins were extracted and Western-Blot method was used to detect nuclear factor-κB,inhibitor of NF-κB,and high-mobility group protein 1,Toll-like receptor 4 etc.The m RNA of brain tissue was extracted for Real-time Quantitative PCR to detect the expression of NF-κB,HMGB-1 and TLR4 m RNA.The neuroprotective effect and pharmacological mechanism of calycosin were investigated through in vivo experiments,.（2）The CCK8 assay was used to determine the drug concentration and oxygen-glucose deprivation time for PC12 cells,and then the oxygen-glucose deprivation model was performed on PC12 cells.The PC12 cells were divided into control group,oxygen-glucose deprivation model group（OGD/R group）and calycosin administration group,the concentration of administration were1μmol/L（low concentration group）,4μmol/L（medium concentration group）,16μmol/L（high concentration group）.Three different concentrations of calycosin in low,medium,and high levels were administered to the administration group for 24 hours.Except for the control group,the remaining groups were subjected to oxygen-glucose deprivation for 4 hours followed by oxygen-glucose recovery for 24 hours.The expression of HMGB-1,TLR4 and other protein levels was detected by Western-Blot method on PC12 cells of each group.The neuroprotective mechanism of calycosin was verified through in vitro experiments.Results:Compared with MCAO group,SD rats pretreated with calycosin for 14 days had lower neurobehavioral scores.The results of TTC staining showed that the volume of cerebral infarction was reduced with the increase of drug concentration.The results of Western Blot showed that phosphorylation activities of NF-κB and IκB in the MCAO group increased compared with the sham operation group,and the expression of HMGB-1 and TLR4 protein increased.Compared with the MCAO group,the NF-κB and IκB phosphorylation activity of the calycosin administered group decreased in a dose-dependent manner,and the protein expression of HMGB-1 and TLR4gradually decreased.The expressions of NF-κB and TLR4 m RNA increased significantly,and the expression of HMGB-1,TLR4 and NF-κB m RNA decreased with the increase of drug concentration after administration of calycosin.The results of CCK8 assay showed that PC12 cells had the highest activity at the calycosin concentration of 10^-5mol/L.In the model of deoxyge glucose deprivation for 4h and recovery of oxygen-glucose for 24h,the activities of PC12 cells were about 60%of those in the control group.The results of Western-Blot showed that the expression levels of HMGB-1 and TLR4 protein in the OGD/R group were significantly increased compared with the control group.Compared with the OGD/R group,the protein expression level of HMGB-1 and TLR4 in the calycosin administration group decreased with the increase of drug concentration.Conclusion:Calycosin have neuroprotective effects on cerebral ischemia-reperfusion injury in vivo and in vitro.The NF-κB signaling pathway mediated by HMGB-1-TLR4 may be the effect target of calycosin.