To Investigate The Protective Effects Of Calycosin On Oxygen-glucose Deprivation(OGD) Model Of Nerve Cells Based On The HMGB1/TLR4/NF-κB Signal Pathway

Objective:Our previous study found that the expression of high mobility group box 1（HMGB1）,Toll-like receptor 4（TLR4）and phosphorylation-nuclear transcription factor-κB（p-NF-κB）was significantly increased in middle cerebral artery occlusion（MCAO）model rats,pretreatment with calycosin can alleviate cerebral ischemia/reperfusion injury,inhibit the expression of HMGB1,TLR4 and p-NF-κB,but its specific mechanism of neuroprotective effect is still unclear.In this study,we established an oxygen-glucose deprivation/reoxygenation（OGD/R）model of PC12 cells and primary fetal rat cortical neurons to observe the effects of calycosin on inflammatory response and apoptosis after OGD/R injury,and to investigate the neuroprotective mechanism of calycosin in cerebral ischemia/reperfusion injury based on HMGB1/TLR4/NF-κB signal pathway.Methods:CCK8 experiment was used to determine the optimal drug concentrations of calycosin and oxygen-glucose deprivation times on PC12 cells and primary cortical neurons of fetal rats.The cells were divided into blank control group,oxygen-glucose deprivation model group（OGD/R group）and calycosin administration group.The m RNA expression of Caspase-3 and other related factors of apoptosis and HMGB1,TLR4 were detected by Quantitative Real-time PCR experiment.The apoptosis rate was detected by flow cytometry.The protein expression of Caspase-3 and other related factors of apoptosis,inflammatory factor IL-18,HMGB1,TLR4 and p-NF-κB were detected by ELISA and Western blot.Then,the optimal stimulation concentration of lipopolysaccharide（LPS）to promote the expression of HMGB1 on PC12 cells was determined by ELISA and CCK8experiment.Set up blank control group,LPS group,LPS+calycosin(8×10^-7mol/L)group,then the expression of HMGB1,TLR4 and p-NF-κB in each group were detected by ELISA and Western blot.Results:Calycosin had the strongest ability to promote the proliferation of PC12 cells and primary cortical neurons of fetal rats at 10^-6 mol/L,and pretreatment with appropriate concentration of calycosin for 24 hours has a dose-dependent protective effect on nerve injury induced by OGD/R.Compared with the blank control group,the m RNA expression of HMGB1,TLR4,Caspase-3 and Bax increased significantly,and the m RNA expression of Bcl-2 decreased absolutely in the OGD/R group,whereas calycosin reversed the above changes in a dose-dependent manner.The protein expression of TLR4,p-NF-κB,Caspase-3,Bax and extracellular HMGB1,IL-18 in OGD/R group increased significantly,while the expression of Bcl-2 decreased.Compared with OGD/R group,the protein expression of TLR4 and p-NF-κB,Caspase-3,Bax and extracellular HMGB1,IL-18 in calycosin administration group decreased in a dose-dependent manner,while the expression of Bcl-2 increased.The apoptosis of cells in OGD/R group increased significantly,while decreased significantly in calycosin administration group.The stimulation concentration of LPS equivalent to the damage caused by OGD/R was selected to promote the release of HMGB1.The results of ELISA and Western blot showed that the protein expression of TLR4,p-NF-κB and extracellular HMGB1 increased significantly in LPS group.Compared with LPS group,the protein expression of TLR4,p-NF-κB and extracellular HMGB1 decreased in LPS+calycosin(8×10^-7 mol/L)group.Conclusion:Calycosin may inhibit HMGB1/TLR4/NF-κB signal pathway to reduce the inflammatory reaction and apoptosis of nerve cells,alleviate the injury caused by OGD/R and produce neuroprotective effect.