Effect And Mechanism Of HMGB1 On Reactive Astrocyte Subtypes After Spinal Cord Injury In Rats

Objective:Based on the in vitro cultured rat astrocytes of spinal cord in oxygen deprivation/after oxygen after sugar sugar(oxygen-glucose deprivation/Restoration,OGD/R)to simulate a rat astrocytes of spinal cord injury.To observe the expression of high mobility group protein B1(HMGB1)and the transformation of normal spinal cord astrocytes into A1 and A2 reactive astrocytes after injury,and to explore the regulatory effect of HMGB1 on the changes of spinal cord astrocytes subtypes after injury in the OGD/R model and its mechanism.Methods:In the first part,the expression of astrocyte subtypes activated after oxygen glucose deprivation / reoxygenation was observed.The model of oxygen sugar deprivation /reoxygenation was established in the neonatal SD rats in 1-2.The experiment was divided into four groups: normal group(cells without oxygen glucose deprivation /reoxygenation),OGD6h/R6h(6 hours of oxygen glucose deprivation / 6 hours of reoxygenation),OGD6h/R12h(6 hours of oxygen glucose deprivation / 12 hours of reoxygenation of sugar),OGD6h/R24h(oxygen glucose deprivation 6 hours / 24 hours of cells of reoxygenation).The release of HMGB1 and glutamate in the four groups of cells were detected by enzyme linked immunoassay.The expression of A1 type astrocyte marker protein C3 and type A2 astrocyte marker protein S100A10 in the four groups were detected by Western blot.In the second part,the effects of HMGB1 inhibition on the subtypes of reactive astrocytes and the function of reactive astrocytes were observed.The first part of the experiment found out the time point of the most expression of type A1 astrocytes.The expression of HMGB1 was inhibited by the transfection of slow virus carrying HMGB1 silencing gene,and the expression of HMGB1 was inhibited by ethyl pyruvate(EP)The experiment was carried out in five groups: normal group,model group(oxygen glucose deprivation / reperfusion group),HMGB1 interference group(sh RNA of HMGB1 was transfected into the expression of astrocyte silencing protein,oxygen glucose deprivation),negative sequence transfection group(sh RNA with negative sequence was transfected into astrocytes,and then oxygen glucose deprivation was performed),HMGB1 + EP group(oxygen glucose deprivation / reperfusion +ep).The levels of HMGB1,glutamate,IL-1 β,TNF-α in the medium of each group were detected by ELISA.The expression of A1 type astrocyte marker protein C3 and type A2 astrocyte marker protein S100A10 and HMGB1 protein were detected by Western blot.In the third part,we observed the regulatory effect of HMGB1 on the subtypes of rat spinal astrocytes after injury through toll like receptor 4(TLR4)/ nuclear transcription factor K-B(nf-kappa B).The experiment was divided into five groups: normal group,model group,HMGB1 inhibitor group,TLR4 inhibitor group,nf-kab inhibitor group.The levels of HMGB1,glutamate,IL-1 β,tnf-α in the medium of each group were detected by enzyme-linked immunosorbent assay.The expression of A1 type astrocyte marker protein C3 and type A2 astrocyte marker protein S100A10 and TLR4 and nf-KB were detected by Western blot.Results:1.In the first part,the expression of astrocyte subtypes activated after oxygen glucose deprivation / reoxygenation was observed.The results of ELISA showed that HMGB1 content in OGD/R cell medium of each group was significantly higher than that of normal group(P < 0.05),and HMGB1 content reached the peak at 12 hours of reoxygenation.The glutamate content in the medium also increased significantly after oxygen glucose deprivation(P < 0.05).The expression of glutamate was the most when the oxygen sugar was deprived of the rehydrated sugar for 12 hours.Western blot showed that the expression of C3 protein was higher than that of normal group(P < 0.05),and the expression was the most after 12 hours of reoxygenation.The expression of S100A10 protein also increased after oxygen sugar deprivation,and there was a significant difference from the normal group(P < 0.05).The time point of the most expression was 6hours after reoxygenation.Because this experiment focused on the study of A1 type astrocytes,we chose OGD6h/R12 h as the typical time point for subsequent experiments.2.In the second part of the experiment,the effects of inhibition of HMGB1 on reactive astrocyte subtypes and the function of reactive astrocytes were observed.ELISA results showed that the expressions of HMGB1,glutamate,IL-1β and TNF-α in cell culture medium were significantly decreased in the sh RNA transfection group and the HMGB1 inhibitor EP group compared with the oxygen-glycogen deprivation/oxygen-glycogen reoxygenation group(P<0.05),there was no significant difference between the model group and the negative sequence transfection group(P>0.05).Western Blot results showed that the expression of C3,S100A10,and HMGB1 decreased in the sh RNA transfection group and the HMGB1 inhibitor EP group compared with the injury group(P<0.05),while the protein expression of the negative sequence transfection group was not significantly different compared with the oxygen-glucose deprivation/oxygen-glucose reoxygen-glucose group(P>0.05).3.In the experiment of the third part: to observe the HMGB1 by toll-like receptor 4(TLR4)/nuclear transcription factor Κ b(the nf-kappa b pathway after injury of spinal cord of rat astrocytes subtype adjustment.ELISA results showed that the expressions of HMGB1,glutamate,IL-1β and TNF-α in cell culture medium were significantly decreased in the EP group of HMGB1 inhibitor,the CLI-095 group of TLR4 inhibitor and the Bay11-7082 group of NF-κB inhibitor compared with the oxygen-glucose deprivation/reoxygen-glucose group(P<0.05),and there was no significant difference between the model group and the negative sequence transfection group(P<0.05).Western Blot results showed that the expression of C3 protein,S100A10 protein,TLR4 protein and NF-κB protein decreased in the HMGB1 inhibitor EP group,the TLR4 inhibitor CLI-095 group and the NF-κB inhibitor BA11-7082 group compared with the injury group(P<0.05),and there was no significant difference in protein expression between the negative sequence transfection group and the oxygen-glucose deprivation/oxygen-glucose reoxygenation group(P<0.05)Conclusion:1.After spinal cord astrocyte injury,normal astrocytes are activated to form reactive astrocytes.Reactive astrocytes can be divided into two subtypes: A1 and A2.Type A1 results in impaired glutamate uptake,depletion of neurotransmitters and damage to the nervous system.2.Inhibition of HMGB1 can reduce the transformation of reactive astrocytes to A1 astrocytes.Reducing the release of glutamate is beneficial to the recovery of the injured nervous system.3.HMGB1 / TLR4 / NF-κ B pathway can promote the transformation of reactive astrocytes to A1 type astrocytes.