The Effect And Mechanism Of High Mobility Group Box-1 Protein On Inflammatory Response Of Cerebral Vaspasm In Rats After Subarachnoid Hemorrhage

PURPOSE:1.To investigate the relationship between tumor necrosis factor-α(TNF-α)and cerebral vasospasm(CVS)after subarachnoid hemorrhage(SAH). 2.To investigate mechanism of expression of High Mobility Group Box-1 Protein (HMGB1)after SAH.3.To explore the signal transduction mechanism of HMGB1 in the development of CVS.METHODS:1.Cerebral vasospasm and inflammatory response after SAH. SAH was produced according to the double-hemorrhage method.We used hematoxylin-eosin(HE)to assess vasospasm in the basilar artery and ELISA and RT-PCR to examine the changes in expression of TNF-αafter SAH.2.Changes and regulate mechanism of expression of HMGB1 in CVS after SAH.We quantitatively measured the expression of HMGB1 in the basilar artery by using ELISA and RT-PCR method.Immunohistochemical staining was performed to evaluate the expression of p-p38MAPK.The expressions ofp38MAPK were examined with RT-PCR.SB203580(SB)was injected into the cisterna magna just before production of the SAH.We measured the expression levels of HMGB1 after SB treatment,using RT-PCR and ELISA.3.The effect and signal transduction mechanism of HMGB1 in the development of CVS.HMGB1(10ng/rat, 100ng/rat,1000ng/rat)was injected into the cistema magna respectively.We assessed vasospasm after HMGB1 injection by H.E.staining.ELISA and RT-PCR technique were used to quantify the expression of TNF-α.HMGB1 rat model was made by injecting HMGB1(100ng/rat)into the cisterna magna.Immunohistochemical staining was performed to evaluate p38MAPK activity.We used Western-blot and RT-PCR to examine the expression of p38MAPK.SB was injected into the cisterna magna just before production of HMGB1 models.We assessed vasospasm after SB treatment by H.E.staining.ELISA and RT-PCR technique were used to quantify the expression level of TNF-α.RESULTS:1.The basilar artery showed obvious vasospasm and reached peak in 5d after SAH.The number of TNF-αpositive endothelial cells started increasing in 1d and reached its peak in 7d.The level of TNF-αin the basilar artery increased, reached the peak in day 7 after SAH.2.The expression of HMGB1 in basilar artery increased 1 day after SAH,reached peak in 7d.The expression of p-p38MAPK started enhancing in ld after SAH,reached its peak in 3d.The level of p38MAPK expressions in the basilar artery was highest in 5d.The expression of HMGB1 in basilar artery increased 1 day after treatment with SB,reached the peak in 7d.They were significantly lower than those of the nontreated SAH group.3.HMGB1 models in rats were made by injecting HMGB1(100ng/rat)into the cistema magna. Prolonged arterial narrowing peaking on 5 day was observed.The level of TNF-αin basilar artery increased 1 day after injection,reached the peak in day 5.The expression of p-p38MAPK started enhancing in 1d,reached its peak in 5d.The level of p-p38MAPK protein and p38MAPK mRNA in the basilar artery was highest in 5d and had lasted till 7d.The basilar artery gradually showed vasospasm from 1d and reachedpeak in 3d after SB treatment.CVS in the HMGB1+ SB group was remarkably attenuated in comparison with that in the HMGB1-only group.The level of TNF-αin the basilar artery was highest in 3d,and returned to baseline levels in 14d.The level of TNF-αin the HMGB1+SB group was remarkably decreased in comparison with that in the HMGB1-only group.CONCLUSIONS:1.The basilar artery show obvious vasospasm after SAH. TNF-αmay lead to the development of CVS after SAH.2.HMGB1 may be induced by the activation of the p38MAPK signaling pathway after SAH.3.The upregulation of TNF-αinduced by HMGB1,through the activation of the p38MAPK signaling pathway,leads to the development of CVS.