Butylphthalide Inhibits Inflammatory Response To Cerebral Ischemia-reper-fusion Injury Via HMGB1/TLR4/NF-κB-p65 Pathway In Rats

Objective To investigate the neuroprotective effect and mechanism of butylphthalide on focal cerebral ischemia-reperfusion injury(CIRI)in rats by studying HMGB1/TLR4/NF-κB p65 Pathway.Methods75 healthy male SD rats were randomly divided into 5 groups: sham operation group,model group,and low,medium and high dose groups of butylphthalide(The doses were 2mg/kg,4mg/kg,8mg/kg,respectively).The middle cerebral artery occlusion/reperfusion model(MCAO/R)was established by the modified suture method,and the neurological deficit of the rats was evaluated by the Longa score.The content of cerebral edema was detected by brainstem wet weight method.The volume of cerebral infarction was detected by 2,3,5-triphenyltetrazolium chloride(TTC)staining;the serum tumor necrosis of the rats was detected by enzyme-linked immunosorbent assay.Expression levels of tumor necrosis factor-α(TNF-α),interleukin-1β(IL-1β),and interleukin-6(IL-6).HE staining was used to observe the pathological changes of brain tissue.Protein expression levels of high mobility group box B1(HMGB1),toll-like receptor 4(TLR4),and phosphorylated Nuclear factorκBp65(p-NF-κBp65)were detected by immunohistochemistry and western blotting.Results1.The behavioral results showed that compared with the sham group,rats,butyrophthalide low-dose group,medium-dose group and high-dose group in the model group had different degrees of neurological deficits caused by cerebral ischemia-reperfusion injury(P<0.01).Compared with the model group,the neurological deficits of each dose group of butyphthalide were significantly reduced(P<0.05).2.TTC brain tissue staining results showed that the brain tissue of the sham surgery group was red and there was no infarction foci.Compared with the sham group,the model group and the butyranide administration group had different areas of white infarct foci,which was significantly different(P<0.01).Compared with the model group,the infarct area of each administration group of butyranphthalide decreased to varying degrees,and the difference was statistically significant(P<0.01).3.The results of brain water content of the model group was significantly higher than that of the sham-operation group(P<0.01).Compared with the model group,the brain water content of butylphthalide treatment groups was significantly decreased(P<0.05 or P<0.01).4.The results of ELISA detection showed that compared with the sham surgery group,the secretion of pro-inflammatory factors IL-1β,IL-6 and TNF-αin the model group was significantly increased,and the expression levels of the above inflammatory factors were reduced to varying degrees after treatment with butyrophthalide(P<0.05 or P<0.01).5.The results of HE staining showed that the brain tissue of the model group had disordered cell arrangement,loose intercellular space,and degeneration and necrosis of some cells.All the above manifestations were significantly improved after butylphthalide treatment.6.The results of immunohistochemical staining showed that there were more yellow or brownish-yellow positive cells of HMGB1,TLR4 and p-NF-κB p65 in the brain tissue of the model group,while the expression of positive cells was significantly reduced after butyranide treatment(P<0.05 or P<0.01).7.The results of western blotting showed that compared with sham surgery,the expression of HMGB1,TLR4 and p-NF-κB p65 proteins in the model group increased significantly,and HMGB1 in brain tissue was significantly increased after treatment with butyrophthalide The expression of TLR4 and p-NF-κB p65 proteins decreased to varying degrees,and the difference was statistically significant(P<0.05 or P<0.01).ConclusionButylphthalide maybe play a protective role in brain injury by inhibiting the HMGB1/TLR4 /NF-κB p65 pathway after CIRI.