薯蓣皂苷抑制HMGB1/TLR4/NF-κB p65通路的活化对老年大鼠急性脑出血模型血灶周围细胞凋亡和炎性因子的影响

Effects of Dioscin on Cell Apoptosis and Inflammatory Factors of around Hemorrhagic Focus in Aged Rats with Acute Cerebral Hemorrhage by Inhibiting Activation of HMGB1/TLR4/NF-κB p65 Pathway

【目的】探讨薯蓣皂苷对老年大鼠急性脑出血模型血灶周围细胞凋亡及炎性因子的影响,并分析其作用机制。【方法】将50只老年SD大鼠随机分为正常对照组,模型对照组,薯蓣皂苷低、中、高剂量组,每组各10只。除正常对照组外,其余各组大鼠通过向脑组织注入自体动脉血构建急性脑出血模型。造模成功后,薯蓣皂苷低、中、高剂量组分别给予20、40、80 mg·kg-1·d-1薯蓣皂苷灌胃,正常对照组和模型对照组灌胃等量溶媒,共给药14 d。给药结束后,计算脑组织含水量与脑指数,进行神经功能损伤评分,采用苏木素-伊红(HE)染色镜下观察脑组织病理学表现,原位末端脱氧核苷酸转移酶(TdT)标记(TUNEL)染色法观察神经细胞凋亡情况,蛋白免疫印迹(Western Blot)法检测Caspase-3、cleaved Caspase-3、Caspase-9、cleaved Caspase-9、Bax、Bcl-2、高迁移率族蛋白B1(HMGB1)、Toll样受体4(TLR4)、核因子(NF)-κB p65、磷酸化NF-κB p65(p-NF-κB p65)表达,酶联免疫吸附分析(ELISA)检测血清中炎症因子白细胞介素(IL)-6、肿瘤坏死因子α(TNF-α)、IL-1β、IL-10含量,免疫组织化学法检测脑组织细胞间黏附分子1(ICAM-1)的表达。【结果】与正常对照组比较,模型对照组的脑含水量、脑指数、神经损伤评分升高(均P<0.05),可见神经组织结构异常,血清IL-6、TNF-α、IL-1β、IL-10含量升高,脑组织细胞凋亡率增加,cleaved caspase-3/Caspase-3、cleaved caspase-9/Caspase-9、Bax/Bcl-2比值上调,ICAM-1阳性表达明显增强,HMGB1、TLR4蛋白相对表达水平与p-NF-κB p65/NF-κB p65比值升高(均P<0.05);与模型对照组比较,薯蓣皂苷中、高剂量组上述指标均得到明显改善(均P<0.05)。【结论】薯蓣皂苷可能通过抑制HMGB1/TLR4/NF-κB p65通路来降低神经细胞的凋亡和炎性反应,进而减轻老年大鼠急性脑出血。

Objective To explore the effects of dioscin on cell apoptosis and inflammatory factors around the hemorrhagic focus in acute cerebral hemorrhage aged rats,and to analyze its mechanisms.Methods Fifty aged SD rats were randomly divided into normal control group,model control group and low-,middle-,high-dose dioscin groups,10 rats in each group.Apart from the normal control group,the rats in the other groups were induced into acute cerebral hemorrhage model by injecting auto logous blood into brain tissue.After successful modeling,the low-,middle-,high-dose dioscin groups were given intragastric administration of 20,40,80 mg·kg-1·d-1 of dioscin,respectively,and the normal control group and model control group were given intragastric administration of the same volume of menstruum,the treatment lasting 14 days.After medication,the brain tissue water content and brain index were calculated,the neurological function scoring was performed,the morphological features of brain tissue was observed under microscope by hematoxylin-eosin(HE)staining,the apoptosis of neurons in brain tissue was observed by terminal deoxyribonucleotidyl transferse(TdT)-mediated biotin-16-dUTP nick-end labelling(TUNEL)assay,and the protein expression levels of Caspase-3,cleaved Caspase-3,Caspase-9,cleaved Caspase-9,Bax,Bcl-2,high mobility group box 1(HMGB1),Toll-like receptor 4(TLR4),nuclear factorκB p65(NF-κB p65),phosphorylated NF-κB p65(p-NF-κB p65)in brain tissue were detected by Western blotting assay,the serum levels of inflammatory factors including interleukin(IL)-6,tumor necrosis factorα(TNF-α),IL-1βand IL-10 were detected by enzyme-linked immunosorbent assay(ELISA),and the intercellular adhesion molecule 1(ICAM-1)expression in brain tissue was determined by immunohistochemistry.Results Compared with the normal control group,the brain water content,brain index and nerve injury score in the model control group were increased(all P<0.05),the abnormal structures in nerve tissue were seen,the serum levels of IL-6,TNF-α,IL-1βand IL-10 were enhanced,the ratio of cell apoptosis in brain tissue was increased,the ratios of cleaved Caspase-3/Caspase-3,cleaved Caspase-9/Caspase-9,Bax/Bcl-2 was up-regulated,the ICAM-1 positive expression was stronger,the protein relative expression levels of HMGB1 and TLR4,and the ratio of p-NF-κB p65/NF-κB p65 was increased(P<0.05),while compared with the model control group,the above indexes were improved in the middle-,high-dose dioscin groups(all P<0.05).Conclusion Dioscin is effective for acute cerebral hemorrhage in aged rats by inhibiting HMGB1/TLR4/NF-κB p65 pathway to reduce apoptosis of brain cells and inflammatory reaction.