天麻钩藤饮加减对脑出血小鼠的神经保护作用及其对高迁移率组蛋白B1/Toll样受体4信号通路的影响

Neuroprotective effect of modified Tianma Gouteng Decoction on mice with intracerebral hemorrhage and its effect on the high-mobility group box 1/Toll-like receptor 4 signaling pathway

摘要:

目的 探讨天麻钩藤饮加减在小鼠脑出血（ICH）急性期对神经炎症和细胞凋亡的影响及其与高迁移率组蛋白B1(HMGB1)/Toll样受体4(TLR4)信号通路的关联。方法 32只雄性小鼠随机分为4组（n=8）：Sham组（假手术组）、ICH组（ICH模型组）、IT组（ICH模型给予天麻钩藤饮干预）、ITG组（ICH模型给予天麻钩藤饮联合HMGB1靶点抑制剂甘草酸干预）。天麻钩藤饮质量由质谱分析控制，同时结果通过网络药理学进行靶点和通路分析。脑内注射胶原酶Ⅶ建立ICH小鼠模型。通过改良神经功能缺损评分、苏木精和伊红（HE）染色分析天麻钩藤饮对小鼠ICH后神经功能缺损和脑组织病理损伤的影响；通过酶联免疫吸附测定法（ELISA）测定肿瘤坏死因子α（TNF-α）、白细胞介素6（IL-6）、白细胞介素1β（IL-1β）水平；通过蛋白质印迹法（Western blotting）分析Bcl-2-Associated X的蛋白质（BAX）、B淋巴细胞瘤-2（Bcl-2）、HMGB1、TLR4、髓样分化因子88（Myd88）、β干扰素TIR结构域衔接蛋白（TRIF）、核因子κB（NF-κB）的表达水平。结果 与Sham组相比，ICH组的神经功能缺损和脑组织病理损伤加剧，炎症表达和细胞凋亡水平上升，HMGB1、TLR4、Myd88、TRIF、NF-κB等通路相关蛋白表达升高；与ICH组相比，IT组和ITG组的神经功能缺损和脑组织病理损伤得到缓解，炎症表达和细胞凋亡水平下降，HMGB1、TLR4、Myd88、TRIF、NF-κB等通路相关蛋白表达在不同程度上降低。此外天麻钩藤饮通过质谱分析和网络药理学分析，得到509种化合物，其中75个成分通过类药性分析并通过靶点预测得到195个可能具备干预效果的分子靶点。基因本体论（GO）功能富集分析及京都基因与基因组百科全书（KEGG）通路富集分析则进一步验证了相关分子靶点与炎症、HMGB1/TLR4通路的相关性。结论 天麻钩藤饮加减治疗缓解了ICH小鼠的神经功能缺损和脑组织病理损伤，并降低了ICH后神经炎症表达和细胞凋亡水平，可能分子机制是抑制HMGB1/TLR4信号通路的表达。

Abstract:

Objective To investigate the effect of modified Tianma Gouteng Decoction on neuroinflammation and cell apoptosis in mice in the acute stage of intracerebral hemorrhage （ICH） and its association with the high-mobility group box 1 （HMGB1）/Toll-like receptor 4 （TLR4） signaling pathway.Methods A total of 32 male mice were randomly divided into sham-operation group （Sham group）， ICH model group （ICH group）， IT group （ICH mice treated with Tianma Gouteng Decoction）， and ITG group （ICH mice treated with Tianma Gouteng Decoction and the HMGB1 target inhibitor glycyrrhizic acid）， with 8 mice in each group. Mass spectrometry was used for drug quality control， and network pharmacology was used to perform target and pathway analyses. Intracerebral injection of collagenase VII was performed to establish a mouse model of ICH. Neurological deficit score and HE staining were used to observe the effect of Tianma Gouteng Decoction on neurological deficit and brain histopathological injury in mice after ICH； ELISA was used to measure the levels of tumor necrosis factor-α （TNF-α）， interleukin-6 （IL-6）， and interleukin-1β （IL-1β）； Western blotting was used to measure the expression levels of Bcl-2-associated X protein （BAX）， B-cell lymphoma-2 （Bcl-2）， HMGB1， TLR4， myeloid differentiation factor 88 （Myd88）， TIR-domain-containing adaptor-inducing interferon-β （TRIF）， and nuclear factor-kappa B （NF-κB）.Results Compared with the Sham group， the ICH group had exacerbation of neurological deficit and brain histopathological injury and increases in the levels of inflammatory markers and cell apoptosis， as well as increases in the expression levels of the pathway-related proteins HMGB1， TLR4， Myd88， TRIF， and NF-κB. Compared with the ICH group， the IT group and the ITG group had alleviation of neurological deficit and brain histopathological injury， reductions in the levels of inflammatory markers and cell apoptosis， and varying degrees of reductions in the expression levels of the pathway-related proteins HMGB1， TLR4， Myd88， TRIF， and NF-κB. In addition， 509 compounds were obtained for Tianma Gouteng Decoction by mass spectrometry and network pharmacology， among which 75 were analyzed in terms of drug-likeness， and 195 molecular targets with a potential interventional effect were identified by target prediction. Gene ontology functional enrichment analysis and Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis further verified the association of related molecular targets with inflammation and the HMGB1/TLR4 pathway.Conclusion Modified Tianma Gouteng Decoction can alleviate neurological deficit and brain histopathological injury in mice with ICH and reduce the levels of neuroinflammatory markers and cell apoptosis after ICH， possibly by inhibiting the expression of the HMGB1/TLR4 signaling pathway.