Guangzhou Hospital of Integrated Traditional and Western Medicine Affiliated to Guangzhou University of Chinese Medicine, No.87 Yingbin Avenue, Huadu District, Guangzhou, 510801, PR China; School of Pharmaceutical Science, Guangzhou University of Chinese Medicine, No. 232, Waihuandong Road, Guangzhou, 510006, PR China.
School of Pharmaceutical Science, Guangzhou University of Chinese Medicine, No. 232, Waihuandong Road, Guangzhou, 510006, PR China.
J Ethnopharmacol. 2024 Nov 15;334:118571. doi: 10.1016/j.jep.2024.118571. Epub 2024 Jul 10.
Jiawei Bai-Hu-Decoction (JWBHD), a prescription formulated with seven traditional Chinese medicinal material has demonstrated clinical efficacy in mitigating brain injury among heat stroke (HS) patients.
This study aimed to evaluate the therapeutic efficacy of JWBHD on rat model of HS and to explore its therapeutic mechanisms by integrating network pharmacology and pharmacodynamic methodologies, which major components were analyzed by using UPLC-MS/MS.
The network pharmacology analysis was firstly conducted to predict the potential active ingredients and therapeutic targets of JWBHD. The anti-HS effectiveness of JWBHD was then evaluated on rats experienced HS. Rat brain tissues were harvested for a comprehensive array of experiments, including Western blot, PCR, H&E staining, Nissl staining, ELISA, transmission electron microscope, flow cytometry and immunofluorescence to validate the protective effects of JWBHD against HS-induced brain damage. Furthermore, the inhibitory effects of JWBHD on TLR4/NF-κB signal and mitophagy of glial were further verified on HS-challenged F98 cell line. Finally, the chemical compositions of the water extract of JWBHD were analyzed by using UPLC-MS/MS.
Network pharmacology has identified fifty core targets and numerous HS-related signaling pathways as potential therapeutic targets of JWBHD. Analysis of protein-protein interaction (PPI) and GO suggests that JWBHD may suppress HS-induced inflammatory signals. In experiments conducted on HS-rats, JWBHD significantly reduced the core temperature, restored blood pressure and alleviated neurological defect. Furthermore, JWBHD downregulated the counts of white blood cells and monocytes, decreased the levels of inflammatory cytokines such as IL-1β, IL-6 and TNF-α in peripheral blood, and suppressed the expression of TLR4 and NF-κB in the cerebral cortex of HS-rats. Besides, JWBHD inhibited the apoptosis of cortical cells and mitigated the damage to the cerebral cortex in HS group. Conversely, overactive mitophagy was observed in the cerebral cortex of HS-rats. However, JWBHD restored the mitochondrial membrane potential and downregulated expressions of mitophagic proteins including Pink1, Parkin, LC3B and Tom20. JWBHD reduced the co-localization of Pink1 and GFAP, a specific marker of astrocytes in the cerebral cortex of HS-rats. In addition, the inhibitory effect of JWBHD on TLR4/NF-κB signaling and overactive mitophagy were further confirmed in F98 cells. Finally, UPLC-MS/MS analysis showed that the main components of JWBHD include isoliquiritigenin, liquiritin, dipotassium glycyrrhizinate, ginsenoside Rb1, ginsenoside Re, etc. CONCLUSIONS: JWBHD protected rats from HS and prevented HS-induced damage in the cerebral cortex by suppressing TLR4/NF-κB signaling and mitophagy of glial.
加味白虎汤（JWBHD）是一种由七种中药组成的方剂，已证明在减轻热射病（HS）患者的脑损伤方面具有临床疗效。
本研究旨在通过整合网络药理学和药效学方法评估 JWBHD 对 HS 大鼠模型的治疗效果，并分析其治疗机制，主要成分采用 UPLC-MS/MS 进行分析。
首先进行网络药理学分析，预测 JWBHD 的潜在活性成分和治疗靶点。然后评估 JWBHD 对 HS 大鼠的抗 HS 效果。采集大鼠脑组织进行一系列实验，包括 Western blot、PCR、H&E 染色、Nissl 染色、ELISA、透射电子显微镜、流式细胞术和免疫荧光，以验证 JWBHD 对 HS 诱导的脑损伤的保护作用。此外，在 HS 挑战的 F98 细胞系上进一步验证了 JWBHD 对 TLR4/NF-κB 信号和神经胶质细胞自噬的抑制作用。最后，采用 UPLC-MS/MS 分析 JWBHD 水提取物的化学成分。
网络药理学已确定五十个核心靶点和许多 HS 相关信号通路作为 JWBHD 的潜在治疗靶点。蛋白质-蛋白质相互作用（PPI）和 GO 分析表明，JWBHD 可能抑制 HS 诱导的炎症信号。在 HS 大鼠实验中，JWBHD 显著降低核心体温、恢复血压并减轻神经缺陷。此外，JWBHD 降低外周血中白细胞和单核细胞的计数，降低白细胞介素-1β、白细胞介素-6 和肿瘤坏死因子-α等炎症细胞因子的水平，并抑制 HS 大鼠大脑皮质中 TLR4 和 NF-κB 的表达。此外，JWBHD 抑制皮质细胞凋亡并减轻 HS 组大脑皮质损伤。相反，在 HS 大鼠的大脑皮质中观察到过度活跃的自噬。然而，JWBHD 恢复了线粒体膜电位并下调了包括 Pink1、Parkin、LC3B 和 Tom20 在内的自噬蛋白的表达。JWBHD 减少了 HS 大鼠大脑皮质中 Pink1 与 GFAP（星形胶质细胞的特定标志物）的共定位。此外，在 F98 细胞中进一步证实了 JWBHD 对 TLR4/NF-κB 信号和过度活跃的自噬的抑制作用。最后，UPLC-MS/MS 分析表明，JWBHD 的主要成分包括异甘草素、甘草素、甘草酸二钾、人参皂苷 Rb1、人参皂苷 Re 等。
JWBHD 通过抑制 TLR4/NF-κB 信号和神经胶质细胞自噬来保护大鼠免受 HS 并防止 HS 引起的大脑皮质损伤。
