Abstract：
BACKGROUND: 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.
OBJECTIVE: 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.
METHODS: 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.
RESULTS: 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.
OBJECTIVE: 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.
METHODS: 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.
RESULTS: 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.
摘要：
背景：加味白虎汤（JWBHD），用七种中药材配制的处方在减轻中暑（HS）患者的脑损伤方面具有临床疗效。
目的：本研究旨在通过整合网络药理学和药效学方法，评价JWBHD对HS大鼠模型的治疗效果，并探讨其治疗机制。使用UPLC-MS/MS分析了哪些主要成分。
方法：首先进行网络药理学分析以预测JWBHD的潜在活性成分和治疗靶标。然后在经历HS的大鼠上评估JWBHD的抗HS有效性。收集大鼠脑组织进行一系列全面的实验，包括westernblot,PCR,H&E染色,尼氏染色,ELISA,透射电子显微镜,流式细胞术和免疫荧光法验证JWBHD对HS诱导的脑损伤的保护作用。此外，在HS攻击的F98细胞系上进一步验证了JWBHD对胶质细胞TLR4/NF-κB信号和线粒体自噬的抑制作用。最后,利用UPLC-MS/MS分析了JWBHD水提取物的化学成分。
结果：网络药理学已经确定了50个核心靶标和许多HS相关信号通路作为JWBHD的潜在治疗靶标。蛋白质-蛋白质相互作用（PPI）和GO的分析表明JWBHD可以抑制HS诱导的炎症信号。在HS大鼠身上进行的实验中，JWBHD显著降低了核心温度，恢复血压，减轻神经功能缺损。此外，JWBHD下调了白细胞和单核细胞的计数,降低炎症细胞因子的水平，如IL-1β，外周血中IL-6和TNF-α,并抑制HS大鼠大脑皮质TLR4和NF-κB的表达。此外,JWBHD抑制大脑皮质细胞凋亡，减轻HS组大脑皮质损伤。相反，在HS大鼠的大脑皮层中观察到过度活跃的线粒体自噬。然而,JWBHD恢复了线粒体膜电位，并下调了包括Pink1，Parkin，LC3B和Tom20。JWBHD减少了Pink1和GFAP的共同定位，HS大鼠大脑皮层星形胶质细胞的特异性标记物。此外,在F98细胞中进一步证实了JWBHD对TLR4/NF-κB信号传导的抑制作用和过度激活的线粒体自噬。最后,UPLC-MS/MS分析表明，JWBHD的主要成分包括异甘草素，甘草苷,甘草酸二钾，人参皂苷Rb1,人参皂苷Re,等。结论：JWBHD通过抑制TLR4/NF-κB信号和胶质细胞的线粒体自噬来保护大鼠免受HS并预防HS诱导的大脑皮质损伤。
目的：本研究旨在通过整合网络药理学和药效学方法，评价JWBHD对HS大鼠模型的治疗效果，并探讨其治疗机制。使用UPLC-MS/MS分析了哪些主要成分。
方法：首先进行网络药理学分析以预测JWBHD的潜在活性成分和治疗靶标。然后在经历HS的大鼠上评估JWBHD的抗HS有效性。收集大鼠脑组织进行一系列全面的实验，包括westernblot,PCR,H&E染色,尼氏染色,ELISA,透射电子显微镜,流式细胞术和免疫荧光法验证JWBHD对HS诱导的脑损伤的保护作用。此外，在HS攻击的F98细胞系上进一步验证了JWBHD对胶质细胞TLR4/NF-κB信号和线粒体自噬的抑制作用。最后,利用UPLC-MS/MS分析了JWBHD水提取物的化学成分。
结果：网络药理学已经确定了50个核心靶标和许多HS相关信号通路作为JWBHD的潜在治疗靶标。蛋白质-蛋白质相互作用（PPI）和GO的分析表明JWBHD可以抑制HS诱导的炎症信号。在HS大鼠身上进行的实验中，JWBHD显著降低了核心温度，恢复血压，减轻神经功能缺损。此外，JWBHD下调了白细胞和单核细胞的计数,降低炎症细胞因子的水平，如IL-1β，外周血中IL-6和TNF-α,并抑制HS大鼠大脑皮质TLR4和NF-κB的表达。此外,JWBHD抑制大脑皮质细胞凋亡，减轻HS组大脑皮质损伤。相反，在HS大鼠的大脑皮层中观察到过度活跃的线粒体自噬。然而,JWBHD恢复了线粒体膜电位，并下调了包括Pink1，Parkin，LC3B和Tom20。JWBHD减少了Pink1和GFAP的共同定位，HS大鼠大脑皮层星形胶质细胞的特异性标记物。此外,在F98细胞中进一步证实了JWBHD对TLR4/NF-κB信号传导的抑制作用和过度激活的线粒体自噬。最后,UPLC-MS/MS分析表明，JWBHD的主要成分包括异甘草素，甘草苷,甘草酸二钾，人参皂苷Rb1,人参皂苷Re,等。结论：JWBHD通过抑制TLR4/NF-κB信号和胶质细胞的线粒体自噬来保护大鼠免受HS并预防HS诱导的大脑皮质损伤。
