目的 通过分子对接、网络药理学及动物实验，探究天然熊胆与人工熊胆在急性咽炎合并发热模型上的解热、抗炎作用及其对瞬时受体电位（TRP） V1和TRPM8通道的调控，以评估人工熊胆替代天然熊胆的可行性并阐释其中药药性。方法 将熊胆中主要化学成分牛磺熊去氧胆酸、牛磺鹅去氧胆酸、鹅去氧胆酸、熊去氧胆酸、牛磺酸、牛磺胆酸钠与TRPV1、TRPM8进行分子对接；应用网络药理学富集分析天然熊胆、人工熊胆作用通路。SD大鼠随机分成7组：对照组，模型组，阳性对照（咽立爽口含滴丸9 mg·kg^-1＋对乙酰氨基酚45 mg·kg^-1，均相当于临床等效剂量）组，天然熊胆低、高剂量（180、360 mg·kg^-1，180 mg·kg^-1相当于临床等效剂量）组，人工熊胆低、高剂量组（180、360 mg·kg^-1，180 mg·kg^-1相当于临床等效剂量），除对照组外，采用辣椒素联合脂多糖建立大鼠急性咽炎合并发热模型；各组造模后持续监测体温9 h（每0.5小时测量1次）并记录； ELISA法检测血清前列腺素E₂（PGE₂）含量；苏木素-伊红（HE）染色观察咽部组织病理变化；实时荧光定量PCR（qRT-PCR）法测定咽部组织、肝脏组织中炎症相关基因的表达；免疫组化染色检测咽部组织、肝脏组织TRPM8蛋白表达；免疫荧光染色检测咽部组织TRPV1蛋白表达； Western blotting检测肝脏组织TRPV1蛋白表达。结果 分子对接显示，除牛磺酸外，熊胆中主要化学成分与TRPV1、TRPM8的结合能均大于7分，提示结合能力良好。网络药理学结果表明，熊胆多种活性成分不仅直接作用于TRPV1/TRPM8，还通过相关靶点连接至多条与炎症、钙信号、类固醇代谢及神经突触传递相关的通路，系统性地支持熊胆“清热、止痛、归肝胆”的传统药性。动物实验结果表明，与模型组比较，天然熊胆与人工熊胆均可有效抑制模型大鼠体温升高，降低血清PGE₂含量，改善咽部组织病理损伤，下调咽部和肝脏组织中促炎因子肿瘤坏死因子（TNF）-α、白细胞介素（IL）-6、IL-1β的mRNA表达，上调抗炎因子IL-10的表达；同时，两者均能减弱咽部组织TRPV1荧光强度，降低肝脏组织TRPV1蛋白表达，并显著升高咽部及肝脏组织TRPM8蛋白表达。结论 天然熊胆与人工熊胆通过抑制TRPV1和上调TRPM8发挥抗炎解热作用，提示其具有寒凉药性与辛味特性；人工熊胆在急性咽炎、发热等炎症性疾病中疗效与天然熊胆相当，可作为天然熊胆的替代治疗药物。

Objective To examine the antipyretic and anti-inflammatory effects of both natural and artificial bear bile in a rat model of acute pharyngitis with fever, as well as their modulation of TRPV1 and TRPM8 channels by molecular docking, network pharmacology, and in vivo experiments. Methods The major chemical constituents of bear bile: tauroursodeoxycholic acid, taurocholic acid, chenodeoxycholic acid, ursodeoxycholic acid, taurine, and sodium taurocholate—were subjected to molecular docking with TRPV1 and TRPM8. Network pharmacology was used to perform enrichment analysis of the pathways involved in natural and artificial bear bile. Sprague-Dawley rats were randomly divided into seven groups: control group, model group, positive control group (Yanli Shuangkou Dropping Pills 9 mg·kg^-1 + acetaminophen 45 mg·kg^-1, both equivalent to clinical doses), low- and high-dose natural bear bile groups (180 and 360 mg·kg^-1, with 180 mg·kg^-1 equivalent to clinical dose), and low- and high-dose artificial bear bile groups (180 and 360 mg·kg^-1, with 180 mg·kg^-1 equivalent to clinical dose). Except for the control group, all other groups were induced with a rat model of acute pharyngitis combined with fever using capsaicin and lipopolysaccharide. Body temperature was continuously monitored for 9 h post-modeling (measured every 0.5 h). Serum prostaglandin E₂ (PGE₂) levels were detected by ELISA. Histopathological changes in pharyngeal tissues were observed via hematoxylin-eosin (HE) staining. Real-time quantitative PCR (qRT-PCR) was used to analyze the expression of inflammation-related genes in pharyngeal and liver tissues; immunohistochemistry was performed to detect TRPM8 protein expression in pharyngeal and liver tissues; immunofluorescence staining was used to assess TRPV1 protein expression in pharyngeal tissues; And Western blotting was used to measure TRPV1 protein expression in liver tissues. Results Molecular docking showed that, except for taurine, all major components of bear bile exhibited binding energies greater than 7.0 with TRPV1 and TRPM8, indicating strong binding affinity. Network pharmacology revealed that multiple active ingredients in bear bile not only directly target TRPV1/TRPM8 but also connect through related targets to multiple pathways associated with inflammation, calcium signaling, steroid metabolism, and synaptic transmission, systematically supporting the traditional Chinese medicine properties of bear bile as “clearing heat, relieving pain, and targeting the liver and gallbladder”. Animal experiment results indicated that compared with the model group, both natural and artificial bear bile effectively suppressed body temperature elevation, reduced serum PGE₂ levels, improved histopathological damage in pharyngeal tissues, downregulated mRNA expression of pro-inflammatory cytokines tumor necrosis factor (TNF)-α, interleukin (IL)-6, and IL-1β in pharyngeal and liver tissues, and upregulated expression of the anti-inflammatory cytokine IL-10. Additionally, both treatments reduced fluorescent intensity of TRPV1 in pharyngeal tissues, decreased TRPV1 protein expression in liver tissues, and significantly increased TRPM8 protein expression in both pharyngeal and liver tissues. Conclusion Natural and artificial bear bile demonstrate antipyretic and anti-inflammatory effects by inhibiting TRPV1 and activating TRPM8, indicating that they embody “cold cool” and “pungent” properties as described in traditional Chinese medicine theory. Furthermore, artificial bear bile exhibits comparable efficacy to natural bear bile in inflammatory conditions such as acute pharyngitis and fever, thereby supporting its use as a viable alternative.

R965

国家中医药管理局《药材替代临床评价的关键技术研究》（GZY-KJS-2021-19）