基于cAMP/PKA通路探讨麻黄-桂枝药对配伍“减毒增效”的作用机制

The Ma Huang-Gui Zhi herb pair is a classic herb pair used for mutual potentiation and mutual assistance according to Chinese records and classic Chinese books, and used to treat various respiratory conditions in China for many years, with few poisoning cases due to overdose reported in the literature. However, several cases of Ma Huang poisoning have been reported, due to overdose and/or long-term consumption of the herb, or by using preparations containing ephedrine without the supervision of qualified healthcare professionals. Due to the adverse effects of Ma Huang, its medicinal use has been restricted. We hypothesized that Ma Huang-Gui Zhi herb pair may be not only used for mutual potentiation and mutual assistance, but also mutual restraint and mutual detoxification. Gui Zhi may exhibit a neuroprotective effect against Ma Huang-induced CNS (central nervous system) toxicity when used in combination. This study is to investigate the material basis and mechanism in preventing neurotoxicity and improving pharmacological efficacy when treated with a Ma Huang-Gui Zhi herb pair. 1) The serum pharmacochemistry of Ma Huang-Gui Zhi herb pair as well as the material basis will be studied through analyzing the constituents absorbed in blood by LC-MS. 2) The neuroprotective effect of Gui Zhi against Ma Huang-induced CNS toxicity will be studied both in vitro and in vivo via the cAMP/PKA signaling pathway. 3) The promotion effect of Gui Zhi in improving Ma Huang-induced sweating will be studied both in vitro and in vivo via the cAMP/PKA signaling pathway. 4) The material basis and mechanism of Ma Huang-Gui Zhi herb pair will be analyzed based on serum pharmacochemistry and serum pharmacology. This study will be a better understanding of the compatibility of Ma Huang-Gui Zhi herb pair.

麻黄-桂枝药对是中医治疗外感风寒表实证相须、相使配伍使用的经典药对。但是，由于麻黄对中枢神经系统的毒副作用，限制了在世界范围内的广泛应用。我们前期探讨了在中医药理论指导下，麻黄通过配伍而起到“减毒增（存）效”作用的可行性，提出麻黄-桂枝药对配伍尚具有相畏、相杀的减毒作用。本研究拟在前期工作基础上，以cAMP/PKA信号通路为着眼点，借助血清药物化学和血清药理学的手段，深入探讨麻黄-桂枝药对配伍的作用机制。具体内容包括：（1）基于血清药物化学研究麻黄-桂枝药对配伍对血中移行成分的影响；（2）基于cAMP/PKA通路研究麻黄-桂枝药对配伍抑制神经毒性的作用机制；（3）基于cAMP/PKA通路研究麻黄-桂枝药对配伍增强发汗药效的作用机制；（4）基于血清药物化学与血清药理学联合分析麻黄-桂枝药对的配伍规律。通过本研究可以进一步揭示麻黄-桂枝药对配伍的客观规律与科学内涵，指导临床遣方用药。

麻黄-桂枝药对是中医用来治疗外感风寒表实证的经典药对，被广泛用于治疗咳嗽、气喘、发热等。本研究基于麻黄-桂枝药对属于相须、相使配伍的传统认识，结合国内外新发现的用药问题，综合探讨麻黄-桂枝药对的配伍规律。通过HPLC、LC-MS等化学分析方法检测，发现麻黄的主要成分去甲基麻黄碱、去甲基伪麻黄碱、麻黄碱、伪麻黄碱、甲基麻黄碱等5个麻黄类生物碱在体外及体内性质相对稳定，桂枝的主要成分桂皮醛、桂皮醇、香豆素等小分子化合物在体外易于氧化，体内变化差异较大，难以控制。通过检测昆明种小鼠后趾肉垫汗腺着色点数和汗腺分泌量，以及测量导管内径，发现麻黄及主要有效成分麻黄碱具有明显的发汗作用，麻黄-桂枝配伍后可以显著增强发汗作用。通过SD大鼠模型，发现麻黄与麻黄碱作用一致，可以升高额叶皮层部位Glu、Asp等兴奋性氨基酸类神经递质含量，并代偿性出现Gly、GABA等抑制类神经递质升高，桂枝可以抑制这种药理作用。通过PC12、HT22和Neuron等细胞模型，发现麻黄碱具有神经细胞毒性，能够显著影响与中枢神经系统功能密切相关的BDNF、PSD95和synapsin1等蛋白表达。通过PC12细胞模型等，发现麻黄碱可以显著升高c-Fos的表达，桂皮醛可以抑制该作用。通过体外PC12细胞模型和体内小鼠模型，发现麻黄及麻黄碱可以激活cAMP/PKA通路，c-Fos、BDNF、PSD95和synapsin1等检测指标均与该通路密切相关，桂枝可以抑制麻黄诱导的差异化反应。据此，推测麻黄的药理作用与cAMP/PKA通路相关，麻黄-桂枝药对存在相畏、相杀的减毒作用。本研究遵循中医药理论指导，兼顾麻黄的发汗药效和中枢神经系统毒副反应，探讨了桂枝与麻黄配伍“减毒增（存）效”的综合作用，拓宽了对麻黄-桂枝药对仅属于相须、相使配伍的传统认识。