基于CYP450酶系探究古方一贯煎中药物配伍的制毒纠偏作用及机制研究

Yiguanjian is famous Liver Yin prescription, which clever contain poison Toosendan. Various drugs restrict Toosendan toxicity. Studies have confirmed Toosendan and its metabolites may cause acute liver injury after short-term application, the mechanism related with liver cell peroxidation and induction of CYP450 enzyme activity.CYP1A2, CYP2E1 and CYP3A4 (rat CYP3A1) are major subtype member of P450 family involved in drug metabolism and toxicology.Research suggests that Toosendan may inhibit CYP3A activity.Another studies suggest that CYP2E1 is a key enzyme involved of hepatic oxidative stress and lipid peroxidation.moreover,toosendan can enhance the overall capacity of CYP1A2 metabolism.So,we Inferred: Toosendan compatibility with various drugs may be through the induction / inhibition of CYP450 enzymes in metabolic capacity and thus attenuated.Studies involving Toosendan compatibility attenuated, yet Yiguanjian compatibility study report lacked.Therefore, this study intends to use orthogonal design, selection of CYP2E1 - / - rats and wild rats, explore and compatibility after Toosendan correlation and NF-κB and liver injury, ICAM-l protein, caspase-3, bel -2 protein change, exploring the best compatibility Toosendan attenuated.Combined with in vitro experiments, observations CYP2E1 and CYP3A, CYP1A2 activity changes clarify Toosendan compatibility attenuated internal mechanism, further clarify Yiguanjian Prescription ideas and mechanisms of action,in addition,to verify compatibility with medicine through drug manufacturing feasibility and scientific correction to provide a scientific basis for the clinical use of drugs.

一贯煎是滋阴疏肝著名方剂，滋阴养血药中少佐一味川楝疏肝理气，补肝与疏肝相结合，以补为主，使肝体得养而无滋腻碍胃之虞，且无伤及阴血之弊。全方组方严谨，配伍精当，体现了“肝体阴用阳”的特点，诚为滋阴疏肝之名方。研究证实川楝子及其代谢产物短期应用能够导致急性肝损伤，机制与肝细胞过氧化及CYP450酶活性诱导有关。对于中药的肝毒性临床上被广泛关注，含有肝毒性药物的川楝子组方在一贯煎中使用确未见有导致肝细胞损伤的报道。故推测：一贯煎中诸药降低了川楝子的毒性，机制可能与诱导/抑制CYP450酶系的代谢能力有关。本研究拟用正交设计，选用CYP2E1-/-大鼠和野生大鼠，探究不同配伍肝损的程度及NF-κB、ICAM-l蛋白、caspase-3、bel-2蛋白改变，探寻最佳组合。体外实验观察CYP酶系活性变化，阐明川楝子配伍减毒内在机制，揭示一贯煎的组方思路和作用机制，验证中药配伍制毒纠偏的可行性与科学性。

药物是一把双刃剑，即可以治疗疾病，也具有毒副作用，肝损伤尤为常见。对于中药的肝毒性临床上被广泛关注，古人运用相杀、相畏的配伍方法降低中药的毒性。一贯煎是滋阴疏肝著名方剂。全方组方严谨，配伍精当，体现了“体阴用阳”的特点，为滋阴疏肝名方。然而，含有肝毒性药物的川楝子在一贯煎中使用确未见有导致肝细胞损伤的报道。关于该方配伍规律及对川楝子制毒纠偏作用的研究鲜有报道。通过本研究，我们证实：川楝素短期大量应用能够导致大鼠急性肝损伤，肝脏细胞出现弥漫脂肪变性，并以1带-3带逐渐递减的趋势分布于全肝。形成机制与肝细胞过氧化及CYP450酶活性诱导有关。一贯煎中沙参、生地减轻川楝素肝毒性作用较弱，麦冬、枸杞、当归及全方配伍后减轻川楝素致肝损害的毒性作用较强，其中以当归及全方最显著，项目组进一步从氧化应激和脂质过氧化的角度入手，检测了TG、MDA和SOD，发现肝损害模型组大鼠的TG和MDA水平明显升高，全方组与当归组TG、MDA含量较低；模型组SOD水平最低，药物与川楝素配伍后均有所上升，以全方组和当归组最为明显，western-blot提示，模型组大鼠caspase-3蛋白表达明显增强，bel-2含量明显下降，川楝素与当归配伍后，bel-2蛋白表达增强，故推断解毒作用可能与诱导/抑制CYP450酶系的代谢能力有关。体外实验观察CYP酶系活性变化，川楝子与麦冬配伍后，大鼠肝脏CYP1A2酶的活性明显增强；川楝子、川楝子+北沙参、川楝子+麦冬均能够诱导CYP2E1，酶活性明显增加，尤以川楝子+麦冬作用较强（P<0.01）；川楝子能够抑制CYP3A4的活性，然而，川楝子+麦冬、全方组能够有效增加CYP3A4酶的活性（P<0.01）。本研究阐明了川楝子配伍减毒内在机制，验证中药配伍制毒纠偏的可行性与科学性，对于合理应用中药，减少药物性肝损害的发生具有重要的临床意义。