“雷公炮炙论”之僵蚕炮制的科学内涵研究

Currently, Bombyx batryticatus (BXB) is commonly processed by stir-baking with wheat bran and boiled by decoction in the clinical application. However, in Lei Gongs Pao Zhi Lun, the earliest book of processing of materia medicinals which wrote in Liu Song period in the Nan Dynasty, recorded that the processing method of BXB was soaked with rice-water and powdered. After that, BXB had been powdered before using in various dynasties. However, the scientific connotation is not clear. In this project, we try to explain the scientific connotation by investigating the material basis and mechanisms of “relieving spasm by calming endogenous wind”. We predicted that the “Bolicha” is also one of active substances of BXB, and the synergy effect of “Bolicha”, beauvericin and ammonium oxalate resulted in the effect of “relieving spasm by calming endogenous wind”. The mechanisms of the effects were by adjusting the content of neurotransmitter in the brain and the expression of receptor proteins. To study the material basis of the effect of “relieving spasm by calming endogenous wind” of “ LeiGong” processed BXB, the active proteins in “Bolicha” and the relationship among proteins, beauvericin and ammonium oxalate were investigated. Meanwhile, combining the experiments in vivo and in vitro, the effects of “LeiGong” BXB on neurotransmitter contents and their receptors expression were studied at the genetic and proteomic level. This project can provide the foundation for revealing the material basis and the mechanisms of BXB. It is beneficial for further inheriting and normalizing the processing and clinical use of BXB . In addition, this project can also provide ideas and reference for the processing and usage of other animal herbal pieces.

目前僵蚕炮制与临床使用主要以麸炒后入煎剂为主，但刘宋时期《雷公炮炙论》收载的僵蚕炮制为“米泔水制、捣粉”，其后的历代以炮制后粉末入药居多，其科学内涵未阐释清楚。本项目以僵蚕“息风止痉”功效相关的物质基础与作用机制研究为切入，阐述其科学内涵。提出工作假说：僵蚕“玻璃碴”也是其药效物质之一，与白僵菌素、草酸铵等协同作用，主要通过调节脑内神经递质含量及其受体蛋白表达而发挥“息风止痉”的功效。通过对僵蚕“玻璃碴”中活性蛋白及其与白僵菌素、草酸铵的关联性研究，阐明“雷公”炮制僵蚕“息风止痉”的物质基础；同时结合体内外实验，从基因和蛋白两个层面研究“雷公”炮制僵蚕对脑内神经递质及其受体的表达的影响，阐明其作用机制。本项目可为阐明僵蚕饮片炮制的物质基础及作用机制提供科学支撑，有利于进一步传承与规范僵蚕的炮制与临床使用，并为动物饮片的炮制与使用提供思路和借鉴。

本项目解决“雷公炮炙论”之僵蚕米泔水炮制的科学性和“捣粉”入药的科学性两个关键科学问题。主要内容如下：.采用SPME-GC-MS法分析发现，僵蚕经米泔炮制后，具有难闻气味的成分相对峰面积明显降低，具有较好气味的成分明显升高。此外，僵蚕经米泔制后，杂质和总灰分含量明显降低，但蛋白含量、蛋白组成无明显区别，HPLC水溶性成分组成也无明显变化。动物实验表明生僵蚕和米泔制僵蚕水煎液均具有明显的“息风止痉”作用，米泔制不会影响僵蚕的功效。.通过分析“雷公”僵蚕仿生液与水煎液化学成分的差异发现，相比水煎液，仿生液的干浸膏得率、蛋白含量、蛋白组成及水溶性成分均明显增加。“雷公”僵蚕粉末具有显著的“息风止痉”作用，且其作用明显强于水煎液。进一步研究发现，“雷公”僵蚕“息风止痉”作用机制与调节大鼠海马中GABA信号通路相关mRNA和蛋白以及Glu相关受体的表达有关，此外还调节IL-1β、TNF-α和GRα水平，使GABA与Glu水平、神经-免疫-内分泌网络趋于平衡。网络药理学结果表明，僵蚕抗癫痫作用靶点包括CNR1和DRD2。.通过DEAE Sepharose FF分离纯化“雷公”僵蚕总蛋白（RBBPs），得到6个组分，其中PF-1、PF-2和PF-3对Glu损伤PC12细胞具有显著的保护作用。通过Sephadex G-75进一步分离纯化活性最强的PF-2，得到具有活性的单一蛋白条带PF-2-2。通过MALDI-TOF-TOF-MS分析发现PF-2中蛋白组成均为家蚕蛋白，主要是家蚕抗胰蛋白酶和30K蛋白。PF-2-2为低分子量30 kDa脂蛋白21G1，也属于家蚕30K蛋白之一。.通过研究“雷公”僵蚕中总蛋白（RBBPs）、白僵菌素和草酸铵抗癫痫作用研究，发现RBBPs具有明显的“息风止痉”作用，而白僵菌素和草酸铵灌胃给药无显著作用，表明RBBPs是“雷公”僵蚕“息风止痉”的主要物质基础之一。通过建立Glu损伤PC12细胞和H2O2损伤PC12细胞模型评价RBBPs的“息风止痉”作用及机制，发现RBBPs对Glu和H2O2损伤PC12细胞的保护作用机制主要为：基于GABA信号通路抗Glu损伤和基于PI3K/Akt信号通路的抗氧化应激。.本项目基本明确了“雷公炮炙论”之僵蚕炮制（“米泔水制、捣粉”）的科学内涵，有利于进一步传承与规范僵蚕的炮制与临床使用。