以動物模式評估比較硃砂單方及含硃砂之複方中藥對於肝臟 和腎臟毒性損傷與安全性研究

黃俊發
中國醫藥大學
硃砂(Cinnabar)，是一種礦物性中藥，是許多複方中藥固有成方中不可缺少之重要成分之ㄧ。然而，由於硃砂之主要成分為硫化汞(HgS)，因此硃砂食入人體內會經由腸胃道將其所含之汞金屬吸收、透過血液運送、進而蓄積在重要器官中，造成損傷。已有研究指出：硃砂及硫化汞的神經毒性大約為一般大家所熟知的劇毒-甲基汞(MeHg)的千分之一。同時，先前研究計畫報告也指出：對於市售傳統水飛硃砂之毒性及安全劑量的研究結果推估出成人使用水飛硃砂的安全劑量為0.05g~0.07 g/day，連續重複使用最好不超過10-14天；且硃砂對於幼鼠鎮靜安神效果較成鼠好，推測幼兒對於硃砂敏感度較成人高。但是，這些研究之結果是根據單方硃砂餵食實驗動物後，經由測試各項神經功能與毒性傷害指標：如鎮靜安神作用、神經與聽力系統毒性反應及偵測汞金屬在腦部各組織蓄積量…等結果所得之綜合結論。由於硃砂進入體內後其主成份硫化汞所含之汞金屬會經吸腸胃收後大量蓄積在肝臟與腎臟組織中(為體內汞金屬最主要且最大量蓄積之器官)，因此我們必須進一步證實：在長期服用硃砂下，是否會引起肝、腎組織毒性反應？並以肝腎毒性指標來探求硃砂在先前研究結果所推估具鎮靜安神作用之硃砂使用安全劑量與期程內對肝腎毒性是否尚未產生？此外，中醫在臨床上使用硃砂作為嬰幼兒鎮靜安神、催眠、抗驚厥作用…等方劑且大多以複方使用，如：八寶散、天王補心丹…等，而硃砂與其他中藥合用時，對於肝腎影響是否會有所改變(加強或降低毒性反應)，也是值得去詳加探討的重要課題！
因此，本計畫所擬定的研究重點如下：(1).探求長時間暴露硃砂下，對於引起肝臟與腎臟毒性反應之時間、效應關係與可能作用機制；(2). 探求暴露含硃砂之複方中藥(成年鼷鼠餵食：天王補心丹；幼年鼷鼠餵食：八寶散)對於肝臟、腎臟是否會產生毒性反應、產生之時間與效應關係、可能作用機轉，並且比較這些相關毒性反應與硃砂單方所引起損傷之間的差異性(加強或降低硃砂對於肝腎組織毒性反應)；(3).分析比較：依據先前研究結果所推估具鎮靜安神作用之硃砂使用安全劑量與安全使用期程內，硃砂單方及含硃砂之複方中藥是否會對幼年與成年鼷鼠之肝、腎組織產生毒性反應，並推估出硃砂使用的確切安全期限。
實驗方法為：在餵食硃砂單方、含硃砂之複方中藥或是氯化汞(HgCl2, 為已知之毒性無機汞化物，作為正向控制組)不同時間後，將鼷鼠犧牲，分析血液、肝臟及腎臟中汞金屬濃變化，以便能比較硃砂單方與含硃砂之複方中藥經腸胃吸收後在肝、腎組織分佈情形的差異性；並且分析實驗鼷鼠：肝、腎功能指標(血漿生化值-GOT, GPO, LDH, BUN, Creatinine的變化、尿液中白蛋白、Creatinine之改變)、肝、腎組織顯微構造改變(組織病理檢查實驗)、以及與肝臟、腎臟毒性損傷相關指標(脂質過氧化(LPO)產生、穀胱甘肽(GSH) 變化、與細胞組織毒性傷害有關之重要傳訊核醣核酸(mRNA))變化情形。透過這一系列實驗執行所獲得之重要結果，能進一步分析比較：(1).長時間暴露硃砂單方下對於肝、腎組織所產生毒性作用起始時間、可能作用機轉；(2).了解含硃砂之複方中藥在長時間暴露下，對於幼年與成年鼷鼠是否會產生肝、腎毒性反應、產生毒性作用起始時間與可能作用機轉，及與硃砂單方所引起的毒性反應是否具有差異性？ (3).釐清含硃砂之複方中藥在短時間暴露下(依據先前研究所推估之硃砂使用安全劑量與期程內)，對於成年與幼年鼷鼠肝、腎組織產生之相關毒性損傷反應程度，並進一步推估出硃砂使用的安全劑量與安全時間。
關鍵字：單方與複方硃砂；肝臟；腎臟；毒性反應；安全劑量; 使用期限

Studies of toxicological effects and safe evaluation of liver and kidney by the single and compound cinnabar used in In Vivo system

Huang CF
Background and Significance: Cinnabar, a mineral traditional Chinese medicine (TCM), is one of the important components in many compound’s TCM. However, cinnabar is composed of more than 95% HgS, mercury metal of cinnabar can be significantly absorbed from gastrointestinal tract following regimen and accumulated in many organs leading to damage. Previous studies indicated that the neurotoxic effects of cinnabar and HgS is about one five-thousandth of methylmercury (MeHg), and estimating the safe dosage regimen of cinnabar to be in the range of 0.05-0.07g/day, once everyday for 10-14 days and the better sedative effect in young than in adult. However, these estimations are based on the restricted conditions performed (to examine the neurobehavior and neurotoxicological tests) in mice. An important question regarding the toxicological effects of cinnabar in liver and kidney, which are the main target and accumulation organs of mercury, is not clear, and to confirm the previous results of the safe dosage and regimen time of cinnabar that not produces liver and kidney toxicity is till awaited to be elucidated. Moreover, the most type of cinnabar in clinical used is the compounds, not the single, it is also a crucial question to explore whether the compound cinnabar can change in the toxic effects of liver and kidney than the single cinnabar. 
In this research project, we propose three specific aims:
(1).to investigate the toxic effects, initiation time, and possible mechanisms of cinnabar-induced in liver and kidney of mice; (2).to explore whether the compound cinnabar (in adult mice fed Tian-Wang-Buu-Shin-Dan; in young mice fed Ba-Bao-San) can induce toxic effects and its possible mechanisms and compare that with the single cinnabar; (3).to estimate the detailed information for the safe dosage and regimen period of cinnabar.
Experimental design and methods:
After oral administration with single, compounds, or HgCl2 (the positive control) for different time intervals, mice will be scarified by deep anesthesia and then collected whole blood, liver, and kidney to detect Hg contents, to analyze biochemical indexes of liver and kidney functions (including: GOT, GPO, LDH, BUN, creatinine, and urine protein and urine creatinine), to observe the change in the pathology, and determine the biomarkers of toxicological damages (such as: lipid peroxidation (LPO) production, change in glutathione (GSH) and nitric oxide (NO) levels, and the importantly related-damage mRNA expressions).
Overall, this research project may yield novel and important insight into: (1) the toxicological effects, initial times, and possible mechanisms of cinnabar-induced liver and kidney damages; (2) the understanding toxicological effects of single and compound cinnabar-induced liver and kidney injuries in young and adult mice, and the compare that difference with single cinnabar; (3) the elucidation of the detailed and important information for the safe dosage and regimen time of cinnabar.
關鍵字：Single and Compound Cinnabar; Liver; Kidney; Toxic effects; Young and Adult; Safe Dosage and Regime Time