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(2-2)

黃俊發
中國醫藥大學
硃砂(Cinnabar)，是一種礦物性中藥，為中醫臨床治療精神障礙之藥物，屬於重鎮寧神類型之常用藥。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、 經由血液運送、蓄積在重要器官中，進而造成損傷。先前研究指出：給予實驗動物餵食大劑量硃砂及硫化汞，會造成神經系統之毒性損傷；而先前對於‘水飛硃砂之毒性及安全劑量’之研究結果推估出：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；而長時間低劑量使用硃砂下，仍是會造成神經功能毒性傷害。由於硃砂進入體內後其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？並以先前研究結果所推估硃砂使用安全劑量(10 mg/kg)與期程內對肝腎毒性是否尚未產生？我們在中醫藥委員會支持下(100年度)，進行硃砂單方對於肝臟和腎臟毒性損傷與安全性研究。初步研究結果顯示：给與實驗動物餵食市售水飛硃砂-10 mg/kg 二星期後，即出現腎臟功能不正常(尿液中蛋白含量上升)；而餵食四星期後，則出現肝功能損傷(肝腎功能損傷亦更加明顯)；並且伴隨著血液、肝腎組織汞金屬大量的蓄積、組織病理構造改變與相關毒性損傷指標產生。然而，值得我們關注的是：中醫在臨床上使用硃砂作為鎮靜安神、抗驚厥作用、中風不語…等藥物時大多以複方使用，亦是許多複方中藥固有成方中不可缺少之重要成分之ㄧ，如：天王補心丹、五寶散…等，而硃砂與其他中藥合用時，對於肝腎毒性作用是否會有所改變(加強或降低毒性反應)，是急需探討的重要課題！
因此，在前一年(100年)度之研究結果為基礎下，本年度之計畫所擬定的研究重點為：(1).探求在較長時間暴露含硃砂之複方中藥(選定含硃砂之複方中藥：天王補心丹與五寶散)，是否會引起肝臟與腎臟毒性反應產生與產生之時間？(2).探討：含硃砂之複方中藥引起毒性損傷之可能作用機轉；(3).並且比較相關毒性反應與硃砂單方所引起損傷之差異性為何(加強或降低硃砂對於肝腎組織毒性反應)？(4). 同時觀察及分析：停止暴露含硃砂之複方中藥後，其所引起肝腎毒性反應是否具有可逆性？
實驗方法為：在餵食硃砂單方、含硃砂之複方中藥不同時間後(2-4週)，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；分析肝、腎功能指標(血漿生化值-GOT, GPT, BUN, Creatinine的變化、尿液中蛋白含量、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、穀胱甘肽變化、與
細胞組織毒性傷害有關之重要傳訊核醣核酸)變化情形。
透過這一系列實驗執行所獲得之重要結果，以便能了解：(1).在較長時間使用含硃砂之複方中藥下對於肝、腎組織所產生毒性作用起始時間與可能相關反應；(2).進一步分析比較使用含硃砂之複方中藥與硃砂單方所引起毒性反應是否具有差異性 (加強或降低硃砂對於肝腎組織毒性影響)？(3).觀察與分析在短時間暴露含硃砂之複方中藥後(引起肝腎毒性傷害初期即停止暴露)，其所引起肝腎毒性反應是否具有可逆性？期望藉由本研究計畫之執行，能夠在以肝臟與腎臟相關毒性反應為基礎下，能更推估出硃砂使用之安全劑量與安全時間。
關鍵字：複方硃砂、天王補心丹、五寶散、肝臟、腎臟、毒性反應、安全期限

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

Huang, C. F.
School of pharmacy, College of Pharmacy China medical University
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, especially in liver and kidney. Previous studies indicated that exposure to high-dose of HgS and cinnabar can cause the neurotoxic effects; and estimating the safe dosage regimen of cinnabar by neurotoxicological properties suggest that the safe dosage of cinnabar in human used is the range of 0.05-0.07g/day, once everyday for 10-14 consecutive days; but that can still induce the impairment of nervous system by long-term exposure. However, an important question regard that the toxicological effects of cinnabar in liver and kidney, which are the main target and accumulation organs of mercury, is still unclear. Thus, it is straved to confirm and elucidate the safe dosage and regimen time of cinnabar involved the toxicological effects in liver and kidney. Furthermore, the most type of cinnabar in clinical used is the compounds (such as Tian-Wang-Buu-Shin-Dan, Wu-Bao-San), not the single. Thus, it is a crucial question to explore whether the compound cinnabar can change the toxic effects of compound TCM with cinnabar in liver and kidney than the single used.
In this research project, we propose specific aims:
(1).to investigate the toxic effect and initiation time of compound TCM with cinnabar (Tian-Wang-Bu-Xin-Dan and Wu-Bao-San)-induced in liver and kidney of mice; (2).to explore the possible mechanisms of compound cinnabar-induced liver and kidney injuries; (3).to compare the different effects between compound and single cinnabar used in liver and kidney; (4). To analysis whether the toxicological effects of compound TCM with cinnabar-induced liver and kidney injuries can be reversed after stop exposing to cinnabar?

Experimental design and methods:
After oral administration with single or compound (Tian-Wang-Buu-Shin-Dan
and Wu-Bao-San) TCM with cinnabar for different time intervals (2-4 weeks), 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 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) 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 compound TCM with cinnabar-induced liver and kidney damages; (2) the distinguishability between single and compound TCM with cinnabar in the toxicological responses; (3) the understanding whether the toxicological effects of compound TCM with cinnabar-induced liver and kidney injuries can be reversed after stop exposing to cinnabar? By this research project executing and finishing, it can elucidate the detailed and important information for the safe dosage and regimen period of cinnabar.
關鍵字：Single and Compound Cinnabar; Liver; Kidney; Toxicological effects; Safe Dosage and Regimen period