傳統中醫藥緩解錳中毒的治療潛力

Therapeutic Potential of Traditional Chinese Medicine in Manganism

徐志翔(Hsu Chih-Hsiang)

指導教授 ： 吳忠信

國立臺灣師範大學/理學院/生命科學系/博士(2016年)

https://doi.org/10.6345/NTNU202204402

摘要

本論文的主要研究目的為探討傳統中醫藥是否具有緩解錳中毒的治療潛力。過去文獻指出錳礦工人會因為吸入大量的錳而引起神經系統中毒以及勃起障礙的症狀，人體如果攝取過量的錳也會造成嚴重的神經系統疾病、行動障礙、心智和情緒的異常，嚴重的錳中毒甚至誘發類巴金森症候群。然而，臨床上利用西方藥物治療人體錳中毒的效果卻相當有限，如果選擇傳統中醫篩選緩解錳中毒的中草藥，則為錳中毒患者帶來替代治療的希望。為此，本研究選擇具有緩解發炎與氧化壓力的中藥配方B401，利用口服餵食錳毒處理的實驗雄鼠，探討中藥配方B401是否具備緩解錳中毒的治療潛力。本實驗選用的中藥配方B401，首先利用高效液相層析儀分析中藥各個配方的有效成分，再利用MTT分析檢視中藥配方B401的半抑制濃度；確定中藥配方B401的有效成分與半抑制濃度後，本實驗選用50隻ICR雄鼠，分為正常飲食(Sham)、錳毒處理(Mn)、餵食中藥配方B401 (B401)、以及餵食中藥配方B401加錳毒處理(B401+Mn)四組。實驗處理過程中，利用滾輪運動實驗檢視四組小鼠的運動與平衡能力，最後四組小鼠分別給予麻醉藥物處理後，採集小鼠血液，利用化學發光分析法檢視並且比較血液中活性氧化物質，同時利用多功能電化學偵測儀‎檢視並且比較陰莖組織一氧化氮的含量；隨後利用心臟灌流方式犧牲，採集四組小鼠的腦部與陰莖組織，再利用免疫組織化學染色以及西方轉漬方法檢視腦部與陰莖組織的相關蛋白質表現。實驗結果顯示ICR雄鼠給予錳毒處理後，會顯著增加血液中活性氧化物質(ROS)表現以及降低陰莖組織中一氧化氮(NO)的含量；從免疫組織化學染色以及西方轉漬方法的結果發現，ICR雄鼠給予錳毒處理後，會顯著降低腦部組織中多巴胺、多巴胺接受器、以及大腦神經生長因子(BDNF)的表現，以及降低陰莖組織中神經型一氧化氮合酶(nNOS)、内皮型一氧化氮合酶(eNOS)的表現；此外，ICR雄鼠給予錳毒處理後，對於腦部與陰莖組織均會增加氧化壓力、發炎反應以及細胞凋亡作用的標記蛋白表現。當ICR雄鼠預先餵食中藥配方B401，再給予錳毒處理，相較於單獨錳毒處理的ICR雄鼠，血液中ROS表現會顯著降低，陰莖組織中NO的含量則是改善增加；從免疫組織化學染色以及西方轉漬方法的結果發現，預先餵食中藥配方B401會使錳毒處理的ICR雄鼠腦部組織中多巴胺、多巴胺接受器、以及BDNF的表現會顯著回升；陰莖組織中nNOS、eNOS的表現也會顯著回升；此外，預先餵食中藥配方B401會使錳毒處理的ICR雄鼠腦部組織與陰莖組織的氧化壓力、發炎反應以及細胞凋亡作用的標記蛋白表現顯著下降。由以上實驗結果發現，ICR雄鼠口服中草藥配方B401可以透過促進腦部組織中多巴胺以及BDNF的表現，以及緩解腦部的氧化壓力、發炎作用與細胞凋亡反應來緩解錳毒引發的神經系統毒性；此外，ICR雄鼠口服中草藥配方B401可以透過增加陰莖組織中NO、nNOS、eNOS的表現，以及緩解陰莖組織的氧化壓力、發炎作用與細胞凋亡反應來緩解錳毒引發的陰莖毒性。因此，本論文結果認為中草藥配方B401應該具有緩解錳毒引起之神經毒性或是生殖毒性的醫療潛力。

關鍵字

錳中毒 ； 類巴金森症候群 ； 陰莖組織 ； 多巴胺 ； 大腦神經生長因子 ； 一氧化氮合成酶 ； 活性氧化物質 ； 氧化壓力 ； 發炎作用 ； 細胞凋亡 ； 草藥配方

並列摘要

The main purpose of my PhD dissertation is to explore whether the traditional Chinese medicine may have therapeutic potential on alleviation. From clinical case reports, welders often have problem with neurological symptoms and erectile dysfunction when they occupationally exposed to excess manganese (Mn) dust. These welders may cause brain, motor and reproductive defects that were known as manganism or Mn-induced parkinsonism. However, the clinical use of the western medicine in manganism alleviation is quite limited. It is possible that alternative therapy of traditional Chinese medicine may be useful in manganism alleviation. In this study, we investigated whether oral treatment of herbal formula B401 has therapeutic potential to alleviate manganism. We used high-performance liquid chromatography to analysis the active ingredients of the herbal formula B401. The half maximal inhibitory concentration (IC50) of the herbal formula B401 was evaluated by MTT assay. Total 50 male ICR mice were divided into four groups: normal diet (sham group), manganism treatment only (Mn group), herbal formula B401 treatment only (B401 group), as well as pretreatment of herbal formula B401 plus manganism treatment B401 (B401+Mn group). Motor coordination of these mice was compared in accelerating mode of rotarod test. Then reactive oxygen species (ROS) of the blood were examined to compare oxidative stress in these mice by using chemiluminescence. The nitric oxide (NO) of the penile cavernous tissue was also examined in these mice by using electrochemical detection instrument. Then these mice were sacrificed by using anesthesia and heart perfusion. We collected brain tissue and penile cavernous tissue from four groups of mice. By using immunohistochemistry staining and western blotting techniques, we examined different protein expressions from brain tissue and penile cavernous tissue in four groups of mice. Our results showed that those mice with Mn treatment showed brain and motor defects in comparison with those mice with sham treatment. As observed from immunohistochemical and western blotting, those mice with Mn treatment reduced dopaminergic and brain-derived neurotrophic factor (BDNF) expressions, while enhanced oxidative stress, inflammation and apoptosis related protein markers in their brain tissue. In addition, those mice with Mn treatment showed significantly decreased NO, neuronal nitric oxide synthase (nNOS) and endothelial nitric oxide synthase (eNOS) levels, while enhanced oxidative stress, inflammation and apoptosis related protein markers in their penile cavernous tissue, In comparison with those mice with Mn treatment only, those Mn treated mice with B401 pretreatment significantly improved their motor coordination, and enhanced dopaminergic and BDNF expressions, but alleviated oxidative stress, inflammation and apoptosis related protein markers in their brain tissue. Furthermore, these mice significantly enhanced NO, nNOS and eNOS levels, but alleviated oxidative stress, inflammation and apoptosis related protein markers in their penile cavernous tissue. Our findings provide evidences that oral B401 treatment may alleviate symptoms of Mn-induced neurotoxicity via enhancing dopaminergic and BDNF expressions, but suppressing oxidative stress, inflammation and apoptosis in their brain tissue. Also, oral B401 treatment may alleviate symptoms of Mn-induced reproductive toxicity via enhancing NO, nNOS and eNOS levels, but suppressing oxidative stress, inflammation and apoptosis in their penile cavernous tissue. Thus we suggest that the herbal formula B401 may have therapeutic potential for ameliorating Mn-induced neurotoxicity and reproductive toxicity

並列關鍵字

Parkinsonism, ； penile cavernous tissue ； brain-derived ； neurotrophic factor ； herbal formula

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