傳統中醫藥緩解錳中毒的治療潛力

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(1)國立臺灣師範大學生命科學系博士論文. 傳統中醫藥緩解錳中毒的治療潛力 Therapeutic Potential of Traditional Chinese Medicine in Manganism. 研究生：徐志翔 (Chih-Hsiang Hsu) 指導教授：吳忠信博士 (Chung-Hsin Wu, Ph.D.). 中華民國 105 年 06 月 08 日.

(2) TABLE OF CONTENTS TABLE OF CONTENTS LIST OF FIGURES ABBREBIATION LIST 中文摘要. I III V VII. ABSTRACT CHAPTER 1. IX 1. INTRODUCTION. 1. What is manganism?. 2. 2. Manganism-induced brain toxicology. 2. 3. Manganism-induced reproductive toxicology. 3. 4. Therapeutic potential of herbal formula B401. 4. 5. Aims of my study. 5. CHAPTER 2. MATERIALS AND METHODS. 7. 1. Chromatographic fingerprint analysis for the herbal formula B401. 8. 2. Cell viability assay. 8. 3. Experimental animal and treatments. 9. 4. Motor performance. 11. 5. Measurements of nitric oxide in penile tissue. 11. 6. Reactive oxygen species (ROS) analysis in the blood. 12. 7. Immunohistochemical (IHC) stain for protein expression. 12. 8. Western blot analysis for protein expressions. 13. 9. Statistical analysis. 14. CHAPTER 3. RESULTS. 15. 1. B401 treatment increases cell viability in glutamate-treated SH-SY5Y cells. 頁I. 16.

(3) TABLE OF CONTENTS 2. Effect of oral B401 treatment on motor coordination in. 16. Mn-treated mice 3. Effect of oral B401 treatment on dopaminergic expressions in the brain. 16. tissue of Mn-treated mice 4. Effect of oral B401 treatment on BDNF expressions in the brain tissue. 17. of Mn-treated mice 5. Effect of oral B401 treatment on the blood ROS in Mn-treated mice. 18. 6. Effect of oral B401 treatment on oxidative stress in the brain tissue. 18. of Mn-treated mice 7. Effect of oral B401 treatment on apoptosis in the brain tissue. 19. of Mn-treated mice 8. Effect of oral B401 treatment on nitric oxide and angiogenesis in. 21. the penile tissue of Mn-treated mice 9. Effect of oral B401 treatment on oxidative stress and inflammation in. 22. the penile tissue of Mn-treated mice 10.Effect of oral B401 treatment on apoptosis in the penile tissue of. 23. Mn-treated mice. CHAPTER 4. DISCUSSION. 25. 1. Oral treatment of the herbal formula B401 effectively alleviates. 26. Mn-induced brain toxicology 2. Oral treatment of the herbal formula B401 effectively alleviates. 29. penile toxicity of Mn-treated mice. CHAPTER 5. CONCLUSION. 33. REFERENCES APPENDIX 1. 35 Curriculum Vitae. i. 頁 II.

(4) LIST OF FIGURES Figure 1. Chromatographic fingerprint analysis and cell viability assay of. 46. Chinese herbal formula B401 Figure 2. Motor coordination of Mn-treated mice was significantly enhance. 48. under oral B401 treatment Figure 3. Expression levels of dopaminergic D1 receptors and dopamine. 50. in the brain tissue of Mn-treated mice were increased significantly under oral B401 treatment Figure 4. Expression levels of brain-derived neurotrophic factor (BDNF). 52. in the brain tissue of Mn-treated mice were increased significantly under oral B401 treatment Figure 5. Expression levels of ROS in the blood of Mn-treated mice was. 54. significantly decreased under oral B401 treatment Figure 6. Expression levels of anti-oxidative stress-related SOD2 in the brain. 56. tissue of Mn-treated mice were increased significantly under oral B401 treatment Figure 7. Expression levels of oxidative stress-related 3-NT in the brain tissue. 58. of Mn-treated mice were decreased significantly under oral B401 treatment Figure 8. The ratio of anti-apoptosis-related Bcl-2/Bax in the brain tissue. 60. of Mn-treated mice were increased significantly under oral B401 treatment Figure 9. Expression levels of apoptosis-related caspase 3 in the brain tissue of Mn-treated mice were decreased significantly under oral B401 treatment. 頁 III. 62.

(5) LIST OF FIGURES Figure 10. Cavernosal nitric oxide (NO) levels and H&E staining in. 64. Mn-treated mice with and without oral B401 treatment Figure 11. Cavernosal levels of nitric oxide synthases between. 66. Mn-treated mice with and without oral B401 treatment Figure 12. Cavernosal levels of VEGF, among Mn-treated mice. 68. with and without oral B401 treatment, and their sham mice Figure 13. Cavernosal levels of catalase, and SOD2 among Mn-treated mice. 70. with and without oral B401 treatment, and their sham mice Figure 14. Cavernosal levels of 4-HNE, an inducer of oxidative stress, between. 72. Mn-treated mice with and without oral B401 treatment Figure 15. Cavernosal levels of TNF-α, a marker of inflammation, between. 74. Mn-treated mice with and without oral B401 treatment Figure 16. Cavernosal levels of anti-apoptotic Bcl-2, and apoptotic inducers of. 76. c-PARP, Bax and Cyt-C between Mn-treated mice with and without oral B401 treatment Figure 17. Cavernosal levels of apoptotic inducers of caspase 12 and caspase 3. 78. among Mn-treated mice with and without oral B401 treatment, and their sham mice Figure 18. The schematic diagram illustrates the possible protective pathways. 80. of B401 treatment in Mn-treated mice Figure 19. The schematic diagram illustrates that oral B401 treatment may alleviate penile toxicity of excess Mn-treated mice in corpus cavernosum. 頁 IV. 82.

(6) Abbreviation List 3-NT. 3-nitrotyrosine. 5-HMF. 5-hydroxymethylfurfural. ANOVA. Analysis of variance. ATP. Adenosine triphosphate. AU. Arbitrary perfusion units. Bax. Bcl-2-associated X protein. Bcl-2. B-cell lymphoma 2. Bcl-2. B-cell lymphoma 2. BDNF. Brain-derived neurotrophic factor. Casp. 3. Caspase 3. c-Casp. 3. Cleaved-caspase 3. CL. Chemiluminescence. c-PARP. Cleaved poly(ADP-ribose) po lymerase. Ctrl. Control. Cyt-C. Cytochrome c. D1R. Dopaminergic D1 receptors. DA. Dopamine β hydroxylase;. ECL. Electrochemiluminescence. EDTA. Ethtlenediaminetetraacetic acid. eNOS. Endothelial nitric oxide synthase. H&E. Hematoxylin and eosin. HPLC. High-performance liquid chromatography. IHC. Immunohistochemistry. 頁V.

(7) Abbreviation List kDa. Kilo Dalton. Mn,. Manganese. MnCl2. Manganese chloride. nNOS. Neuronal nitric oxide synthase. NO. Nitric oxide. NTNU. National Taiwan Normal University. PBS. Phosphate buffered solution. ROS. Reactive oxygen species. Rpm. Revolutions per minute. SEM. Standard error of the mean. SOD2. Superoxide dismutase 2. TNF-α. Tumor necrosis factor alpha. VEGF. Vascular endothelial growth factor. 頁 VI.

(8) 中文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頁 VII.

(9) 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。. 關鍵詞：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。. 頁 VIII.

(10) ABSTRACT 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 頁 IX.

(11) 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.. KEYWORDS: manganism, Parkinsonism, penile cavernous tissue, dopamine, brain-derived neurotrophic factor, nitric oxide synthase, reactive oxygen species, oxidative stress, inflammation, apoptosis, herbal formula.. 頁X.

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(13) CHAPTER 1 INTRODUCTION. 頁1.

(14) 1. What is manganism ? Manganese (Mn), an common elements in the modern Industry, commonly used inthe production of ferromanganese alloys and other industrial products, such as dry-cell batteries, paints, glazes, electronic parts, and chemicals for coloring glasses and tiles. Excessive exposure to Mn may cause central nervous system (CNS) symptoms known as manganism. Chronic manganese poisoning was reported in a manganese miner that mainly via occupational inhalation the manganese (Mn) dust (Couper, 1837; Cersosimo & Koller, 2006). A lack intake in Mn can defer growth, cause epilepsy initiation, impair fertility. At the other hand, excessive exposure to Mn is associated with an irreversible brain disease with prominent psychological and neurological disturbances. Furthermore, ataxia ensues followed by an extrapyramidal syndrome, resembling several clinical disorders collectively described as "extrapyramidal motor system dysfunction," and in particular, Parkinson's-like disease and dystonia ( Massaro, 2001). From clinical case reports, welders often have problem with impotence when they occupationally exposed to excess manganese (Mn) dust (Emara et al., 1971; Mena et al., 1967; Rodier, 1995). In addition, male workers may reduce the rate of births when they exposed to Mn dust (Lauwerys et al., 1985). From animal experiments, male rabbits often caused testicular degeneration and sterility when they exposed to a high dose of manganism (Chandra et al., 1973; Seth et al., 1973). Until now, possible mechanisms for Mn-induced reproductive toxicology are rarely explored.. 2. Manganism-induced brain toxicology Manganese (Mn) is an essential nutrient that functions as a cofactor in numerous enzymes critical to metabolic homeostasis in the brain. However, excessive exposure to Mn, mainly via occupational inhalation, may cause brain and motor defects that were 頁2.

(15) known as manganism or Mn-induced Parkinsonism (Couper, 1837; Huang et al., 1989; Kawamura et al., 1941; Mergler et al., 1994). In addition, those people in the manufacturing of dry batteries, steel, aluminum, and welding metals are at a higher risk of manganism intoxication (Hauser et al., 1994; Krieger et al., 1995). The brain is the major target organ for Mn toxicity. It retains Mn much longer than other tissues. Following chronic overexposure, Mn can produce a progressive, permanent neurodegenerative disorder, with few options for treatment and no cure (Crossgrove & Zheng, 2004). The clinical symptoms of patients with Mn-induced parkinsonian exhibit monotone speech, tremor, rigidity, decreased hand dexterity, gait and balance problem (Calne et al., 1999; Olanow, 2004; Pal et al., 1999). As observed in animal and human studies, Mn-induced parkinsonian is clearly associated with damage to basal ganglia structures, (Bernheimer et al., 1973; Eriksson et al., 1987; Yamada et al., 1986). It has been reported that patients with Mn-induced Parkinsonism show markedly decreased dopaminergic levels in striatum (Mena et al., 1970). In addition, Mn-induced Parkinsonism has been reported that is mediated by excessive oxidative stress via enhancing free radical reactive oxygen species (ROS) formation, NMDA mediated excitotoxicity, and neuronal apoptosis (Brouillet et al., 1993; Maynard and Cotzias, 1955). Although there are many studies explore the possible pathology of manganism in human and animal, quite a few pharmacological researches focus on clinical treatment of Mn-induced neurotoxicity.. 3. Manganism-induced reproductive toxicology Activity of nitric oxide in corpus cavernosum plays a key role for penile erection (Burnett et al., 1992; Andersson and Wagner, 1995). In penile tissue, nitric oxide is released in the corpus cavernosum by regional expression of neuronal (nNOS), inducible (iNOS), and endothelial nitric oxide synthases (eNOS) (Moncada, 1997; Podlasek et al., 頁3.

(16) 2001). Reduction of nitric oxide release in penile tissue was often observed in aging and specific diseases such as diabetes mellitus, hyperlipidemia, and hypertension that often caused the problem with erectile dysfunction (Saenz and Goldstein, 1988; Sullivan et al., 1999; Bakircioglu et al., 2001). In addition, oxidative stress and down-regulation of vascular endothelial growth factor (VEGF) expression were often observed in corpus cavernosum of impotence animals (Jiang et al., 2005; Rajasekaran et al., 2002; Toblli et al., 2000). Oxidative stress and VEGF deficit may be involved in manganism-induced reproductive toxicology.. 4. Therapeutic potential of herbal formula B401 In Taiwan, traditional Chinese medicines are the composite formula of herbs those have been used historically for treating wide variety of brain disorders. The herbal formula B401 is a Taiwan-US patent formula and consists of six herbal ingredients. Currently, the herbal formula B401 has been widely used as a health supplement in supporting healthy neurovascular and cardiovascular function. The neuroprotective function of the herbal formula B401 has been reported in Huntington’s disease, such that it protects the brain atrophy of R6/2 mice by enhancing the level of brain-derived neurotrophic factor (BDNF) and vascular endothelial growth factor (VEGF), but suppressing marker expressions of excitotoxicity, inflammation, oxidative stress and apoptosis (Wang et al., 2015a; Wang et al., 2015b ). We reasonably infer that the herbal formula B401 may be a potential dietotherapic supplement for ameliorating Mn-induced reproductive toxicology in mice via enhancing NOS activity, and reducing oxidative stress, inflammation, and apoptosis signaling pathways. In this sense, the herbal formula B401 may be served as new insights or new leads in advancing Mn-induced neurotoxicity.. 頁4.

(17) 5. Aims of my study In the present study we elucidated the role of NOS activity, oxidative stress, inflammation and apoptosis in penile toxicity of ICR mice associated with manganism and investigated the effect of oral treatment with a herbal formula B401 in this respect. The other side, we focused on the protective potential of the herbal formula B401 against brain damages in mice with Mn-induced neurotoxicity. We used behavioral, immunohistochemical, and biochemical studies to assess the beneficial use of the herbal formula B401 in complimentary or integrated therapy for brain and motor defects in manganism.. 頁5.

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(19) CHAPTER 2. MATERIALS AND METHODS. 頁7.

(20) 1. Chromatographic fingerprint analysis for the herbal formula B401 The herbal formula B401 has a Taiwan-US patent (No. US 7,838,048 B2). It contains six herbal ingredients, including extracts from Panax ginseng, Astragalus membranaceus, Angelica sinensis, Rehmannia glutinosa, Ligustri Fructus, and Eclipta prostrata in specific ratios, and has been developed as a health supplement. High-performance liquid chromatography (HPLC) charts of the six marker compounds (ginsenosides Rb1, formononetin, 5-hydroxymethylfurfural, ferulic acid, oleanolic acid, and wedelolactone) of the herbal formula B401 with different wavelengths were shown in Figure 1. All compounds used in this analysis were solubilized in distilled H2O/MeOH. High-performance liquid chromatography grade acetonitrile from Burdick & Jackson (Gyeonggi-do, Korea) and methanol from Avantor (Center Valley, USA) were used with these herbal ingredients. A Milli-Q water purification system (EDM Millipore, Billerica, USA) provided purified water. For further information, experiments would not be affected by the vehicle.. 2. Cell viability assay A total of human neuroblastoma SH-SY5Y cells at a density of 3.0 x 104 cells/well in 100 μl culture medium were grown in a 96-well plate for 24 hours to reach ~60% confluence. Then neuroblastoma SH-SY5Y cells were treated with vehicle, the herbal formula B401 (5, 10, 20 40 and 80 mg/ml, Brion Research Institute of Taiwan, Taipei, Taiwan). The number of viable cells was compared between the control and treated conditions.. The. culture. medium. was. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium. supplemented bromide. (MTT,. with 0.5. the mg/ml,. Sigma-Aldrich Corporation) for 3 hours, and then 10% SDS/HCl buffer was added to each well. The number of viable cells was determined by the measurement of MTT absorbance at 570 nm using a microplate reader (Bio-Tek u-Quant Instrument, Winooski, 頁8.

(21) Vermont, USA). The half maximal inhibitory concentration (IC50) of the herbal formula B401 was evaluated by MTT assay. In this study, cell viability assay was approved by the Committee on Biological Research of National Taiwan Normal University and implemented under the guidelines of the Committee.. 3. Experimental animal and treatments In the brain toxicology experimental, a total of 32 ICR mice at 4-month of age were divided into the sham treatment group (n=8), Mn treatment group (n=8), the herbal formula B401 treatment group (n=8), and the herbal formula B401 plus Mn treatments group (n=8). All mice were housed and maintained on a 12:12 h light–dark cycle with water and food ad libitum. Those mice with the sham treatment were fed with the same amount of distilled water without the herbal formula B401 and Mn treatment. Those mice with Mn treatment were given an intraperitoneal injection with a high single dose of manganese chloride (100 mg/kg, the pH value was close to 7.0; Sigma-Aldrich Corporation) per day for 5 days without oral B401 treatment. Those mice with the herbal formula B401 plus Mn treatments were orally fed with the B401 formulation (50 mg/kg, the pH value was close to 7.0, Brion Research Institute of Taiwan) twice per day for 5 days and then treated with a high single dose of manganese chloride (100 mg/kg, the pH value was close to 7.0; Sigma-Aldrich Corporation) per day for 5 days by intraperitoneal injection. During intraperitoneal injection of manganese chloride, the herbal formula B401 was continuously fed for those mice with the herbal formula B401 treatment. All doses of the herbal formula B401 were adjusted according to individual weight and water consumption, which was much lower than the dosage of half-maximal inhibitory concentration (IC50). Flowing treatments with different conditions, the motor performance, the blood ROS, the immunohistochemistry (IHC) staining and western blotting for the brain tissue were examined at the same time. All protocols in this study 頁9.

(22) were executed according to the international guidelines for care and use of laboratory animals. Animal experiment and drug trials on animals was approved by the Committee on Animal Research of National Taiwan Normal University (NTNU-104022) and implemented under the guidelines of the Committee. In the penile toxicity experimental, a total of 18 ICR mice at 24 months of age were divided into the sham group (n=6), Mn group (n=6), and the B401+Mn group (n=6). Those mice of the sham group were fed with the same amount of distilled water without B401 and Mn treatment. Those of the Mn group were given an intraperitoneal injection with a high single dose of manganese chloride (MnCl 2, 100 mg/kg, the pH value was closed to the 7.0) without oral B401 treatment. Those mice of the B401+Mn group were orally fed with the B401 formulation (50 mg/kg, the pH value was closed to the 7.0) twice every day for 5 days and then treated with a high single dose of MnCl 2 (100 mg/kg, the pH value was closed to the 7.0) by intraperitoneal injection. All doses of the herbal formula B401 were adjusted according to individual weight and water consumption, which was much lower than the dosage of IC50. One day after MnCl2 treatment, the levels of cavernosal nitric oxide of the mice of the Mn group and B401+Mn group were examined with a nitric oxide electrochemical sensor meter. Also, the level of cavernosal nitric oxide in the mice of the sham group was examined at the same time. All protocol in the experiment were executed according to the international guidelines for care and use of laboratory animals. This animal experiment was approved by the Committee on Animal Research of National Taiwan Normal University (NTNU) and implemented under the guidelines of the Committee (protocol number: NTNU/ Animal Use & Care/ No 103017).. 頁 10.

(23) 4. Motor performance Rotarod performance was used to examine the motor coordination of those mice with different treatments. The mice were tested on a rotarod apparatus (Ugo Basile, Varese, Italy) starting at 5 rpm and accelerating to 10 and 20 rpm over a period of two minutes, and then maintaining a constant speed of 5, 10, and 20 rpm. The mice were rested for 30 minutes between trials, and they were trained at five rpm for ten minutes per day for three days after treatments to become acquainted with the rotarod apparatus. The mice were tested three times per day for three days, and we recorded the latency period before the mice fell off the rotating rod.. 5. Measurements of cavernosal nitric oxide in the penile tissue One day after MnCl2 treatment, the expression of cavernosal nitric oxide in the penile tissue of the mice was measured with a nitric oxide electrochemical sensor meter (Model IMEC-601; Inter Medical Co, Tokyo, Japan). Like the previous study, the nitric oxide-selective microelectrode was measured by the polarographic spectrum (Majid et al., 1998). The counter electrode was made of carbon fiber (OD 0.5 mm) and surrounded the working electrode. The working electrode consisted of a platinum/iridium alloy wire (OD 0.2 mm) coated with a 3-layer membrane that included KCl resin, a nitric oxide-selective membrane, and a normal silicone membrane. The electrode was touched to the surface of the penis, and the tip of the electrode was positioned in the mid-dorsal artery. Electrochemical oxidation of nitric oxide at the electrode surface measured the current polarographic electrode. The output current from the electrodes were monitored on the monitor and were recorded in the Lab Chart (AD Instrument Pty Ltd, New South Wales, Australia). The change percentage provided an index of changes in tissue nitric oxide concentration on the basis of the changes in output currents.. 頁 11.

(24) 6. Reactive oxygen species (ROS) analysis in the blood For determining blood ROS levels in the mice with different treatments, lucigeninand luminol-amplified chemiluminescence (CL) methods were used to measure O2radical and H2O2 activity. As described previously,17 the lucigenin-enhanced CL method provides a reliable assay for ROS. A heparinized 0.2 ml sample of whole blood was taken from the left femoral artery of each mouse. The ROS blood levels in the mice were measured in a CL analyser (CLA-ID3 chemiluminescence analyzer; Tohoku Electronic Industrial, Japan) after administration of 1.0 ml of 0.1 mM lucigenin in phosphate-buffered saline (pH 7.4) into the tested samples. The assay was duplicated for each sample, and total CL counts in 180 seconds were calculated by integrating the area under the curve.. 7. Immunohistochemical (IHC) stain for protein expression One day after the last time MnCl2 treatment, both corpus cavernosal tissues and brain tissue of Mn-treated mice and their controls were simultaneously sampled to examine their IHC staining. After formalin fixing, the cavernosal tissue section (5μ) immersed in a citrate buffer was applied to a heart-induced epitope retrieval for 15 minutes; it was next reacted with the primary antibody. Anti-eNOS (Cell Signaling Technology Inc., Danvers, Massachusetts, USA), anti-nNOS (Cell Signaling Technology Inc.), D1 dopaminergic receptor (Abcam Inc., Cambridge, USA), superoxide dismutase 2 (SOD2) (Cell Signaling Technology Inc., Danvers, USA), 3-Nitrotyrosine (3-NT) (Cell Signaling Technology Inc.), B-cell lymphoma 2 (Bcl-2) (Santa Cruz Biotechnology Inc., Santa Cruz, USA), Bcl-2 associated X protein (Bax) (Thermo Fisher Scientific Inc., Waltham, USA), caspase 3 (Cell Signaling Technology Inc., Danvers, USA). The slide was kept at room temperature for 60 minutes. The slide was washed with phosphate buffered solution (PBS) for 10 minutes, reacted with the secondary antibody attached with biotin for 30 minutes, 頁 12.

(25) and then reacted with DAB chromogen for 5 minutes. The slide was then reacted with NoVolink polymer (Leica, polymer detection system) for 30 minutes, and it was washed with PBS three times. The IHC staining was examined by one researcher.. 8. Western blot analysis for protein expressions One day after the last time MnCl2 treatment, the corpus cavernosal tissue and a brain tissue of Mn-treated mice and their controls were simultaneously sampled to examine their western blots. The removed penile tissue was homogenized in a buffer solution that included 0.05 M tris (hydroxymethyl) aminomethane (Tris, pH 8.0, Bionovas, Washington DC, USA), 0.15 M sodium chloride (NaCl, Bionovas, Washington DC, USA), 0.02 M ethylenediaminetetraacetic acid (EDTA, Bionovas, Washington DC, USA), 1 % deoxycholic acid (Bionovas, Washington DC, USA), 1 % nonidet P40 (Bionovas, Washington DC, USA), 0.1 % sodium dodecyl sulfate (SDS, Bionovas, Washington DC, USA), 1% protease inhibitor cocktail for full range (Bionovas, Washington DC, USA), 1% serine/threonine phosphatase inhibitor cocktail (Bionovas, Washington DC, USA), and 1% tyrosine phosphatase inhibitor cocktail (Bionovas, Washington DC, USA). The homogenized buffer solution was placed on ice for an hour and then centrifuged at 4 °C for 13,000 rpm for another 20 minutes, and the supernatant solution was separated. The separated solution was quantitated by using a BCA protein assay kit (Thermo Fisher Scientific Inc., Waltham, USA). Thirty micrograms of the total protein was denatured at 95 °C for 5 minutes with 5X sample dye, that included 0.25 M tris (hydroxymethyl) aminomethane hydrochloride (Tris-HCl, pH 6.8, Bionovas, Washington DC, USA), 10 % SDS (Bionovas, Washington DC, USA), 0.5 % bromophenol blue (Bionovas, Washington DC, USA), 50 % glycerol (Bionovas, Washington DC, USA), and 5 % beta-mercaptoethanol (Bionovas, Washington DC, USA). The electrophoresis was done with 12.5% discontinuous sodium dodecylsulfate (SDS-PAGE)-polyacrylamide gel. The 頁 13.

(26) proteins were then electroblotted onto a 0.2 μm polyvinylidenedifluoride (PVDF, GE Healthcare Life Sciences, Barrington, Illinois, USA) membrane for 120 minutes at 100 volts. The membranes were reacted with a blocking buffer (5% skim milk in TBS-T buffer) for an hour at the ambient temperature, and then they were blocked. The 3-NT, 4-HNE, Cyt-C, dopamine, D1 dopaminergic receptor (Abcam, Cambridge, Massachusetts, USA), alpha-tubulin, beta-actin, Bax (Thermo Fisher Scientific Inc. , Waltham, USA), Bcl-2 (Santa Cruz Biotechnology Inc., Santa Cruz, California, USA), catalase, caspase 12, caspase 3, eNOS, nNOS, c-PARP, SOD2 and TNF-α (Cell Signaling Technology Inc. , Danvers, USA) antibodies were reacted for two hours at the ambient temperature (or overnight at 4 centigrade). The membrane was washed three times using TBS-T at intervals of 10 minutes. As the secondary antibodies, anti-rabbit IgG-HRP (1:5000 dilution), anti-mouse IgG-HRP (1:5000 dilution), and anti-goat IgG-HRP (1:5000 dilution, PerkinElmer, Waltham, Massachusetts, USA) were reacted at the ambient temperature for one hour, and the membrane was washed again with TBS-T three times with an interval of 10 minutes between each washing. Each band was visualized by utilizing ECL Western blotting detection reagents (GE Healthcare Life Sciences, Barrington, Illinois, USA), and the chemiluminescence was detected using LAS-4000 (GE Healthcare Life Sciences, Barrington, Illinois, USA). Densitometric assessments of the bands were performed using Image J software (version 1.48t, Wayne Rasnabd, Washington DC, USA).. 9 Statistical analysis All data was given as mean ± standard error of the mean (SEM) from at least six independent experiments. Statistical analysis was performed using one-way or two-way ANOVA followed by Student-Newman-Keuls multiple comparisons post-test. The p-values of at least < 0.05 were considered significant. 頁 14.

(27) CHAPTER 3 RESULTS. 頁 15.

(28) 1. B401 treatment increases cell viability in glutamate-treated SH-SY5Y cells Effects of the herbal formula B401 in excitotoxicity were studied by examining glutamate-treated SH -SY5Y cells viability. From Figure 1B, the herbal formula B401 has very few cytotoxicity in SH-SY5Y cells under treatment at a dose of less than 80 mg/mL.. 2. Effect of oral B401 treatment on motor coordination in Mn-treated mice Effects of the herbal formula B401 in motor coordination were studied by examining fall time of rotarod test records in accelerating mode. As observed from the rotarod test with accelerated speed gradually (5, 10, 20 revolutions per minute, rpm), all mice with different treatments shorted their fall time gradually. Furthermore, we observed that fall time of rotarod test records of Mn-treated mice was significantly shorter than those mice with sham treatment (Figure 2, Mn vs Sham, p<0.01 for rotarod test of 5, 10, and 20 rpm), but longer than those mice with oral B401 treatment (Figure 2, Mn vs B401+Mn, p<0.01 for rotarod test of 5, 10, and 20 rpm).. 3. Effect of oral B401 treatment on dopaminergic expressions in the brain tissue of Mn-treated mice We examined effects of the herbal formula B401 in brain dopaminergic expressions of Mn-treated mice in Figure 3. As observed from IHC staining, D1 receptors expressions were obvious in the substantia nigra and the striatum of mice with the sham, B401, and B401+Mn treatments individually, while they were not obvious in the same areas of Mn-treated mice (Figure 3A). As analyzed from western blotting analysis (Figure 3Ba), the quantified brain D1 receptors expression levels of Mn-treated mice were significantly lower than those mice with sham, B401, and B401+Mn treatments 頁 16.

(29) individually (Figure 3Bb, Mn vs Sham, Mn vs B401, Mn vs B401+Mn, p<0.01). Also, we observed that quantified brain D1 receptors expression levels in B401-treated mice were significantly greater than those mice with sham and B401+Mn treatments individually (Figure 3Bb, B401 vs sham, B401 vs B401+Mn, p<0.01). We further compared brain dopamine expressions in those mice with different treatments in Figure 3B. As analyzed from western blotting analysis (Figure 3Ba), quantified brain dopamine expression levels in Mn-treated mice were significantly lower than those mice with sham, B401, and B401+Mn treatments individually (Figure 3Bb, Mn vs Sham, Mn vs B401, Mn vs B401+Mn, p<0.01). Also, we observed that quantified brain dopamine expression levels in B401-treated mice were significantly greater than those mice with sham and B401+Mn treatments individually (Figure 3Bb, B401 vs sham, B401 vs B401+Mn, p<0.05).. 4. Effect of oral B401 treatment on BDNF expressions in the brain tissue of Mn-treated mice We evaluated effects of the herbal formula B401 in brain BDNF expressions of Mn-treated mice Figure 4. As observed from IHC staining, expressions of BDNF were obvious in the substantia nigra and the striatum of mice with the sham, B401, and B401+Mn treatments individually, while they were not obvious in the same areas of mice with the Mn treatments (Figure 4A). As analyzed from western blotting analysis (Figure 4Ba), quantified brain BDNF expression levels of Mn-treated mice were significantly lower than those mice with sham, B401, and B401+Mn treatments individually (Figure 4Bb, Mn vs Sham, Mn vs B401, Mn vs B401+Mn, p<0.01). Also, we observed that quantified brain BDNF expression levels of B401-treated mice were significantly higher than those mice with sham and B401+Mn treatments individually (Figure 4Bb, B401 vs sham, B401 vs B401+Mn, p<0.01). 頁 17.

(30) 5. Effect of oral B401 treatment on the blood ROS in Mn-treated mice In comparing effects of oral B401 treatment on oxidative stress, we examined blood ROS levels in Mn-treated mice with oral B401 treatment in Figure 5. We observed that blood ROS levels of Mn-treated mice were greatly increased than those mice with the sham, B401, and B401+Mn treatments individually (Figure 5A). Furthermore, total counts of blood ROS of Mn-treated mice were significantly increased than those mice with the sham, B401, and B401+Mn treatments individually (Figure 5B, Mn vs Sham, Mn vs B401, Mn vs B401+Mn, p<0.01). Also, we observed that total counts of blood ROS of B401-treated mice were significantly decreased than those mice with the sham, and B401+Mn treatments individually (Figure 5B, B401 vs sham, B401 vs B401+Mn, p<0.05).. 6. Effect of oral B401 treatment on oxidative stress in the brain tissue of Mn-treated mice Effects of the herbal formula B401 in brain oxidative stress were studied by comparing brain SOD2 and 3-NT expressions in Mn-treated mice between with and without oral B401 treatment in Figures 6 and 7. We compared brain SOD2 expression levels of those mice with the sham, B401, Mn, and B401+Mn treatments individually. As observed from IHC staining, SOD2 expressions were obvious in the substantia nigra and the striatum of mice with the sham, B401, and B401+Mn treatments individually, while they were not obvious in the same areas of Mn-treated mice (Figure 6A). As analyzed from western blotting analysis (Figure 6Bb), quantified brain SOD2 expression levels of Mn-treated mice were significantly lower than those mice with sham, B401, and B401+Mn treatments individually (Figure 6Bb, Mn vs Sham, Mn vs B401, Mn vs B401+Mn, p<0.01). Also, we observed that quantified brain SOD2 expression levels 頁 18.

(31) of sham- and B401-treated mice were significantly higher than those mice with B401+Mn treatment (Figure 6Bb, sham vs B401+Mn, B401 vs B401+Mn, p<0.05). We further compared brain 3-NT expression levels of those mice with different treatments in Figure 7. As observed from IHC staining, 3-NT expressions were obvious in the substantia nigra and the striatum of mice with the Mn treatments, while they were not obvious in the same areas of mice with the sham, B401, and B401+Mn treatments individually (Figure 7A). As analyzed from western blotting analysis (Figure 7Ba), quantified brain 3-NT expression levels of Mn-treated mice were significantly greater than those mice with sham, B401, and B401+Mn treatments individually (Figure 7Bb, Mn vs Sham, Mn vs B401, Mn vs B401+Mn, p<0.01). Also, we observed that quantified brain 3-NT expression levels of B401-treated mice were significantly less than those mice with sham and B401+Mn treatments (Figure 7Bb, B401 vs sham, B401 vs B401+Mn, p<0.05).. 7. Effect of oral B401 treatment on apoptosis in the brain tissue of Mn-treated mice We examined effects of the herbal formula B401 in apoptosis by comparing the ratio of Bcl-2/BAX and expression levels of caspase 3 in the brain of Mn-treated mice in Figures 8 and 9. As observed from IHC staining, Bcl-2 expressions were obvious in the substantia nigra and the striatum of mice with the sham, B401, and B401+Mn treatments individually, while they were not obvious in the same areas of mice with the Mn treatment (Figure 8A). On the contrary, BAX expressions were obvious in the substantia nigra and the striatum of mice with the Mn treatment, while they were not obvious in the same areas of mice with the sham, B401, and B401+Mn treatments individually (Figure 8A). As analyzed from western blotting analysis (Figure 8Bb), the quantified brain Bcl-2/BAX 頁 19.

(32) ratio of Mn-treated mice were significantly less than those mice with sham, B401, and B401+Mn treatments individually (Figure 8Bb, Mn vs Sham, Mn vs B401, Mn vs B401+Mn, P<0.01). In addition, the quantified brain Bcl-2/BAX ratio of B401-treated mice were significantly greater than those mice with sham and B401+Mn treatments individually (Figure 8Bb, sham vs B401+Mn, B401 vs B401+Mn, P<0.01). We further compared brain caspase 3 expression levels of those mice with different treatments in Figure 9. As observed from IHC staining, caspase 3 expressions were obvious in the substantia nigra and the striatum of Mn-treated mice, while they were not obvious in the same areas of those mice with the sham, B401, and B401+Mn treatments individually (Figure 9A). As analyzed from western blotting analysis(Figure 9Ba), the quantified brain caspase 3 expression levels of Mn-treated mice were significantly greater than those mice with sham, B401, and B401+Mn treatments individually (Figure 9Bb, Mn vs Sham, Mn vs B401, Mn vs B401+Mn, P<0.01). In addition, the quantified brain caspase 3 expression levels of B401+Mn-treated mice were significantly greater than those mice with sham and B401 treatments individually (Figure 9Bb, B401+Mn vs sham, B401+Mn vs B401, P<0.05). We further observed that quantified brain cleaved caspase 3 expression levels were significantly greater than those mice with sham, B401, and B401+Mn treatments individually (Figure 9Bb, Mn vs Sham, Mn vs B401, Mn vs B401+Mn, P<0.01). The quantified brain cleaved caspase 3 expression levels of B401+Mn-treated mice were significantly greater than those mice with B401 treatments (Figure 9Bb, B401+Mn vs B401, P<0.05).. 頁 20.

(33) 8. Effect of oral B401 treatment on synthesis of nitric oxide and angiogenesis in the penile tissue of Mn-treated mice Effect of B401 treatment on cavernosal synthesis of nitric oxide was shown in Figure 10A. As detected by the electrochemical se3ntnsor meter, the value of cavernosal platelet-derived nitric oxide in Mn-treated mice was significantly lower than in the sham mice (Figure 10Ab, Mn group:. 450.2±12.2 pA, vs Sham group: 477.8 ±14.4 pA,. p<0.05). The cavernosal nitric oxide value in Mn-treated mice under B401 treatment was significantly higher than those Mn-treated mice without B401 treatment (Figure 10Ab, B401+Mn group:. 502.2±17.8 pA vs Mn group: 450.2±12.2 pA, p<0.05). The. cavernosal nitric oxide value in Mn-treated mice was reduced to ∼94 % of the value of sham mice, but was ∼105 % of the value of sham mice with those Mn-treated mice under B401 treatment (Fig. 10Ab). Interestingly, the cavernosal nitric oxide value in the cavernosal tissues of Mn-treated mice under oral B401 treatment were also significantly higher than those in the sham mice (Fig. 10Ab, B401+Mn vs Sham, p<0.05). From hematoxylin and eosin (H&E) histological aspects, we observed that cavernosal tissues between Mn-treated mice with and without oral B401 administration were very similar (Fig. 10Aa). The effect of B401 treatment on expression levels of cavernosal nitric oxide synthases was shown in Figure 11. Our immunohistochemical staining study revealed that cavernosal eNOS expression levels of Mn-treated mice under B401 treatment (B401+Mn group) were visibly enhanced beyond those of Mn-treated mice without B401 treatment (Mn group) (Fig. 11A, B401+Mn vs Mn). We quantified cavernosal eNOS expression levels of Mn-treated mice by Western blotting analysis as shown in Figure 11B. Both cavernosal nNOS and eNOS expression levels of Mn-treated mice under B401 treatment were significantly higher than those in the Mn-treated mice without B401 treatment (Fig. 11A, B401+Mn vs Mn, p<0.01). 頁 21.

(34) We further quantified the expression levels of cavernosal VEGF expression levels by Western blotting analysis as shown in Figure 12A. VEGF is a marker protein that stimulates angiogenesis. We found that the expression levels of VEGF in the cavernosal tissues of Mn-treated mice was not significant when compared with those mice under sham treatment (Fig. 12B, Mn vs Sham, p>0.05). However, the expression levels of VEGF in the cavernosal tissues of Mn-treated mice under B401 treatment were significantly higher than those in the Mn-treated mice without B401 treatment (Fig. 12B, B401+Mn vs Mn, p<0.01). Interestingly, expression levels of VEGF in the cavernosal tissues of Mn-treated mice under oral B401 treatment were significantly higher than those in the sham mice (Fig. 12B, B401+Mn vs Sham, p<0.05).. 9. Effect of oral B401 treatment on oxidative stress and inflammation in the penile tissue of Mn-treated mice The effect of B401 treatment on expression levels of marker proteins of oxidative stress in the cavernosal tissues was shown in Figures 12 and 13. We quantified the expression levels of catalase and SOD2 by Western blotting analysis as shown in Figure 13A. Both expression levels of catalase and SOD2 in the cavernosal tissues of Mn-treated mice were significantly weaker than those of the sham mice (Fig. 12B, Mn vs Sham, p<0.05), but were significantly increased in the cavernosal tissues of Mn-treated mice under oral B401 treatment (Figure. 12B, B401+Mn vs Mn, p<0.01). Interestingly, both expression levels of catalase and SOD2 in the cavernosal tissues of Mn-treated mice under oral B401 treatment were significantly increased beyond those of the sham mice (Fig. 12B, B401+Mn vs Sham, p<0.01). We further quantified the expression levels of 4-HNE by Western blotting analysis as shown in Figure 14. The 4-HNE has been hypothesized to play a key role in oxidative stress due to the increase in the lipid peroxidation chain reaction. We found that the expression levels of 4-HNE in the 頁 22.

(35) cavernosal tissues of Mn-treated mice under oral B401 treatment were significantly weaker than those of the Mn-treated mice without B401 treatment (Fig. 13B, B401+Mn vs Mn, p<0.01). The effect of B401 treatment on inflammation in the cavernosal tissues was shown in Figure 15. We quantified the expression levels of TNF-α by Western blotting analysis as shown in Figure 15. TNF-α is a signaling protein that is involved in systemic inflammation. We found that the expression levels of TNF-α in the cavernosal tissues of Mn-treated mice under oral B401 treatment were significantly weaker than those of the Mn-treated mice without B401 treatment (Figure 15B, B401+Mn vs Mn, p<0.01).. 10. Effect of oral B401 treatment on apoptosis in the penile tissue of Mn-treated mice It has been described that apoptosis is a downstream event in erectile dysfunction (Yamanaka et al., 2003). Thus we examined and compared expressions of apoptosis related proteins in cavernosal tissues of Mn-treated mice with and without oral B401 treatment. As suggested in previous studies, Bcl-2 is a marker of anti-apoptosis, while Bax and Cyt-C are two markers of mitochondrial apoptosis (Machado-Vieira et al., 2011). The ratio of Bcl-2 protein to Bax protein determines survival or death after an apoptotic stimulus. We quantified the ratio of Bcl-2/Bax by Western blotting analysis as shown in Figure 16B. The ratio of Bcl-2/Bax in the cavernosal tissues of Mn-treated mice under oral B401 treatment had significantly increased compared with the Mn-treated mice without oral B401 treatment (Figure 16B, B401+Mn vs Mn, p<0.01). We further quantified expression levels of Cyt-C by Western blotting analysis. Expression levels of Cyt-C in the cavernosal tissues of Mn-treated mice under oral B401 treatment were significantly weaker than those of Mn-treated mice without oral B401 treatment (Fig. 15B, B401+Mn vs Mn, p<0.05). Moreover, c-PARP is involved in programmed cell 頁 23.

(36) death (Yu et al., 2006). We quantified expression levels of c-PARP by Western blotting analysis. Expression levels of c-PARP in the cavernosal tissues of Mn-treated mice under oral B401 treatment were significantly weaker than those of Mn-treated mice without oral B401 treatment (Fig. 15B, B401+Mn vs Mn, p<0.05). Apoptosis in the cavernosal tissues of Mn-treated mice was further assessed by expression levels of caspase-12 and caspase-3, two important activators for apoptosis signaling. We quantified expression levels of caspase-12 and caspase-3 by Western blotting analysis as shown in Figure 17. Both expression levels of caspase-12 and caspase-3 in the cavernosal tissues of Mn-treated mice were significantly increased beyond those of the sham mice (Fig. 16B, Mn vs Sham, p<0.01-0.05), but were significantly decreased in the cavernosal tissues of Mn-treated mice under oral B401 treatment (Fig. 16B, B401+Mn vs Mn, p<0.01-0.05). Interestingly, expression levels of caspase-12 in the cavernosal tissues of Mn-treated mice under oral B401 treatment were significantly higher than those of the sham mice (Fig. 16B, B401+Mn vs Sham, p<0.05).. 頁 24.

(37) CHAPTER 4 DISCUSSION. 頁 25.

(38) 1. Oral treatment of the herbal formula B401 effectively alleviates Mn-induced brain toxicology In this study, we demonstrated that chronic exposure to Mn induced oxidative and apoptotic signaling pathways, and triggered behavioral dysfunction. Moreover, we showed that the herbal formula B401, a potent antioxidant, significantly attenuated the blood ROS generation, alleviated expressions of oxidative stress and apoptosis in the substantia nigra and the striatum. Therefore, our study could be profitably directed to determine the therapeutic efficacy of this agent in reducing Mn-induced neurotoxicity. Metal Mn is an essential metal for living organisms, but excessive Mn may cause pathological processes characterized by irreversible damage in the brain (Lazrishvili et al., 2009). Mn is often accumulated in the basal ganglia, and in adults it may cause parkinsonism (Cordova et al., 2013). Compared to adults, younger people may accumulate more Mn in the brain and are more vulnerable to its toxicity (Keen et al., 2000; Mergler et al., 1994). In this study, we found that Mn-treated mice worsened the motor coordination by reducing the fall time of the rotarod test recordings. It was reported that Mn-induced neurotoxicity is characterized by extrapyramidal symptoms resembling idiopathic parkinsonism (Roth, 2009). Results of our studies demonstrated that mice following Mn exposure showed a significant reduction of dopaminergic D1 receptors and dopamine levels in the substantia nigra and the striatum. These findings are consistent with the previous report that parkinsonism patients show a significant decrease in dopamine synthesis and dopamine transporter levels in the substantia nigra (Cummings et al., 2011). It has been reported that BDNF has potent effects on the survival and morphology of dopaminergic neurons, and BDNF loss may contribute to the death of dopaminergic neurons in the substantia nigra of parkinsonism patients (Howells et al., 2000). We also observed that the mice with Mn-induced neurotoxicity reduced BDNF expression levels 頁 26.

(39) in the substantia nigra. and the striatum significantly, and the reduction of BDNF. expressions might be part of the reason of reducing dopaminergic D1 receptors and dopamine levels in the substantia nigra and the striatum of the mice with Mn-induced neurotoxicity. The reduction of dopaminergic receptors and dopamine levels in the substantia nigra and the striatum may cause motor dysfunction. Previous study has reported that rats with Mn-induced neurotoxicity showed motor impairment by reducing fall time of the rotarod test, and enhancing oxidative stress by increasing ROS generation (Cordova et al., 2012). Even though mechanisms are not fully understood, Mn-induced parkinsonism may be associated with mitochondrial dysfunction that causes ROS generation and oxidative stress in dopaminergic cells (Pennathur et al., 1999; Tamm et al., 2008; Yin et al., 2008; Zhang et al., 2008a). Here we observed that the mice with Mn-induced parkinsonism enhanced the blood ROS level, and reduced SOD2 expressions, but elevated 3-NT expressions in the substantia nigra and striatum. SOD2 is an important antioxidant enzyme for oxidative stress, but 3-NT is a marker of oxidative damage (Chong et al., 2005). Therefore, the oxidative stress is accepted as the critical factor in pathogenesis of Mn-induced neurotoxicity. Mn-induced oxidative stress may cause the mitochondrial permeability transition and then leads to cell death in dopaminergic neurons (Tatton et al., 2003). In the present study, we characterized apoptotic mechanisms associated with Mn-induced parkinsonism in the brain tissue. We observed that Mn treatment targets the mitochondria and shifts the balance of Bcl-2 family proteins to favor the apoptotic death in neurons of the substantia nigra and the striatum. Immunoblotting and western blotting analysis revealed that chronic exposure to Mn causes a decrease in the antiapoptotic Bcl-2 protein, but increase in the pro-apoptotic Bax protein in the substantia nigra and the striatum. Furthermore, the Bcl-2/BAX ratio was also decreased significantly. To determine if the mitochondrial dysfunction would activate effector caspases, we assayed caspases 3 activities in the 頁 27.

(40) brain tissue of the mice following chronic exposure to Mn. As shown in Figure 9, chronic exposure to Mn causes a significant increase of caspase 3 expressions in the substantia nigra and the striatum of the mice. Our results may provide evidences that mitochondria-mediated apoptosis involves caspases cascades in the brain tissue of mice with Mn-induced parkinsonism. It has been reported that more active caspase 3 positive neurons were observed among dopaminergic neurons in parkinsonism patients (Yu et al., 2005). Alternative. medical. applications. of. traditional. Chinese. medicines. in. neurodegenerative disease remission are becoming popular because of its reputed safety and clinical efficacy for chronic conditions. The most widely used type of herbal treatment is the formula that is comprised of specific mixtures of several herbs. It is believed that complex interactions produce synergistic effects and a reduction of side effects and toxicity. For example, the herbal formula B401 contains six herbal ingredients, including extracts from Panax ginseng, Astragalus membranaceus, Angelica sinensis, Rehmannia glutinosa, Ligustri fructus, and Eclipta prostrata in specific ratios. By MTT assay, we found that quite few cytotoxicity was detected in the herbal formula B401-containing SH-SY5Y cell culture at any concentration no more than 80 mg/mL. In addition, we also found that the herbal formula B401 may alleviate excitotoxicity by increasing their cells viability in B401-containing glutamate-treated SH-SY5Y cell culture (Figure 1B). As illustrated from HPLC fingerprint in Figure 1A, active ingredients of the herbal formula B401 have ginsenosides Rb1 from Panax ginseng, formononetin from Astragalus membranaceus, 5-HMF from Rehmannia glutinosa, ferulic acid from Ligustri Fructus, and wedelolactone from Eclipta prostrata. Some of these compounds have been verified those have neuroprotective properties. For example, ginsenoside Rb1 has been widely used as a traditional herbal medicine that have anti-oxidative and anti-apoptotic effects in brain function (Chen et al., 2003). 頁 28.

(41) Furthermore, formononetin has been used to protect brain function by suppressing cellular excitotoxicity (Yu et al., 2005). It is unknown whether the mixtures of the herbal formula B401 have more advantages than a single ingredient. Therefore, further studies may be required to clarify the underlying molecular mechanism of components interaction and to develop an appropriate system to allow the evaluation of the ameliorative potential of such herbal formula against Mn-induced neurotoxicity.. 2. Oral treatment of the herbal formula B401 effectively alleviates penile toxicity of Mn-treated mice In this study, we evaluated the effects of the Chinese herbal formula B401 in the cavernosal tissues of Mn-treated mice. Our results demonstrated that oral treatment of the herbal formula B401 effectively enhances synthesis of nitric oxide (Figures 10 and 11) and angiogenesis (Figure 12), and reduces oxidative stress (Figures 13 and 14), inflammation (Figure 15), and apoptosis (Figures 16 and 17) in penile corpus cavernosum of Mn-treated mice. We demonstrated ingredients of the herbal formula B401 as shown in Figure 1A, the extract from Panax ginseng has been proven to reduce oxidative stress (Chen et al., 2003) and inflammation (Lee et al., 2011; Lin et al., 2007; Yu et al., 2000). Furthermore, Rg1 from Panax ginseng can induce rapid nitric oxide production from eNOS via the PI3K/Akt pathway (Leung et al., 2006). The extract from Astragalus membranaceus has been reported to reduce oxidative stress (Ji et al., 2014) and enhanced nitric oxide production (Meng et al., 2007). The extract from Angelica sinensis has been reported to have angiogenic effects in zebrafish via enhancing VEGF mRNA expression (Lam et al., 2008). The extract from Rehmannia glutinosa has long been used in age-related diseases and its therapeutic efficacy via attenuating oxidative damage in the brain of mice (Zhang et al., 2008b). The extract from Ligustri fructus has been used to protect against hydrogen 頁 29.

(42) peroxide-induced oxidative damage (Ju et al., 2012). The extract from Eclipta prostrata has been has been reported to have cerebroprotective and antioxidant effect via reducing global cerebral ischemia in rat (Mansoorali et al., 2012). Taken together, the herbal formula B401 might have protective effect via enhancing nitric oxide and VEGF production, and reducing oxidative stress and inflammation. As for traditional Chinese medicine, we know that none of these herbs have much therapeutic value. To use any of them alone may encounter problems. That is why we use combined herbal formula to treat penile toxicity of mice with acute manganism. As suggested in the Introduction, exposure to excessive Mn exposuremay lead to erectile dysfunction in human and other animal models (Emara et al., 1971; Mena et al., 1967; Rodier, 1995; Lauwerys et al., 1985; Chandra et al., 1973; Seth et al., 1973). The nitric oxide play a key mediator in penile erection (Rajfer et al., 1992). Impaired nitric oxide formation may be considered pathological mechanisms in erectile dysfunction (Kim et al., 1991). Nitric oxide can be synthesized and released from non-adrenergic, non-cholinergic nerve endings by specific nNOS and from the endothelium by eNOS (Burnett, 1997). It has been suggested that rapid, brief activation of nNOS initiates the erectile process, whereas PI3-kinase/Akt-dependent phosphorylation and activation of eNOS by augmented blood flow and endothelial shear stress lead to sustained nitric oxide production and maximal erection (Hurt et al., 2002). Inhibition of nNOS attenuated erectile responses (Ignarro et al., 1990). Thus, it is very likely that physiologic penile erection is mediated by both nNOS and eNOS. Furthermore, it has been suggested that VEGF induced penile erection and corrected alterations in eNOS phosphorylation (Musicki et al., 2005). Excess expression of VEGF may induce synthesis eNOS and iNOS in the penis of rats (Lin et al., 2002). As suggested from our observations in this study, we found that Mn-treated mice with oral B401 treatment (ICR mice) were significantly reduced synthesis of nitric oxide (Figure 10) and expression levels of nNOS, 頁 30.

(43) eNOS (Figure 11) and VEGF (Figure 12) in penile corpus cavernosum. Taken together, excessive Mn exposure may lead to erectile dysfunction via inhibiting nitric oxide-mediated signaling pathways. In Taiwan, the Chinese herbal formula B401 has been developed as a health supplement for promoting blood circulation and enhancing brain function. In this study, we observed that synthesis of nitric oxide, and expression levels of nNOS, eNOS and VEGF were significantly enhanced in penile corpus cavernosum of Mn-treated mice under oral the herbal formula B401treatment (Figures 10-12). Thus, we assumed that B401 might may have protective effects on penile toxicity of mice with acute manganism via promoting nitric oxide-mediated signaling pathways. Several evidences have indicated that oxidative stress, mitochondrial dysfunction, inflammation and apoptosis were involved in underlying pathological mechanisms of excessive Mn-induced neurodegeneration (Milatovic et al., 2009). As suggested from our results, we found that ICR mice were significantly reduced SOD2, catalase (Figure 13), and Bcl-2 (Figure 16) in penile corpus cavernosum, but increased expression levels of 4-HNE (Figure 14), TNF-α (Figure 15), Bax, Cyt-C( Cyt. C) (Figure 16), caspase-12 and caspase-3 (Figure 17) in penile corpus cavernosum. Previous studies have suggested that oxidative stress plays a causative role in erectile dysfunction (Agarwal et al., 2006; Jeremy et al., 2000). Enhanced expression of 4-HNE, an oxidative marker, has been observed in a rat model of radiation-induced erectile dysfunction (Kimura et al., 2012). Oxidative stress and inflammation lead to endothelium dysfunction that plays a key event in the pathophysiology of erectile dysfunction (Berk et al., 2001). Administration of the antioxidants catalase and SOD2 may restore diabetes-induced oxidative stress and erectile dysfunction (Kawakami et al., 2009). Inflammatory conditions were also observed in patients with erectile dysfunction via increasing plasma levels of TNF-α (Giugliano et al., 2004). Furthermore, apoptosis is a downstream event in erectile dysfunction (Yamanaka et al., 2003). The Bcl-2 protein plays an important role in 頁 31.

(44) inhibiting apoptosis, while the Bax is a pro-apoptotic member that releases Cyt-C from the mitochondria (Jürgensmeier et al., 1998; Reed, 1994). The PARP is cleaved by caspase-3 that is a key executor of apoptosis (Boulares et al., 1999; Jiang et al., 2001). In this study, we observed that expression levels of anti-oxidative catalase and SOD2, and anti-apoptotic Bcl-2 were significantly enhanced, but expression levels of 4-HNE, c-PARP, Bax, Cyt-C, caspase-12 and caspase-3 were significantly reduced in penile corpus cavernosum of Mn-treated mice under oral the herbal formula B401treatment (Figures 12-16). In other words, B401 might may have protective effects on penile toxicity of ICR mice with acute manganism via reducing oxidative stress, mitochondrial dysfunction, inflammation and apoptosis.. 頁 32.

(45) CHAPTER 5 CONCLUSION. 頁 33.

(46) Here we used oral treatment with the herbal formula B401 as alternative medical applications in remission of Mn-induced neurotoxicity. As summarized in Figure 18, we found that Mn-treated mice with oral B401treatment significantly improves the motor coordination, reduces blood ROS, and enhances expression levels of dopaminergic receptors, dopamine and BDNF, but suppressing oxidative stress (by up-regulating SOD2 expressions, and down-regulating 3-NT expressions) and apoptosis (by up-regulating Bcl-2/BAX ratio, and down-regulating caspase 3 expressions) in the brain, especially in the substantia nigra and the striatum. As summarized in Figure 19, we have suggested that oral B401 treatment could alleviate penile toxicity of excess Mn-treated mice via enhancing synthesis of nitric oxide and angiogenesis, and reducing oxidative stress, inflammation, and apotosis in corpus cavernosum. We suggested that the herbal formula B401 might be developed as a potential health supplement for ameliorating Mn-induced neurotoxicity, and it is highly possible that the herbal formula B401 may also be useful dietotherapic supplement for penile toxicity or dysfunction.. 頁 34.

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