臺灣博碩士論文加值系統

勃起功能障礙（Erectile Dysfunction, ED）問題的定義是指持續、且不能獲得並保持 足以令人滿意的勃起表現，屬於性功能障礙最重要的一環。雖然 ED 是一種良性疾病，但 它會對身體和社會心理健康產生不利的效應，並對患者及其伴侶的生活質量產生重大影 響。因此，為了研究針對 ED 的治療措施，我們進行了 3 個部分的研究，採用不同的中醫 藥方式（TCM）：1. 電針對大鼠性功能的影響 2. 電針併用威而鋼的藥動藥效的研究，以 及 3. 枸杞多醣（LBP）對性功能的影響以及機轉。具體來說，在針灸對性功能的影響研究 中，我們設計了電針（EA）刺激不同頻率的穴位：關元（CV4）和中極（CV3），藉此研 究對雄性大鼠性行為的影響。以 Long-Evans 大鼠分別接受低頻 2Hz 和高頻 80Hz 的電針 ( Electro-acupuncture, EA )治療。低頻和高頻 EA 的強度分別為 1.5 mA（30 分鐘/天， 持續 5 天）和 1.5 mA（30 分鐘一次）。普通針灸組則是在沒有加上電刺激的情況下僅接 受 CV4 和 CV3 的針刺。在穴位刺激後，進行大鼠交配行為實驗，其中低頻 EA 處理的大 鼠表現出插入頻率和射精潛伏期的增加，而高頻 EA 處理的大鼠表現出插入和射精頻率的 抑制。這些結果表明，低頻 EA 可能有效改善性行為，進而治療早洩，而高頻 EA 則會降 低性行為的表現。
Sildenafil（商品名 Viagra, 威而鋼），一種磷酸二酯酶抑製劑，是一種廣泛認可的 藥物，在 ED 治療方面有顯著的效果。然而，電針和威而鋼並用的情形如何仍未有研究。 因此，我們試驗了電針同時併用 sildenafil 對 ED 的治療。我們特別評估了 EA 對大鼠血漿 中 sildenafil 的藥物動力學和藥效學的影響。在大鼠電針治療後之後給予 sildenafil （10mg / Kg），使用超高效液相色譜法測定藥物動力學參數。藥物動力學研究表明，經 過給予低頻 EA 之後顯著增加了 sildenafil 的血中濃度。之後，使用下半身和陰莖的都普 勒血流顯影圖像模式研究藥效學。在都普勒成像的藥效學研究顯示，在 sildenafil 和低頻 EA 的合併使用，與下半身相比，大鼠陰莖中的血流有增強的效果。這些數據表明 EA 和 sildenafil 用於治療 ED有相輔相成效果。除了 EA 研究，我們還設計了枸杞多醣（LBP） 對雄性大鼠性行為的影響的實驗。從過去文獻已知 LBP 對生殖功能有促進作用，但其在由大腦區域，包括內側視前區（MPOA）和室旁核（PVN）區域引發的性行為和陰莖的非接 觸性勃起（NCE）的作用尚未被研究。我們的研究設計為，讓成年雄性大鼠口服 100 mg / kg 的 LBP 或媒介物。在使用 14 天之前和之後，記錄大鼠交配行為和非接觸性勃起 （NCE）的次數。在最後的性行為實驗記錄之後，取大鼠的腦以免疫螢光染色測量 MPOA 和 PVN 區域中的 nNOS 表達。結果顯示，服用過 LBP14 天後顯著增加了大鼠性 行為的插入和射精頻率。同組若與服用前相比，我們觀察到射精間隔的減少。此外，經 LBP 治療後，插入頻率、插入間隔和 MPOA nNOS 免疫反應細胞（nNOS-ir）的數量都顯著增加。另外，我們還觀察到射精頻率與 MPOA nNOS-ir 細胞之間的顯著正相關。值 得注意的是，LBP 的治療對 NCE 和 PVN nNOS-ir 表達沒有影響。這些發現表明，LBP 是透過增加雄性大鼠腦內 MPOA 中的 nNOS 表達來增強性行為。我們的研究表明，電針與威而鋼並用以及枸杞多醣等中醫藥的治療對性功能的改善，有顯著的助益。

ABSTRACT
Sexual problems such as erectile dysfunction (ED) is a persistent inability to attain and maintain an erection sufficient to permit satisfactory sexual performance. Though ED is a benign disorder, it adversely influences physical and psychosocial health and has a significant impact on the quality of life of sufferers and their partners. Therefore, in order to investigate therapeutic measure against ED, we conducted 3 studies employed different modalities of Traditional Chinese Medicines (TCM). Specifically, in study 1, we investigated the effect of electro-acupuncture (EA) stimulation of different frequencies at Guanyuan (CV4) and Zhongji (CV3) acupoints on sexual behavior in male rats. Animals were subjected to low and high EA at 2Hz and 80Hz, respectively. The intensities at low and high frequency EA were 3 mA (30 min/day for 5days) and 1.5mA (30 min once), respectively. The only acupuncture-administered group received acupunctures at CV4 and CV3 without electrical stimulation. After acupuncture stimulation, copulatory behavior test was conducted in which low frequency EA-treated rats exhibited increasing intromissions and ejaculation latency, while high frequency EA-treated rats displayed lower intromissions and ejaculation frequency. These results indicated that the low frequency EA might be efficacious in treatment of premature ejaculation thereby improving the sexual behavior, whereas, high frequency EA might decline sexual functions. In addition to EA therapy, the sildenafil citrate (Viagra), a phosphodiesterase 5 inhibitor, is a widely recognized drug which has achieved significant success in the treatment of ED. However, the combined effect of EA and sildenafil remains to be examined. Therefore, in study 2, we tested a combined EA and sildenafil citrate treatment against ED. We specifically assessed the effect of EA on the pharmacokinetics and pharmacodynamics of sildenafil in rat plasma. The pharmacokinetic parameters were determined by using ultra performance liquid chromatography after EA and sildenafil administration (10 mg/Kg). The pharmacokinetic studies demonstrated significantly increased levels of sildenafil by administration of low-frequency EA. Thereafter, the pharmacodynamics was studied via blood flow pattern using developing Doppler images of lower body and penis. Further, the Doppler imaging-based pharmacodynamic studies using revealed an enhanced blood flow in rat penis compared to lower body during combined treatment of sildenafil and low-frequency EA. These data indicate a synergistic therapeutic effect of EA and sildenafil for the treatment of ED. Besides EA studies, we also inspected the effect of Lycium barbarum polysaccharide (LBP) on male sexual behavior. Though the reproductive function-promoting nature of LBP is known, its role in noncontact erection (NCE) of penis initiated by brain regions including medial preoptic area (MPOA) and paraventricular nucleus (PVN) regions, responsible for sexual behavior has not been investigated. In this study, the adult male rats were treated with 100 mg/kg of LBP or vehicle by oral gavage. Before and after 14 days of treatment, copulatory behavior and noncontact erection (NCE) was recorded. After the last behavioral test, the brain was isolated to measure nNOS expression in MPOA and PVN. Results showed that LBP significantly increased both the frequencies of intromission as well as ejaculation. Whereas, a reduced post-ejaculatory interval was observed compared to same group on day 0. Furthermore, the treatment also increased intromission ratio, inter-intromission interval, and the number of MPOA nNOS-immunoreactive cells (nNOS-ir). Additionally, a significantly positive correlation between ejaculation frequency and MPOA nNOS-ir cells was recorded. Of note, LBP treatment had no effects on NCE and PVN nNOS-ir expression. These findings suggest that LBP enhances sexual behaviour through increased nNOS expression in the MPOA in male rats. Taken together, study including Traditional Chinese Medicines such as acupuncture and its combination with sildenafil, as well as LBP showed a significant improvement on sexual dysfunction.

Table of Contents
Chinese Abstract .................................. viii
English Abstract ......................................x
List of Tables .....................................xiii
List of Figures .....................................xiv
Abbreviations........................................xvi
1. Introduction........................................1
1.1 Sexual function....................................2
1.2 Sexual dysfunction: The definition.................3
1.2.1 Sexual dysfunction and Western medicine............4
1.2.2 Sexual dysfunction and Traditional Chinese Medicine..6
1.3 Acupuncture in sexual dysfunction..................9
1.4 Acupuncture and electro-acupuncture.................9
1.5 Therapeutic acupuncture and Guanyuan (CV4) and Zhongji (CV3) acupoints.....11
1.6 Lycium Barbarum, a Traditional Chinese Herb.......12
1.7 Aims & objectives of the study....................14
2. Materials and methods..............................15
2.1 Investigation of Effect of Low Frequency Electro-acupuncture at Acupoints
Guanyuan (CV4) and Zhongji (CV3) for Improving Sexual Function.....16
2.1.1 Animals grouping and their maintenance........17
2.1.2 Stimulation of the female rats.................17
2.1.3 Copulation screening of test male rats..........17
2.1.4. Copulatory behavior testing....................18
2.1.5 Electro-acupuncture (EA)-dependent stimulation....18
2.1.5.1 EA at low frequency ........................18
2.1.5.2 EA at high frequency.........................19
2.1.6 Statistical analysis............................19
2.2 Determining Interactive Activity of Electro-Acupuncture on Pharmacokinetics of
Sildenafil and Their Synergistic Effect on Penile Blood Flow in rats...........21
2.2.1 Animals and study design .......................22
2.2.2 HPLC -UV Instrumentation........................22
2.2.3 Sildenafil and Electro-Acupuncture (EA) administration.......24
2.2.4 Pharmacokinetics parameters.....................24
2.2.5 Measurement of blood flow in lower body and rat penis........24
2.2.6 Statistical analysis............................25
2.3 Effect of Lycium Barbarum Polysaccharides on Sexual Performance...........26
2.3.1 Animals................................................27
2.3.2 Behavioral testing..............................27
2.3.2.1 Copulatory screening test for male rats.......27
2.3.2.2 Noncontact erection (NCE) test................28
2.3.2.3 Copulatory behavior testing...................28
2.3.3 Immunohistochemical staining of nNOS in medial preoptic area (MPOA) and paraventricular nucleus (PVN)..29
2.3.3.1 Perfusion and tissue preparation...............29
2.3.3.2 Immunohistochemical staining of nNOS...........30
2.3.3.3 Quantification of nNOS immunohistochemistry.....31
2.3.3.4 Determination of Testicular weight and testosterone level.31
2.3.3.5 Statistical analysis...........................31
3. Results ...........................................33
3.1 Low frequency electro-acupuncture at acupoints Guanyuan (CV4) and Zhongji
(CV3) lengthen ejaculatory latency and improves sexual function in male rats...34
3.1.1 Effect of low frequency electroacupuncture (EA)..35
3.1.2 Effect of high frequency electroacupuncture (EA)..35
3.2 Investigation of Interactive Activity of Electro-Acupuncture on Pharmacokinetics
of Sildenafil and Their Synergistic Effect on Penile Blood Flow in Rats........36
3.2.1 Analytical Method Validation for Sildenafil .......37
3.2.2 Effect of sildenafil and EA on the pharmacokinetic parameters.37
3.2.3 Efficacy of sildenafil and EA on Pharmacodynamics parameters..38
3.3 Effect of Lycium Barbarum Polysaccharides on Sexual Performance...........40
3.3.1 Effect of LBP on male sexual behavior............41
3.3.2 Effect of LBP on non-contact erection (NCE) .....42
3.3.3 Immunoreactivity of nNOS (NOS-ir) in the MPOA and PVN.42
3.3.4 Correlation between sexual behavior parameters and distribution of MPOA nNOS-ir cells.42
3.3.5 Testis weight and serum testosterone (T) concentration.43
4. Discussion.........................................44
4.1 Low frequency electro-acupuncture at acupoints Guanyuan (CV4) and Zhongji
(CV3) lengthen ejaculatory latency and improves sexual function in male rats...45
4.2 Investigation of Interactive Activity of Electro-Acupuncture on Pharmacokinetics
of Sildenafil and Their Synergistic Effect on Penile Blood Flow in Rats........49
4.3 Effect of Lycium Barbarum Polysaccharides on Sexual Performance...........52
5. Conclusion.........................................57
6. Tables and Figures.................................60
Table 1. Precision (%R.S.D.) and accuracy (% Bias) data for sildenafil.........61
Table 2. The dosage regimens of sildenafil and electro-acupuncture administration...62
Table 3. Pharmacokinetics parameters of sildenafil.......62
Table 4. Effect of LBP on sexual behavior in male rats...63
Table 5. Effect of LBP on NCE............................63
Table 6. Correlational analysis between sexual behavior measures and the number of nNOS-ir cells in the MPOA of male rats.............64
Table 7. Effect of LBP on the body weights, testes, and serum testosterone levels.64
Figure 1. Schematic of overall study investigating the effect of Traditional Chinese Medicines, including, electro-acupuncture and Lycium Barbarum polysaccharide..........65
Figure 2. Experimental design and procedure illustrating effect of electro-acupuncture on sexual behavior........66
Figure 3. Comparative effect of pre- and post-low frequency EA treatment on sexual behaviour of male rats...........67
Figure 4. Effect of high frequency EA treatment on intromission and ejaculation frequency of male rats...68
Figure 5. Summary of results demonstrating the effect of low and high EA at acupoints Guanyuan (CV4) and Zhongji (CV3) on sexual behavior..69
Figure 6. HPLC chromatograms of intravenous sildenafil after
administration of electro-acupuncture (EA) ............70
Figure 7. Pharmacokinetics experimental design of EA and sildenafil treatment..71
Figure 8. Blood plasma concentration-time profile of intravenous sildenafil in the blood ....................72
Figure 9. The schematic of pharmacodynamics study with Doppler imaging after sildenafil and EA administration...73
Figure 10. Doppler images of penile and lower body blood flow in electro-acupunctured rat at 1.5 mA and 2Hz......74
Figure 11. Blood flow and time profile in penis and lower body of Rats after combined treatment of both EA and sildenafil......75
Figure 12. Schematic of study showing effect of lycium barbarum................76
Figure 13. Effect of LBP on inter-intromission interval and
intromission ratio in male rats........................77
Figure 14. Photomicrographs of nNOS immunoreactivity in the MPOA of the male rats following LBP treatment..........78
Figure 15. nNOS immunoreactivity in PVN of the male rats following LBP treatment................................79
7. References..........................................80
8. Publications........................................91

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