開環透明質酸攜帶瘦肉精緩釋用於高脂餵食的肥胖小鼠治療

Abstract

全球肥胖患病率逐年遞增，肥胖及肥胖相關疾病的治療及控制的醫療成本隨之增加。儘管治療肥胖的方法很多，如：物理性的飲食控制或運動、化學性的健康食品攝取、藥理性使用減肥藥、或是最激烈的手術，上述沒有一種策略既安全有效又易於維持。瘦肉精一直是充滿爭議的藥物，由於其副作用（特別是肝腎毒性），克倫特羅在健美運動中的使用和職業運動員的使用存在爭議，受限於副作用及有效窗口僅在某些地區被准許用在氣喘治療上。這項研究以顯著低於安全水平劑量給予克崙特羅的原位注射脂肪組織緩釋作為治療肥胖的療法研究。研究顯示在更低的劑量和原位注射脂肪組織緩釋的方式下，能降低副作用發生的風險，提高安全性，對於克崙特羅真正使用在對抗肥胖方面的應用上相當有幫助。本研究以開環玻尿酸的溫感水凝膠為載體，負載克倫特羅以實現緩釋的效果。本實驗分為三個部分：材料合成、體外測試與動物實驗。在材料合成的實驗裡，利用傅立葉轉換紅外光譜確認玻尿酸確實開環後，加入高碘酸鈉氧化後的克倫特羅以製作含有克倫特羅的緩釋溫感水凝膠。在合成含有克倫特羅的緩釋溫感水凝膠後，使用流變儀確認在標準溫度及體溫下的成膠時間，並確認溶脹率與降解時間符合安全性及有效性需求。此外，利用可見光分光光度計檢測溫感水膠緩釋平台中的克倫特羅釋放曲線。在體外研究中，新開發的系統顯示在以開環玻尿酸的溫感水凝膠為載體的攜帶的克崙特羅，不僅對3T3-L1細胞無細胞毒性，並且可有效抑制體外培養細胞內的脂肪生成。在動物研究中，小鼠被餵食高脂肪飲食，並通過口服克崙特羅或定期注射攜帶克倫特羅的透明質酸溫感水凝膠 (HAC) 進行治療。實驗顯示全身、內臟和性腺脂肪含量和體重都有所下降。在使用脂肪模式和水模式下的 MRI比對成像分析腹部脂肪，我們發現脂肪內注射 HAC 的小鼠的腹部脂肪比率在試驗組中最低，甚至接受正常飲食治療的小鼠，而用高脂肪飲食 (HFD) 治療的小鼠顯示出 53.78% 的最高值。在血球和血清學分析中，確認了克倫特羅口服給藥造成的急性與慢性體內毒性在HAC組別試驗中並無觀察到，證明了有別於傳統口服給藥，每天 2 μg – 10 μg 克倫特羅的控釋注射劑是安全的。這項研究開發了一種新的和有潛力的抗肥胖治療方法，使用每月脂肪內控釋注射 HAC水凝膠。新研發的鹽酸克倫特羅配方不僅有效降低體重和體脂含量，還能抑制內臟組織的脂肪生成，減少性腺周圍的脂肪組織。本研究未觀察到傳統口服克崙特羅引起的副作用；這項研究極有可能成為未來肥胖治療甚至是第二型糖尿病的有效治療手段，而無需擔心安全問題。

The prevalence of obesity around the world is rising yearly, as are the associated costs. Although there are many ways to combat obesity, there isn't a single strategy that is both secure and effective. The use of Clenbuterol in bodybuilding and by professional athletes is controversial owing to its side effects, including hepatotoxicity. This study administered Clenbuterol at a much lower dose than the established safety level, and rather than through oral administration, the treatments were delivered through controlled-release intra-adipose injection; and a thermo-sensitive hydrogel was used as the carrier uploaded with Clenbuterol to achieve controlled-release. On the other hand, this study also demonstrates that Clenbuterol which at the different dosing and mode of administration will lower the risk of side effects, increase the safety profile, and could facilitate use in the anti-obesity market. A thermo-sensitive hydrogel was used as the carrier uploaded with Clenbuterol to achieve controlled-release. The HAC hydrogel was prepared and then checked using Fourier-transform infrared spectroscopy (FTIR), and rheometer to confirm functional groups, and gelation time and temperature, respectively. The release profile in vitro was used to soak HAC in PBS solution for some time and then check the Clenbuterol level in the supernatant by UV-Vis. Then, the results of the following: cell viability, cytotoxicity, lipolysis on a cellular level, chronic toxicity, body weight control, whole-body adipose tissue, gonadal fat tissue, blood analysis, serological analysis, and sectioning examination of internal organs. These would be used to evaluate the safety and efficacy of the synthesized HAC, both in vitro and in vivo. The HAC group was not cytotoxic to 3T3-L1 cells and could inhibit lipogenesis effectively. In the animal study, the mice were fed a high-fat diet and treated with Clenbuterol by oral administration or injected with Clenbuterol-modified hyaluronate hydrogel (HAC) regularly. The HAC group showed reduction in whole-body, visceral, and gonadal fat contents and body weight. The abdominal fat was analyzed using MRI imaging in adipose mode and water mode. The abdominal fat ratio in the mice treated with normal diet and those given intra-adipose injections with HAC had the lowest value among the test groups. The mice treated with high-fat diet (HFD) showed the highest value of 53.78 %. The chronic toxicity in-vivo tests proved that controlled-release injections of 2 g – 10 g Clenbuterol daily were safe, as demonstrated in the blood elements and serological analyses. This study developed a new and promising method for anti-obesity treatment, using a monthly intra-adipose controlled-release injection of HAC hydrogel. The developed new treatment of Clenbuterol only effectively decreased body weight and body fat content but also inhibited lipogenesis on the harvested visceral tissue and reduced adipose tissue around the gonadal area. The side effects induced by traditional oral administration of Clenbuterol was not observed in this research; this has excellent potential to be a useful tool for future obesity treatment without safety concerns.