Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/90315
標題: Regulation of sphingolipid pathway in muscle growth; studies using in vivo animal models
神經鞘脂質途徑影響肌肉生長;活體動物模式之研究
作者: Wei-Kai Chen
陳威凱
關鍵字: 神經鞘脂質
生肌作用
伏馬鐮孢毒素
肌肉再生
體增重
sphingolipids
myogenesis
muscle regeneration
Fumonisin B1
body weight gain
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摘要: 黴菌毒素(mycotoxins) 廣泛存在於被污染穀類和玉米中,台灣因氣候常處於高溫潮溼的環境下,禽畜飼料與原料亦易受黴菌毒素污染。伏馬鐮孢毒素(fumonisins)為污染率最高黴菌毒素,其在玉米的污染率高80%,嚴重影響禽畜生長性能與經濟效益。神經鞘脂質 (sphingolipid) 家族成員複雜,多為富有生物活性之分子且兼具形成細胞膜結構與訊號傳遞功能角色,能調控細胞增生、分化、移動、存活等功能。Fumonisins之毒性因其結構類似 sphingoid bases,故其會阻斷 ceramide synthase 的活性,進而干擾神經鞘脂質途徑。其中fumonisin B1 (FB1) 為含量最多且毒性最強之伏馬鐮孢毒素,故本試驗藉由餵飼或注射 FB1以及抑制劑來干擾體內神經鞘脂質代謝途徑,並以外源性加入神經鞘脂質途徑之下游產物,觀察活體生長性狀以及對生肌作用之影響。試驗一為肌肉再生實驗,分為兩部分,皆採用滿8週齡之公鼠,以注射 cardiotoxin (CTX) 誘發其肌肉再生後進行試驗。第一部分為注射FB1、D609 (tricyclodecan-9-yl-xanthogenate.K)、PDMP (d-l-threo-1-phenyl-2-decanoyl amino-3-morpholino-1-propanol) 等抑制劑到受傷部位;另外再打入 ganglioside GM3、sphingomyelin (SM)、sphingosine-1 phosphate (S1P) 等下游產物來看是否能改善再生情形。第二部分則是餵飼小鼠含有FB1 (100 ppm) 及其下游產物神經鞘磷脂 (sphingomyelin, 500 ppm) 的飼糧。皆在第5天和第10天取比目魚肌 (soleus) 做組織切片 (H&E) 以及組織免疫染色 (Immunohistochemistry) 來觀察生肌蛋白之表現量進而判斷生肌作用程度。結果顯示: 在組織型態上,FB1等抑制劑之處理組其肌肉再生情形皆較控制組差,而以神經鞘脂質下游產物處理能一定程度改善FB1等抑制劑對肌肉再生的負面作用。生肌蛋白 embryonic MHC (myosine heavy chain)表現結果類似,在有抑制劑處理下表現量較低,但在加入下游產物後其表現量有較高之現象。試驗二則為生長實驗,取5週齡之公鼠用上述之飼糧餵飼四週後記錄體重、計算肌肉纖維直徑大小。結果顯示: 餵飼FB1之小鼠其體增重以及肌纖維大小皆小於控制組 (P<0.05),而以SM+FB1飼糧餵飼小鼠則能明顯改善其體增重且增大肌纖維直徑。骨骼肌中 insulin growth factor-1 (IGF-1) 的表現量 FB1+SM 處理組也略比 FB1 組來的高,而骨骼肌及肝臟中FB1及SM含量消長之情形與體增重以及肌纖維大小結果一致。綜合以上之結果顯示: 受到伏馬鐮孢毒素或抑制劑干擾神經鞘脂質途徑後,活體動物肌肉受傷後再生,正常肌肉生長以及生長性狀確實是會受到影響,而在飼糧中加入神經鞘脂質之下游代謝物能夠改善FB1對動物肌肉生長與生長性狀的負面作用。此外,本研究也釐清神經鞘脂質彼此間的消長對於活體動物生肌作用過程之影響。
Due to the hot weather temperature and high humidity in Taiwan, very high contamination of mycotoxins in feedstuffs and raw materials tend to seriously threatens not only the production of domestic animals but the farmer profits. Fumonisins are classified as a family of fungus toxins that were frequently found in contaminated cereals and corn. Sphingolipid family comprises numerous members, some of which function as an architecture component of membranous structures or act as a messenger molecule in cell signaling relay. These bioactive lipids mediate the signaling cascade to regulate cell functions such as proliferation, differentiation, mobility and survival. Due to its similar structure to sphingoid bases, fumonisins can interrupt ceramide synthase activity and then perturbs sphingolipid metabolic pathway. Fumonisin B1 (FB1) is the most common and toxic fumonisins. Therefore, in this study we used FB1 and several pharmacological inhibitors to interrupt sphingolipid pathway in vivo through oral or muscular injection. In the study of muscle regeneration model, the soleus muscle of 8 weeks old male mice were injected with cardiotoxin (CTX) alone or in combination with inhibitors including FB1, D609 (tricyclodecan-9-yl-xanthogenate. K), PDMP (d-l-threo-1-phenyl-2-decanoyl amino-3-morpholino-1-propanol) to the damage site to examine muscle regeneration. Then we also injected ganglioside GM3, sphingomyelin (SM), sphingosine-1 phosphate (S1P), the downstream products of ceramide synthase, into the solei damaged by CTX to examine regenerative progression. In the study of oral administration of FB1 (100 ppm) and SM (500 ppm), CTX-injured soleus were excised at 5 and 10 day and used for histological analysis and myogenic protein expression through immunohistochemical study. Results showed that muscle regeneration in FB1, D609, or PDMP treated mice was much worse than that of control and treatment of downstream product, GM3, SM, or S1P could improve the regeneration process to some degrees. Besides, in situ injection of pharmacological inhibitor, FB1, D609, or PDMP hampered embryonic MHC expression and exogenous treatment of GM3, SM, or S1P, promoted embryonic MHC expression. In the normal muscle growth model, 5 weeks old mice were fed with diet supplemented with FB1 alone or with FB1+SM for 4 weeks. At the end, mice were weighted and sacrificed for myofiber diameter measurement and FB1 and SM determination. Results suggested that FB1-toxified mice had lower body mass and smaller myofiber diameters than those of control (P<0.05). Dietary supplementation of SM significantly rescued body mass and increased myofiber diameters in FB1-toxified mice (P<0.05). Mice fed diet with FB1+SM had higher muscle insulin growth factor-1 (IGF-1) expression when compared with FB1 treated mice. Besides, FB1 and SM content in the muscle and liver showed consistent results in response to FB1 treatment alone or in combination with SM (P<0.05). In conclusion, results in the study suggest that muscle regeneration and growth are impaired by mycotoxin FB1 and other pharmacological inhibitors that perturb sphingolipid pathway. Treatment of GM3, SM, or S1P, the downstream products of sphingolipid pathway rescue muscle regeneration impaired by FB1 and the inhibitors. Oral provision of SM can alleviate the impact of postnatal muscle growth in FB1-toxified mice. Moreover, the approaches through regeneration and normal postnatal muscle growth modle in combination with pharmacological inhibitors clarify the role of various members of sphingolipid pathway in myogenic process and subsequent muscle growth.
URI: http://hdl.handle.net/11455/90315
文章公開時間: 2015-01-28
Appears in Collections:動物科學系

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