砷誘發肌肉萎縮之作用及分子機制探討

Abstract

砷 (arsenic, As)為自然界中廣泛存在之有毒類金屬，並且為國際癌症研究署 (International Agency for Research on Cancer, IARC)所認定之一級致癌物質，其中又以無機三價砷化合物：三氧化二砷 (arsenic trioxide, As2O3)最具代表性並被廣為研究。已知長期飲用含無機砷化物之地下水易造成烏腳病、皮膚癌與肺癌等多種病症，另外，也觀察到新生兒體重減輕與肌肉組成減縮的現象。臨床研究發現三氧化二砷可用於治療急性前骨髓細胞白血病 (acute promyelocytic leukaemia, APL)，但持續使用此療法卻會產生多發性神經病變、肌肉萎縮 (muscle atrophy)等不良影響。以上文獻皆顯示出三氧化二砷具有肌肉毒性，且近來的研究亦指出三氧化二砷會抑制肌肉分化 (myogenic differentiation)與肌肉再生 (muscle regeneration)作用，故本實驗欲以動物及細胞模式探討三氧化二砷是否造成肌肉萎縮及其分子機制。本實驗使用剔除坐骨神經之ICR小鼠進行實驗，並餵予0.05或0.5 ppm之三氧化二砷飲水達四週進行肌肉功能之探討，以去除神經源性肌萎縮之影響。首先，以滾輪測試儀 (rota-rod)測試小鼠之肌肉耐受力，實驗結果發現去神經合併餵食砷飲水之小鼠停留於滾輪上的時間顯著地縮短，此外，其下肢肌肉(比目魚肌 (soleus muscle)、脛前肌 (tibialis anterior muscle)、腓腸肌 (gastrocnemius muscle))的重量與肌纖維束大小皆顯著地下降，並藉由免疫組織化學染色法 (immunohistochemistry, IHC)觀察到Atrogin-1與Noggin的大量表現。另外，本實驗亦利用C2C12肌小管細胞 (C2C12 myotube)處理0.25-1 μM三氧化二砷達48小時，以探討其誘發肌肉萎縮之分子機制。實驗結果證實肌肉萎縮指標蛋白 (Atrogin-1、MuRF1)的表現皆明顯被砷所誘發，並以蘇木素-伊紅染色 (hematoxylin and eosin stain, H&E stain)發現肌小管直徑顯著地減縮，且其訊號上游之FoxO1、FoxO3a、Akt磷酸化蛋白表現皆受三氧化二砷抑制。此外，亦發現三氧化二砷所誘發之肌肉萎縮是經由抑制骨塑型蛋白 (bone morphogenetic protein, BMP)訊號途徑 (BMP2、BMP7、BMPR1A、BMPR2、p-Smad1/5/9 and Smad4)而使Akt磷酸化蛋白下降所導致。除此之外，免疫沉澱法分析 (immunoprecipitation, IP)顯示三氧化二砷藉由干擾磷酸化Smad1/5/9和Smad4的結合，並促進BMP2和Noggin結合達到抑制BMP訊號傳導的結果。最後，實驗發現處理Akt活化劑可明顯抑制三氧化二砷造成之肌小管萎縮。綜合上述，本實驗證實三氧化二砷為誘發肌肉萎縮之危險因子，並可藉由抑制BMP/p-Smad1/5/9/Akt此訊號傳遞途徑誘發Atrogin-1、MuRF1的表現，最終導致肌肉萎縮。

Arsenic (As) is a widely distributed poisonous metalloid in the environment and is classified as a Class I carcinogen by International Agency for Research on Cancer (IARC). Arsenic trioxide (As2O3), one of the most toxic forms of inorganic As, is most representative and well studied. Chronic exposure to groundwater containing inorganic As is known to cause black foot disease, cancers and many diseases. Besides, As also has been found to be associated with the low-birth-weight infants and the impairment of muscle regenerative capacity in areas with high levels of As in drinking water. Clinically, Arsenic trioxide (As2O3) is used as an effective salvage therapy for acute promyelocytic leukemia (APL), but it has some side-effects, such as polyneuropathy and distal muscular atrophy. All of these studies indicate that As2O3 has muscle toxicity. Recent studies also have found that As2O3 inhibits myogenic differentiation and muscle regeneration. Therefore, the aim of the present study is to investigate the action and molecular mechanism of As2O3 on muscle atrophy in vivo and in vitro. In this study, we used a sciatic nerve denervation model to avoid the neural interference caused by As2O3. And the mice were exposed to drinking water containing 0.05 or 0.5 ppm As2O3 for 4 weeks. First, we tested the muscle endurance by rota-rod, and the results showed that combined denervation and As2O3 exposure significantly shorten the time on the rota-rod and caused muscle fatigue. Besides, the muscle weighs of the lower limb (soleus, tibialis anterior and gastrocnemius muscles) as well as the cross-sectional area of these muscles were significantly decreased. Also, As2O3 induced the expression of Atrogin-1 and Noggin in muscle tissues by immunohistochemistry (IHC). On the other hand, to investigate the action and molecular mechanism of As2O3 on muscle atrophy induction, we treated C2C12 myotubes with As2O3 (0.25-1 μM) for 48 hours. The protein expressions of atrogenes (Atrogin-1, MuRF1) were significantly induced by As2O3. As2O3 notably reduced the myotube diameters by hematoxylin and eosin stain (H&E stain). And the upstream proteins of atrogenes (p-FoxO1, p-FoxO3a, p-Akt) were also inhibited by As2O3. Furthermore, As2O3 decreased the bone morphogenetic protein (BMP) signaling pathway (BMP2, BMP7, BMPR1A, BMPR2, p-Smad1/5/9 and Smad4) to inhibit the expression of phosphorylated Akt. Using immunoprecipitation (IP), As2O3 interfered with protein interaction between p-Smad1/5/9 and Smad4, and promote the binding between BMP2 and Noggin. Finally, we found that Akt activator can reverse the As2O3 induced muscle atrophy. Taken together, these results suggested that As2O3 is a potential risk factor for skeletal muscle atrophy and dysfunction and the putative mechanism of As2O3 induced muscle atrophy is through BMP/p-Smad1/5/9/Akt signaling pathway.