三氧化二砷對於骨骼肌細胞的增生、分化及再生之影響

Abstract

砷是在環境中發現的毒性類金屬物質，主要以無機和有機的形式存在於自然界。人類暴露到無機砷的主要來源是飲水中的高濃度砷和工業污染。在水源中，砷主要是以無機的形式存在。根據之前的研究發現，當母親飲水中含較高砷濃度會增加新生兒低體重的發生率。骨骼肌母細胞增生和分化在胚胎之肌肉發育及成體的肌肉新生和維持中扮演重要的角色。但到目前為止，無機砷對於肌肉生長和分化的影響尚未被探討。因此，我們假設砷導致新生兒低體重可能是經由影響肌肉的生長或分化所造成的。 本研究分為兩個部份，第一部分探討砷對於骨骼肌母細胞增生和毒性的影響。根據實驗結果，我們發現處理3和10 μM三氧化二砷 (arsenic trioxide)24小時後，小鼠肌母細胞株C2C12存活率會顯著下降；而處理0.25-1 μM三氧化二砷72小時後，細胞增生也顯著降低。由BrdU實驗發現，0.5和1 μM 三氧化二砷能夠顯著抑制C2C12細胞株DNA的合成，且細胞核中PCNA蛋白表現也明顯下降。利用流式細胞儀分析細胞週期的實驗結果指出，三氧化二砷會讓細胞週期停滯在G1和G2/M。在觀察調控細胞週期的蛋白中結果發現，cyclin D1、cyclin B1、CDK2和CDK4會顯著降低。此外，處理C2C12肌母細胞3和10 μM 三氧化二砷24小時後，會顯著增加細胞凋亡 (apoptosis)以及PARP和caspase-3蛋白。流式細胞儀分析結果顯示，三氧化二砷會增加C2C12細胞活性氧化物 (reactive oxygen species; ROS)的產生。使用抗氧化劑NAC會顯著降低三氧化二砷所造成的活性氧化物和細胞凋亡。本部份證明了無機砷在肌母細胞生長中扮演負向的角色，其主要機制是為在低濃度主要經由抑制細胞週期而抑制增生；在高濃度則經由ROS所引起細胞凋亡。 在第二部份研究中，我們也進一步探討砷對於肌母細胞的分化 (myogenic differentiation)和對動物體肌肉再生之影響。在體外實驗，我們採用C2C12細胞株以及人類和小鼠初代培養的肌母細胞，經過處理分化培養液並同時加入三氧化二砷四天後，評估肌肉分化指標蛋白: myogenin和myosin heavy chain以及多核肌管 (multinucleated myotube)的形成。在動物實驗，我們使用甘油造成小鼠肌肉壞死-新生模式來觀察砷對於骨骼肌新生的影響。根據實驗結果發現，低於毫莫爾濃度 (submicromolar)的三氧化二砷可抑制肌母細胞的分化，且具有劑量效應關係。在分化過程中，三氧化二砷顯著的抑制Akt和p70s6k蛋白磷酸化，並也存在著劑量效應關係。我們轉殖持續活化的Akt (constitutively active form of Akt )到C2C12細胞，三氧化二砷抑制肌肉分化的蛋白和肌管的形成可以被逆轉。在動物實驗方面，ICR小鼠局部注射甘油後，由肌肉的切片觀察到典型的肌肉傷害和再生反應。在給予小鼠飲水砷的組別，再生的肌肉中myogenin的表現和Akt的磷酸化被顯著抑制。因此我們的結果證明三氧化二砷抑制肌肉的分化主要是經由抑制Akt相關的訊息傳遞所導致。 綜合這些結果猜測三氧化二砷能夠抑制骨骼肌細胞增生、分化和延遲肌肉再生。此外，我們的結果提供新的証據顯示: 三氧化二砷可能影響肌肉新生並且指出發育中的肌肉對於砷的污染是相當敏感的。

Arsenic is a naturally occurring toxic metalloid found in the environment in both inorganic and organic forms. Human exposure to arsenic occurs primarily through the consumption of contaminated drinking water and occupational exposures. The incidence of low birth weights is increased in offspring of women who are exposed to high concentrations of arsenic in drinking water compared with other women. Both the myoblast cell proliferation and differentiation are essential for embryonic development of muscle and muscle regeneration in adult. However, the effects of inorganic arsenic on myoblast growth and differentiation have not been investigated. Here, we hypothesized that effects of arsenic on birth weight may be related to effects on myogenic proliferation and/or differentiation. In the first part of this study, we investigated the in vitro effect of As2O3 on C2C12 myoblast growth and toxicity. We found that the myoblast cell growth was slightly but significantly decreased by 0.25-1 μM As2O3 after 72 h treatment. In addition, the cell viability was markedly decreased by 3 and 10 μM As2O3 after 24 h treatment. Low-concentration As2O3 (0.5 and 1 μM) suppressed cell proliferative activity, showing a low proportion of bromodeoxyuridine (BrdU) incorporation, indicating a low DNA synthesis rate. The expression of proliferating cell nuclear antigen (PCNA) protein was also significantly decreased in low-concentration As2O3-treated C2C12 myoblasts. Cell cycle analysis indicated that low-concentration As2O3 inhibits cell proliferation via the cell cycle arrest at G1 and G2/M phases. The expressions of cyclin D1, cyclin B1, CDK2, and CDK4 were significantly decreased in As2O3-treated C2C12 myoblasts. On the other hand, C2C12 myoblasts treated with high-concentration As2O3 (3 and 10 μM) for 24 h showed the induction of apoptosis and the cleavages of caspase-3 and poly (ADP-ribose) polymerase (PARP) proteins. High-concentration As2O3 could also induce the intracellular reactive oxygen species (ROS) generation in C2C12 myoblasts. Anti-oxidant N-acetyl-L-cysteine (NAC) effectively reversed the As2O3-induced ROS generation and apoptosis. These results indicate that As2O3 possesses a negative influence on myoblast growth via the suppression of proliferation at low concentration levels and the induction of apoptosis at high concentration levels. In the second part of this study, we investigated the effects of As2O3 on the myogenic differentiation of myoblasts in vitro and muscle regeneration in vivo. C2C12 myoblasts and primary mouse and human myoblasts were cultured in differentiation medium with or without As2O3 (0.1–0.5 μM) for 4 days. Myogenic differentiation was assessed by myogenin and myosin heavy chain expression and multinucleated myotube formation in vitro; skeletal muscle regeneration was tested using an in vivo mouse model with experimental glycerol myopathy. A submicromolar concentration of As2O3 dose-dependently inhibited myogenic differentiation without apparent effects on cell viability. We next demonstrated that As2O3 significantly and dose-dependently decreased phosphorylation of Akt and p70s6k proteins during myogenic differentiation. As2O3-induced inhibition in myotube formation and muscle-specific protein expression was reversed by transfection with the constitutively active form of Akt. Sections of soleus muscles stained with hematoxylin and eosin showed typical changes of injury and regeneration after local glycerol injection in mice. Regeneration of glycerol-injured soleus muscles, myogenin expression, and Akt phosphorylation were suppressed in muscles isolated from As2O3-treated mice compared with untreated mice. In addition, our results suggest that As2O3 inhibits myogenic differentiation by inhibiting Akt-regulated signaling. Taken together, these results suggest that As2O3 is capable of inhibiting skeletal muscle cells proliferation, differentiation and retarding muscle regeneration. In addition, our results provide the novel evidence that As2O3 may affect myogenesis and point out that developing muscle is a sensitive target of this contaminant.