探討肌肉萎縮及失能之危險因子：以苯并芘與高糖化終產物為例

Abstract

隨著全球健康意識日益提升，生理及疾病狀態所衍生之骨骼肌損傷危險因子亦受到重視，例如老化、肥胖、糖尿病等慢性生理及疾病變化進展所誘發之肌肉減少亦或經環境危險因子暴露誘發肌肉生長遲滯之情形。骨骼肌的減損會弱化骨骼肌之生理功能，導致肌肉無力、疲乏，甚者進而無法行動，而肌肉生長遲滯則可能導致低出生體重之早產兒。目前已知孕婦若暴露於香菸中多環芳香烴物質，如苯并芘 (benzo(a)pyrene, BaP)，則會產生肌肉生長遲滯之低出生體重早產兒。此外，患有老化肌肉衰減症(sarcopenia)與許多肌肉耗損性疾病之肌纖維萎縮為最顯著的病理組織現象。在老化與糖尿病進程中，體內形成高糖化終產物(advanced glycation end products, AGEs)的量會明顯增加並被堆積起來，且AGEs的堆積已被證實與糖尿病併發症和年齡相關性疾病有關；另外，亦有研究指出糖尿病狀態與年長者的肌肉組織會大量增加AGEs的形成與堆積。然而，目前對於BaP與AGEs是否為於肌肉萎縮與功能失調中扮演重要角色則仍待釐清。本論文首先利用人類骨骼肌前驅細胞 (human skeletal muscle progenitor cells, HSMPCs)，探討BaP與其代謝物(benzo(a)pyrene-7,8-dihydrodiol-9,10-epoxide, BPDE)是否影響HSMPCs之分化及其作用機轉。另者，本論文亦探討糖尿病狀態下AGEs在肌肉萎縮與失能中所扮演的角色，並找尋將來可以用於臨床肌肉萎縮及失能之治療方向。首先，實驗結果顯示BaP與BPDE皆會抑制HSMPCs之分化，並正向調控磷酸化neuclear factor (NF)-κB的表現與負向調控肌肉特定分化蛋白(myogenin及myosin heavy chain)及磷酸化Akt的表現。給予芳香烴受體(aryl hydrocarbon receptor, AhR)抑制劑、雌激素受體(estrogen receptor, ER)抑制劑及NF-κB抑制劑能夠有效回復由BaP與BPDE所引發抑制分化相關的訊息傳遞。然而，當停止持續暴露BaP及BPDE時，則僅BaP具回復性作用，而BPDE則反之。另一方面，本篇研究發現糖尿病患者較一般正常人呈現大量AGEs累積於肌肉組織，並且肌肉萎縮相關蛋白表現(Atrogin-1與AMP-activated protein kinase (AMPK))亦大量表現於其中。本篇研究亦發現以streptozotocin誘導之糖尿病小鼠會增加血液與肌肉中AGEs含量，並造成肌肉含量衰減、肌肉萎縮、肌肉再生能力弱化及增加肌肉萎縮與失能相關蛋白表現(RAGE、Atrogin-1與AMPK)。給予AGEs阻斷劑 (alagebrium chloride, Ala-Cl)可有效緩減糖尿病小鼠所引發肌肉功能受損現象及其相關蛋白表現。此外，細胞研究結果顯示AGEs可促發小鼠肌母細胞(mouse myoblasts)與HSMPCs之肌小管萎縮及抑制其肌肉分化。同時，AGEs可藉由RAGE正向調控AMPK，進而負向調控Akt之訊息傳遞，導致肌小管萎縮與肌肉分化抑制。綜合上述，本論文研究成果證實BaP與AGEs皆為肌肉萎縮與失能之潛在危險因子，結果顯示BaP與其代謝物BPDE可藉由AhR或ER調控NF-κB/Akt 訊息傳遞途徑，進而抑制肌肉分化；另一方面，人類、動物及細胞研究成果亦證實AGEs可經RAGE正向調控AMPK，進而負向調控Akt之訊息傳遞，導致肌肉萎縮與失能，並提供Ala-Cl可做為糖尿病或老年引發肌肉萎縮與失能之治療用藥之新選擇。未來仍需進一步探討BaP與BPDE如何造成低出生體重早產兒之詳細機轉，而亦須進一步探尋老年與糖尿病所造成肌肉萎縮與失能之臨床有效與專一性之診斷生物指標。

There is a growing global health awareness of the risk factors of skeletal muscle in many physiological and disease processes, including skeletal muscle mass declines with aging, obesity, diabetes and other chronic diseases, and in environmental risk factors-inducing muscle growth retardation. Loss of skeletal muscle has been related to the frailty of skeletal muscle function by weakness, fatigue and disability, and muscle growth retardation may result in low birth weight infants. Exposure to cigarette smoke or its polycyclic aromatic hydrocarbons component (eg, benzo(a)pyrene, BaP) during pregnancy has been to give birth to low birth weight and lower peripheral muscle area neonates. Muscle fibres atrophy has been reported as an obvious pathohistological phenomenon of people with sarcopenia and muscle wasting diseases. The aging and diabetic process has been ultimately associated with elevated advanced glycation end products (AGEs) presence contributing to chronic diabetic complications and age-related diseases. Accumulation AGEs in the skeletal muscle tissue has been also increasingly identified in diabetic animals as well as in elderly human subjects. However, it still needs to be certified BaP and AGEs play important roles in muscle atrophy and dysfunction. Therefore, the first aim of this study is to investigate the action and cellular mechanism of BaP and its metabolite (benzo(a)pyrene-7,8-dihydrodiol-9,10-epoxide, BPDE) on myogenic differentiation in HSMPCs. The other aim of this study is to characterize the action and molecular mechanism of AGEs-induced skeletal muscle atrophy and regeneration impairment and find the therapeutic potential for muscle atrophy and dysfunction in the clinic. First, both BaP and BPDE inhibited myogenic differentiation and showed upregulation of phosphorylated nuclear factor (NF)-κB and downregulation of the muscle-specific protein expressions (myogenin and myosin heavy chain) and phosphorylation of Akt, which could be significantly reversed by the inhibitors for aryl hydrocarbon receptor (AhR), estrogen receptor (ER), and NF-κB. The inhibitory effects of BaP and BPDE on myogenesis were reversed after withdrawing BaP exposure, but not after BPDE withdrawal. On the other hand, the muscle fiber atrophy and immunoreactivities for AGEs, Atrogin-1 (a muscle atrophy marker), and phosphorylated AMP-activated protein kinase (AMPK) expressions were markedly increased in the human skeletal muscles from diabetic patient as compared with the normal human subject. Moreover, there were increased blood AGEs, less muscle mass, lower muscular endurance, atrophic muscle size, poor regenerative capacity, and increased expressions of muscle AGEs, receptor for AGE (RAGE), Atrogin-1, and phosphorylated AMPK in streptozotocin-induced diabetic mice. AGEs cross-link breaker alagebrium chloride (Ala-Cl) significantly ameliorated the muscular malfunction observed in diabetic mice. Furthermore, AGEs also induced atrophic myotubes and inhibited myogenesis in mouse myoblasts and HSMPCs. We further demonstrated that AGEs induced muscle atrophy/myogenesis impairment via a RAGE-mediated AMPK-down-regulated Akt pathway. Taken together, the present study has been demonstrated BaP and AGEs are the potential risk factor for muscle atrophy and dysfunction. These results suggest that both BaP and BPDE are capable of inhibiting myogenesis via an AhR or/and ER-regulated NF-κB/Akt signaling pathway. On the other hand, these findings suggest that AGEs elicit skeletal muscle atrophy and regeneration impairment in the models of the models of the patient sample, diabetic mice and cultured muscle cells, and Ala-Cl may be a new therapeutic strategy for management of diabetic or aging muscle atrophy and dysfunction. However, the causation of BaP/BPDE-related low birth weight needs to be clarified in the future. We also need to explore specific and efficient biomarkers for clinical diagnosis of aging- and diabetes-induced skeletal muscle atrophy and dysfunction.