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標題: | 細胞自噬在硫酸吲哚酚誘發尿毒肌少症的角色強調細胞機轉 The Roles of Autophagy in Indoxyl Sulfate-induced Uremic Sarcopenia Emphasize in Cellular Mechanisms |
作者: | Ting-wei Mai 麥庭威 |
指導教授: | 姜至剛(Chih- Kang Chiang) |
關鍵字: | 硫酸??酚,慢性腎臟病,肌少症,骨骼肌,分化,細胞自噬,粒線體,粒線體自噬, Chronic kidney disease,Sarcopenia,Indoxyl sulfate,Differentiation,autophagy,Mitophagy,Mitochondria, |
出版年 : | 2018 |
學位: | 碩士 |
摘要: | 尿毒肌少症(Uremic sarcopenia)是常見於慢性腎臟病(Chronic kidney disease, CKD)患者的肌肉耗損症狀,經研究證實會增加CKD患者的死亡率。因此,發展治療尿毒症肌少症的方法顯得格外重要。然而,目前對於CKD造成肌少症的致病分生機制仍未明確。先前研究顯示,硫酸吲哚酚(Indoxyl sulfate, IS)為一種親蛋白質尿毒素,會抑制骨骼肌細胞分化。研究指出IS會使肌肉細胞的粒線體功能受損,並藉由增加氧化壓力改變細胞的代謝進而使骨骼肌細胞萎縮,最終造成肌少症。近期研究證實,骨骼肌細胞在分化初期會活化細胞自噬作用,在分化後期則會回復至一般生理狀態。然而,細胞自噬受損是否為尿毒症肌少症的致病機轉仍不清楚。在本篇研究中,我們推測尿毒素可能藉由損害細胞自噬而抑制肌肉生成。為了證實此假設並闡明其中的機制作用,我們使用IS處理小鼠肌纖維母細胞(C2C12 myoblast),探討其對於分化過程中細胞自噬的影響。本研究主要利用西方點墨法(Western Blot)和即時定量聚合酶連鎖反應(Real-time Quantitative PCR)分析與分化、細胞自噬及粒線體功能相關的指標。研究結果顯示,透過LC3 turnover assay可證實,在C2C12分化過程中IS的處理會誘發完整的細胞自噬作用。基於初步的結果,我們進而探討IS誘導的細胞自噬是否會進一步影響到粒線體自噬、粒線體功能及粒線體動態平衡。結果顯示C2C12粒線體內的粒線體自噬指標p62表現量在IS處理後會上升,粒線體功能指標PGC-1α、TOM70以及粒線體動態平衡指標OPA1的表現量則會下降。綜合上述,本研究指出IS的暴露可能會造成細胞自噬失調而干擾骨骼肌細胞的分化,且IS造成的粒線體功能與粒線體動態平衡損害,可能與分化過程中誘發的粒線體自噬有關。然而,未來仍需要更多研究去釐清IS誘導之細胞自噬與粒線體自噬的詳細分子機轉。 Uremic sarcopenia is a muscle wasting syndrome that often observed in chronic kidney disease (CKD) patients, which is associated with increased mortality. Therefore, developing therapeutic strategy for treating CKD-associated sarcopenia is important. However, the underlying molecular mechanism of CKD-associated sarcopenia is still poorly understood. Our previous study demonstrated that indoxyl sulfate (IS), one of the protein-bound uremic toxins, dysregulates myogenic differentiation. Other studies also showed that IS strongly accelerates skeletal muscle atrophy through oxidative stress-mediated metabolism alteration, which in turn adversely contribute to sarcopenia. It has been demonstrated that IS also induces mitochondria dysfunction in skeletal muscle cells. Currently, physiologic activation of autophagy has been reported in skeletal muscles. However, whether impaired autophagy involved in uremic sarcopenia remains unclear. In this study, we hypothesized that uremic toxins may exert its anti-myogenesis by impaired autophagy. To verify this hypothesis and elucidate the mechanism, we examined the effects of IS on skeletal muscle differentiation in mouse C2C12 myoblast cells with a focus on alterations in autophagic process. The expression of myogenic differentiation markers, autophagy-related markers, mitochondria -related markers were determined by western blotting and quantitative PCR. Our results indicated that IS treatment induces a complete autophagy as demonstrated by LC3 turnover assay. Based on the preliminary data, we further explore whether the IS-induced autophagy interferes the mitophagy, mitochondrial functions and dynamic. We observed that the expression of p62 levels, which is a mitophagy marker, were increased in the mitochondria extracts from IS-treated C2C12 cells, which accompanied with the decrease of the levels of mitochondrial function markers, PGC-1α, TOM70 and dynamic marker, OPA1. In conclusion, the present study indicated that IS exposure might interfere with myoblast differentiation by dysregulation of autophagy. And the exposure of IS might impair the mitochondrial functions and dynamics, which associated with the induction of mitophagy in C2C12 differentiation. However, further experiments are indicated to clarify the molecular mechanisms, which are responsible for IS-induced autophagy and mitophagy. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/70229 |
DOI: | 10.6342/NTU201803584 |
全文授權: | 有償授權 |
顯示於系所單位: | 毒理學研究所 |
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