白术内酯1对TNF-α诱导的C2C12骨骼肌细胞凋亡及自噬的抑制作用

发布时间：2018-07-27 15:54

【摘要】：研究背景骨骼肌由肌细胞组成,约占人体总质量的50%,人体通过骨骼肌的收缩来完成各种活动。骨骼肌萎缩是指骨骼肌的质量和功能(力量和性能)的丧失,生理状态下如年老等可出现骨骼肌萎缩的状况,病理状态下各种慢性消耗性疾病如慢性肾功能衰竭、慢性心力衰竭、慢性阻塞性肺病、白血病、癌症、糖尿病、艾滋病、自身免疫性疾病等都可导致骨骼肌萎缩。研究发现炎症因子如肿瘤坏死因子-α(TNF-α)等在骨骼肌的萎缩过程中发挥重要作用,它可影响骨骼肌肌细胞的生长、分化及坏死等,以往研究认为TNF-α水平的病理性升高与骨骼肌萎缩密切相关,其可以抑制肌细胞的生长、增殖、分化,且诱导肌细胞凋亡和自噬。凋亡是细胞为维护细胞内环境的稳定而发生的自主有序的死亡。细胞凋亡的途径主要包括线粒体途径、死亡受体介导的凋亡途径和内质网应激途径。细胞凋亡能够去除体内多余或异常的细胞,对维持机体内环境的稳定及生长发育起着重要作用,但研究证实凋亡过度会导致机体内环境紊乱,出现肌肉萎缩。研究证实自噬过度与骨骼肌萎缩密切相关,在骨骼肌组织中,自噬是肌蛋白分解代谢的首要途径,它降解细胞内储存的蛋白质,直接诱导骨骼肌蛋白质分解代谢,自噬还可反作用于Akt/mTOR信号影响合成代谢,这些过程均与骨骼肌萎缩相关。因此,预防和延缓骨骼肌细胞凋亡和自噬是治疗肌萎缩相关性疾病的关键。白术内酯1(Atractylenolidel,ATL-1)是从中药白术中提取获得的挥发油成份,它具有明显的延缓衰老、防止老年痴呆、抗肿瘤、抗氧化、抗炎和调节免疫功效,但其在骨骼肌细胞凋亡和自噬方面的研究目前尚无报道,本研究旨在探究白术内酯1对TNF-α诱导的C2C12小鼠骨骼肌细胞的凋亡和自噬的抑制作用及其潜在机制,为营养不良导致的骨骼肌萎缩相关性疾病的治疗提供新的理论依据。目的:本课题旨在探索白术内酯1对TNF-α诱导的C2C12小鼠骨骼肌细胞凋亡及自噬的抑制作用及其潜在机制。方法:通过CCK-8法检测白术内酯1对C2C12小鼠骨骼肌细胞毒性以确定合适的药物干预浓度,分组干预(正常对照组、DMSO对照组、20ng/mITNF-α模型组、TNF-α+白术内酯1不同剂量组(10、20、30μg/mL))后,分别用AnnexinV/PI法和Hoechst33342染色法评估细胞凋亡率,流式细胞术检测凋亡细胞,细胞活性氧检测试剂盒检测细胞活性氧水平,共聚焦荧光显微镜检测细胞凋亡、自噬情况,免疫印迹法检测C2C12凋亡及自噬相关蛋白水平的表达。结果:1.CCK-8法检测结果提示浓度为40μg/mL的白术内酯1明显抑制C2C12骨骼肌细胞生长,差异有统计学意义(P0.05),浓度在3(μg/mL以下的白术内酯1对细胞活力影响不明显。2.与DMSO对照组相比,TNF-α模型组细胞活性氧水平显著升高,白术内酯1不同剂量(10、20、30μg/mL))干预组细胞活性氧水平显著降低,差异具有统计学意义(P0.05)。3.白术内酯1通过抑制ASK1-JNK/p38信号通路抑制TNF-α诱导的C2C12小鼠成肌细胞凋亡。4.白术内酯1通过激活Akt信号通路抑制TNF-α诱导的C2C12小鼠成肌细胞自噬。结论:白术内酯1通过抑制ASK1-JNK/p38信号通路、激活Akt信号通路,抑制抑制TNF-α诱导的C2C12小鼠骨骼肌细胞凋亡和自噬。
[Abstract]:The skeletal muscle is composed of muscle cells, which accounts for about 50% of the total mass of the human body. The human body completes various activities through the contraction of the skeletal muscle. Skeletal muscle atrophy refers to the loss of the quality and function (strength and performance) of the skeletal muscle. In the physiological state, the state of skeletal muscle atrophy can occur as a year old, and various chronic consumptive diseases in the pathological state. Such as chronic renal failure, chronic heart failure, chronic obstructive pulmonary disease, leukemia, cancer, diabetes, AIDS, autoimmune diseases and so on, can cause skeletal muscle atrophy. It is found that inflammatory factors such as tumor necrosis factor - alpha (TNF- alpha) play an important role in the atrophy of skeletal muscle, which can affect the birth of skeletal muscle myocytes. Long, differentiation and necrosis, the previous study suggests that the histopathological elevation of TNF- alpha level is closely related to skeletal muscle atrophy. It can inhibit the growth, proliferation and differentiation of muscle cells, and induce apoptosis and autophagy. Apoptosis is an autonomous and orderly death of cells in order to maintain the stability of the intracellular environment. The main pathways of cell apoptosis include the pathway of cell apoptosis. Mitochondrial pathway, death receptor mediated apoptosis pathway and endoplasmic reticulum stress pathway. Apoptosis can remove superfluous or abnormal cells in the body. It plays an important role in maintaining the stability and growth of the body environment. However, the study confirms that excessive apoptosis may lead to the internal environment disorder and muscle atrophy. The study confirmed the excessive autophagy. It is closely related to skeletal muscle atrophy. Autophagy is the primary pathway for myosin metabolism in skeletal muscle tissue. It degrades the protein stored in the cells and directly induces the protein catabolism of skeletal muscle. Autophagy can also reverse the effect of Akt/mTOR signal on the metabolism. These processes are related to skeletal muscle atrophy. Therefore, the prevention and delay are prevented and delayed. Apoptosis and autophagy of skeletal muscle cells is the key to the treatment of amyotrophic related diseases. Atractylode 1 (Atractylenolidel, ATL-1) is a volatile oil extracted from Chinese traditional Chinese medicine Atractylodes macrocephala. It has a significant delay in aging, prevention of Alzheimer's, anti-tumor, antioxidation, anti-inflammatory and immune regulation, but it is in skeletal muscle cell apoptosis and autophagy. The study has not yet been reported. The purpose of this study is to explore the inhibitory effect of Atractylodes 1 on the apoptosis and autophagy of skeletal muscle cells in C2C12 mice induced by TNF- alpha and its potential mechanism, and to provide a new theoretical basis for the treatment of skeletal muscle atrophy related diseases caused by malnutrition. Objective: the purpose of this study is to explore the 1 pairs of alba in TNF. - alpha induced C2C12 mouse skeletal muscle cell apoptosis and autophagy inhibition and its potential mechanism. Methods: CCK-8 method was used to detect the toxicity of Atractylodes 1 on C2C12 mouse skeletal muscle cells to determine the appropriate drug intervention concentration, group intervention (normal control group, DMSO control group, 20ng/mITNF- alpha model group, TNF- alpha + atractylode lactone different dosage) After the group (10,20,30 mu g/mL)), the apoptosis rate was evaluated by AnnexinV/PI and Hoechst33342 staining, the apoptotic cells were detected by flow cytometry, the activity oxygen level of cell was detected by the cell active oxygen detection kit. The apoptosis, autophagy and autophagy related protein water of C2C12 were detected by confocal fluorescence microscopy. Results: the results of 1.CCK-8 assay showed that Atractylodes 1 significantly inhibited the growth of C2C12 skeletal muscle cells with a concentration of 40 mu g/mL. The difference was statistically significant (P0.05). The concentration of Atractylodes 1 on cell viability was not significantly higher than that of the DMSO control group, and the level of reactive oxygen species in the TNF- a model group was significantly higher than that in the TNF- a model group. 1 different doses of lactone (10,20,30 mu g/mL) the level of reactive oxygen species in the cells of the intervention group decreased significantly, the difference was statistically significant (P0.05).3. Atractylodes 1 by inhibiting the ASK1-JNK/p38 signaling pathway to inhibit TNF- a induced apoptosis of the myoblast of C2C12 mice,.4. Baizhu lactone 1 inhibited TNF- alpha induced C2C12 murine myoblasts by stimulating the Akt signaling pathway Conclusion: Atractylodes 1 activates the Akt signaling pathway by inhibiting the ASK1-JNK/p38 signaling pathway and inhibits the apoptosis and autophagy of skeletal muscle cells in C2C12 mice induced by TNF- alpha.
【学位授予单位】：南方医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R692.5

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