白術(shù)內(nèi)酯1對(duì)TNF-α誘導(dǎo)的C2C12骨骼肌細(xì)胞凋亡及自噬的抑制作用
發(fā)布時(shí)間：2018-07-27 15:54
【摘要】：研究背景骨骼肌由肌細(xì)胞組成,約占人體總質(zhì)量的50%,人體通過(guò)骨骼肌的收縮來(lái)完成各種活動(dòng)。骨骼肌萎縮是指骨骼肌的質(zhì)量和功能(力量和性能)的喪失,生理狀態(tài)下如年老等可出現(xiàn)骨骼肌萎縮的狀況,病理狀態(tài)下各種慢性消耗性疾病如慢性腎功能衰竭、慢性心力衰竭、慢性阻塞性肺病、白血病、癌癥、糖尿病、艾滋病、自身免疫性疾病等都可導(dǎo)致骨骼肌萎縮。研究發(fā)現(xiàn)炎癥因子如腫瘤壞死因子-α(TNF-α)等在骨骼肌的萎縮過(guò)程中發(fā)揮重要作用,它可影響骨骼肌肌細(xì)胞的生長(zhǎng)、分化及壞死等,以往研究認(rèn)為TNF-α水平的病理性升高與骨骼肌萎縮密切相關(guān),其可以抑制肌細(xì)胞的生長(zhǎng)、增殖、分化,且誘導(dǎo)肌細(xì)胞凋亡和自噬。凋亡是細(xì)胞為維護(hù)細(xì)胞內(nèi)環(huán)境的穩(wěn)定而發(fā)生的自主有序的死亡。細(xì)胞凋亡的途徑主要包括線粒體途徑、死亡受體介導(dǎo)的凋亡途徑和內(nèi)質(zhì)網(wǎng)應(yīng)激途徑。細(xì)胞凋亡能夠去除體內(nèi)多余或異常的細(xì)胞,對(duì)維持機(jī)體內(nèi)環(huán)境的穩(wěn)定及生長(zhǎng)發(fā)育起著重要作用,但研究證實(shí)凋亡過(guò)度會(huì)導(dǎo)致機(jī)體內(nèi)環(huán)境紊亂,出現(xiàn)肌肉萎縮。研究證實(shí)自噬過(guò)度與骨骼肌萎縮密切相關(guān),在骨骼肌組織中,自噬是肌蛋白分解代謝的首要途徑,它降解細(xì)胞內(nèi)儲(chǔ)存的蛋白質(zhì),直接誘導(dǎo)骨骼肌蛋白質(zhì)分解代謝,自噬還可反作用于Akt/mTOR信號(hào)影響合成代謝,這些過(guò)程均與骨骼肌萎縮相關(guān)。因此,預(yù)防和延緩骨骼肌細(xì)胞凋亡和自噬是治療肌萎縮相關(guān)性疾病的關(guān)鍵。白術(shù)內(nèi)酯1(Atractylenolidel,ATL-1)是從中藥白術(shù)中提取獲得的揮發(fā)油成份,它具有明顯的延緩衰老、防止老年癡呆、抗腫瘤、抗氧化、抗炎和調(diào)節(jié)免疫功效,但其在骨骼肌細(xì)胞凋亡和自噬方面的研究目前尚無(wú)報(bào)道,本研究旨在探究白術(shù)內(nèi)酯1對(duì)TNF-α誘導(dǎo)的C2C12小鼠骨骼肌細(xì)胞的凋亡和自噬的抑制作用及其潛在機(jī)制,為營(yíng)養(yǎng)不良導(dǎo)致的骨骼肌萎縮相關(guān)性疾病的治療提供新的理論依據(jù)。目的:本課題旨在探索白術(shù)內(nèi)酯1對(duì)TNF-α誘導(dǎo)的C2C12小鼠骨骼肌細(xì)胞凋亡及自噬的抑制作用及其潛在機(jī)制。方法:通過(guò)CCK-8法檢測(cè)白術(shù)內(nèi)酯1對(duì)C2C12小鼠骨骼肌細(xì)胞毒性以確定合適的藥物干預(yù)濃度,分組干預(yù)(正常對(duì)照組、DMSO對(duì)照組、20ng/mITNF-α模型組、TNF-α+白術(shù)內(nèi)酯1不同劑量組(10、20、30μg/mL))后,分別用AnnexinV/PI法和Hoechst33342染色法評(píng)估細(xì)胞凋亡率,流式細(xì)胞術(shù)檢測(cè)凋亡細(xì)胞,細(xì)胞活性氧檢測(cè)試劑盒檢測(cè)細(xì)胞活性氧水平,共聚焦熒光顯微鏡檢測(cè)細(xì)胞凋亡、自噬情況,免疫印跡法檢測(cè)C2C12凋亡及自噬相關(guān)蛋白水平的表達(dá)。結(jié)果:1.CCK-8法檢測(cè)結(jié)果提示濃度為40μg/mL的白術(shù)內(nèi)酯1明顯抑制C2C12骨骼肌細(xì)胞生長(zhǎng),差異有統(tǒng)計(jì)學(xué)意義(P0.05),濃度在3(μg/mL以下的白術(shù)內(nèi)酯1對(duì)細(xì)胞活力影響不明顯。2.與DMSO對(duì)照組相比,TNF-α模型組細(xì)胞活性氧水平顯著升高,白術(shù)內(nèi)酯1不同劑量(10、20、30μg/mL))干預(yù)組細(xì)胞活性氧水平顯著降低,差異具有統(tǒng)計(jì)學(xué)意義(P0.05)。3.白術(shù)內(nèi)酯1通過(guò)抑制ASK1-JNK/p38信號(hào)通路抑制TNF-α誘導(dǎo)的C2C12小鼠成肌細(xì)胞凋亡。4.白術(shù)內(nèi)酯1通過(guò)激活A(yù)kt信號(hào)通路抑制TNF-α誘導(dǎo)的C2C12小鼠成肌細(xì)胞自噬。結(jié)論:白術(shù)內(nèi)酯1通過(guò)抑制ASK1-JNK/p38信號(hào)通路、激活A(yù)kt信號(hào)通路,抑制抑制TNF-α誘導(dǎo)的C2C12小鼠骨骼肌細(xì)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.
【學(xué)位授予單位】：南方醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R692.5
本文編號(hào)：2148333
[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.
【學(xué)位授予單位】：南方醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R692.5
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