虾青素通过AMPK/mTOR信号通路促进鸡肌肉干细胞增殖与分化

doi:10.13560/j.cnki.biotech.bull.1985.2024-0228

摘要/Abstract

摘要：

【目的】 研究虾青素（astaxanthin，AST）对鸡肌肉干细胞（chicken muscle stem cells，Ch-MuSCs）增殖的影响及其潜在机制。【方法】 原代分离Ch-MuSCs，不同浓度AST（0、0.3、0.6、1.25、2.5、5 μmol/L）处理，利用7-AAD联合Calcein AM染色法、MTT法、EdU法等检测细胞活力与增殖，免疫荧光染色检测肌管发育变化，Western blot分析AMPK/mTOR信号通路关键蛋白PI3k、AKT、p-PI3k、p-AKT以及mTOR、AMPK、p-mTOR、p-AMPK的蛋白表达。【结果】 0.6、1.25 μmol/L AST处理后均可显著增加细胞活性，减少坏死细胞（P < 0.01），其中1.25 μmol/L AST处理后细胞增殖最为显著（P < 0.01）；Titin与MyoD免疫荧光染色显示，1.25 μmol/L AST处理后Titin阳性肌管长度增加显著（P < 0.05），且每条肌管内MyoD阳性细胞核增多（P < 0.05）。Western blot结果显示，1.25 μmol/L AST处理组p-PI3K和p-AKT水平显著升高（P < 0.05），AMPK及P-AMPK蛋白增加，mTOR蛋白表达水平降低（P < 0.05），加入mTOR抑制剂雷帕霉素后，AMPK、mTOR蛋白表达量显著降低（P < 0.05），但p-AMPK蛋白表达水平无明显变化；若同时采用AST处理，则AMPK及P-AMPK蛋白表达水平显著增加（P < 0.05），但mTOR蛋白及p-mTOR表达水平显著降低（P < 0.05）。【结论】 AST可通过激活AMPK/mTOR信号通路中AMPK及其上游信号分子PI3K与Akt促进Ch-MuSCs的增殖与分化，通过抑制mTOR来调控Ch-MuSCs的生长速度。

关键词: 鸡, 肌肉干细胞, 虾青素, 细胞增殖, 细胞分化

Abstract:

【Objective】 To study the effect of astaxanthin（AST）on the proliferation and differentiation of chicken muscle stem cells（Ch-MuSCs）and its potential mechanism. 【Method】 Primary Ch-MuSCs were isolated and treated with different concentrations of AST（0, 0.3, 0.6, 1.25, 2.5, and 5 μmol/L）for 24 h. Cell viability and proliferation were analyzed using 7-AAD and Calcein AM staining, MTT assay and EdU assay. Myotube development was evaluated through immunofluorescence staining. Western blot analysis was performed to examine the expression of key proteins in the AMPK/mTOR signaling pathway, including PI3k, AKT, phosphorylated PI3k, phosphorylated AKT, mTOR, AMPK, phosphorylated mTOR, and phosphorylated AMPK. 【Result】 Both 0.6 and 1.25 μmol/L AST treatments significantly increased cell activity and reduced necrotic cells（P < 0.01）, with the 1.25 μmol/L AST treatment exhibiting the most significant effect on cell proliferation（P < 0.01）. Immunofluorescence staining of Titin and MyoD revealed that treatment with 1.25 μmol/L AST significantly increased the length of Titin-positive muscle tubes（P < 0.05）and the number of MyoD-positive nuclei per muscle tube（P < 0.05）. Western blot demonstrated that the levels of p-PI3K and p-AKT were significantly elevated in the 1.25 μmol/L AST treatment group（P < 0.05）. The expressions of AMPK and p-AMPK proteins increased, while the expression of mTOR protein decreased（P < 0.05）. After adding the mTOR-selective rapamycin, the expressions of AMPK protein and mTOR protein significantly decreased（P < 0.05）, but the expression of p-AMPK protein did not change significantly. If AST was used simultaneously, it increased the expressions of AMPK and P-AMPK proteins（P < 0.05）, but the expressions of mTOR protein and p-mTOR significantly decreased（P < 0.05）. 【Conclusion】 AST can promote the proliferation and differentiation of Ch-MuSCs by activating AMPK and its upstream signaling molecules PI3K and Akt in the AMPK/mTOR signaling pathway, and regulate the growth rate of Ch-MuSCs by inhibiting mTOR.

Key words: chicken, muscle stem cells, astaxanthin, cell proliferation, cell differentiation

段子朋, 孙缦利, 陈彦锋, 邓同兴, 金少举, 范文娟, 陈旭东. 虾青素通过AMPK/mTOR信号通路促进鸡肌肉干细胞增殖与分化[J]. 生物技术通报, 2024, 40(11): 312-320.

DUAN Zi-peng, SUN Man-li, CHEN Yan-feng, DENG Tong-xing, JIN Shao-ju, FAN Wen-juan, CHEN Xu-dong. Astaxanthin Promotes the Proliferation and Differentiation of Chicken Muscle Stem Cells via AMPK/mTOR Signaling Pathway[J]. Biotechnology Bulletin, 2024, 40(11): 312-320.

图/表 5

图1 不同浓度AST对Ch-MuSCs细胞活性与细胞活力的影响 A: Ch-MuSCs经不同浓度AST处理48 h后，荧光显微镜下活细胞被Calcein AM染色成绿色荧光，坏死细胞的细胞核被7-AAD染色成红色荧光。标尺=100 μm；B：AST处理48 h后，各组Calcein AM阳性细胞的相对百分比；C：AST处理48 h后，各组7-AAD染色阳性细胞的相对百分比。与对照组（0 μmol/L）相比，*P < 0.05，# P <0.01，## P <0.001，ns表示无统计学意义，下同

Fig. 1 Effects of different concentrations of AST on Ch-MuSCs cell activity and cell viability A: Under fluorescence microscope, live cells were stained with Calcein AM（green）and dead cells were stained with 7-AAD（red）. Scale =100 μm. B: Relative percentage of Calcein AM positive cells in Ch-MuSCs after treatment with AST for 48 h. C: Relative percentage of 7-AAD-stained positive cells in Ch-MuSCs after treatment with AST for 48 h. Compared with control（0）, *P < 0.05，# P <0.01，## P <0.001, ns indicates not statistically significant, the same below

图2 不同浓度AST对Ch-Muscs增殖与细胞毒性的影响 A: EdU标记（红色）表示增殖的细胞, DAPI标记细胞核；B: EdU阳性细胞的百分比；C：MTT法测定AST作用48 h后Ch-MuSCs的细胞增殖与细胞毒性

Fig. 2 Effects of different concentrations of AST on the proliferation and cytotoxicity of Ch-Muscs A: EdU-labeled（red）indicates the proliferation of cells, DAPI marks nuclei. B: Percentage of EdU positive cells. C: The cell proliferation were estimated by MTT assay with AST for 48 h

图3 AST对Ch-Muscs成肌分化能力的影响 A: Titin（红色）与MyoD（绿色）免疫荧光双标记，示分化的肌管与肌细胞核，标尺=100 μm；B：Titin阳性肌管的长度；C：每根肌管细胞核数

Fig. 3 Effect of AST on the myogenic differentiation ability of Ch-Muscs A: Titin（red）and MyoD（green）immunofluorescence double labeling, showing differentiated muscle tubes and muscle nuclei. Bar=100 μm. B: Quantitative analysis of myotube length; C: number of nuclei per muscular duct

图4 通过Western blot分析PI3K/Akt信号通路关键蛋白表达水平 A: Western blot结果图；B: AKT、p-AKT 相对蛋白表达量；C：PI3K、p-PI3K相对蛋白表达量

Fig. 4 Key protein levels in the PI3K/Akt signaling pathway measured by Western blot analysis A: Results of Western blot. B: Quantitative analysis of Western blots for AKT and p-AKT. C: Quantitative analysis of Western blots forPI3K and p-PI3K

图5 AMPK、p-AMPK、mTOR、p-mTOR的表达水平 A: Western blot结果图；B: AMPK、p-AMPK 相对蛋白表达量；C：mTOR、p-mTOR相对蛋白表达量

Fig. 5 Expressions of AMPK, p-AMPK, mTOR, and p-mTOR A: Results of Western blot. B: Quantitative analysis of Western blot for AMPK and p-AMPK. C: Quantitative analysis of Western blot for mTOR and p-mTOR

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