Astaxanthin Promotes the Proliferation and Differentiation of Chicken Muscle Stem Cells via AMPK/mTOR Signaling Pathway

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

Abstract

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

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.

Figures/Tables 5

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

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

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

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

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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