虾青素通过AMPK/mTOR信号通路促进鸡肌肉干细胞增殖与分化  

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

作　　者：段子朋[1,2] 孙缦利 陈彦锋[1,2] 邓同兴 金少举[1,2] 范文娟 陈旭东[1,2] DUAN Zi-peng;SUN Man-li;CHEN Yan-feng;DENG Tong-xing;JIN Shao-ju;FAN Wen-juan;CHEN Xu-dong(Luohe Medical College,Luohe 462002;Henan Provincial Nutrition and Health Engineering Research Center,Luohe 462002)

机构地区：[1]漯河医学高等专科学校,漯河462002 [2]河南省营养与健康工程研究中心,漯河462002

出　　处：《生物技术通报》2024年第11期312-320,共9页Biotechnology Bulletin

基　　金：河南省科技厅科技攻关项目(212102310896,232102310501);河南省自然科学基金项目(222300420246);漯河医专博士后科研项目(PR20210001)。

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、5μmol/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、1.25μmol/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1.25μmol/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,1.25μmol/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,1.25μmol/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。【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 5μmol/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 1.25μmol/L AST treatments significantly increased cell activity and reduced necrotic cells(P<0.01),with the 1.25μmol/L AST treatment exhibiting the most significant effect on cell proliferation(P<0.01).Immunofluorescence staining of Titin and MyoD revealed that treatment with 1.25μmol/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 1.25μmol/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 ChMuSCs by inhibiting mTOR.

关 键 词：鸡 肌肉干细胞 虾青素 细胞增殖 细胞分化 

分 类 号：S831.5[农业科学—畜牧学]