Department of Animal and Poultry Sciences, Virginia Tech, Blacksburg, VA 24061, USA.
Department of Animal and Poultry Sciences, Virginia Tech, Blacksburg, VA 24061, USA.
Domest Anim Endocrinol. 2019 Apr;67:28-36. doi: 10.1016/j.domaniend.2018.12.006. Epub 2018 Dec 19.
Many studies have shown positive effects of prostaglandins (PGs) on various steps of skeletal muscle formation such as myoblast proliferation and myotube hypertrophy. In animals, PGs are synthesized through the action of the rate-limiting enzymes cyclooxygenase (COX) -1 and COX-2 from arachidonic acid (AA), a conditionally essential fatty acid. As a step toward exploring the possibility of using dietary supplementation of AA to improve skeletal muscle growth in cattle, which are major meat-producing animals, we determined the effects of AA and its major PG derivatives PGE, PGF, and PGI on proliferation, differentiation, and fusion of primary bovine myoblasts in vitro. In the proliferation experiment, myoblasts were cultured in a growth medium to which was added 10 μM AA, 1 μM PGE, 1 μM PGF, 1 μM PGI, or vehicle control for 24 h, and the proliferating cells were identified by 5-ethynyl-2'-deoxyuridine (EdU) labeling. This experiment revealed that AA, PGE, PGF, and PGI each increased the number of proliferating cells by 13%, 24%, 16%, and 16%, respectively, compared to the control (n = 7, P < 0.05). In the differentiation and fusion test, myoblasts were induced to differentiate and fuse into myotubes in the presence of the aforementioned treatments for 0, 24, 48, and 72 h. Based on quantitative reverse transcription PCR analyses of mRNAs of myoblast differentiation and fusion markers (myogenin; myosin heavy chain 3; creatine kinase, muscle; myomaker) at 0, 24, and 48 h of differentiation, AA, PGE, and PGF promoted myoblast differentiation (n = 6, P < 0.05). Based on Giemsa staining and counting the number of myotubes at 72 h of differentiation, PGE enhanced the number of formed myotubes by 14% (P < 0.05) compared to the control. Treating the myoblasts with AA and either the COX-1 and COX-2 common inhibitor indomethacin or the COX-2-specific inhibitor NS-398 reversed the stimulatory effect of AA on myoblast proliferation (n = 4, P < 0.05). Overall, this study demonstrates that exogenous AA stimulates bovine myoblast proliferation and differentiation in culture. The results of this study suggest that AA stimulates myoblast proliferation through its metabolites PGE, PGF, or PGI, and that AA stimulates myoblast differentiation through PGE.
许多研究表明前列腺素（PGs）对骨骼肌形成的各个步骤都有积极影响，如成肌细胞增殖和肌管肥大。在动物中，PGs 是由花生四烯酸（AA）通过限速酶环氧化酶（COX）-1 和 COX-2 合成的，AA 是一种条件必需脂肪酸。为了探索通过膳食补充 AA 来改善牛（主要的肉用动物）骨骼肌生长的可能性，我们确定了 AA 及其主要 PG 衍生物 PGE、PGF 和 PGI 对原代牛成肌细胞增殖、分化和融合的影响。在增殖实验中，将成肌细胞在生长培养基中培养，向其中添加 10μM AA、1μM PGE、1μM PGF、1μM PGI 或载体对照 24 小时，并通过 5-乙炔基-2'-脱氧尿苷（EdU）标记鉴定增殖细胞。该实验表明，与对照组相比，AA、PGE、PGF 和 PGI 分别使增殖细胞数量增加了 13%、24%、16%和 16%（n=7，P<0.05）。在分化融合试验中，在上述处理存在的情况下，将成肌细胞诱导分化融合为肌管 0、24、48 和 72 小时。基于 0、24 和 48 小时分化时成肌细胞分化和融合标志物（肌生成素；肌球蛋白重链 3；肌酸激酶，肌肉；肌动蛋白）mRNA 的定量逆转录 PCR 分析，AA、PGE 和 PGF 促进成肌细胞分化（n=6，P<0.05）。通过 Giemsa 染色和计数分化 72 小时后的肌管数量，与对照组相比，PGE 使形成的肌管数量增加了 14%（P<0.05）。用 COX-1 和 COX-2 共同抑制剂吲哚美辛或 COX-2 特异性抑制剂 NS-398 处理成肌细胞，逆转了 AA 对成肌细胞增殖的刺激作用（n=4，P<0.05）。总的来说，这项研究表明外源性 AA 刺激牛成肌细胞在培养中增殖和分化。这项研究的结果表明，AA 通过其代谢物 PGE、PGF 或 PGI 刺激成肌细胞增殖，AA 通过 PGE 刺激成肌细胞分化。
