Co-culture system of C26 colon cancer cells and RAW264.7 macrophages induces myotube atrophy in C2C12 cells and protective effect of glycyrrhiza polysaccharides

LI Yajing; SHEN Qiang; KUANG Jixia; LIU Xuan

doi:10.16306/j.1008-861x.2021.04.007

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Co-culture system of C26 colon cancer cells and RAW264.7 macrophages induces myotube atrophy in C2C12 cells and protective effect of glycyrrhiza polysaccharides

Experiment Research | 更新时间：2022-09-09

- Co-culture system of C26 colon cancer cells and RAW264.7 macrophages induces myotube atrophy in C2C12 cells and protective effect of glycyrrhiza polysaccharides
- Academic Journal of Shanghai University of Traditional Chinese Medicine Vol. 35, Issue 4, Pages: 45-53(2021)
- 作者机构：
  
  1.上海中医药大学交叉科学研究院（上海　201203）
- 作者简介：
- 基金信息：
- DOI：10.16306/j.1008-861x.2021.04.007
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LI Yajing, SHEN Qiang, KUANG Jixia, et al. Co-culture system of C26 colon cancer cells and RAW264.7 macrophages induces myotube atrophy in C2C12 cells and protective effect of glycyrrhiza polysaccharides. [J]. Academic Journal of Shanghai University of Traditional Chinese Medicine 35(4):45-53(2021)
DOI：

LI Yajing, SHEN Qiang, KUANG Jixia, et al. Co-culture system of C26 colon cancer cells and RAW264.7 macrophages induces myotube atrophy in C2C12 cells and protective effect of glycyrrhiza polysaccharides. [J]. Academic Journal of Shanghai University of Traditional Chinese Medicine 35(4):45-53(2021) DOI： 10.16306/j.1008-861x.2021.04.007.

摘要

目的：,2,基于C26结肠癌细胞与RAW264.7巨噬细胞共培养体系，构建C2C12细胞肌管萎缩模型，利用该模型研究甘草多糖对肌管的保护作用及机制。,方法：,2,构建C26细胞和RAW264.7细胞共培养体系，分别制备单一细胞和共培养细胞的条件培养液。加入诱导分化液使C2C12成肌细胞形成肌管。①将已分化的C2C12细胞分为对照组、C26模型组、RAW264.7模型组、共培养模型组。各组分别给予相应的条件培养液，孵育48 h，HE染色观察肌管形态，并测定肌管直径。②将已分化的C2C12细胞分为对照组及甘草多糖不同浓度（0.312 5、0.625、1.25、2.5、5 mg/ml）组，分别给予相应浓度的甘草多糖，孵育48 h后，测定细胞活力。③将已分化的C2C12细胞分为对照组、C26模型组、C26+甘草多糖组。除对照组外，其他各组均给予C26结肠癌细胞条件培养液；同时，甘草多糖干预组分别给予0.5、1、2 mg/ml甘草多糖。孵育48 h后，HE染色观察肌管形态，并测定肌管直径。④将已分化的C2C12细胞分为对照组、共培养模型组、共培养+甘草多糖组。除对照组外，其他各组均给予共培养细胞条件培养液；同时，甘草多糖干预组分别给予0.5、1、2 mg/ml甘草多糖。孵育48 h后，HE染色观察肌管形态，并测定肌管直径。⑤不同的条件培养液或共培养条件培养液与甘草多糖（2 mg/ml）协同干预后，Western blot检测信号传导与转录激活因子3（STAT3）蛋白表达及磷酸化水平。,结果：,2,①3种条件培养液均可诱导肌管直径减小（,P,<,0.01），且共培养模型组的相对肌管直径较C26模型组显著降低（,P,<,0.05）。②≤2.5 mg/ml甘草多糖对细胞活力无明显影响。③与C26模型组相比，C26+甘草多糖（0.5、1和2 mg/ml）组细胞的相对肌管直径显著升高（,P,<,0.01）。④与共培养模型组相比，共培养+甘草多糖（0.5、1和2 mg/ml）组细胞的相对肌管直径显著升高（,P,<,0.01）。⑤3种条件培养液干预后均可诱导C2C12肌管细胞STAT3磷酸化水平升高，且共培养模型组的p-STAT3/STAT3蛋白表达比值明显高于C26模型组（,P,<,0.01）。与共培养模型组相比，共培养+甘草多糖（2 mg/ml）组p-STAT3/STAT3蛋白表达比值显著下调（,P,<,0.01）。,结论：,2,甘草多糖可能通过抑制STAT3信号通路减轻C26结肠癌细胞与RAW264.7巨噬细胞共培养体系诱导的肌管萎缩。

Abstract

Objective:,2,To establish the myotube atrophy model of C2C12 cells based on co-culture system of C26 colon cancer cells and RAW264.7 macrophages, and study the protective effect of glycyrrhiza polysaccharides on myotubes and its mechanism.,Methods:,2,The co-culture system of C26 cells and RAW264.7 cells was constructed, and the conditioned medium of single cells and co-culture cells was prepared, respectively.The differentiation of myotubes was induced from C2C12 myoblasts.①The differentiated C2C12 cells were divided into the control group, C26 model group, RAW264.7 model group and co-culture model group.Each group was treated with the corresponding conditioned medium.After incubation for 48 hours, the morphology of myotubes was observed after HE staining, and the diameter of myotubes was determined.②The differentiated C2C12 cells were divided into the control group and glycyrrhiza polysaccharides groups with different concentrations(0.312 5, 0.625, 1.25, 2.5 and 5 mg/ml) .Each group was treated with glycyrrhiza polysaccharides at the corresponding concentration.After incubation for 48 hours, the cell viability was measured.③The differentiated C2C12 cells were divided into the control group, C26 model group and C26+glycyrrhiza polysaccharides groups with different concentrations.Except the control group, the other groups were treated with the conditioned medium of C26 colon cancer cells.Meanwhile, the glycyrrhiza polysaccharides intervention groups were treated with glycyrrhiza polysaccharides at concentration of 0.5, 1, 2 mg/ml, respectively.After incubation for 48 hours, the morphology of myotubes was observed after HE staining, and the diameter of myotubes was determined.④The differentiated C2C12 cells were divided into the control group, co-culture model group and co-culture+glycyrrhiza polysaccharides groups with different concentrations.Except the control group, the other groups were treated with the conditioned medium of co-culture cells.Meanwhile, the glycyrrhiza polysaccharides intervention groups were treated with glycyrrhiza polysaccharides at concentration of 0.5, 1, 2 mg/ml, respectively.After incubation for 48 hours, the morphology of myotubes was observed after HE staining, and the diameter of myotubes was determined.⑤After treatment with different conditioned medium or co-treatment of co-culture conditioned medium and glycyrrhiza polysaccharides(2 mg/ml), the protein expression and phosphorylation of signal transducer and activator of transcription 3(STAT3) were detected by Western blot.,Results:,2,①All three kinds of conditioned medium could induce the decrease of myotube diameter(,P,<,0.01), and the relative myotube diameter of the co-culture model group was significantly lower than that of the C26 model group(,P,<,0.05) .②Glycyrrhiza polysaccharides≤2.5 mg/ml showed no significant effect on the cell viability.③Compared with C26 model group, the relative myotube diameter of C26+glycyrrhiza polysaccharides(0.5, 1 and 2 mg/ml) groups was significantly increased(,P,<,0.01) .④Compared with co-culture model group, the relative myotube diameter of co-culture+glycyrrhiza polysaccharides(0.5, 1 and 2 mg/ml) groups was significantly increased(,P,<,0.01) .⑤All three conditioned medium could induce the increase of STAT3 phosphorylation level in myotubes, and the protein expression ratio of p-STAT3/STAT3 in the co-culture model group was obviously higher than that in the C26 model group(,P,<,0.01) .Compared with co-culture model group, the protein expression ratio of p-STAT3/STAT3 was significantly down-regulated in the co-culture+glycyrrhiza polysaccharides(2 mg/ml) group(,P,<,0.01) .,Conclusion:,2,Glycyrrhiza polysaccharides may alleviate the myotube atrophy induced by co-culture system of C26 colon cancer cells and RAW264.7 macrophages via inhibiting STAT3 signaling pathway.

关键词

甘草多糖肿瘤恶病质肿瘤微环境共培养肌肉萎缩

Keywords

glycyrrhiza polysaccharidescancer cachexiatumor microenvironmentco-culturemuscle atrophy

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