C26结肠癌细胞与RAW264.7巨噬细胞共培养体系诱导C2C12细胞肌管萎缩及甘草多糖的保护作用

李亚静; 沈强; 邝计霞; 刘璇

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

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C26结肠癌细胞与RAW264.7巨噬细胞共培养体系诱导C2C12细胞肌管萎缩及甘草多糖的保护作用

实验研究 | 更新时间：2022-09-09

- C26结肠癌细胞与RAW264.7巨噬细胞共培养体系诱导C2C12细胞肌管萎缩及甘草多糖的保护作用
- Co-culture system of C26 colon cancer cells and RAW264.7 macrophages induces myotube atrophy in C2C12 cells and protective effect of glycyrrhiza polysaccharides
- 上海中医药大学学报 2021年35卷第4期页码：45-53
- 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：
  
  李亚静，女，在读硕士生，主要从事中药多糖抗肿瘤恶病质作用研究。沈强，男，在读博士生，主要从事肿瘤恶病质的病理机制研究（本文贡献与第一作者等同）
  刘璇，研究员，博士生导师；E-mail：xuanliu@shutcm.edu.cn
- 基金信息：
  
  国家自然科学基金资助项目(81873056);上海中医药大学研究生创新创业能力培养项目(A1-GY20-204-0144)
- DOI：10.16306/j.1008-861x.2021.04.007
  中图分类号：
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李亚静, 沈强, 邝计霞, 等. C26结肠癌细胞与RAW264.7巨噬细胞共培养体系诱导C2C12细胞肌管萎缩及甘草多糖的保护作用[J]. 上海中医药大学学报, 2021,35(4):45-53.

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, 2021,35(4):45-53.
李亚静, 沈强, 邝计霞, 等. C26结肠癌细胞与RAW264.7巨噬细胞共培养体系诱导C2C12细胞肌管萎缩及甘草多糖的保护作用[J]. 上海中医药大学学报, 2021,35(4):45-53. DOI： 10.16306/j.1008-861x.2021.04.007.

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, 2021,35(4):45-53. 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

references

MIAO C X，LV Y Y，ZHANG W L，et al. Pyrrolidine Dithiocarbamate（PDTC）Attenuates Cancer Cachexia by Affecting Muscle Atrophy and Fat Lipolysis[J]. Front Pharmacol，2017，8：915.

BROWN J L，ROSA-CALDWELL M E，LEE D E，et al. Mitochondrial degeneration precedes the development of muscle atrophy in progression of cancer cachexia in tumor-bearing mice[J]. J Cachexia Sarcopenia Muscle，2017，8(6)：926-938.

FEARON K，STRASSER F，ANKER S D，et al. Definition and classification of cancer cachexia：an international consensus[J]. Lancet Oncol，2011，12(5)：489-495.

ZHANG W L，LI N，SHEN Q，et al. Establishment of a mouse model of cancer cachexia with spleen deficiency syndrome and the effects of atractylenolide Ⅰ[J]. Acta Pharmacol Sin，2020，41(2)：237-248.

MARTIN L，BIRDSELL L，MACDONALD N，et al. Cancer Cachexia in the Age of Obesity：Skeletal Muscle Depletion Is a Powerful Prognostic Factor，Independent of Body Mass Index[J]. J Clin Oncol，2013，31(12)：1539-1547.

BOSSOLA M，PACELLI F，TORTORELLI A，et al. Skeletal Muscle in Cancer Cachexia：The Ideal Target of Drug Therapy[J]. Curr Cancer Drug Targets，2008，8(4)：285-298.

FEARON K，ARENDS J，BARACOS V. Understanding the mechanisms and treatment options in cancer cachexia[J]. Nat Rev Clin Oncol，2013，10(2)：90-99.

ARGILÉS J M，BUSQUETS S，STEMMLER B，et al. Cancer cachexia：understanding the molecular basis[J]. Nat Rev Cancer，2014，14(11)：754-762.

ZIMMERS T A，FISHEL M L，BONETTO A. STAT3 in the Systemic Inflammation of Cancer Cachexia[J]. Semin Cell Dev Biol，2016，54：28-41.

GENG Z，WEI L，ZHANG C，et al. Astragalus polysaccharide，a component of traditional Chinese medicine，inhibits muscle cell atrophy（cachexia）in an in vivo and in vitro rat model of chronic renal failure by activating the ubiquitin-proteasome pathway[J]. Exp Ther Med，2017，14(1)：91-96.

AYEKA P A，BIAN Y，MWITARI P G，et al. Immunomodulatory and anticancer potential of Gan cao（Glycyrrhiza uralensis Fisch.）polysaccharides by CT-26 colon carcinoma cell growth inhibition and cytokine IL-7 upregulation in vitro[J]. BMC Complement Altern Med，2016，16：206.

AYEKA P A，BIAN Y，GITHAIGA P M，et al. The immunomodulatory activities of licorice polysaccharides（Glycyrrhiza uralensis Fisch.）in CT26 tumor-bearing mice[J]. BMC Complement Altern Med，2017，17(1)：536.

HINSHAW D C，SHEVDE L A. The Tumor Microenvironment Innately Modulates Cancer Progression[J]. Cancer Res，2019，79(18)：4557-4566.

SEGATTO M，FITTIPALDI R，PIN F，et al. Epigenetic targeting of bromodomain protein BRD4 counteracts cancer cachexia and prolongs survival[J]. Nat Commun，2017，8(1)：1707.

CHEN L，YANG Q，ZHANG H，et al. Cryptotanshinone prevents muscle wasting in CT26-induced cancer cachexia through inhibiting STAT3 signaling pathway[J]. J Ethnopharmacol，2020，260：113066.

SHUKLA S K，MARKOV S D，ATTRI K S，et al. Macrophages potentiate STAT3 signaling in skeletal muscles and regulate pancreatic cancer cachexia[J]. Cancer Lett，2020，484：29-39.

BONETTO A，RUPERT J E，BARRETO R，et al. The Colon-26 Carcinoma Tumor-bearing Mouse as a Model for the Study of Cancer Cachexia[J]. J Vis Exp，2016，117：54893.

MOLINARI F，PIN F，GORINI S，et al. The mitochondrial metabolic reprogramming agent trimetazidine as an “exercise mimetic” in cachectic C26-bearing mice[J]. J Cachexia Sarcopenia Muscle，2017，8(6)：954-973.

VILLARS F O，PIETRA C，GIULIANO C，et al. Oral Treatment with the Ghrelin Receptor Agonist HM01 Attenuates Cachexia in Mice Bearing Colon-26（C26）Tumors[J]. Int J Mol Sci，2017，18(5)：986.

GE Z J，LIU D，SHANG Y，et al. Magnolol inhibits myotube atrophy induced by cancer cachexia through myostatin signaling pathway in vitro[J]. J Nat Med，2020，74(4)：741-749.

LIU D，QIAO X R，GE Z J，et al. IMB0901 inhibits muscle atrophy induced by cancer cachexia through MSTN signaling pathway[J]. Skelet Muscle，2019，9(1)：8.

ZHANG G H，LIN R K，KWON Y T，et al. Signaling mechanism of tumor cell-induced up-regulation of E3 ubiquitin ligase UBR2[J]. FASEB J，2013，27(7)：2893-2901.

CHANMEE T，ONTONG P，KONNO K，et al. Tumor-associated macrophages as major players in the tumor microenvironment[J]. Cancers（Basel），2014，6(3)：1670-1690.

FEARON K C，GLASS D J，GUTTRIDGE D C. Cancer cachexia：mediators，signaling，and metabolic pathways[J]. Cell Metab，2012，16(2)：153-166.

WU Q，SUN S，LI Z，et al. Breast cancer-released exosomes trigger cancer-associated cachexia to promote tumor progression[J]. Adipocyte，2019，8(1)：31-45.

BONETTO A，AYDOGDU T，KUNZEVITZKY N，et al. STAT3 activation in skeletal muscle links muscle wasting and the acute phase response in cancer cachexia[J]. PLoS One，2011，6(7)：e22538.

ESKILER G G，BEZDEGUMELI E，OZMAN Z，et al. IL-6 mediated JAK/STAT3 signaling pathway in cancer patients with cachexia[J]. Bratisl Lek Listy，2019，66(11)：819-826.

LU S，ZHANG Y，LI H，et al. Ginsenoside Rb1 can ameliorate the key inflammatory cytokines TNF-α and IL-6 in a cancer cachexia mouse model[J]. BMC Complement Med Ther，2020，20(1)：11.

KANDARIAN S C，NOSACKA R L，DELITTO A E，et al. Tumour-derived leukaemia inhibitory factor is a major driver of cancer cachexia and morbidity in C26 tumor-bearing mice[J]. J Cachexia Sarcopenia Muscle，2018，9(6)：1109-1120.

CHEN J L，WALTON K L，QIAN H W，et al. Differential Effects of IL6 and Activin A in the Development of Cancer-Associated Cachexia[J]. Cancer Res，2016，76(18)：5372-5382.

MA J F，SANCHEZ B J，HALL D T，et al. STAT3 promotes IFNγ/TNFα-induced muscle wasting in an NF-κB-dependent and IL-6-independent manner[J]. EMBO Mol Med，2017，9(5)：622-637.

SILVA K A，DONG J，DONG Y，et al. Inhibition of Stat3 activation suppresses caspase-3 and the ubiquitin-proteasome system，leading to preservation of muscle mass in cancer cachexia[J]. J Biol Chem，2015，290(17)：11177-11187.

ZHANG Y，HAN X，OUYANG B，et al. Chinese Herbal Medicine Baoyuan Jiedu Decoction Inhibited Muscle Atrophy of Cancer Cachexia through Atrogin-1 and MuRF-1[J]. Evid Based Complement Alternat Med，2017，2017：6268378. doi：10.1155/2017/6268378http://doi.org/10.1155/2017/6268378.

KANG H J，JEONG M K，PARK S J，et al. Efficacy and safety of Yukgunja-Tang for treating anorexia in patients with cancer：The protocol for a pilot，randomized，controlled trial[J]. Medicine（Baltimore），2019，98(40)：e16950.

CHOI Y K，JUNG K Y，WOO S M，et al. Effect of Sipjeondaebo-tang on cancer-induced anorexia and cachexia in CT-26 tumor-bearing mice[J]. Mediators Inflamm，2014，2014：736563. doi：10.1155/2014/736563http://doi.org/10.1155/2014/736563.

OHBUCHI K，NISHIUMI S，FUJITSUKA N，et al. Rikkunshito Ameliorates Cancer Cachexia Partly through Elevation of Glucarate in Plasma[J]. Evid Based Complement Alternat Med，2015，2015：871832. doi：10.1155/2015/871832http://doi.org/10.1155/2015/871832.

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