保元解毒汤通过细胞因子-泛素-蛋白酶体途径干预癌因性肌管萎缩机制的研究
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摘要
目的观察保元解毒汤对癌性环境下C2C12小鼠成肌细胞分化的影响,探讨其缓解肌肉萎缩的可能机制。方法先分别用1∶2、1∶4、1∶8的Lewis肺腺癌细胞上清液诱导C2C12细胞,确定癌因性肌管萎缩模型建立的最佳浓度和培养时间。造模成功后,将细胞分为模型组,保元解毒汤高剂量组(125 g/L)、中剂量组(62.5 g/L)、低剂量组(31.25 g/L),光学显微镜下观察肌管形成情况;ELISA法检测C2C12细胞上清液中肿瘤坏死因子-α(TNF-α)和白介素-6(IL-6)含量;Western blot、Real-Time PCR法检测肌肉萎缩盒F蛋白(Atrogen-1)和肌肉特异性环指蛋白1(MuRF-1)的表达。结果 1∶2的Lewis肺腺癌细胞上清液诱导C2C12细胞96 h,TNF-α和IL-6含量明显增多,为构建癌因性肌管萎缩模型的最佳条件。高、中、低剂量的保元解毒汤干预后,肌管横径增加(P<0.05);上清液中TNF-α和IL-6含量减少(P<0.05),Atrogin-1和MuRF-1蛋白及mRNA表达下调(P<0.05)。结论保元解毒汤可能通过降低细胞因子含量,抑制泛素-蛋白酶体途径(UPP),减少肌蛋白分解而改善癌因性肌管萎缩。
Objective To investigate the effect of Baoyuan Jiedu( BYJD) decoction on C2 C12 cells differentiation in cancer-induced myotube atrophia model and explore its mechanism. Methods Lewis lung carcinoma cell supernatant at different concentrations( 1 ∶ 2,1 ∶ 4,and 1 ∶ 8) were injected into C2 C12 cells to induce cancer-induced myotube atrophy and identify the optimal concentration and culture duration. Four groups was divided after the model was built: normal group,high-dose BYJD group( 125 g/L),mid-dose BYJD group( 62. 5 g/L),and low-dose BYJD group( 31. 25 g/L). The condition of myotube was observed under electro-microscope; levels of TNF-α and IL-6 were measured with ELISA; muscle fiber transverse diameter of C2 C12 cells were observed by using inverted microscopy at every 24 h;the expressions of Atrogin-1 and MuRF-1 were detected by using Real-time PCR and Western blot assay.Results TNF-α and IL-6 in C2 C12 indicated that the optimal condition to induce cancer-induced myotube atrophy was Lewis lung carcinoma cell supernatant at concentration of 1∶ 2 for 96 hours. TNF-α and IL-6 in BYJD groups decreased significantly( P < 0. 05); transverse diameter of myotubes in BYJD groups were prolonged after intervention of BYJD decoction at high-dose,mid-dose and low-dose( P < 0.05); the expression of Atrogin-1,MuRF-1 protein and mRNA was down-regulated( P < 0. 05). Conclusion BYJD decoction could effectively reduce cancer-induced myotube atrophy by inhibiting UPP through lowering cytokine levels.
Objective To investigate the effect of Baoyuan Jiedu( BYJD) decoction on C2 C12 cells differentiation in cancer-induced myotube atrophia model and explore its mechanism. Methods Lewis lung carcinoma cell supernatant at different concentrations( 1 ∶ 2,1 ∶ 4,and 1 ∶ 8) were injected into C2 C12 cells to induce cancer-induced myotube atrophy and identify the optimal concentration and culture duration. Four groups was divided after the model was built: normal group,high-dose BYJD group( 125 g/L),mid-dose BYJD group( 62. 5 g/L),and low-dose BYJD group( 31. 25 g/L). The condition of myotube was observed under electro-microscope; levels of TNF-α and IL-6 were measured with ELISA; muscle fiber transverse diameter of C2 C12 cells were observed by using inverted microscopy at every 24 h;the expressions of Atrogin-1 and MuRF-1 were detected by using Real-time PCR and Western blot assay.Results TNF-α and IL-6 in C2 C12 indicated that the optimal condition to induce cancer-induced myotube atrophy was Lewis lung carcinoma cell supernatant at concentration of 1∶ 2 for 96 hours. TNF-α and IL-6 in BYJD groups decreased significantly( P < 0. 05); transverse diameter of myotubes in BYJD groups were prolonged after intervention of BYJD decoction at high-dose,mid-dose and low-dose( P < 0.05); the expression of Atrogin-1,MuRF-1 protein and mRNA was down-regulated( P < 0. 05). Conclusion BYJD decoction could effectively reduce cancer-induced myotube atrophy by inhibiting UPP through lowering cytokine levels.
引文
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[17]Marchione R,Leibovitch SA,Lenormand JL.The translational factor e IF3f:the am bivalent e IF3f subunit[J].Cell Mol Life Sci,2013,70(19):3603-3613.
[18]Yuan L,Han J,Meng Q,et al.Muscle-specific E3 ubiquitin ligases are involved in muscle atrophy of cancer cachexia:an in vitro and in vivo study[J].Oncol Rep,2015,33(5):2261-2268.
[2]Zhang G,Liu Z,Ding H,et al.Tumor induces muscle wasting in mice through releasing extracellular Hsp70 and Hsp90[J].Nat Commun,2017,8(1):589-599.
[3]Stene GB,Helbostad JL,Amundsen T,et al.Changes in skeletal muscle mass during palliative chemotherapy in patients with advanced lung cancer[J].Acta Oncol,2015,54(3):340-348.
[4]张蕴超,张欣,杨佩颖,等.中西医对癌性恶病质干预作用的研究概况[J].肿瘤学杂志,2014,20(6):504-507.Zhang YC,Zhang X,Yang PY,et al.The study of the interventional effect of Chinese and Western medicine on cancer[J].Journal of Chinese Oncology,2014,20(6):504-507.
[5]王文苹,欧阳兵,吴智春,等.保元解毒汤改善癌性恶病质模型小鼠骨骼肌萎缩的实验研究[J].山东中医药大学学报,2013,37(5):423-425.Wang WP,Ouyang B,Wu ZC,et al.The experimental study on the improvement of skeletal muscle atrophy in mice with cancer cachexia[J].Journal of Shandong University of Traditional Chinese Medicine,2013,37(5):423-425.
[6]王文苹,季旭明,刘超,等.癌性恶病质模型生活质量评价体系的建立及保元解毒汤的干预作用研究[J].微循环学杂志,2012,22(2):91.Wang WP,Ji XM,Liu C,et al.Study on the establishment of quality evaluation system of cancer cachexia model and the intervention effect of prophylactic decoction[J].Chinese Journal of Microcirculation,2012,22(2):91.
[7]于华芸,王文苹,吴智春,等.保元解毒汤对癌性恶病质小鼠骨骼肌蛋白质降解及MAFbx、MuRF-1基因表达的影响[J].时珍国医国药,2015,26(1):226-229.Yu HY,Wang WP,Wu ZC,et al.Bao yuan jiedu decoction on cancer cachexia mice skeletal muscle protein degradation and MAFbx MuRF-1 gene expression[J].Lishizhen Medicine and Materia Medica Research,2015,26(1):226-229.
[8]Mc Lean JB,Moylan JS,Andrade FH.Mitochondria dysfunction in lung cancer-induced muscle wasting in C2C12myotubes[J/OL].Front Physiol,2014,5:503[2014-12-18].http://www.frontiersin.org/articles/10.3389/fphys.2014.00503/full.
[9]Fearon KC,Glass DJ,Guttridge DC.Cancer cachexia:mediators,signaling,and metabolic pathways.[J].Cell Metab,2012,16(2):153-166.
[10]Zhang G,Liu Z,Ding H,et al.Toll-like receptor 4 mediates Lewis lung carcinoma-induced muscle wasting via coordinate activation of protein degradation pathways[J/OL].Sci Rep,2017,7(1):2273[2017-05-23].http://www.nature.com/articles/s41598-017-02347-2.
[11]Vander Veen BN,Fix DK,Carson JA.Disrupted Skeletal Muscle Mitochondrial Dynamics,Mitophagy,and Biogenesis during Cancer Cachexia:A Role for Inflammation[J/OL].Oxid Med Cell Longev,2017,2017:3292087[2017-07-13].http://www.hindawi.com/journals/omcl/2017/3292087.
[12]Chen JL,Colgan TD,Walton KL,et al.The TGF-beta signaling network in muscle development,adaptation and disease[J].Adv Exp Med Biol,2016,900:97-131.
[13]Patel HJ,Patel BM.TNF-alpha and cancer cachexia:molecular insights and clinical implications[J].Life Sci,2017,170:56-63.
[14]White JP,Puppa MJ,Sato S,et al.IL-6 regulation on skeletal muscle mitochondrial remodeling during cancer cachexia in the ApcMin/+mouse[J].Skelet Muscle,2012,6(2):14[2012-07-06].http://www.skeletalmusclejournal.biomedcentral.com/articles/10.1186/2044-5040-2-14.
[15]Bilodeau PA,Coyne ES,Wing SS.The ubiquitin proteasome system in atrophying skeletal muscle:roles and regulation[J].Am J Physiol Cell Physiol,2016,311(3):392-403.
[16]Llovera M,CarbóN,López-Soriano J,et al.Different cytokines modulate ubiquitin gene expression in rat skeletal muscle[J].Cancer Lett,1998,133(1):83-87.
[17]Marchione R,Leibovitch SA,Lenormand JL.The translational factor e IF3f:the am bivalent e IF3f subunit[J].Cell Mol Life Sci,2013,70(19):3603-3613.
[18]Yuan L,Han J,Meng Q,et al.Muscle-specific E3 ubiquitin ligases are involved in muscle atrophy of cancer cachexia:an in vitro and in vivo study[J].Oncol Rep,2015,33(5):2261-2268.