Effects of a traditional Chinese medicine formula and its extraction on muscle fiber characteristics in finishing pigs, porcine cell proliferation and isoforms of myosin heavy chain gene expression in myocytes

  • Yu, Qin Ping (College of Animal Science, South China Agricultural University) ;
  • Feng, Ding Yuan (College of Animal Science, South China Agricultural University) ;
  • He, Xiao Jun (College of Animal Science, South China Agricultural University) ;
  • Wu, Fan (College of Animal Science, South China Agricultural University) ;
  • Xia, Min Hao (College of Animal Science, South China Agricultural University) ;
  • Dong, Tao (College of Animal Science, South China Agricultural University) ;
  • Liu, Yi Hua (College of Animal Science, South China Agricultural University) ;
  • Tan, Hui Ze (Guangdong Wen's Foodstuffs Group Co., Ltd.) ;
  • Zou, Shi Geng (Guangdong Wen's Foodstuffs Group Co., Ltd.) ;
  • Zheng, Tao (Nong Zhi Dao Co., Ltd.) ;
  • Ou, Xian Hua (Nong Zhi Dao Co., Ltd.) ;
  • Zuo, Jian Jun (College of Animal Science, South China Agricultural University)
  • Received : 2016.11.13
  • Accepted : 2017.05.23
  • Published : 2017.11.01


Objective: This study evaluated the effects of a traditional Chinese medicine formula (TCMF) on muscle fiber characteristics in finishing pigs and the effects of the formula's extract (distilled water, ethyl acetate and petroleum ether extraction) on porcine cell proliferation and isoforms of myosin heavy chain (MyHC) gene expression in myocytes. Methods: In a completely randomized design, ninety pigs were assigned to three diets with five replications per treatment and six pigs per pen. The diets included the basal diet (control group), TCMF1 (basal diet+2.5 g/kg TCMF) and TCMF2 (basal diet+5 g/kg TCMF). The psoas major muscle was obtained from pigs at the end of the experiment. Muscle fiber characteristics in the psoas major muscle were analyzed using myosin ATPase staining. Cell proliferation was measured using 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) dye and cytometry. Isoforms of MyHC gene expression were detected by real-time quantitative polymerase chain reaction. Results: The final body weight and carcass weight of finishing pigs were increased by TCMF1 (p<0.05), while the psoas major muscle cross-sectional area was increased by TCMF (p<0.05). The cross-sectional area and diameter of psoas major muscle fiber Ι, IIA, and IIB were increased by TCMF2 (p<0.05). The cross-sectional area and fiber diameter of psoas major muscle fiber IIA and IIB were increased by diet supplementation with TCMF1 (p<0.05). Psoas major muscle fiber IIA and IIB fiber density from the pigs fed the TCMF1 diet and the type IIB fiber density from the pigs fed the TCMF2 diet were lower compared to pigs fed the control diet (p<0.05). Pigs fed TCMF2 had a higher composition of type Ι fiber and a lower percentage of type IIB fiber in the psoas major muscle (p<0.05). The expression levels of MyHC Ι, MyHC IIa, and MyHC IIx mRNA increased and the amount of MyHC IIb mRNA decreased in the psoas major muscle from TCMF2, whereas MyHC Ι and MyHC IIx mRNA increased in the psoas major muscle from TCMF1 (p<0.05). Peroxisome proliferator-activated receptor ${\gamma}$ $coactivator-1{\alpha}$ and CaN mRNA expression in the psoas major muscle were up-regulated by TCMF (p<0.05). Porcine skeletal muscle satellite cell proliferation was promoted by $4{\mu}g/mL$ and $20{\mu}g/mL$ TCMF water extraction (p<0.05). Both $1{\mu}g/mL$ and $5{\mu}g/mL$ of TCMF water extraction increased MyHC IIa, MyHC IIb, and MyHC IIx mRNA expression in porcine myocytes (p<0.05), while MyHC Ι mRNA expression in porcine myocytes was decreased by $5{\mu}g/mL$ TCMF water extraction (p<0.05). Porcine myocyte MyHC Ι and MyHC IIx mRNA expression were increased, and MyHC IIa and MyHC IIb mRNA expression were down-regulated by $5{\mu}g/mL$ TCMF ethyl acetate extraction (p<0.05). MyHC Ι and MyHC IIa mRNA expression in porcine myocytes were increased, and the MyHC IIb mRNA expression was decreased by $1{\mu}g/mL$ TCMF ethyl acetate extraction (p<0.05). Four isoforms of MyHC mRNA expression in porcine myocytes were reduced by $5{\mu}g/mL$ TCMF petroleum ether extraction (p<0.05). MyHC IIa mRNA expression in porcine myocytes increased and MyHC IIb mRNA expression decreased by $1{\mu}g/mL$ in a TCMF petroleum ether extraction (p<0.05). Conclusion: These results indicated that TCMF amplified the psoas major muscle cross-sectional area through changing muscle fiber characteristics in finishing pigs. This effect was confirmed as TCMF extraction promoted porcine cell proliferation and affected isoforms of MyHC gene expression in myocytes.


Muscle Fiber;Cell Proliferation;Traditional Chinese Medicine Formula;Pigs


  1. Jeong DW, Choi YM, Lee SH, et al. Correlations of trained panel sensory values of cooked pork with fatty acid composition, muscle fiber type, and pork quality characteristics in Berkshire pigs. Meat Sci 2010;86:607-15.
  2. Lefaucheur L, Ecolan P, Plantard L, Gueguen N. New insights into muscle fiber types in the pig. J Histochem Cytochem 2002;50:719-30.
  3. Adamovic I, Vitorovic D, Blagojevic M, Nesic I, Brkic Z. Histological and histochemical properties of M. semitendinosus in German Landrace pigs at birth and market weight. Acta Vet-Beograd 2014;64:319-26.
  4. Biswas AK, Keshri RC, Bisht GS. Effect of enrobing and antioxidants on quality characteristics of precooked pork patties under chilled and frozen storage conditions. Meat Sci 2004;66:733-41.
  5. Koreleski J, Swiatkiewicz S. Effect of dietary supplementation of vitamin E, antioxidants and a synthetic carotenoid on changes in chicken breast meat quality during storage. Ann Anim Sci 2008;8:167-74.
  6. Vandghanooni S, Forouharmehr A, Eskandani M, et al. Cytotoxicity and DNA fragmentation properties of butylated hydroxyanisole. DNA Cell Biol 2013;32:98-103.
  7. Qiao GH, Shao T, Yang X, et al. Effects of supplemental Chinese herbs on growth performance, blood antioxidant function and immunity status in Holstein dairy heifers fed high fibre diet. Italian J Anim Sci 2013;12:e20.
  8. Huang CW, Lee TT, Shih YC, Yu B. Effects of dietary supplementation of Chinese medicinal herbs on polymorphonuclear neutrophil immune activity and small intestinal morphology in weanling pigs. J Anim Physiol Anim Nutr 2012;96:285-94.
  9. Liu FX, Sun S, Cui ZZ. Analysis of immunological enhancement of immunosuppressed chickens by Chinese herbal extracts. J Ethnopharmacol 2010;127:251-6.
  10. Qiao GH, Zhou XH, Li Y, et al. Effect of several supplemental Chinese herbs additives on rumen fermentation, antioxidant function and nutrient digestibility in sheep. J Anim Physiol Anim Nutr 2012;96:930-8.
  11. Li S, Zhang B, Jiang D, Wei YY, Zhang NB. Herb network construction and co-module analysis for uncovering the combination rule of traditional Chinese herbal formulae. BMC Bioinformatics 2010;11(Suppl 11):S6.
  12. Bentzinger CF, Romanino K, Cloetta D, et al. Skeletal muscle-specific ablation of raptor, but not of rictor, causes metabolic changes and results in muscle dystrophy. Cell Metab 2008;8:411-24.
  13. Schuler M, Ali F, Chambon C, et al. PGC1 alpha expression is controlled in skeletal muscles by PPAR beta, whose ablation results in fiber-type switching, obesity, and type 2 diabetes. Cell Metabol 2006;4:407-14.
  14. Lin J, Wu H, Tarr PT, et al. Transcriptional co-activator PGC-1 alpha drives the formation of slow-twitch muscle fibres. Nature 2002;418:797-801.
  15. Miura S, Kai Y, Ono M, Ezaki O. Overexpression of peroxisome proliferator-activated receptor gamma coactivator-1 alpha down-regulates GLUT4 mRNA in skeletal muscles. J Biol Chem 2003;278:31385-90.
  16. Handschin C, Chin S, Li P, et al. Skeletal muscle fiber-type switching, exercise intolerance, and myopathy in PGC-1alpha muscle-specific knock-out animals. J Biol Chem 2007;282:30014-21.
  17. Sakuma K, Nishikawa J, Nakao R, et al. Calcineurin is a potent regulator for skeletal muscle regeneration by association with NFATc1 and GATA-2. Acta Neuropathol 2003;105:271-80.
  18. Calabria E, Ciciliot S, Moretti I, et al. NFAT isoforms control activitydependent muscle fiber type specification. Proc Natl Acad Sci USA 2009;106:13335-40.
  19. Talmadge RJ, Otis JS, Rittler MR, et al. Calcineurin activation influences muscle phenotype in a muscle-specific fashion. BMC Cell Biol 2004;5:28.
  20. Song MY. The Effects of Astragali Radix Extracts on mitochondrial function in C2C12 myotubes. Korean Med Obesity Res 2014;14:55-62.
  21. Jeon WJ, Lee DS, Shon SY, et al. Effects of ethanol extract of Polygonatum sibiricum rhizome on obesity-related genes. Korean Food Sci Technol 2016;48:384-91.
  22. Sang HK. Skeletal muscle glycogen breakdown according to duration of endurance training. Korea Sports Med 2016;34:101-6.
  23. Kim K, Kim SJ, Cho NC, et al. Reactive oxygen species-dependent transcriptional regulation of peroxisome proliferator-activated receptor gamma coactivator 1 alpha in a human hepatocarcinoma cell line. Genes Genomics 2012;34:709-13.
  24. Soudani N, Ghantous CM, Farhat Z, et al. Calcineurin/NFAT activation- dependence of Leptin synthesis and vascular growth in response to mechanical stretch. Front Physiol 2016;7:433.
  25. Mu LH, Huang ZX, Liu P, et al. Acute and subchronic oral toxicity assessment of the herbal formula Kai-Xin-San. J Ethnopharmacol 2011;138:351-7.
  26. NRC. Committee on Nutrient Requirements of Swine, National Research Council. Nutrient requirements of swine. 11th ed. Washington, DC: National Academy Press; 2012.
  27. Brook MH, Kaiser KK. Three myosin adenosine triphosphatase systems: The nature of their pH lability and sulfhydryl dependence. Histochem Cytochem 1970;18:670-2.
  28. Lind A, Kernell D. Myofibrillar ATPase histochemistry of rat skeletal muscles: A "two- dimensional" quantitative approach. Histochem Cytochem 1991;39:589-97.
  29. Patterson J, Mura C. Rapid colorimetric assays to qualitatively distinguish RNA and DNA in biomolecular samples. J Vis Exp 2013:e50225.
  30. Peuker H, Pette D. Non-radioactive reverse transcriptase/polymerase chain reaction for quantification of myosin heavy chain mRNA isoforms in various rabbit muscles. FEBS Lett 1993;318:253-8.
  31. Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C (T)) Method. Methods 2001;25:402-8.
  32. Mwanza M, Kametler L, Bonai A, et al. The cytotoxic effect of fumonisin B1 and ochratoxin A on human and pig lymphocytes using the Methyl Thiazol Tetrazolium (MTT) assay. Mycotoxin Res 2009;25:233-8.
  33. Gomez L A, Alekseev AE, Aleksandrova LA, et al. Use of the MTT assay in adult ventricular cardiomyocytes to assess viability: Effects of adenosine and potassium on cellular survival. J Mol Cell Cardiol 1997;29:1255-66.
  34. Misawa K, Hashizume K, Yamamoto M, et al. Ginger extract prevents high-fat diet-induced obesity in mice via activation of the peroxisome proliferator-activated receptor delta pathway. Nutri Biochem 2015;26:1058-67.
  35. Bakhtiari N, Hosseinkhani S, Soleimani M, et al. Short-term ursolic acid promotes skeletal muscle rejuvenation through enhancing of SIRT1 expression and satellite cells proliferation. Biomed Pharmacother 2016;78:185-96.
  36. Hatazawa Y, Senoo N, Tadaishi M, et al. Metabolomic analysis of the skeletal muscle of mice overexpressing PGC-1alpha. PLOS ONE 2015;10:e0129084.
  37. Xu R, Andres-Mateos E, Mejias R, et al. Hibernating squirrel muscle activates the endurance exercise pathway despite prolonged immobilization. Exp Neurol 2013;247:392-401.
  38. Zhang C, Luo JQ, Zheng P, et al. Differential expression of lipid metabolism-related genes and myosin heavy chain isoform genes in pig muscle tissue leading to different meat quality. Animal 2015;9:1073-80.
  39. Pandorf CE, Jiang WH, Qin AX, et al. Calcineurin plays a modulatory role in loading-induced regulation of type I myosin heavy chain gene expression in slow skeletal muscle. Am J Physiol Regul Integr Comp Physiol 2009;297:R1037-48.
  40. Seyer P, Grandemange S, Rochard P, et al. P43-dependent mitochondrial activity regulates myoblast differentiation and slow myosin isoform expression by control of Calcineurin expression. Exp Cell Res 2011;317:2059-71.
  41. da Costa N, Edgar J, Ooi PT, et al. Calcineurin differentially regulates fast myosin heavy chain genes in oxidative muscle fibre type conversion. Cell Tissue Res 2007;329:515-27.
  42. Men XM, Deng B, Tao X, Qi KK, Xu ZW. Association analysis of myosin heavy-chain genes mRNA transcription with the corresponding proteins expression of Longissimus muscle in growing pigs. Asian-Australas J Anim Sci 2016;29:457-63.
  43. Pette D, Staron RS. Transitions of muscle fiber phenotypic profiles. Histochem Cell Biol 2001;115:359-72.
  44. Li BJ, Li PH, Huang RH, et al. Isolation, culture and identification of porcine skeletal muscle satellite cells. Asian-Australas J Anim Sci 2015;28:1171-7.
  45. Wilschut KJ, Jaksani S, Van Den Dolder J, Haagsman HP, Roelen BA. Isolation and characterization of porcine adult muscle-derived progenitor cells. J Cell Biochem 2008;105:1228-39.
  46. Singh M, Sharma AK. Outgrowth of fibroblast cells from goat skin explants in three different culture media and the establishment of cell lines. In Vitro Cell Dev Biol Anim 2011;47:83-8.
  47. Sebastian S, Goulding L, Kuchipudi SV, Chang KC. Extended 2D myotube culture recapitulates postnatal fibre type plasticity. BMC Cell Biol 2015;16:23.
  48. Chung WT, Lee SH, Kim JD, et al. Effect of the extracts from Glycyrrhiza uralensis Fisch on the growth characteristics of human cell lines: Anti-tumor and immune activation activities. Cytotechnol 2001;37:55-64.
  49. Lau KM, Lai KK, Liu CL, et al. Synergistic interaction between Astragali Radix and Rehmanniae Radix in a Chinese herbal formula to promote diabetic wound healing. J Ethnopharmacol 2012;141:250-6.
  50. Paduch R, Wozniak A, Niedziela P, Rejdak R. Assessment of eyebright (Euphrasia Officinalis L.) extract activity in relation to human corneal cells using in vitro tests. Balkan Med J 2014;31:29-36.
  51. Yao Y, Zhang X, Wang Z, et al. Deciphering the combination principles of traditional Chinese medicine from a systems pharmacology perspective based on Ma-huang Decoction. J Ethnopharmacol 2013;150:619-38.