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

Abstract

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 I, 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 I fiber and a lower percentage of type IIB fiber in the psoas major muscle (p<0.05). The expression levels of MyHC I, MyHC IIa, and MyHC IIx mRNA increased and the amount of MyHC IIb mRNA decreased in the psoas major muscle from TCMF2, whereas MyHC I and MyHC IIx mRNA increased in the psoas major muscle from TCMF1 (p<0.05). Peroxisome proliferator-activated receptor γ coactivator-1α 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 μg/mL and 20 μg/mL TCMF water extraction (p<0.05). Both 1 μg/mL and 5 μg/mL of TCMF water extraction increased MyHC IIa, MyHC IIb, and MyHC IIx mRNA expression in porcine myocytes (p<0.05), while MyHC I mRNA expression in porcine myocytes was decreased by 5 μg/mL TCMF water extraction (p<0.05). Porcine myocyte MyHC I and MyHC IIx mRNA expression were increased, and MyHC IIa and MyHC IIb mRNA expression were down-regulated by 5 μg/mL TCMF ethyl acetate extraction (p<0.05). MyHC I and MyHC IIa mRNA expression in porcine myocytes were increased, and the MyHC IIb mRNA expression was decreased by 1 μg/mL TCMF ethyl acetate extraction (p<0.05). Four isoforms of MyHC mRNA expression in porcine myocytes were reduced by 5 μg/mL TCMF petroleum ether extraction (p<0.05). MyHC IIa mRNA expression in porcine myocytes increased and MyHC IIb mRNA expression decreased by 1 μg/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.

Keywords: Cell Proliferation; Muscle Fiber; Pigs; Traditional Chinese Medicine Formula.

Figure 1

Effects of the traditional Chinese medicine formula (TCMF) on finishing pig carcass traits. (A) Effects of TCMF on the final body weight of finishing pigs. (B) Effects of TCMF on the carcass weight of finishing pigs. (C) Effects of TCMF on the psoas major muscle cross-sectional area of finishing pigs. CON, Control group, basal diet; TCMF1, basal diet+2.5 g/kg TCMF; TCMF2, basal diet+5 g/kg TCMF. Compared to CON, * represents p<0.05. n = 5.

Figure 2

Effects of the traditional Chinese medicine formula (TCMF) on muscle fiber type characteristics in the psoas major muscle in finishing pigs. (A) Effects of TCMF on the cross-sectional area of the muscle fiber in the psoas major muscle of finishing pigs. (B) Effects of TCMF on muscle fiber diameter in the psoas major muscle of finishing pigs. (C) Effects of TCMF on muscle fiber density in the psoas major muscle of finishing pigs. (D) Effects of TCMF on muscle fiber types in the psoas major muscle of finishing pigs. (E, F, G) Enzyme histochemical staining of muscle fiber types in the psoas major muscle from pigs fed CON, TCMF1, and TCMF2 (100×), respectively. (H, I) Separate sections stained for mATPase and SDH, respectively. CON, control group, basal diet; TCMF1, basal diet+2.5 g/kg TCMF; TCMF2, basal diet+5 g/kg TCMF. Compared to CON, * represents p<0.05. n = 5. Bar = 100 μm.

Figure 3

Effects of traditional Chinese medicine formula (TCMF) on gene expression levels in the psoas major muscle in finishing pigs. (A) Effects of TCMF on the four isoforms of the myosin heavy chain (MyHC) gene expression level in the psoas major muscle in finishing pigs. (B) Effects of TCMF on expression levels of key genes (PGC-1α and CaN) in the regulation pathways of muscle fiber type in the psoas major muscle of finishing pigs. CON, control group, basal diet; TCMF1, basal diet+2.5 g/kg TCMF; TCMF2, basal diet+5 g/kg TCMF. Compared to CON, * represents p<0.05. n = 5. PGC-1α, peroxisome proliferator-activated receptor γ coactivator-1α; CaN, calcineurin.

Figure 4

Morphology and identification of porcine skeletal muscle satellite cells (SCs) (100×). (A) Morphology of SCs after incubation for 2 h. (B) Morphology of SCs after incubation for 24 h. (C) Morphology of SCs after incubation for 48 h. (D) Morphology of SCs after incubation for 120 h. (E) Identification of SCs using Desmin immunochemistry. (F) Identification of fibroblasts with Desmin immunochemistry. Fibroblasts were used as a negative control for the identification of SCs.

Figure 5

Porcine skeletal muscle satellite cells (SCs) proliferation curve from day 2 to day 6. (A) Porcine SCs proliferation curve was determined by the MTT method. n = 6. (B) Porcine SCs proliferation curve was measured by cell counting.

Figure 6

Induction of porcine skeletal muscle satellite cells (SCs) (100×). (A) Morphology of SCs after 2% horse serum induction for 24 h. (B) Morphology of SCs after 2% horse serum induction for 48 h. (C) Morphology of SCs after 2% horse serum induction for 72 h. (D) Morphology of SCs after 2% horse serum induction for 96 h.

Figure 7

Effects of traditional Chinese medicine formula (TCMF) water extraction on porcine skeletal muscle satellite cells (SCs) proliferation. (A) Effects of different levels (0, 0.0064, 0.032, 0.16, 0.8, 4, and 20 μg/mL) of TCMF water extraction on porcine SCs proliferation from day 1 to day 5. n = 6. (B) Comparison of the effects of different levels of TCMF water extraction on porcine SCs proliferation on the same day.

Figure 8

Effects of different levels of traditional Chinese medicine formula (TCMF) extractions (water extraction, ethyl acetate extraction and petroleum ether extraction) on four isoforms of the myosin heavy chain (MyHC) gene expression in porcine myocytes. (A) Effects of different levels (0, 1, and 5 μg/mL) of TCMF extractions (water extraction, ethyl acetate extraction, and petroleum ether extraction) on the MyHC I expression level. (B) Effects of different levels (0, 1, and 5 μg/mL) of TCMF extractions (water extraction, ethyl acetate extraction and petroleum ether extraction) on MyHC IIa expression level. (C) Effects of different level (0, 1, and 5 μg/mL) of TCMF extractions (water extraction, ethyl acetate extraction and petroleum ether extraction) on the MyHC IIb expression level. (D) Effects of different levels (0, 1, and 5 μg/mL) of TCMF extractions (water extraction, ethyl acetate extraction and petroleum ether extraction) on the MyHC IIx expression level. Compared to 0 μg/mL, ^* represents p<0.05. n = 6.