Systematic Myostatin Expression Screening Platform for Identification and Evaluation of Myogenesis-Related Phytogenic in Pigs

Abstract

Skeletal muscle growth in livestock impacts meat quantity and quality. Concerns arise because certain feed additives, like beta-agonists, may affect food safety. Skeletal muscle is a specialized tissue consisting of nondividing and multinucleated muscle fibers. Myostatin (MSTN), a protein specific to skeletal muscle, is secreted and functions as a negative regulator of muscle mass by inhibiting the proliferation and differentiation of myoblasts. To enhance livestock muscle growth, phytogenic feed additives could be an alternative as they inhibit MSTN activity. The objective of this study was to establish a systematic screening platform using MSTN activity to evaluate phytogenics, providing scientific evidence of their assessment and potency. In this study, we established a screening platform to monitor myostatin promoter activity in rat L8 myoblasts. Extract of Glycyrrhiza uralensis (GUE), an oriental herbal medicine, was identified through this screening platform, and the active fractions of GUE were identified using a process-scale liquid column chromatography system. For in vivo study, GUE as a feed additive was investigated in growth-finishing pigs. The results showed that GUE significantly increased body weight, carcass weight, and lean content in pigs. Microbiota analysis indicated that GUE did not affect the composition of gut microbiota in pigs. In summary, this established rodent myoblast screening platform was used to identify a myogenesis-related phytogenic, GUE, and further demonstrated that the active fractions and compounds inhibited MSTN expression. These findings suggest a novel application for GUE in growth performance enhancement through modulation of MSTN expression. Moreover, this well-established screening platform holds significant potential for identifying and assessing a diverse range of phytogenics that contribute to the process of myogenesis.

Keywords: Glycyrrhiza uralensis; feed additive; myogenesis; myostatin; skeletal muscle.

Figure 1

Effect of various herb extracts (a–j) on L8 MSTN-Luc cells proliferation. L8 MSTN-Luc cells were treated with various herbal extracts, Andrographis paniculate (a), Centella asiatica (b), Glycyrrhiza uralensis (c), Gynostemma pentaphyllum (d), Platycodon grandifloras (e), Polygonum chinense (f), Portulaca oleracea (g), Saururus chinensis (h), Smilax china (i) and Taraxacum campylodes (j), in different concentrations or with 1% ethanol as vehicle control. L8 MSTN-Luc cell proliferation was measured using MTT assay. Different letters (a–d) denote significant differences (p < 0.05) among treatments. Each data point represents the mean ± SEM derived from three separate and independent experiments.

Figure 2

Effect of G. uralensis extract (GUE) on MSTN mRNA level and MSTN promoter activity in L8 MSTN-Luc cells. The effect of GUE on MSTN mRNA level were determined by reverse transcription-polymerase chain reaction (RT-PCR) (a). The relative ratio of MSTN mRNA level to internal control was calculated (b). The effect of GUE on cell proliferation was investigated by MTT assay (Figure 1c). MSTN promoter activity was measured by luciferase activity assay (c). The ratio of luciferase activity to cell proliferation (d) was used to evaluate GUE inhibitory effect. Different letters (a–d) denote significant differences (p < 0.05) among treatments. Each data point represents the mean ± SEM derived from three separate and independent experiments.

Figure 3

Systematic analysis of major active fractions of G. uralensis extract. The ethanolic extract of G. uralensis was purified using process-scale liquid column chromatography and separated into 10 fractions (a). The bioactive fraction was identified by the MSTN gene reporter assay in L8 MSTN-Luc cells treated with various GUE concentrations (b). The results are indicated as the ratio of luciferase activity to cell proliferation.

Figure 4

Chemical profile of the MSTN-inhibitory fraction (G9) in G. uralensis extract. The chemical profile of G. uralensis active fraction (G9) was analyzed using the RP-18 column and detected with DAD detection at 210–540 nm (a). Peaks of bioactive compounds and resulting chemical structures were identified as shown (b). Dose-response curves of three individual phytocompounds and the G9 fraction for MSTN suppression activity (c). Each data point represents the mean ± SEM.

Figure 5

Effect of GUE as a feed additive on body weight in growth-finishing pigs. Flow charts show the timeline for evaluating of GUE effect as a feed additive on growth-finishing pigs (a). After weaning, pigs were randomized into four groups. Standard control diet (CTL), low GUE concentration (LGUE), medium GUE concentration (MGUE), and high GUE concentration (HGUE) treatment of 10 animals in each group. GUE was used as a feed additive for 18 weeks. Body weight (b) and body gain weight (c) in each treatment group was monitored before and after 18-week treatment. Asterisks indicate the significant difference (p < 0.05) in body gain weight between HGUE and CTL groups in the same treatment week. Each data point represents the mean ± SEM.

Figure 6

The effect of GUE as a feed additive on carcass characteristics of pigs. Four groups of weaned pigs were fed standard control diet (CTL), or standard diet containing low GUE concentration (LGUE), medium GUE concentration (MGUE), or high GUE concentration (HGUE) for 18 weeks. Carcass weight (a), carcass percentage (b), carcass length (c), as well as the percentage of lean (d), fat (e), bone (f) and skin (g) of six representative pigs from each groups were measured and calculated at the end of 18 weeks. Each data point represents the mean ± SEM. Different letters denote statistical significance (p < 0.05) among groups.

Figure 7

The effect of GUE as a feed additive on gut microbiota in pigs. The bacterial composition at the genus level in the guts of three representative pigs fed control diet (CTL) or the diet containing low GUE concentration (LGUE), medium GUE concentration (MGUE), and high GUE concentration (HGUE) for 18 weeks were pyrosequenced, and the data were analyzed at the end of the 18 weeks.