Matrine improves skeletal muscle atrophy by inhibiting E3 ubiquitin ligases and activating the Akt/mTOR/FoxO3α signaling pathway in C2C12 myotubes and mice

Abstract

Skeletal muscle wasting is a feature of cancer cachexia that increases patient morbidity and mortality. Matrine, the main bioactive component of Sophora flavescens, has been approved for the prevention and therapy of cancer cachexia in China. However, to the best of our knowledge, its mechanism in improving muscle wasting remains unknown. The present study demonstrated that matrine increases muscle fiber size and muscle mass in an in vivo CT26 colon adenocarcinoma cachexia mouse model. Concurrently, other cachexia symptoms, including body and organ weight loss, were alleviated. In in vitro experiments, matrine substantially improved C2C12 myoblast differentiation with or without dexamethasone treatment. In addition, matrine reduced C2C12 myotube atrophy and apoptosis induced by dexamethasone, tumor necrosis factor α and conditioned medium. Two E3 ubiquitin ligases, muscle RING‑finger containing protein‑1 and muscle atrophy F-box protein, which are specifically expressed in wasting skeletal muscle, were also significantly downregulated (P<0.05) by matrine both in C2C12 myotubes and skeletal muscle. Furthermore, matrine increased the phosphorylation of Akt, mTOR and FoxO3α in the atrophying C2C12 myotube induced by dexamethasone. In conclusion, matrine can alleviate muscle atrophy and improve myoblast differentiation possibly by inhibiting E3 ubiquitin ligases and activating the Akt/mTOR/FoxO3α signaling pathway.

Figure 1.

Effect of matrine on CT26-induced cancer cachexia symptoms, and CT26 tumor growth and cell proliferation. (A) Chemical structure of matrine. (B) Cumulative food intake of mice with different treatments in each group (n=10). (C) Mice tumor free bodyweight during the experiment (n=10). Levels of (D) TNFα, (E) IL-6 and (F) IL-1β in mice serum (n=10). (G) Calculated weight of CT26 tumors (n=10). (H) Effect of matrine on the cell viability of CT26 cells (n=4). (I) Inhibition curve of matrine to CT26 cell proliferation. Data are presented as the mean ± standard deviation. Statistical significance was determined by one-way ANOVA. ^###P<0.001 vs. NC. *P<0.05, **P<0.01, ***P<0.001 vs. CC. NC, negative control; Mat, matrine; CC, cancer cachexia.

Figure 2.

Matrine alleviates mice myofibers atrophy by down-regulating the expression of E3 ubiquitin ligase. (A) Hematoxylin-eosin staining of myofibers in mice gastrocnemius. (B) The statistical results of myofiber CSA distribution in each group (n>180 per group). (C) Mean muscle fibers CSA. (D) Reverse transcription-quantitative polymerase chain reaction detected MAFbx and MuRF1 mRNA expression in mice Ga (upper panels) and TA (lower panels) (n=6). (E) Representative western blot images of MuRF1 and MAFbx in mice Ga. (F and G) Statistical results of the western blot analysis (n=3). Data are presented as the mean ± standard deviation. Statistical significance was determined by one-way ANOVA. ^##P<0.01, ^###P<0.001, vs. NC. *P<0.05, **P<0.01, ***P<0.001 vs. CC. NC, negative control; Mat, matrine; CC, cancer cachexia; CSA, cross-sectional area; Ga, gastrocnemius; TA, tibialis anterior; MuRF1, muscle RING-finger containing protein-1; MAFbx, muscle atrophy Fbox protein.

Figure 3.

Matrine exhibits no significant cytotoxic effect to C2C12 myocytes and myotubes. (A) Inhibition rate and (B) IC₁₀ of matrine to C2C12 myocytes for 48 h determined by CCK-8 assay (n=4). (C-F) The mRNA levels of MHC subfamily proteins, including (C) MHC Ib, (D) MHC IIa, (E) MHC IIb and (F) MHC IIx, in myotubes following treatment with matrine (0.025, 0.05, 0.1, 0.2 and 0.4 mM) were analyzed by reverse transcription-quantitative polymerase chain reaction (n=3). Data are presented as the mean ± standard deviation. Statistical significance was determined by one-way ANOVA. **P<0.01, ***P<0.001 vs. untreated control. MHC, myosin heavy chain.

Figure 4.

Matrine promotes C2C12 myoblasts differentiation and fusion. (A) Immunofluorescence staining of MHC (green), MyoD (red) and DAPI (blue) in C2C12 myotubes. After getting close to 80% confluence, C2C12 myoblasts were treated with or without the indicated concentrations of matrine in differential medium for 72 h. Quantification of C2C12 myotubes (B) mean diameter (3–5 measures per myotube; n>150 per group) and (C) fusion index. Myotubes fusion index was calculated by the proportion of nuclei in myotubes to the total nuclei (n=6). Data are presented as the mean ± standard deviation. Statistical significance was determined by one-way ANOVA. ^#P<0.05 vs. 0.1 mM Mat. *P<0.05, **P<0.01, ***P<0.001 vs. NC. MHC, major histocompatibility complex; NC, negative control; Mat, matrine; MyoD, myogenic differentiation antigen.

Figure 5.

Matrine improves the inhibitory effect of dexamethasone on C2C12 myoblasts differentiation. (A) Immunofluorescence staining of MHC (green), MyoD (red) and DAPI (blue) in C2C12 myotubes. C2C12 myoblasts were treated with Dex combined with or without Mat in differential medium for 72 h. Quantification of C2C12. (B) Mean myotubes diameter (3–5 measures per myotube; n>150 per group) and (C) Fusion index (n=6). Data are presented as the mean ± standard deviation. Statistical significance was determined by one-way ANOVA. ^#P<0.05 vs. Dex + 0.1 mM Mat. *P<0.05, **P<0.01, ***P<0.001 vs. Dex. ^&&P<0.01, ^&&&P<0.001 vs. NC. MHC, myosin heavy chain.

Figure 6.

Anti-atrophic effect of matrine on C2C12 myotubes. (A) Immunofluorescence staining of MHC in C2C12 myotubes. C2C12 myotubes (B) diameter distribution and (C) mean diameter in each group. Ten images were selected from each group and each myotube was measured three to five times along the axis. In total, >50 myotubes were determined and ≥150 sets of data were collected. Data are presented as the mean ± standard deviation. Statistical significance was determined by one-way ANOVA. ^#P<0.001 vs. NC. *P<0.001 vs. Dex. ^&P<0.001 vs. TNFα. ^§P<0.001 vs. CM. NC, negative control; Dex, dexamethasone; Mat-L, 100 µM matrine; Mat-H, 200 µM matrine; CM, conditioned medium.

Figure 6.

Anti-atrophic effect of matrine on C2C12 myotubes. (A) Immunofluorescence staining of MHC in C2C12 myotubes. C2C12 myotubes (B) diameter distribution and (C) mean diameter in each group. Ten images were selected from each group and each myotube was measured three to five times along the axis. In total, >50 myotubes were determined and ≥150 sets of data were collected. Data are presented as the mean ± standard deviation. Statistical significance was determined by one-way ANOVA. ^#P<0.001 vs. NC. *P<0.001 vs. Dex. ^&P<0.001 vs. TNFα. ^§P<0.001 vs. CM. NC, negative control; Dex, dexamethasone; Mat-L, 100 µM matrine; Mat-H, 200 µM matrine; CM, conditioned medium.

Figure 7.

Matrine inhibits the expression of MAFbx and MuRF1 in C2C12 myotubes. (A) Representative western blot images of MuRF1 and MAFbx. (B) Quantitation of western blot analysis (n=3). (C) Immunofluorescence staining of MAFbx (red) and MuRF1 (green) in C2C12 myotubes (n=3). Data are presented as the mean ± standard deviation. Statistical significance was determined by one-way ANOVA. ^#P<0.001 vs. NC. *P<0.001 vs. Dex. NC, negative control; Dex, dexamethasone; Mat-L, 100 µM matrine; Mat-H, 200 µM matrine; MuRF1, muscle RING-finger containing protein-1; MAFbx, muscle atrophy Fbox protein.

Figure 8.

Effect of matrine on the Akt/mTOR/FoxO3α signalling pathway in C2C12 myotubes. Representative western blot images and densitometric quantification of the associated phosphorylated levels of (A) FoxO3α, Akt, (B) mTOR, (C) MAFbx and (D) MuRF1 in C2C12 myotubes. Dex, matrine and wortmannin were added to culture medium for 48 h at 100 µM, 0.1 mM and 10 nM, respectively. Data are presented as the mean ± standard deviation (n=3). Statistical significance was determined by one-way ANOVA. *P<0.05, **P<0.01 vs. Dex. ^##P<0.01, ^###P<0.001 vs. NC. ^§P<0.05 vs. Dex + Mat. NC, negative control; Dex, dexamethasone; Mat, matrine; Wor, wortmannin; p, phosphorylated; MuRF1, muscle RING-finger containing protein-1; MAFbx, muscle atrophy Fbox protein.

Figure 9.

Possible mechanism of matrine on inhibiting skeletal muscle atrophy. Through increasing phosphorylation of key proteins, matrine activates the Akt/mTOR/FoxO3α signaling pathway; therefore, increased muscle protein synthesis and decreased protein degradation occurs.