Effects of Alnus japonica Hot Water Extract and Oregonin on Muscle Loss and Muscle Atrophy in C2C12 Murine Skeletal Muscle Cells

Abstract

Background/Objectives: Sarcopenia is characterized by the loss of muscle mass and function, increases in mortality rate, and risk of comorbidities in the elderly. This study evaluated the effects of Alnus japonica hot water extract (AJHW) and its active compound, oregonin, on muscle atrophy and apoptosis in vitro. Methods: AJHW underwent phytochemical analysis. C2C12 cells were subjected to H₂O₂ and dexamethasone to induce oxidative stress and muscle loss, after which AJHW and oregonin were administered to assess their impacts on cell viability, apoptosis, muscle protein synthesis stimulation, and muscle protein degradation inhibition. Cell viability was assessed via an MTT assay, and apoptosis was analyzed by measuring Bcl-2, Bax, cleaved caspase-3, and cleaved PARP through Western blotting. Western blotting and RT-PCR were utilized to analyze MyoD, Myogenin, Atrogin-1, and MuRF1 protein and gene expression in a muscle atrophy model, as well as the Akt/mTOR and FoxO3α pathways. Results: AJHW was confirmed to contain oregonin, an active compound. AJHW and oregonin significantly increased cell viability and reduced apoptosis by upregulating Bcl-2 and downregulating Bax, cleaved caspase-3, and cleaved PARP. They significantly enhanced muscle protein synthesis through the upregulation of MyoD and Myogenin, while diminishing muscle degradation by downregulating Atrogin-1 and MuRF1. The activation of the Akt/mTOR pathway and inhibition of the FoxO3α pathway were also observed. Conclusions: AJHW and oregonin effectively prevented muscle cell apoptosis, promoted muscle protein synthesis, and inhibited muscle protein degradation in vitro. These results suggest that AJHW and oregonin could serve as therapeutic agents to prevent and treat sarcopenia.

Keywords: Alnus japonica; apoptosis; hot water extract; muscle atrophy; oregonin; sarcopenia.

Figure 1

TLC chromatograms of each sample compound and the references: (A) UV 254 nm, (B) 10% H₂SO₄, (C) ρ-anisaldehyde H₂SO₄, and (D) FeCl₃. The eluent system employed was chloroform/methanol/water = 70:30:4 (v/v/v). ① Oregonin, ② Alnus japonica ethanol extract (AJE), and ③ Alnus japonica hot water extract (AJHW).

Figure 2

Negative mode LC-MS/MS analysis of oregonin (1000 μg/mL). (A) Extracted ion chromatogram of oregonin and (B) product ion mass spectrum of oregonin.

Figure 3

Calibration curve and equation of oregonin (100, 50, 25, 10, 5, and 1 μg/mL of oregonin).

Figure 4

HPLC chromatogram of oregonin (100, 50, 25, 10, 5, and 1 μg/mL of oregonin).

Figure 5

HPLC chromatogram of (A) Alnus japonica ethanol extract (AJE) (1000 μg/mL) and (B) Alnus japonica hot water extract (AJHW) (1000 μg/mL).

Figure 6

Cytotoxicity of Alnus japonica hot water extract (AJHW) on C2C12 myoblasts. (A) Treatment concentrations: 0, 25, 50, 100, 200, 400, 600, 800, and 1000 μg/mL. (B) Treatment concentrations: 0, 2.5, 5, 10, 20, and 25 μg/mL. Cell viability was calculated as described in Section 4. Values are expressed as the mean ± S.E.M. (n = 5). *** p < 0.001 significantly different from that of the 0 μg/mL group.

Figure 7

Cytotoxicity of oregonin (ORE) on C2C12 myoblasts. Cell viability was calculated as outlined in the Section 4. Values are expressed as the mean ± S.E.M. (n = 4). *** p < 0.001 indicating a significant difference from the 0 μg/mL group.

Figure 8

Protective effect of (A) Alnus japonica hot water extract (AJHW) and (B) oregonin (ORE) on cell viability in H₂O₂-treated C2C12 myoblasts. Values are expressed as the mean ± S.E.M. (n = 4). *** p < 0.001 significantly different from that of [H₂O₂ (−)/AJHW (−)] and [H₂O₂ (−)/ORE (−)] group. ^# p < 0.05, ^## p < 0.01, ^### p < 0.001 significantly different from that of the [H₂O₂ (+)/AJHW (−)] and [H₂O₂ (+)/ORE (−)] group.

Figure 9

Protective effect of (A) Alnus japonica hot water extract (AJHW) and (B) oregonin (ORE) on cell viability in DEX-treated C2C12 myotubes. Values are expressed as the mean ± S.E.M. (n = 4). * p < 0.05, ** p < 0.01 significantly different from that of [DEX (−)/AJHW (−)], [DEX (−)/ORE (−)] group. ^# p < 0.05, ^## p < 0.01 significantly different from that of [DEX (+)/AJHW (−)], [DEX (+)/ORE (−)] group.

Figure 10

Protective effect of (A) Alnus japonica hot water extract (AJHW) and (B) oregonin (ORE) on apoptosis in H₂O₂-treated C2C12 myoblasts. Values are expressed as the mean ± S.E.M. (n = 4). *** p < 0.001 significantly different from that of [H₂O₂ (−)/AJHW (−)], [H₂O₂ (−)/ORE (−)] group. ^# p < 0.05, ^## p < 0.01, ^### p < 0.001 significantly different from those of the [H₂O₂ (+)/AJHW (−)] and [H₂O₂ (+)/ORE (−)] groups.

Figure 11

Anti-apoptotic effects of Alnus japonica hot water extract (AJHW) and oregonin (ORE) on H₂O₂-induced oxidative damage in C2C12 myoblasts. Western blotting was used to analyze the levels of (A–D) Bax, (E–H) Bcl-2, and β-actin, as described in Section 4. Values are expressed as the mean ± S.E.M. (n = 3). * p < 0.05, ** p < 0.01, *** p < 0.001 significantly different from that of [H₂O₂ (−)/AJHW (−)], [H₂O₂ (−)/ORE (−)] group. ^# p < 0.05, ^## p < 0.01 significantly different from those of the [H₂O₂ (+)/AJHW (-)] and [H₂O₂ (+)/ORE (−)] groups.

Figure 12

Anti-apoptotic effects of Alnus japonica hot water extract (AJHW) and oregonin (ORE) on H₂O₂-induced oxidative damage in C2C12. Western blotting was used to analyze the levels of (A–D) cleaved caspase-3, (E–H) cleaved PARP, and β-actin, as outlined in Section 4. Values are expressed as the mean ± S.E.M. (n = 3). ** p < 0.01, *** p < 0.001 significantly different from [H₂O₂ (−)/AJHW (−)], [H₂O₂ (−)/ORE (−)] group. ^# p < 0.05, ^## p < 0.01 significantly different from [H₂O₂ (+)/AJHW (−)] and [H₂O₂ (+)/ORE (−)] groups.

Figure 13

Effects of (A,C) Alnus japonica hot water extract (AJHW) and (B,D) oregonin (ORE) on dexamethasone-induced muscle atrophy in C2C12 myotubes. Values are expressed as the mean ± S.E.M. (n = 5). The scale bar represents 100 μm. * p < 0.05, *** p < 0.001 significantly different from that of [DEX (−)/AJHW (−)], [DEX (−)/ORE (−)] group. ^# p < 0.05, ^### p < 0.001 significantly different from that of [DEX (+)/AJHW (−)], [DEX (+)/ORE (−)] group.

Figure 14

Effect of Alnus japonica hot water extract (AJHW) and oregonin (ORE) on the muscle-degradation-related protein expression levels of (A–D) Atrogin-1, (E–H) MuRF1 in DEX-treated C2C12 myotubes. AJHW and ORE were added to DEX-treated C2C12 myotubes and cultured for 24 h. Protein expression levels were determined using Western blotting. The expression levels were normalized to that of β-actin and expressed relative to those in the CON group. Values are expressed as the mean ± S.E.M. (n = 4). ** p < 0.01, *** p < 0.001 significantly different from [DEX (−)/AJHW (−)], [DEX (−)/ORE (−)] group. ^# p < 0.05, ^## p < 0.01, ^### p < 0.001 significantly different from the [DEX (+)/AJHW (−)], [DEX (+)/ORE (−)] group.

Figure 15

Effect of Alnus japonica hot water extract (AJHW) and oregonin (ORE) on the muscle-synthesis-related protein expression levels of (A–D) MyoD, and (E–H) Myogenin in DEX-treated C2C12 myotubes. AJHW and ORE were added to DEX-treated C2C12 myotubes and cultured for 24 h. Protein expression levels were determined using Western blotting. The protein expression levels were normalized to β-actin and expressed relative to those in the CON group. Values are expressed as the mean ± S.E.M. (n = 4). * p < 0.05, ** p < 0.01 significantly different from that of [DEX (−)/AJHW (−)], [DEX (−)/ORE (−)] group. ^# p < 0.05, ^## p < 0.01 significantly different from the [DEX (+)/AJHW (−)], [DEX (+)/ORE (−)] group.

Figure 16

Effect of Alnus japonica hot water extract (AJHW) and oregonin (ORE) on the protein expression levels of (A–D) phospho-Akt and Akt, and (E–H) phospho-mTOR and mTOR in DEX-treated C2C12 myotubes. AJHW and ORE were separately added to these cells and incubated for 24 h. Protein expression was assessed via Western blotting, with β-actin as the normalization standard, and expressed relative to the CON group. Values are reported as the mean ± S.E.M. (n = 3). * p < 0.05, ** p < 0.01, *** p < 0.001, indicating significant differences from the [DEX (−)/AJHW (−)], [DEX (−)/ORE (−)] group. ^# p < 0.05, ^## p < 0.01, ^### p < 0.001 indicating significant differences from the [DEX (+)/AJHW (−)], [DEX (+)/ORE (−)] group.

Figure 16

Effect of Alnus japonica hot water extract (AJHW) and oregonin (ORE) on the protein expression levels of (A–D) phospho-Akt and Akt, and (E–H) phospho-mTOR and mTOR in DEX-treated C2C12 myotubes. AJHW and ORE were separately added to these cells and incubated for 24 h. Protein expression was assessed via Western blotting, with β-actin as the normalization standard, and expressed relative to the CON group. Values are reported as the mean ± S.E.M. (n = 3). * p < 0.05, ** p < 0.01, *** p < 0.001, indicating significant differences from the [DEX (−)/AJHW (−)], [DEX (−)/ORE (−)] group. ^# p < 0.05, ^## p < 0.01, ^### p < 0.001 indicating significant differences from the [DEX (+)/AJHW (−)], [DEX (+)/ORE (−)] group.

Figure 17

Effect of Alnus japonica hot water extract (AJHW) and oregonin (ORE) on the protein expression levels of (A–D) phospho-FoxO3α and FoxO3α in DEX-treated C2C12 myotubes. AJHW and ORE were separately added to these cells and incubated for 24 h. Protein expression was measured using Western blotting, normalized to β-actin, and reported relative to the CON group. Values are expressed as the mean ± S.E.M. (n = 3). ** p < 0.01, *** p < 0.001 indicating significant differences from the [DEX (−)/AJHW (−)], [DEX (−)/ORE (−)] group. ^# p < 0.05, ^## p < 0.01, ^### p < 0.001 indicating significant differences from the [DEX (+)/AJHW (−)], [DEX (+)/ORE (−)] group.

Figure 18

The structure of oregonin. The chemical structure was illustrated using ChemDraw Ultra 7.0 (CambridgeSoft, Cambridge, MA, USA).