Effect of pinoresinol and vanillic acid isolated from Catalpa bignonioides on mouse myoblast proliferation via the Akt/mTOR signaling pathway

doi:10.21203/rs.3.rs-1770207/v1

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Effect of pinoresinol and vanillic acid isolated from Catalpa bignonioides on mouse myoblast proliferation via the Akt/mTOR signaling pathway

https://doi.org/10.21203/rs.3.rs-1770207/v1

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Sarcopenia is a disease in which skeletal muscle decreases with age. Stimulating the proliferation and differentiation of muscle cells may help prevent sarcopenia. To discover effective natural substances enabling to treat muscle loss without side effects, we evaluated muscle growth with several compounds extracted from Catalpa bignonioides Walt. Among these compounds, pinoresinol and vanillic acid increased C2C12, a mouse myoblast cell line, proliferation the most without cytotoxicity. These substances activated the Akt/mammalian target of rapamycin (mTOR) pathway, which positively regulates the proliferation of muscle cells. In addition, they strongly bound to insulin-like growth factor 1 receptor (IGF-1R), which is an upstream of the Akt/mTOR pathway, indicating that both pinoresinol and vanillic acid stimulate myoblast proliferation through direct interaction with IGF-1R. These results suggest that pinoresinol and vanillic acid may improve the proliferation of skeletal muscle via IGF-1R/Akt/mTOR signaling and thus alleviate diseases such as sarcopenia.

Catalpa bignonioides

pinoresinol

vanillic acid

sarcopenia

IGF-1 signaling.

Sarcopenia refers to the gradual loss of skeletal muscle mass and strength associated with age. Sarcopenia is inevitable in most people and is considered a major cause of disability and fragility in older adults, reducing their quality of life. It begins at the age of 40 years, with a decrease in muscle mass of up to 8% decennially; this rate may double by the age of 70 years [1]. Loss of skeletal muscle mass and function is caused by impaired myogenesis [2]. Skeletal muscle formation is achieved through proliferation and differentiation of muscle fibers, and the insulin-like growth factor 1 (IGF-1)/Akt/mammalian target of rapamycin (mTOR) signaling pathway, which stimulates them, is considered to be a master regulator of skeletal myogenesis [3, 4]. Although IGF-1 upregulation inhibited sarcopenia in animal studies, the administration of IGF-1 itself had a minor effect in older adults [5], encouraging further studies on other compounds that can achieve the desired effect. Therefore, compounds that stimulate skeletal myogenesis and IGF-1 can help overcome age-related skeletal muscle loss.

Steroidal androgens of the protein anabolic steroid class are drugs typically used to stimulate muscle enhancement. The biological efficacy of these drugs is demonstrated by muscle mass increase, growth spurts in premature children, and bone loss attenuation in older adults [6, 7], These compounds are prescribed for various therapeutic purposes. However, long-term or excessive usage may cause side effects, such as skin diseases [7] or reproductive and endocrine functional deterioration [8, 9]. Aiming to treat sarcopenia via metabolic changes, Belli et al. reported that trimetazidine, a metabolic modulator drug, may induce myoblast differentiation in a cell line and increase muscle strength in mice [10]. Although trimetazidine is used in the treatment of angina, its long-term use has been associated with gastrointestinal disturbances, vomiting, and nausea [11]. Therefore, there is a need for a compound that prevents skeletal muscle mass loss without increasing side effect risks.

Catalpa bignonioides Walt. (Bignoniaceae) is a bean tree native to southeastern America. It has been used in traditional medicinal practices for respiratory diseases, scrofulous ulcers, and helminthic infections, among others. A previous study on the bioactivity of C. bignonioides extracts [12] has shown that its flowers, leaves, and capsule valves have antioxidant activity [13]. Other studies revealed that catalpic acid, a conjugated triene fatty acid abundant in C. bignonioides, may improve insulin homeostasis by decreasing fat accumulation in the adipose tissue of mice [14]. Recently, we identified the constituents of the methanol extract of C. bignonioides fruits through phytochemical analysis, revealing that some compounds had properties stimulating α-glucosidase inhibition and insulin secretion [15]. Although studies evaluating the biological activities of compounds isolated from C. bignonioides are on-going, the impact of C. bignonioid-derived substances on muscle diseases, such as sarcopenia, remains unclear. Herein, we aimed to identify components from the fruits of C. bignonioides extracts that promote muscle proliferation and differentiation via Akt/mTOR signaling.

2.1. Materials

Dulbecco modified eagle medium (DMEM) was purchased from Welgene (Gyeongsangbuk-do, Korea). Fetal bovine serum (FBS) was purchased from Omega Scientific, Inc. (Tarzana, CA, USA). Penicillin and streptomycin were purchased from Invitrogen (Carlsbad, CA). Horse serum (HS), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), dimethyl sulfoxide (DMSO), and radioimmunoprecipitation assay buffer were purchased from Sigma-Aldrich (St. Louis, MO, USA). NuPAGE 4–12% Bis-Tris gel was purchased from Life Technologies (Carlsbad, CA, USA). Ployvinylidine fluoride membranes were purchased from Bio-Rad Laboratories (Hercules, CA). Rabbit anti-mouse phospho-Smad2(Ser465/467)/3(Ser423/425) (Cat#8828), rabbit anti-mouse Smad4 (Cat#46535), rabbit anti-mouse phospho-Akt (Ser473, Cat#4060), rabbit anti-mouse phospho-mTOR (Ser2448, Cat#5536), rabbit anti-mouse phospho-ribosomal protein S6 kinase (p70S6K) (Thr421/Ser424, Cat#9204), rabbit anti-mouse phospho-eukaryotic initiation factor 4E-binding protein 1 (4E-BP1) (Thr37/46, Cat#2855), goat anti-rabbit IgG (Cat#7074), and goat anti-mouse IgG (Cat#7076) were purchased from Cell Signaling Technology (Danvers, MA, USA). Mouse anti-mouse myoblast determination protein 1 (MyoD) (Cat#sc-377460), mouse anti-mouse myogenin (Cat#sc-12732), and mouse anti-mouse β-actin (Cat#sc-47778) were purchased from Santa Cruz Biotechnology (Dallars, TX, USA).

2.2. Plant material

C. bignonioides fruits were collected from the Arboretum of Seoul National University in Suwon, Korea in 2021 and authenticated by Dr. Rack-Seon Seong, a director of the Center of Natural Resources Research, Jeonnam Bioindustry Foundation. A voucher specimen (CB202106) was deposited at the Korea Basic Science Institute (Chuncheon, Korea).

2.3. Extraction and isolation

The dried fruits of C. bignonioides (1.3 kg) were extracted with MeOH (5 L × 3 times) under sonication at 30 ℃ for 4 h to yield an extract (91.0 g), which was then dissolved in H₂O and successively partitioned using CHCl₃ and EtOAc to obtain CHCl₃ (CB1, 16.0 g), EtOAc (CB2, 2.5 g), and H₂O (CB3, 71.0 g) extracts after removing the solvents in vacuo.

The CHCl₃ fraction was subjected to silica gel CC and eluted with a gradient of hexane : acetone (40:1 → 2.5:1, v/v) and CHCl₃ : MeOH (20:1 → 2.5:1, v/v) to yield nine sub-fractions, CB1A (3.0 g), CB1B (2.4 g), CB1C (1.0 g), CB1D (1.5 g), CB1E (1.0 g), CB1F (1.2 g), CB1G (0.8 g), CB1H (1.0 g), and CB1I (0.5 g). The CB1F fraction was applied to a YMC RP-18 column, which was eluted with MeOH : H₂O (1.3:1, v/v), yielding four smaller fractions, CB1F1 (58.2 mg), CB1F2 (41.5 mg), CB1F3 (18.4 mg), and CB1F4 (16.5 mg). The CB1F1 fraction was subjected to HPLC using a J’sphere ODS H-80 250 mm × 20 mm column, eluted with 28% MeCN in H₂O at a flow rate of 3 mL/min to yield 1 (6.8 mg) and 2 (7.1 mg). The CB1F2 fraction was subjected to the same HPLC conditions, except that the elution solvent was 40% MeCN in H₂O, to afford 3 (8.1 mg).

The H₂O fraction (CB3, 71.0 g) was chromatographed on a Diaion HP-20 column and eluted with H₂O containing increasing concentrations of MeOH (25, 50, and 100%) to obtain three subfractions, CB3A (10.0 g), CB3B (13.0 g), and CB3C (6.0 g). The CB3B fraction was subjected to silica gel CC and eluted with a gradient of CHCl₃ : MeOH (10:1 → 2.5:1, v/v) to yield three sub-fractions, CB3B1 (2.0 g), CB3B2 (2.7 g), and CB3B3 (2.0 g). The CB3B1 fraction was applied to a silica gel column and eluted with CHCl₃ : MeOH : H₂O (5:1:0.1, v/v), CB3B11 (31.0 mg), CB3B12 (96.0 mg), CB3B13 (82.0 mg), CB3B14 (150.4 mg), CB3B15 (66.7 mg), and CB3B16 (213.8 mg). The CB3B14 fraction was subjected to HPLC purification under 40% MeCN to yield 13 (26.2 mg) and 14 (6.8 mg). The CB3B16 fraction was subjected to the same HPLC conditions, except that elution with 23% MeCN in H₂O afforded 15 (140.0 mg). The CB3C fraction was subjected to silica gel CC and eluted with a gradient of CHCl₃ : MeOH (10:1 → 2.5:1, v/v) to yield three sub-fractions, CB3C1 (0.4 g), CB3C2 (1.5 g), and CB3C3 (1.0 g). The CB3C1 fraction was applied to a YMC RP-18 column and eluted with MeOH : H₂O (1:1, v/v), yielding three smaller fractions, CB3C11 (0.2 g), CB3C12 (55.5 mg), and CB3C13 (14.0 mg). The CB3C11 fraction was subjected to HPLC using a J’sphere ODS H-80 250 mm × 20 mm column, eluted with MeCN : H₂O (18:82), and a flow rate of 3 mL/min to yield 4 (55.9 mg), 5 (7.3 mg), and 6 (14.3 mg). The CB3C2 fraction was applied to a YMC RP-18 column, which, when eluted with MeOH : H₂O (1:1, v/v), yielded three smaller fractions, CB3C21 (0.2 g), CB3C22 (0.6 g), and CB3C23 (0.2 g). The CB3C21 fraction was subjected to HPLC using a J’sphere ODS H-80 250 mm × 20 mm column, eluted with MeCN : H₂O (30:70), and a flow rate of 3 mL/min to yield 7 (35.5 mg), whereas the CB3C23 fraction gave 8 (30.1 mg), 9 (18.5 mg), and 10 (6.3 mg). The CB3C3 fraction was applied to a YMC RP-18 column, which when eluted with MeOH : H₂O (1.4:1, v/v), yielded four smaller fractions, CB3C31 (42.8 mg), CB3C32 (0.1 g), CB3C33 (30.8 mg), and CB3C34 (18.6 g). The CB3C32 fraction was subjected to HPLC using a J’sphere ODS H-80 250 mm × 20 mm column, eluted with MeCN : H₂O (25:75), at a flow rate of 3 mL/min to yield 11 (11.3 mg). The CB3C34 fraction was subjected to the same HPLC conditions, except that the eluding solvent was MeCN : H₂O (23:77), to afford 12 (7.1 mg).

2.4. Cell culture and differentiation

C2C12 cells (mouse myoblast cell line) were maintained in DMEM supplemented with 10% FBS, penicillin (100 U/mL), and streptomycin (100 µg/mL). For differentiation, when the cell confluence reached approximately 80%, the medium was replaced with DMEM containing 2% HS. DMEM containing 2% FBS was replaced every other day, and differentiation proceeded for 6 days. All cell cultures were maintained at 37 ℃ in a 5% CO₂ incubator.

2.5. Cytotoxicity assay

The cytotoxicity of compounds extracted from C. bignonioides was assessed using a colorimetric assay. In total, 1 × 10⁵ cells/mL were seeded in 96-well plates and incubated with the test compounds for 24 h. Thereafter, 100 µg/mL MTT was added to each well. After 2.5 h incubation at 37 ℃, the supernatants were aspirated, and cells were treated with DMSO to dissolve the formazan crystals. The absorbance of the colored solution was determined at 540 nm using a SpectraMax M2/M2e spectrophotometer (Molecular Devices, San Jose, CA, USA).

2.6. Cell proliferation activity

To measure skeletal muscle cell proliferation activity, C2C12 cells were seeded at a concentration of 5 × 10⁴ cells/mL in a 96-well plate, and the medium was replaced with DMEM containing 2% HS two days later to induce differentiation. The compounds were added whenever the medium (DMEM with 2% HS) was changed every other day. Six days after differentiation induction, cell proliferation was assessed using a 5-bromo-2’-deoxyuridine (BrdU) assay kit (Millipore, Billerica, MA, USA).

2.7. Western blot

Cell lysates were prepared using radioimmunoprecipitation assay buffer. Quantified protein lysates were loaded onto NuPAGE 4–12% Bis-Tris gels, which were then blotted onto a polyvinylidene fluoride membrane. Primary antibodies, including rabbit anti-mouse phospho-Smad2/3, rabbit anti-mouse Smad4, rabbit anti-mouse phospho-Akt, rabbit anti-mouse phospho-mTOR, rabbit anti-mouse phospho-p70S6K, rabbit anti-mouse phospho-4E-BP1, mouse anti-mouse MyoD, mouse anti-mouse myogenin, and mouse anti-mouse β-actin antibodies were diluted at 1:1000 and incubated overnight at 4 ℃. Secondary antibodies, including goat anti-rabbit IgG and goat anti-mouse IgG, were diluted at 1:3000 and incubated for 1.5 h at 25 ℃. Signals were developed using the SuperSignal West Femto Trial Kit (Thermo Fisher Scientific; Waltham, MA, USA), and images were acquired using Fusion FX (Vilber Lourmat Ste, Collegien, France) or VISQUE^® InVivo Smart-LF (Vieworks. Co, Ltd., Anyang-si, Korea).

2.8. In silico molecular docking simulation

To verify the potential active chemicals that could act as IGF-1 receptor (IGF-1R) agonists, a molecular docking study was performed. First, the crystal structure of IGF-1R (PDB ID: 1IGR) was obtained from the Protein Data Bank (PDB, http://www.pdb.org; accessed on January 1, 2022). A docking simulation was performed using AUTODOCK VINA to investigate whether the potential active chemicals bind to IGF-1R [16] and LIGPLOT to analyze IGF-1R and chemical interactions [17]. The 2-dimensional interaction map shows hydrogen bonds in green and labeled non-ligand residues involved in hydrophobic contact in red.

2.9. Statistical analysis

Variables were compared using two-tailed one-way ANOVA with Tukey’s post-hoc test using Prism software (Version 4.00; GraphPad Inc.; La Jolla, CA, USA). Findings were considered statistically significant at p-values of < 0.05.

3.1. Pinoresinol and vanillic acid facilitated C2C12 cell proliferation

To examine the effect of 15 constituents from C. bignonioides fruit extracts on skeletal muscle growth, we measured cell proliferation activity in cell line C2C12 (Fig. 1) [15]. Prior to examining the effect on cell proliferation, the viability of C2C12 myoblasts was evaluated using MTT colorimetric assay. We confirmed that none of the compounds, except compound 9 (6-O-trans-feruloyl catalpol), showed toxicity to C2C12 cells at a concentration of 50 µM (Fig. 2A).

Next, the proliferation effect of skeletal muscle cells was measured during the differentiation period by BrdU cell proliferation assay, except for compound 9, which was excluded because of its toxicity. Compared to the control conditions, most compounds triggered approximately 1.2-fold increase in cell proliferation (Fig. 2B). Among them, compound 3 (pinoresinol) and compound 5 (vanillic acid) showed a 1.8-fold increase.

To determine effects at low concentrations, cell proliferation was measured in a dose-dependent manner using pinoresinol and vanillic acid, which had the greatest effect among the compounds. Pinoresinol and vanillic acid showed cell proliferation activity during the differentiation phase even at concentrations of 6.25 µM and 12.5 µM, respectively (Fig. 3).

3.2. Pinoresinol and vanillic acid stimulated Akt/mTOR signaling pathway in C2C12 cells

Myogenesis, differentiation, and maturation of skeletal muscle cells are regulated by signaling pathways activated by the transforming growth factor beta (TGF-β) superfamily [18]. To determine whether cell proliferation activity of pinoresinol and vanillic acid was mediated by TGF-β signaling, we performed western blotting for Smad proteins in C2C12 differentiated cells treated with either compound. We found that p-Smad2 and p-Smad3 were not significantly downregulated by pinoresinol or vanillic acids (Fig. 4). Furthermore, the expression level of Smad4 was mildly decreased by these compounds.

IGF-1 signaling is a positive regulator of muscle cell proliferation and differentiation [19]. To examine the activation of IGF-1 signaling by pinoresinol and vanillic acid, we analyzed the phosphorylation levels of Akt, mTOR, and p70S6K using western blotting. We found that p-Akt, p-mTOR, and p-p70S6K levels were increased by these compounds (Fig. 4). In addition, these compounds decreased the phosphorylation of 4E-BP1, indicating suppression of its growth inhibitory function. Furthermore, essential regulators that induce muscle differentiation, such as MyoD and myogenin, were significantly increased in C2C12 myoblasts treated with pinoresinol or vanillic acid. These results suggest that pinoresinol and vanillic acid stimulate myogenic differentiation by regulating Akt/mTOR signaling in mouse muscle cells.

3.3. Pinoresinol and vanillic acid were docked into IGF-1 receptor through in silico analysis

We showed that the levels of protein expression involved in IGF-1 signaling were highly upregulated in pinoresinol- or vanillic-acid-treated C2C12 cells. To determine the potential of these two active compounds to bind to the IGF-1R, we simulated the docking of pinoresinol, vanillic acid, and IGF-1R. Docking of the ligand-protein complexes was successful, as both compounds stably posed to the active sites of IGF-1R (Fig. 5A to 5C). In addition, dihydrotestosterone (DHT), used as a positive control [20], was shown in the docking simulation to stably pose the active site of IGF-1R with a similar low binding energy value as pinoresinol or vanillic acid (Fig. 5D and Table 1).

Table 1

Results of docking simulations of two active chemicals (pinoresinol and vanillic acid) with IGF-1R (PDB ID: 1IGR). DHT is dihydrotestosterone used as positive control.
Receptor (Binding site)	Ligand	Binding energy (kcal/mol)
IGF-1R (1IGR-active site)	Pinoresinol Vanillic acid DHT	-6.7 -5.2 -7.0

In this study, we demonstrated the effects on myoblast proliferation of two compounds extracted from C. bignonioides: pinoresinol and vanillic acid. Pinoresinol is a biologically active ligand mainly found in medicinal plants, such as Styrax sp. and Forsythia suspense, and in olive oil [21, 22]. Pinoresinol possesses antioxidant, anti-inflammatory, and antifungal activities, and has been used in traditional medicine for a long time [21–23]. A recent study demonstrated that the effect of defatted sesame seeds in alleviating hypoglycemia is mediated by the inhibitory function of α-glucosidase activity of pinoresinol [24]. In a study on anticancer activity, pinoresinol was found to induce apoptosis and to suppress migration in human liver cancer cells [25]. Vanillic acid, an oxidative form of vanillin and phenolic compound, has also been used in folk medicine. Vanillic acid is abundant in the root of Angelica sinensis, also known as female ginseng, and exhibits antioxidant, anticancer, and cardioprotective activities [26–28]. However, no studies have examined the effect of vanillic acid or pinoresinol on muscle cells or muscle-related diseases. This study is first to show that pinoresinol and vanillic acid extracted from an edible plant promote proliferation in myoblasts.

Skeletal muscles play pivotal roles in physical activity and energy metabolism. Growth and maintenance of skeletal muscle are essential for the management of sarcopenia, an age-related disease in which skeletal muscle decreases. The growth and differentiation of skeletal muscle are regulated by negative and positive regulators, specifically, TGF-β and IGF-1/Akt/mTOR pathways, respectively. TGF-β family members, including myostatin, are known to inhibit myogenic differentiation in cultured primary myoblasts or myoblast cell lines, such as C2C12 [29–31]. C2C12 myoblasts lacking the TGB-β1 signal due to mutation of TβR II, a component of the TGF-β receptor, cannot form myotubes [32]. IGF-1 binds to IGF-1R, phosphorylates insulin receptor substrate-1 (IRS-1), an adaptor protein inside the cell, and sequentially phosphorylates phosphoinositide 3-kinase (PI3K) and Akt. Meanwhile, mTOR, which is a downstream target of Akt, phosphorylates p70S6K to promote protein synthesis and 4E-BP1 to induce translation initiation [33]. The Akt/mTOR/p706K pathway mediates downstream signaling of IGF-1 to promote protein synthesis and body growth and promotes both cell proliferation and differentiation in cultured myoblasts [34]. IGF-1 also induces cell differentiation by inducing the expression of myogenic regulatory factors (MEFs) such as MyoD and myogenin during myogenic differentiation [35]. In the L6E9 cell line (rat-derived myoblast) in the process of differentiation, when IGF-1 was overexpressed, myogenin levels increased, and myotubes became enlarged [36]. We demonstrated that natural substances with muscle proliferation activity, pinoresinol and vanillic acid, increased the expression of MyoD and myogenin, and phosphorylation of downstream targets of IGF-1, including Akt, mTOR, and p70S6K. Furthermore, through in silico docking analysis, we showed the potential of these substances to bind to the active site of IGF-1R.

Although TGB-β is known to negatively regulate myoblast differentiation by suppressing the expression of two MRFs (MyoD and myogenin) through Smad3 [18, 37–39], our study confirmed that the myogenic effects of pinoresinol and vanillic acid were unlikely achieved through the TGB-β/Smad pathway.

In summary, this study demonstrated that pinoresinol and vanillic acid isolated from C. bignonioides stimulated the Akt/mTOR pathway to promote proliferation and differentiation of myoblasts, suggesting that they bind to IGF-1R, upstream of Akt/mTOR. These findings may provide fundamental data for pinoresinol and vanillic acid as novel therapeutic agents to treat muscle-related diseases such as sarcopenia by inhibiting muscle loss.

Funding

This research was financially supported by the Ministry of Small and Medium-sized Enterprises (SMEs) and Startups (MSS), Korea, under the “Regional Specialized Industry Development Plus Program (R&D+, S3092691)” supervised by the Korea Institute for Advancement of Technology (KIAT).

Data availability

Data is available upon request.

Authors' contributions

Seo-Young Kim: Conceptualization, Data curation, Formal analysis. Sung-Pil Kwon: Conceptualization, Funding acquisition, Investigation. SeonJu Park: Formal analysis. Su-Hyeon Cho: Formal analysis, Resources. Youngse Oh: Formal analysis, Resources. Seung Hyun Kim: Formal analysis. Yoon Ho Park: Formal analysis, Visualization. Hyun Suk Jung: Formal analysis, Visualization. Deug-chan Lee: Funding acquisition. Hoibin Jeong: Data curation, Investigation, Project administration, Validation, Writing - original draft. Kil-Nam Kim: Conceptualization, Data curation, Funding acquisition, Project administration, Supervision.

Ethics approval

Not applicable.

Consent to participate

Not applicable.

Consent for publication

Not applicable.

Conflicts of Interest

The authors declare no conflict of interest.

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No competing interests reported.

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Effect of pinoresinol and vanillic acid isolated from Catalpa bignonioides on mouse myoblast proliferation via the Akt/mTOR signaling pathway

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Abstract

Figures

1. Introduction

2. Materials And Methods

2.1. Materials

2.2. Plant material

2.3. Extraction and isolation

2.4. Cell culture and differentiation

2.5. Cytotoxicity assay

2.6. Cell proliferation activity

2.7. Western blot

2.8. In silico molecular docking simulation

2.9. Statistical analysis

3. Results

3.1. Pinoresinol and vanillic acid facilitated C2C12 cell proliferation

3.2. Pinoresinol and vanillic acid stimulated Akt/mTOR signaling pathway in C2C12 cells

3.3. Pinoresinol and vanillic acid were docked into IGF-1 receptor through in silico analysis

4. Discussion

5. Conclusions

Declarations

References

Additional Declarations

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