Handelin alleviates cachexia- and aging-induced skeletal muscle atrophy by improving protein homeostasis and inhibiting inflammation

Abstract

Background: Handelin is a bioactive compound from Chrysanthemum indicum L. that improves motor function and muscle integrity during aging in Caenorhabditis elegans. This study aimed to further evaluate the protective effects and molecular mechanisms of handelin in a mouse muscle atrophy model induced by cachexia and aging.

Methods: A tumour necrosis factor (TNF)-α-induced atrophy model was used to examine handelin activity in cultured C2C12 myotubes in vitro. Lipopolysaccharide (LPS)-treated 8-week-old model mice and 23-month-old (aged) mice were used to examine the therapeutic effects of handelin on cachexia- and aging-induced muscle atrophy, respectively, in vivo. Protein and mRNA expressions were analysed by Western blotting, ELISA and quantitative PCR, respectively. Skeletal muscle mass was measured by histological analysis.

Results: Handelin treatment resulted in an upregulation of protein levels of early (MyoD and myogenin) and late (myosin heavy chain, MyHC) differentiation markers in C2C12 myotubes (P < 0.05), and enhanced mitochondrial respiratory (P < 0.05). In TNF-α-induced myotube atrophy model, handelin maintained MyHC protein levels, increased insulin-like growth factor (Igf1) mRNA expression and phosphorylated protein kinase B protein levels (P < 0.05). Handelin also reduced atrogin-1 expression, inhibited nuclear factor-κB activation and reduced mRNA levels of interleukin (Il)6, Il1b and chemokine ligand 1 (Cxcl1) (P < 0.05). In LPS-treated mice, handelin increased body weight (P < 0.05), the weight (P < 0.01) and cross-sectional area (CSA) of the soleus muscle (P < 0.0001) and improved motor function (P < 0.05). In aged mice, handelin slightly increased the weight of the tibialis anterior muscle (P = 0.06) and CSA of the tibialis anterior and gastrocnemius muscles (P < 0.0001). In the tibialis anterior muscle of aged mice, handelin upregulated mRNA levels of Igf1 (P < 0.01), anti-inflammatory cytokine Il10 (P < 0.01), mitochondrial biogenesis genes (P < 0.05) and antioxidant-related enzymes (P < 0.05) and strengthened Sod and Cat enzyme activity (P < 0.05). Handelin also reduced lipid peroxidation and protein carbonylation, downregulated mRNA levels of Fbxo32, Mstn, Cxcl1, Il1b and Tnf (P < 0.05), and decreased IL-1β levels in serum (P < 0.05). Knockdown of Hsp70 or using an Hsp70 inhibitor abolished the ameliorating effects of handelin on myotube atrophy.

Conclusions: Handelin ameliorated cachexia- and aging-induced skeletal muscle atrophy in vitro and in vivo, by maintaining homeostasis of protein synthesis and degradation, possibly by inhibiting inflammation. Handelin is a potentially promising drug candidate for the treatment of muscle wasting.

Keywords: Aging; Cachexia; Handelin; Inflammation; Protein homeostasis; Skeletal muscle atrophy.

Figure 1

Effects of handelin on C2C12 myotubes differentiation. (A) Schematic diagram of the experimental design of C2C12 myoblasts treated with handelin for 6 days. (B, C) C2C12 myoblasts were cultured to 70–80% confluent, then were treated with differentiation medium and handelin (0–125 nM) together every day for 6 days. The protein expressions of early differentiation marker myogenin and MyoD (B), and late differentiation marker MyHC (C) were determined by western blotting and quantified by ImageJ. n = 3–4. (D) Schematic diagram of the experimental design of C2C12 myotubes treated with handelin for 48 h. (E, F) The differentiated C2C12 myotubes were treated with handelin (0–250 nM) for 48 h. the protein expressions of myogenin, MyoD (E), and MyHC (F) were determined by western blotting and quantified by ImageJ. n = 3–4. Values are mean ± SD. Significance was determined using one‐way ANOVA (B–F). *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001 versus vehicle‐treated group (0 nM handelin). Data are representative of at least two independent experiments.

Figure 2

Effects of handelin on TNF‐α treated C2C12 myotubes. (A) Schematic diagram of the experimental design of C2C12 myotubes treated with TNF‐αand handelin together for 48 h. (B, C) The differentiated C2C12 myotubes were treated with TNF‐α (20 ng/mL) and handelin (0–125 nM) together for 48 h. The protein expressions of MyHC were determined by western blotting and quantified by ImageJ (B). n = 3. The myotubes structure was visualize by immunostaining. Scale bar: 100 μm (C). The myotubes diameter was quantified by ImageJ (D). Values are mean ± SD. Significance determined using one‐way ANOVA (B, D). *P < 0.05 and ****P < 0.0001 versus vehicle‐treated group (0 nM handelin). Data are representative of at least two independent experiments.

Figure 3

Effects of handelin on LPS‐challenged mice. (A) Schematic diagram of the experimental design of 8‐week‐old mice treated with LPS and handelin for 10 days. (B–F) Mice were administrated LPS (2 mg/kg) and handelin (0, 10 and 20 mg/kg) by intraperitoneal injection every day for 10 days, and control group mice received equal volume solvent. n = 7–8 per group. (B) Body weight of each group mice. (C) Average accumulative food intake of each group mice. (D) Representative images of the soleus and gastrocnemius muscles of each group mice and the weight of the soleus muscle of each group mice were normalized to body weight. (E) Representative photomicrographs of H&E‐stained soleus muscle sections and cross‐sectional area (CSA) from each group mice. Scale bar: 50 μm. (F) Functional tests including grip strength, rotarod test and electric shocks. Values are mean ± SD. Significance determined using one‐way ANOVA (B, E and F). **P < 0.01, ***P < 0.001 and ****P < 0.0001 versus LPS‐treated group.

Figure 4

Effects of handelin on aged mice. (A) Schematic diagram of the experimental design of 23‐month‐old mice treated with vehicle or handelin for 2 months. (B–F) Aged mice were administrated handelin (10 mg/kg) or equal volume solvent (vehicle control) by intraperitoneal injection every day for 1 months. (B) Body weight of each group mice. n = 7. (C) Representative images of the soleus, gastrocnemius and tibialis anterior muscles of each group mice. (D) The weights of the tibialis anterior and gastrocnemius muscle of each group mice were normalized to body weight. n = 3. (E) Representative photomicrographs of H&E‐stained tibialis anterior muscle sections and CSA from each group mice. Scale bar: 100 μm. n = 3. (F) Representative photomicrographs of H&E‐stained gastrocnemius muscle sections and CSA from each group mice. Scale bar: 100 μm. n = 3. Values are mean ± SD. Significance determined using unpaired t test (B, D, E and F). ****P < 0.0001 versus vehicle‐treated group.

Figure 5

Effects of handelin on protein synthesis and degradation pathways in TNF‐α treated C2C12 myotubes and aged mice. (A–E) C2C12 myotubes were treated with TNF‐α (20 ng/mL) and handelin (0–125 nM) together for 48 h. n = 3. The mRNA expression of Igf1 was measured by qPCR (A). The protein expressions of phosphorylated AKT and total AKT, phosphorylated mTOR and total mTOR were measured by western blotting and quantified by ImageJ (B). The global protein synthesis was assayed by puromycin incorporation and western blotting (C). The mRNA expression of Fbxo32 and Trim63 was measured by qPCR (D). Protein expression of MAFbx and global protein degradation were measured by western blotting analysis and quantified by ImageJ (E). (F) Aged mice were administrated handelin (10 mg/kg) or equal volume solvent (vehicle control) by intraperitoneal injection every day for 1 months. The mRNA expression of Igf1, Fbxo32, Trim63 and Mstn in tibialis anterior muscle was measured by qPCR. n = 3. Values are mean ± SD. Significance determined using one‐way ANOVA (A, B, D and E) and unpaired t test (F). *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001 versus vehicle‐treated group.

Figure 6

Effects of handelin on NF‐κB pathways and related inflammatory cytokines production in TNF‐α treated C2C12 myotubes and aged mice. (A, B) The differentiated C2C12 myotubes were treated with TNF‐α (20 ng/mL) and handelin (0–125 nM) together for 48 h. n = 3. (A) The protein expressions of phosphorylated IκBα and total IκBα, phosphorylated p65 and total p65 were measured by western blotting and quantified by ImageJ. (B) The mRNA expression of proinflammatory cytokines Il6, Cxcl1, IL‐1β, and anti‐inflammatory cytokine Il10 were measured by qPCR. (C) Aged mice were administrated handelin (10 mg/kg) or equal volume solvent (vehicle control) by intraperitoneal injection every day for 1 months. The mRNA expression of proinflammatory cytokines Il6, Cxcl1, Il1b, Tnf and anti‐inflammatory cytokine Il10 in tibialis anterior muscle was measured by qPCR. (D) The concentration of IL‐1β in serum of aged mice. n = 3. Values are mean ± SD. Significance determined using one‐way ANOVA (A, B) and unpaired t test (D). *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001 versus vehicle‐treated group.

Figure 7

Effects of handelin on mitochondrial function and biogenesis and antioxidant activity. (A) The protein expression of myoglobin in tibialis anterior muscle of aged mice which administrated handelin (10 mg/kg) or equal volume solvent (vehicle control) by intraperitoneal injection every day for 1 months. (B) The oxygen consumption rate (OCR) in C2C12 cells treated with handelin was analysed using a Seahorse XF‐24 analyser. Rates of basal respiration, maximal respiratory capacity, and ATP production were quantified by normalization of OCR levels to total protein levels. (C) Protein levels of OXPHOS complex subunits in C2C12 myotubes which were treated with TNF‐α or handelin (0‐125 nM) were detected by western blotting. (D) The antioxidant enzymes activity of SOD and CAT in aged mice's tibialis anterior muscle. (E) Lipid peroxidation (thiobarbituric acid reactive substances) and (F) protein carbonylation in aged mice's tibialis anterior muscle. n = 3. Values are mean ± SD. Significance determined using unpaired t test (A, D, E and F), *P < 0.05 and **P < 0.01 and versus vehicle‐treated group.

Figure 8

Handelin improves muscle atrophy by targeting Hsp70. (A) The myotube atrophy was induced by TNF‐α with or without the presence of handelin, VER155008 (10 μM) for 48 h. The protein expressions of phosphorylated p65 and total p65, phosphorylated mTOR and total mTOR, phosphorylated AKT and total AKT, phosphorylated mTOR and total mTOR, HSP70, MAFbx, OXPHOS were detected by western blotting. (B) The myotube atrophy was induced by TNF‐α with or without the presence of handelin, VER155008 (10 μM) or hsp70 siRNA for 48 h. The protein expressions of MyHC were detected by western blotting. (C) Proposed protective mechanisms of handelin against skeletal muscle atrophy in vitro and in vivo.