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. 2020 Aug 12:11:988.
doi: 10.3389/fphys.2020.00988. eCollection 2020.

Isoquercitrin Delays Denervated Soleus Muscle Atrophy by Inhibiting Oxidative Stress and Inflammation

Yuntian Shen  1 , Qiuyu Zhang  1 , Ziwei Huang  1 , Jianwei Zhu  2 , Jiayi Qiu  3 , Wenjing Ma  1 , Xiaoming Yang  1 , Fei Ding  1 , Hualin Sun  1
Affiliations

Isoquercitrin Delays Denervated Soleus Muscle Atrophy by Inhibiting Oxidative Stress and Inflammation

Yuntian Shen et al. Front Physiol. .

Abstract

Although denervated muscle atrophy is common, the underlying molecular mechanism remains unelucidated. We have previously found that oxidative stress and inflammatory response may be early events that trigger denervated muscle atrophy. Isoquercitrin is a biologically active flavonoid with antioxidative and anti-inflammatory properties. The present study investigated the effect of isoquercitrin on denervated soleus muscle atrophy and its possible molecular mechanisms. We found that isoquercitrin was effective in alleviating soleus muscle mass loss following denervation in a dose-dependent manner. Isoquercitrin demonstrated the optimal protective effect at 20 mg/kg/d, which was the dose used in subsequent experiments. To further explore the protective effect of isoquercitrin on denervated soleus muscle atrophy, we analyzed muscle proteolysis via the ubiquitin-proteasome pathway, mitophagy, and muscle fiber type conversion. Isoquercitrin significantly inhibited the denervation-induced overexpression of two muscle-specific ubiquitin ligases-muscle RING finger 1 (MuRF1) and muscle atrophy F-box (MAFbx), and reduced the degradation of myosin heavy chains (MyHCs) in the target muscle. Following isoquercitrin treatment, mitochondrial vacuolation and autophagy were inhibited, as evidenced by reduced level of autophagy-related proteins (ATG7, BNIP3, LC3B, and PINK1); slow-to-fast fiber type conversion in the target muscle was delayed via triggering expression of peroxisome proliferator-activated receptor γ coactivator 1α (PGC-1α); and the production of reactive oxygen species (ROS) in the target muscle was reduced, which might be associated with the upregulation of antioxidant factors (SOD1, SOD2, NRF2, NQO1, and HO1) and the downregulation of ROS production-related factors (Nox2, Nox4, and DUOX1). Furthermore, isoquercitrin treatment reduced the levels of inflammatory factors-interleukin (IL)-1β, IL-6, and tumor necrosis factor-α (TNF-α)-in the target muscle and inactivated the JAK/STAT3 signaling pathway. Overall, isoquercitrin may alleviate soleus muscle atrophy and mitophagy and reverse the slow-to-fast fiber type conversion following denervation via inhibition of oxidative stress and inflammatory response. Our study findings enrich the knowledge regarding the molecular regulatory mechanisms of denervated muscle atrophy and provide a scientific basis for isoquercitrin as a protective drug for the prevention and treatment of denervated muscle atrophy.

Keywords: denervated muscle atrophy; inflammation; isoquercitrin; mitophagy; oxidative stress; proteolysis.

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Figures

Figure 1
Figure 1
Isoquercitrin reduces soleus muscle mass loss caused by denervation (n = 6). (A) The ratio of muscles wet weight in each group. (B) Representative images of laminin-stained muscles cross-sections in each group. Green indicates laminin staining. Scale bar: 50 μm. (C) The histogram shown the cross-sectional area (CSA) of muscles in each group. Ctrl, control group; Den, denervation group; ISO-L, denervated target muscle plus low-dose isoquercitrin (10 mg/kg/d); ISO-M, denervated target muscle plus middle-dose isoquercitrin (20 mg/kg/d); ISO-H, denervated target muscle plus high-dose isoquercitrin (40 mg/kg/d). **p < 0.01 and ****p < 0.0001 vs. Ctrl; #p < 0.05, ##p < 0.01, ###p < 0.001, and ####p < 0.0001 vs. Den; &&p < 0.01 vs. ISO-L.
Figure 2
Figure 2
Isoquercitrin inhibits proteolysis via the ubiquitin-proteasome pathway (n = 6). (A) Western blot of MyHC, MAFbx, and MuRF1. (B–D) Quantification of the expression of MyHC, MAFbx, and MuRF1. Ctrl, control group; Den, denervation group; ISO, denervated target muscle plus middle-dose isoquercitrin (20 mg/kg/d) group. *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001 vs. Ctrl; ##p < 0.01vs. Den. MyHC, myosin heavy chain; MuRF1, muscle RING finger 1; MAFbx, muscle atrophy F-box.
Figure 3
Figure 3
Isoquercitrin reduces mitochondrial autophagy in denervated soleus muscles. (A) Ultrastructure of muscle fibers observed using transmission electron microscopy (n = 3). The white arrow indicates mitochondria between muscle fibers. The black arrow indicates an autophage or an autophagic vesicle. (B) Western blot and quantification of the autophagy-related proteins ATG7, BNIP3, PINK1, and LC3B (n = 6). Ctrl, control group; Den, denervation group; ISO, denervated target muscle plus isoquercitrin (20 mg/kg/d) group. *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001 vs. Ctrl; #p < 0.05 and ##p < 0.01 vs. Den.
Figure 4
Figure 4
Isoquercitrin delays the slow-to-fast fiber type conversion caused by denervation. (A) Immunofluorescence staining of fast myosin skeletal heavy chain in soleus muscle. (B) Quantification of the positive proption of fast muscle fibers (n = 3). (C) Western blot of PGC-1α, Tn I-FS, and Tn I-SS related to the slow-to-fast fiber type conversion. (D) Quantification of PGC-1α, Tn I-FS, and Tn I-SS. Ctrl, control group; Den, denervation group; ISO, denervated target muscle plus isoquercitrin (20 mg/kg/d) group. *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001 vs. Ctrl; #p < 0.05 and ##p < 0.01 vs. Den. PGC-1α, peroxisome proliferator-activated receptor γ coactivator 1α; TnI-FS, fast skeletal muscle troponin I; TnI-SS, slow skeletal muscle troponin I.
Figure 5
Figure 5
Isoquercitrin inhibits the inflammatory response in denervated soleus muscles (n = 6). (A) Quantitative polymerase chain reaction detection of changes in the expression of the inflammatory factors interleukin (IL)-1β, IL-6, and tumor necrosis factor (TNF)-α. (B) Enzyme-linked immunosorbent assay (ELISA) detection of the expression of IL-1β, IL-6, and TNF-α; (C) Western blot analysis of JAK/STAT3 activation. Ctrl, control group; Den, denervation group; ISO, denervated target muscle plus isoquercitrin (20 mg/kg/d) group. **p < 0.01, ***p < 0.001, and ****p < 0.0001 vs. Ctrl; #p < 0.05, ##p < 0.01, and ###p < 0.001 vs. Den.
Figure 6
Figure 6
Isoquercitrin inhibits oxidative stress in denervated soleus muscles (n = 6). (A) Fluorescence diagram of reactive oxygen species (ROS) level and quantification of relative fluorescence intensity of each group in the dihydroethidium (DHE) probe detection experiment. (B) Quantitative polymerase chain reaction detection of the relative expression of antioxidant-related genes and ROS production-related genes. (C) Western blot of ROS-related and antioxidant-related proteins and quantification of the relative expressions. ROS, reactive oxygen species; Ctrl, control group; Den, denervation group; ISO, denervated target muscle plus isoquercitrin (20 mg/kg/d) group. *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001 vs. Ctrl; #p < 0.05, ##p < 0.01, ###p < 0.001, and ####p < 0.0001 vs. Den.
Figure 7
Figure 7
A schematic diagram illustrating the proposed mechanism by which peripheral nerve injury induces soleus muscle atrophy. Denervation-induced skeletal muscle atrophy is associated with oxidative stress and inflammation. The inhibition of oxidative stress and inflammation through Isoquercitrin alleviated denervation-induced skeletal muscle atrophy by reducing proteolysis, inhibiting mitophagy, and reversing the slow-to-fast fiber type conversion following denervation.
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