Salidroside mitigates skeletal muscle atrophy in rats with cigarette smoke-induced COPD by up-regulating myogenin and down-regulating myostatin expression

Abstract Objectives: The present study aimed at investigating the therapeutic effect of Salidroside on skeletal muscle atrophy in a rat model of cigarette smoking-induced chronic obstructive pulmonary disease (COPD) and its potential mechanisms. Methods: Male Wistar rats were randomized, and treated intraperitoneally (IP) with vehicle (injectable water) or a low, medium or high dose of Salidroside, followed by exposure to cigarette smoking daily for 16 weeks. A healthy control received vehicle injection and air exposure. Their lung function, body weights and gastrocnemius (GN) weights, grip strength and cross-section area (CSA) of individual muscular fibers in the GN were measured. The levels of TNF-α, IL-6, malondialdehyde (MDA), superoxide dismutase (SOD), glutathione (GSH) in serum and GN tissues as well as myostatin and myogenin expression in GN tissues were measured. Results: In comparison with that in the healthy control, long-term cigarette smoking induced emphysema, significantly impaired lung function, reduced body and GN weights and CSA values in rats, accompanied by significantly increased levels of TNF-α, IL-6 and MDA, but decreased levels of SOD and GSH in serum and GN tissues. Furthermore, cigarette smoking significantly up-regulated myostatin expression, but down-regulated myogenin expression in GN tissues. Salidroside treatment decreased emphysema, significantly ameliorated lung function, increased antioxidant, but reduced MDA, IL-6 and TNF-α levels in serum and GN tissues of rats, accompanied by decreased myostain, but increased myogenin expression in GN tissues. Conclusion: Salidroside mitigates the long-term cigarette smoking-induced emphysema and skeletal muscle atrophy in rats by inhibiting oxidative stress and inflammatory responses and regulating muscle-specific transcription factor expression.


Introduction
Chronic obstructive pulmonary disease (COPD) is a commonly chronic inflammatory disease and it will be the third most dead disease worldwide in 2030 [1]. Long-term heavy smoking is a risk factor for the development of COPD. This, together with air pollution, increases the incidence of COPD in smoking population. Many harmful components in cigarettes can damage epithelial cells in the respiratory tract and vascular endothelial cells, and cause chronic inflammation, leading to respiratory and cardiovascular diseases [2]. Furthermore, long-term heavy cigarette smoking and COPD can also result in skeletal muscular

Lung function
The lung function of individual rats was measured by the ratios of forced expiratory volume (FEV) at 0.2 s (FEV 0.2 ) to forced vital capacity (FVC) and peak expiratory flow (PEF) [28]. Briefly, individual rats were injected IP with 1% pentobarbital sodium (40 mg/kg) and maintained in an appropriate anesthesia. The trachea of each rat was cut, intubated, and attached to a ventilator (Res3020, Bestlab High-Tech, Beijing, China) at a respiratory rate of 75 beats/min with a tidal volume (5 ml/kg). The FEV 0.2 /FVC and PEF were measured using the AniRes2005 pulmonary mechanics analyzer (version 3.0, Bestlab High-Tech).

Sample collection
Individual rats were subjected to chest surgery and their blood samples were obtained from the inferior vena cava. After coagulation, their sera were prepared by centrifugation. Their left lungs were perfused with 4% paraformaldehyde at a constant pressure of 25 cm H 2 O [27] and fixed in 4% paraformaldehyde for 48 h, followed by paraffin-embedded for histology and immunohistochemistry. Their right lungs were snap-frozen in liquid nitrogen. The gastrocnemius (GN) of each rat was dissected out. The lateral head and medial head of each GN were cut-fixed in 4% paraformaldehyde, and snap-frozen in liquid nitrogen, respectively.

Histological analysis
The paraffin-embedded lung and the middle part of lateral head of GN tissue sections (4 μm) were stained with Hematoxylin and Eosin (HE) and photoimaged under a light microscope. The mean linear intercept (MLI) and mean alveolar number (MAN) of individual samples were measured [28,29]. Similarly, the cross-section area (CSA) of individual muscular fibers in the middle of the lateral head of GN was measured using the image proplus software (Media Cybernetics, Silver Spring, U.S.A.). A total of 27 fields selected randomly from three sections of each rat and three rats per group were measured.

Assessment of grip strength, body and skeletal muscle weights
The total power of four limbs of each rat was measured for its grip power 1 day before anesthesia using an YLS-13A grip strength meter (Yiyan Technology Development, Shandong, China), according to the manufacturer's instructions. The rats were food-fasted overnight and their body weights were measured before and 16 weeks after smoking. The body weight change was calculated. Their GN tissues were wet-weighed.

Measurement of inflammatory cytokines and oxidant stress in serum and muscle homogenate samples
Individual GN tissues were homogenized and centrifuged, followed by quantifying the concentrations of proteins in their supernatants using bicinchoninic acid (BCA, Beyotime). The concentrations of TNF-α and IL-6 in individual sera and GN tissue homogenates were determined by enzyme-linked immunosorbent assay (ELISA, Cloud Clone, Wuhan, China), according to the manufacturer's protocol. The levels of malondialdehyde (MDA), GSH and superoxide dismutase (SOD) inhibition ratio in individual sera and GN tissue homogenates were measured by chemical reactions using specific kits (Nanjing Jiancheng Bioengineering Institute, China), according to the manufacturer's instructions. The SOD activity were calculated with this formula: SOD activity = SOD inhibition ratio × 24 × Sample dilution factor before test.

Immunohistochemistry
The paraffin-embedded muscular tissue sections (5 μm) were deparaffinized, rehydrated and subjected to antigen retrieval in citrate buffer, pH 6.0 in a microwave for 20 min. The sections were treated with 3% H 2 O 2 in methanol for 15 min and blocked with 10% goat sera. After being washed, the sections were incubated with anti-myostatin (1:400, Abcam), anti-myogenin (1:50, Santa Cruz Biotechnology, Santa Cruz, U.S.A.). The bound antibodies were detected using horseradish peroxidase (HRP)-conjugated secondary antibodies (Maixin, Fuzhou, China) and visualized with DAB, followed by photoimaging. The staining intensity of individual sections was evaluated using the Image-Pro Plus software (Media Cybernetics). The intensity of anti-myostatin and the percentages of nuclear anti-myogenin stained cells were measured in a blinded manner.

RNA extraction and quantitative real-time PCR
Total RNA was extracted from individual GN tissue samples and reverse transcribed into cDNA using a reverse transcription kit (Takara, Japan). The relative levels of myostatin and myogenin to control β-actin mRNA transcripts in individual muscle samples were assessed by quantitative RT-PCR using SYBR Premix Ex Taq™ (Takara, Japan) and specific primers in LightCycler 480 (Roche, Switzerland). The sequences of primers were forward

Statistical analysis
Data are present as the means + − standard deviation (SD). The difference among the groups was analyzed by one-way ANOVA and post hoc Bonferroni's test using the GraphPad Prism 5.0 software (San Diego, CA, U.S.A.). A P-value of less than 0.05 was considered statistically significant.

Salidroside treatment ameliorates lung function in rats with cigarette smoking-induced COPD
Long-term heavy cigarette smoking is the highest risk factor for the development of COPD, which usually affects the bronchioles and alveoli, leading to emphysema. To determine the therapeutic effect of Salidroside, Wistar rats were subjected to cigarette smoking, randomized and treated with vehicle or different doses of Salidroside daily for 16 weeks. Histological examination indicated that compared with the healthy control, there was obvious emphysema and enlarged alveoli in the lung tissues of the COPD group of rats while Salidroside treatment mitigated and abrogated in the COPD-related emphysema and alveolus enlargement in the lungs of rats in a dose-dependent manner ( Figure  1A). Further analyses indicated that the ratios of FEV 0.2 /FVC and the values of PEF and MAN were significantly reduced in the COPD group of rats (P<0.001) while Salidroside treatment at a medium or high dose significantly mitigated the COPD-decreased ratios of FEV 0.2 /FVC in the rats ( Figure 1B-D). Similarly, Salidroside treatment at a high dose also significantly improved the values of PEF and MAN in the rats, compared with that in the COPD group (P<0.001, P<0.01, Figure 1C,D). Moreover, while the values of MLI in the COPD group were significantly higher than the healthy control (P<0.01), the values of MLI in the rats that had been treated with a high dose of Salidroside were significantly reduced (P<0.05, Figure 1E). Hence, Salidroside treatment significantly mitigated the heavy smoking-related lung damage and ameliorated the lung function in rats with COPD.

Salidroside treatment mitigates the COPD-mediated skeletal muscle atrophy in rats
Long-term smoking-induced COPD can cause skeletal muscle atrophy, reduce skeletal muscle weights, and change body weight and grip strength [30]. To determine the consequence of COPD and Salidroside treatment, the grip strengths of individual groups of rats were measured. The grip strengths in the rats that had been treated with a medium or high dose of Salidroside were significantly higher than that in the COPD group, but remained lower than that in the healthy group (P<0.05, P<0.001, Figure 2A). Similarly, the body and GN weights in the rats received a high dose of Salidroside were significantly greater than that in the COPD group, but were less than that in the healthy group (P<0.05, P<0.01, Figure 2B,C). Furthermore, histological examination and quantitative analyses revealed that the CSA values in the rats received a high dose of Salidroside were significantly larger than that in the COPD group (P<0.001 for both, Figure 2D,E). Thus, Salidroside treatment significantly mitigated the COPD-mediated skeletal muscle atrophy in rats.

Salidroside treatment mitigates the oxidative stress-related pro-inflammatory cytokine production in rats
Long-term cigarette smoking can induce oxidative stress, which induces pro-inflammatory cytokine production in the skeletal muscles. To understand the pathogenesis of the COPD-related skeletal muscle atrophy, the levels of MDA, SOD and GSH in serum and GN tissues of individual rats were measured. The levels of MDA in serum and GN tissues of the rats that had been treated with Salidroside at a medium or high dose were similar to that of the healthy control, but significantly lower than that of the COPD group (P<0.01, P<0.001, Figure 3A). In contrast, the levels of SOD and GSH in serum and GN tissues of the rats that had been treated with Salidroside at a medium or high dose were similar to that of the healthy control, but were significantly higher than that in the COPD group ( Figure 3B,C). Further analysis indicated that the levels of TNF-α and IL-6 in serum and GN tissues of the rats that had been treated with Salidroside at a medium or high dose were comparable with that in the healthy group, but were significantly lower than that in the COPD group (P<0.01, P<0.001, Figure 3D,E). Collectively, such data demonstrated that Salidroside treatment at a medium or higher dose significantly mitigated the COPD-related oxidative stress and inflammatory cytokine production in rats.

Salidroside treatment alters the myostatin and myogenin expression in GN tissues of rats with COPD
It is well known that myostatin can inhibit myogenesis while myogenin is a muscular transcription factor to promote myogenesis [31][32][33][34]. To further understand the action of Salidroside treatment in regulating the COPD-related skeletal muscle atrophy, the relative levels of myostatin and myogenin expression in the GN tissue samples were determined by Western blot ( Figure 4A). Quantitative analyses revealed that the relative levels of myostatin in the GN tissues from the rats that had been treated with Salidroside at a medium or high dose were comparable with that in the healthy group, but significantly lower than that in the COPD group (P<0.001 for all, Figure 4B). In contrast, the relative levels of myogenin expression in GN tissues from the rats received a medium or high dose of Salidroside were significantly higher than that in the COPD group (P<0.05, P<0.01, Figure 4B). Similar patterns of myostain and myogenin mRNA transcripts and protein expression were detected by quantitative RT-PCR and immunohistochemistry in different groups of rats ( Figure 4C-F). Therefore, Salidroside treatment significantly decreased myostatin expression, but increased myogenin expression in the GN tissues of COPD rats, contributing to protection from the COPD-induced skeletal muscle atrophy in rats.

Discussion
Previous studies have shown that long-term cigarette smoking can cause COPD, which is characterized by emphysema and impaired lung function [29,35]. In this study, we found that cigarette smoking for 16 weeks decreased the ratios of FEV 0.2 /FVC, the values of PEF and MAN, but increased MLI values, consistent with characteristics of emphysema and COPD [28]. Furthermore, long-term cigarette smoking significantly decreased body and GN weights by reducing CSA of muscular fibers in the GN and functionally imparing grip strengths in rats, hallmarks of COPD-related skeletal muscle atrophy. Conceivably, the skeletal muscle atrophy also occurred in other muscles, particularly in the diaphragm and quadriceps muscles, leading to its fatigue, contributing to poor quality of life and respiratory failure [4,36]. More importantly, we demonstrated that Salidroside treatment, particularly with a high dose, significantly mitigated and abrogated the cigarette smoking-induced emphysema and COPD-impaired lung function and decreased the COPD-related skeletal muscle atrophy in rats in a dose-dependent manner. Such novel data suggest that Salidroside may be a promising candidate for design of new therapies for intervention of smoking-related COPD.
Long-term cigarette smoking can cause oxidative stress and inflammation, contributing to skeletal muscle atrophy [37]. In this study, we found that while cigarette smoking significantly increased the levels of MDA, TNF-α and IL-6, but decreased levels of SOD and GSH in serum and GN tissues of rats. Salidroside treatment significantly mitigated smoking-related oxidative stress and pro-inflammatory cytokine production in rats, consistent with its antioxidant and anti-inflammatory activity, which may contribute to its therapeutic effect on inhibiting the COPD-related skeletal muscle atrophy [10,11,18]. Given that Salidroside has been demonstrated to be relatively safe for humans Salidroside may be valuable for intervention of other inflammatory diseases [38].
Myogenic regulatory factors (MRFs) in the MyoD family are mainly muscle-specific transcription factors, including MyoD, myogenin, myf-5 and MRF4, and are crucial for regulating myogenesis [31,32]. Myogenin is expressed in all skeletal muscles and is a key factor to promote the terminal differentiation of myocytes. MRFs can promote muscle damage repair in animal models of trauma [39,40], neurogenic and myogenic myopathy [41]. On the other hand, myostatin has structure similar to TGF-β and can inhibit the myogenesis [33,34]. Myostatin −/− mice display increased body weights and larger CSA of muscular fibers [34]. In this study, we found that long-term cigarette smoking significantly increased levels of myostatin expression, but decreased myogenin expression in the GN tissues of rats. Treatment with Salidroside at a medium or high dose completely abrogated the cigarette smoking up-regulated myostatin expression and significantly mitigated the COPD-decreased myogenin expression in GN tissues of rats. These data extended previous findings on the pathogenic role of myostatin in tumors [17,[42][43][44], sarcopenia [45], neuromuscular diseases [46,47] and COPD [48][49][50], and support the notion that myostatin participates in the pathogenic process of skeletal muscle damage. Actually, recent studies reveal a three-fold increase in myostatin mRNA transcripts in the vastus lateralis muscles in COPD patients with significant quadriceps weakness [51,52]. Furthermore, higher plasma myostatin levels are detected in COPD patients with corpulmonale complication [53,54]. It is possible that Salidroside and its metabolites may inhibit oxidative stress, inflammation and myostatin expression in COPD rats. These, together with promoting myogenin expression, promote the repair of damaged muscles to inhibit the COPD-related skeletal muscle atrophy. We are interested in further investigating the molecular mechanisms underlying the action of Salidroside in regulating the expression of these molecules during the development and progression of COPD.
In conclusion, our data indicated that Salidroside treatment significantly reduced the cigarette smoking-induced emphysema and ameliorated lung function in rats. Furthermore, Salidroside treatment significantly mitigated the COPD-induced skeletal muscle atrophy by reducing oxidative stress and the production of pro-inflammatory cytokines in serum and the GN tissues and altering myostatin and myogenin expression in the GN tissues. Therefore, our findings may provide a basis for design of new therapies for COPD-related skeletal muscle atrophy.