A Review of Complementary and Alternative Medicine Therapies on Muscular Atrophy: A Literature Review of In Vivo/In Vitro Studies

Objective The objective of this review is to evaluate the recent treatment and study trends of complementary and alternative medicine (CAM) treatments on muscular atrophy by reviewing in vivo/in vitro studies. Materials and Methods The searches were conducted via electronic databases including PubMed, the Cochrane Library, China National Knowledge Infrastructure (CNKI), Wanfang MED, and five Korean databases. Only in vivo and in vitro studies were included in this study. Results A total of 44 studies (27 in vivo studies, 8 in vitro studies, and 9 in vivo with in vitro) were included. No serious maternal or fetal complications occurred. There were various animal models induced with muscular atrophy through “hindlimb suspension”, “nerve damage”, ‘alcohol or dexamethasone treatment', “diabetes”, “CKD”, “stroke”, “cancer”, “genetic modification”, etc. In 28 of 36 articles measuring muscle mass, CAM significantly increased the mass. Additionally, 10 of them showed significant improvement in muscle function. In most in vitro studies, significant increases in both the diameter of myotubes and muscle cell numbers were reported. The mechanisms of action of protein synthesis, degradation, autophagy, and apoptotic markers were also investigated. Conclusions These results demonstrate that CAM could prevent muscular atrophy. Further studies about CAM on muscular atrophy are needed.


Introduction
Muscle atrophy is defined as a reduction in muscle mass, which is a concept that covers partial or complete wasting away from muscle. In general, functional and morphological changes caused by muscular atrophy usually result in decreased muscle fiber cross-sectional area, protein content, muscle strength, and increased insulin resistance [1]. Muscle atrophy occurs in cachexia, a comorbidity of abnormal conditions such as cancer, AIDS, congestive heart failure, and chronic obstructive pulmonary disease (COPD). It also occurs in sarcopenia, a decrease in muscle mass and strength associated with aging [2].
Although the causes of muscle atrophy are not fully understood, numerous factors contributing to the deterioration of muscle atrophy have been found through recent studies. Among these, decreased alpha motor neuron numbers, increased inflammatory cytokinesis, and decreased hormonal function are considered important factors in its pathogenesis [3].
Meanwhile, according to research by Alfonso et al. [4], the prevalence of sarcopenia in senior citizens aged 60 to 70 years was found to be 5-13% and 50% in patients over 80 years. Around the world, the population over 60 years was estimated to be about 60 million in the year 2000 and has been projected to be about 1.2 billion by 2025 and 2 billion by 2050. On condition that the current average prevalence of muscle loss is maintained, at least 50 million people to date and about 200 million people in the next 40 years will be affected by sarcopenia.
In complementary and alternative medicine, symptoms of muscular atrophy are treated using acupuncture, chuna 2 Evidence-Based Complementary and Alternative Medicine treatment, and herbal medicine. In addition to these clinical treatments, many studies on complementary and alternative medicine (CAM) therapies for muscular atrophy have been conducted recently, but the results in literature have been insufficient. Against this background, we performed a literature review of CAM on muscular atrophy to establish a research model and suggest a direction for future research.

Method
. . Search Strategies. We conducted this literature review of in vivo/in vitro studies through Korean and foreign electronic database searches. Foreign databases used included PubMed, Cochrane Library, China National Knowledge Infrastructure (CNKI), and Wanfang MED. Domestic databases searched included Korean Traditional Knowledge Portal (KTKP), Oriental Medicine Advanced Searching Integrated System (OASIS), Research Information Services (RISS), National Assembly Library, and the Korean studies information Service System (KISS). In the Korean databases, "muscular atrophy", "muscle loss", and "complementary and alternative medicine" were used as search terms. In CNKI and Wanfang MED, we used cross-language searches and related search terms which were "muscle atrophy", "muscle loss", and "肌 肉減少".
The search strategy used in the searches in PubMed and the Cochrane Library was based on a combination of medical subject headings outlined in Table 1. The searches were conducted independently by two investigators (SM Jeong, YC Park) who retrieved relevant studies published before August 2018. After retrieving the articles, we extracted and classified the data by animal or cell models, intervention type (herbal medicine, acupuncture, and moxibustion), and outcome measures (muscle mass, strength, and histological and/or biochemical measurements). The extracted data was then verified by the third author (YH Baek). Disagreements were resolved by consensus through face-to-face discussions among the three authors.
. . Study Selection. The inclusion criteria for the literature review were as follows: (1) articles on complementary and alternative medicine treatments for muscular atrophy and (2) in vivo or in vitro studies, while the exclusion criteria included the following: (1) clinical trial; (2) articles that were not original; (3) articles that were not in English, Korean, or Chinese; (4) duplicate studies. We also excluded studies that did not focus on muscle loss or muscle cell loss and those which did not use complementary and alternative medicine treatment. Full articles meeting the inclusion criteria were retrieved and read carefully.

Results
. . Literature Search. The search initially yielded 973 articles out of which duplicates were eliminated. After screening the titles, 872 articles were excluded due to a lack of adequate coverage of the topic of interest, and after full text assessment, 57 additional articles were excluded for one or more of the following reasons: (1) articles that were not based on original research; (2) articles that did not involve in vivo or in vitro studies; and (3) abstracts and comments. Finally, 44 articles were included in our literature review (Figure 1).
. . In Vivo Studies. A total of 36 articles (27 of which were in vivo only and 9 of which were in vivo with in vitro studies) were classified as in vivo studies in our review. . . . Animal Model Species. The most commonly used model was the Sprague-Dawley rat which was used by 22 out of the 36 studies. C57BL/6 mice were used in 6 studies, BALB/c mice were used in 2 studies, and DBA/1 J mice, Kunming mice, and SPF/VAF mice were used in one study each. Three papers did not mention which animal models were used. There were variations in the ages of the animals used in the studies, with 16-, 12-, 10-, 9-, 8-, 7-, and 6-weekold animals being used in 1, 2, 11, 1, 6, 1, and 7 studies, respectively. In one study, the ages of animals used in the test group differed from that of the control group, being 5 and 19 months, respectively. With regard to the sex of the animals, male rats were predominantly used, featuring in 30 studies, while one study used only female rats and one study used a combination of male and female animals. The other studies did not mention sex of the animals.
In order to induce muscular atrophy, various disease models were used in the studies. The most commonly used model for muscular atrophy was the hindlimb suspension (HS) method, developed by Morey-Holton and Wronski [49]. HS rat model was used in 7 of the 36 studies. The next commonly used model was nerve damage-induced muscular atrophy model which was used in 6 studies. Among these studies, direct injury to the sciatic nerve or the peroneal nerve was used to cause atrophy. The chronic kidney disease (CKD) model, in which induction was done by 5/6 nephrectomy was used in 5 studies. The alcohol-induced muscular atrophy model, transgenic model, and the cancer-induced cachexia model (C26-colon-adenocarcinoma implanted) were used, and each of these models was used in three of the 36  studies reviewed. In two studies, diabetes was induced by the administration of streptozotocin to evaluate muscular dystrophy due to diabetes. There were 2 studies using stroke models (middle cerebral artery occlusion, MCAO) and one study for each of the following models: chronic obstructive pulmonary disease (COPD), collagen-induced arthritis (CIA), dexamethasone-induced atrophy, spinal cord injury, and the aging models ( Figure 2).
. . . Interventions. The most common types of intervention were herbal medicines which were used in 28 studies. In addition, there were 7 studies using electroacupuncture and one study using moxibustion ( Figure 3). The treatment periods were 12 days in 2 studies, 2-4 weeks in 24 studies, 1-6  months in 8 studies, and 38 weeks in 1 study. One study did not limit the treatment period.
. . . Outcomes. Muscle mass, the most common primary outcome, was investigated in 28 out of the 36 studies. All except one reported a significant increase in muscle mass. Ten articles investigated muscle function, all of which reported significant increases in muscle strength (Tables 2 and 3).
(A) Hindlimb Suspension Model. In the study conducted by Akiko Onda et al., electroacupuncture (E-AT) inhibited the expression of the MuRF-1 gene. E-AT also elevated the expression of Akt1 and TRPV4 [5]. In the study by Zhang ZK et al., icaritin extract was used which inhibited reduced phosphorylation of PI1K-p110, thereby promoting protein synthesis. In addition, the extract inhibited muscle atrophy through modulation of the FoxO3a and FoxO1, the O subclasses of the forkhead family of transcription factors [33]. In the study by Soh KS et al., after using KangwhalSokdantang (KWSDT), the ratio of type I muscle fiber/type II fiber was increased showing that KWSDT inhibited early muscular atrophy. IGF-1 and MyoD were also measured and increasing of these key myogenic factors was involved in the improvement of muscle mass [21]. In Cho SG et al., the combination of Schisandra chinensis Baill, Lycium chinense Mill, and Eucommia ulmoides Oliv inhibited muscular atrophy and increased muscle strength and cross-sectional area of muscle fiber [36]. Zhu M et al. also reported the similar effects on muscular atrophy by using Bu Zhong Yi Qi decoction [37].
Bax and Bcl-2 were measured in Kim BH's study. Daeyeoung-jeon extract significantly reduced the immunoreactivity of Bax and increased Bcl-2 in gastrocnemius muscle [23]. In another study by Kim BH, Dangguibohyultang (Dangguibuxuetang) extract significantly enhanced the antioxidant enzyme, Cu/Zn-SOD which inhibited disuse muscular atrophy by suppressing oxidative damage [24].
(B) Nerve Damaged Model. In the studies using nerve damaged model, one study provided treatment with Radix Puerariae extract and the other used Radix Dipsaci extract. Both studies measured Bcl-2 and Bax. While Radix Puerariae extract was found to significantly inhibit Bax but not Bcl-2 [15], Radix Dipsaci extract significantly inhibited both Bax and Bcl-2 expression [17].
In the study by Zhou L. et al., the result indicated that Buyang Huanwu Tang (BYHWT) decreased inflammatory cells through the activation of angiopoietin-like protein 4 (ANGPTL4) and inhibited muscle cell apoptosis in the denervated-dependent skeletal muscle atrophy model. The effect of BYHWT was mediated by increased expression of NF-B and MuRF-1 [27]. Another study by Zhou L. et al., BYHWT increased the NF-B p65 and MuRF1, too [30].
Yu J. et al. applied E-AT on penicillin injection-induced sciatic nerve injury model and the treatment alleviated muscular atrophy by upregulating agrin and acetylcholine receptor-(AChR-), and downregulating AChR-associated in neuromuscular transmission [28]. In Cao R. et al., E-AT on sciatic nerve injury inhibited muscle cell apoptosis by suppressing Bax, cytochrome C, and caspase-3 [31].
(C) Stroke-Induced Model. In the studies by Lee et al. and Han et al., the Radix Dipsaci extract inhibited the "slow-to-fast shift" phenomenon in muscle fibers and MHC-II expression while increasing MyoD expression and thereby significant efficacy in muscle recovery [18,19].
In Wang D. et al., the administration of Jian-Pi-Yi-Shen (JPYS) decoction prevented muscle loss, muscle protein degradation, and increased muscle protein synthesis. In addition, JPYS decoction not only inhibited FoxO3a activation and ubiquitin-proteasome system (UPS), but also increased Cox IV, NRF-1, and PGC-1 [25]. Atrogin-1 and MuRF-1 were also measured in the study by Geng Z et al., and astragalus polysaccharide significantly reduced the expression of atrogin-1 and ubiquitin in rat skeletal muscle [38].
In CKD-induced mice, the levels of autophagy-related proteins including Bnip3 and Beclin-1 were increased. Likewise, the ratio of the autophagy proteins LC3II-to-LC3I, associated with autophagosome formation, was increased. Su Z. et al. suggested that using Acu/LFES suppressed the CKD-induced upregulation of autophagy by inhibiting those proteins [26].
Evidence-Based Complementary and Alternative Medicine 5               (F) Tumor-Induced Model. All the tumor model studies measured markers of the immune system, such as TNFand interleukins. In the study by Kim AY et al., Sosihotang suppressed the production of cachexia-inflammatory cytokines (IL-6, IL-1, and TNF-) and these inhibited the activation of nitric oxide and p38, NF-B, and STAT3, thereby reducing muscle loss [32]. Another study by Kim AY et al., a novel herbal formula, SGE, also decreased inflammatory cytokines [39] and Zhuang et al. found that the Zhimu-Huangbai (1:1) herb pair reduced tumor-induced muscle loss and inhibited cachectic cytokines [8]. (H) e Others. In a study by Yuki Kishida et al., Gosha-jinki-Gan (GJG) was administered to a senescenceaccelerated mouse which promoted pAMPK and PGC-1 , thereby activating mitochondrial neogenesis and oxidative metabolism [9].
Yuya Nakashima et al. demonstrated that significant decreases in muscle loss and oxidative stress due to the reduction of malondialdehyde (MDA) and 4-hydroxyalkenals (HAE) of muscle cells occurred after treating with Chlorella extract [11].
In Zhang YT et al., spinal cord injury-induced (SCI) muscular atrophy model was treated with E-AT [29]. E-AT increased the expression levels of choline acetyltransferase (ChAT), which is a transferase enzyme responsible for the synthesis of the excitatory neurotransmitter acetylcholine [50]. Likewise, E-AT increased the protein neurotrophin-3 (NT-3), a member of the neurotrophin family, and has an important role in neuroprotection and axonal regeneration [51]. Through these mechanisms, E-AT on SCI rat protected the motor neurons as well as alleviated muscular atrophy.
In Tseng YT et al. using spinal muscular atrophy rat model, Liuwei dihuang water extract (LWDH-WE) improved muscle strength and body weight accompanied by upregulation of survival motor neuron protein in spinal cord and gastrocnemius muscle tissues [40].
. . In Vitro Studies. A total of 17 (8 in vitro only, 9 in vivo with in vitro) articles were classified as in vitro studies in this review.
. . . Muscle Cell Model. C2C12 cells (myoblasts and myotubes) were used in 15 of the 17 studies. There was 1 study using L6 myoblasts and 1 using NSC34 motor neuronlike cells. The TNF--induced apoptosis and autophagy model were used in 4 studies, while the oxidative stress model was used in 3 studies. In addition to these, 3 studies utilized the no treatment model, 2 using cancer model. The dexamethasone-treated, PI3K-p110-suppressed, AICARtreated, starvation-induced, and transgenic models were used in 1 study each (Figure 4).
. . . Interventions. All 17 studies used herbal medicine as the intervention method. The treatment period spanned 24 hours in 8 studies, 48 hours in 7 studies, and 72 hours in 1 study. One study did not report the treatment period.
. . . Outcomes. Myotube diameter and muscle cell number were set as the primary outcomes. In most studies, significant increases in both the diameter of myotubes and muscle cell numbers were reported (Tables 3 and 4).

(A) Oxidative Stress Model.
In the three studies using the oxidative stress model, Jung et al. found that idesolide extract protected muscle cells from oxidative stress by promoting the activation of heat shock protein 70 (HSP70) in the cells [42]. HSP70 protects cells from a number of apoptotic stimuli, including oxidative stress and is significantly downregulated in various models of skeletal muscle atrophy [52]. They also found the same effects when sauchinone (root of Saururus chinensis) was used as the mode of treatment [44].
In Choe YH., isorhamnetin (ISO) extract was treated to hydrogen peroxide-(H2O2-) induced oxidation damaged muscle cells. In general, H2O2 causes cell apoptosis with mitochondrial damage and ROS generation by upregulation of Bax and downregulation of Bcl-2 [53]. The ISO extract inhibited the apoptosis by the restoration of Bax, Bcl-2 proteins, and decreasing of caspase-9 and caspase-3. In addition, the ISO activated the antioxidant protein heme oxygenase-1 (HO-1) and its transcription factor, nuclear factor erythroid derived 2-related factor 2 (Nrf2), thereby suppressing oxidative stress [47].
(B) AICAR-Induced Model. Kang et al. found that the administration of Schisandrae Fructus (SF) extract to AICAR (5-aminoimidazole-4-carboxamide-ribonucleotide)induced muscle atrophy C2C12 myotubes significantly counteracted AICAR-induced muscle cell atrophy and reversed the increased expression of MuRF-1 and FoxO3a [46]. AICAR is an enzyme that upregulates MuRF-1 and stimulates the levels of the FoxO3a transcription factor [54]. SF extract could reverse the muscle cell atrophy caused by AICAR through regulation of the AMPK and FoxO3a signaling pathways, followed by inhibition of MuRF-1.    [38].
(D) Untreated Model. In the studies using the untreated C2C12 myoblast model, Takeda et al. showed that administration of Hachimijiogan (HJG) to untreated C2C12 cells promoted muscle cell proliferation through activation of ERK1/2 signaling pathway without affecting the Akt signaling pathway [43]. In a study by Sung BK et al., the expression levels of myogenic proteins (MyoD and myogenin) and functional myosin heavy chain (MyHC) were measured to evaluate the myogenic potential of loquat leaf extract (LE). LE enhanced those proteins and also activated the PI3K-Akt/mTOR signaling pathway [34]. In Zhu M. et al., the results indicated that Bu Zhong Yi Qi decoction (BZ) significantly downregulated NCoR1 expression and further induced muscle differentiation and metabolism by regulating NCoR1-associated gene expression [37].
Lu L. et al. reported in a study using the dexamethasoneinduced muscle cell injury model that APS increased p-Akt, p-mTOR, p-P70s6k, and p-rsS6 and inhibited FoxO3a/FoxO1 [41].
In Tseng YT et al., inducible survival motor neuronknockdown NSC34 motor neuron-like cells were used to mimic survival motor neuron-(SMN-) deficient condition. LWDH-WE significantly increased mitochondrial membrane potential and suppressed apoptosis by attenuating the SMN deficiency-induced downregulation of Bcl-2 and upregulation of cytosolic cytochrome c and cleaved caspase-3 [40].
In Li F et al., starvation-induced muscle protein degradation model by incubation of the cells with serum-free basal medium was treated with ginsenoside Rg1. The Rg1 inhibited MuRF-1 and atrogin-1 and also activated the p-mTOR, Akt, p-FoxO1, and p-FoxO3a [48].

Discussion
As the threat of the global aging phenomenon rises, there has been a growing interest in healthy aging, which in turn has led to an increased interest in muscular atrophy and sarcopenia within the medical community.
Recently, many clinical cases, trials, and preclinical studies have been conducted on the effect of CAM on muscular atrophy; however, a review of the study results and treatment mechanisms reported has been inadequate. We performed the review to establish clinical evidence and suggest a direction for future research in finding treatment mechanisms.
As the results showed, the hindlimb suspension and nerve damaged models were most commonly used in the in vivo studies, which are preferred because they can be induced muscle atrophy by directly and mechanically. CKD model, cancer model, stroke model, and diabetes model were useful for studying muscle atrophy that results from comorbidity of several common diseases.
In many studies, to find the mechanisms of action of muscular atrophy treatment, various markers of muscle protein synthesis and muscular atrophy inhibitors were measured.
In the majority of studies including our review, PI3K-Akt-mTOR signaling pathway factors were investigated. The activation of PI3K leads to activation of the serine/threonine kinase Akt, which in turn phosphorylates and activates its downstream proteins (mTOR, p70S6K, 4EBP1, and eIF4E) and resulted in increased protein synthesis and led to skeletal muscle hypertrophy [55,56]. Mammalian target of rapamycin (mTOR) which is activated by PI3K-Akt signaling pathway functions as a protein kinase that directly influence cell growth and protein synthesis [57]. As the above markers are included in the major pathways in muscular atrophy, these are suggested to be measured in the related studies.
In addition, other cell proliferation pathway proteins like TRPV4, ERK1/2 could be investigated. TRPV4, a member of the TRP channel superfamily, is known to promote physiological functions in muscle [58], and ERK1/2 is member of the mitogen-activated protein kinase super family that can mediate cell proliferation and apoptosis in the Ras-Raf-MEK-ERK signaling cascade [59].
MuRF1 and FoxO3/atrogin-1 are proteins leading to upregulation of the UPS pathway to catalyze the myofibrillar proteins [60]. FoxO3 can be phosphorylated by Akt, and deactivation of Akt would lead to relocation of FoxO into the nucleus and activate atrophy-induced genes. Activated FoxO3 stimulates lysosomal proteolysis in muscle by activating the autophagy process by transcriptional regulation [61]. So, these proteins are needed to find the activation of UPS pathway in muscle atrophy.
In the cell apoptotic markers, Bax, a proapoptotic factor that promotes cell death, and Bcl-2, an antiapoptotic factor, were mainly used. Caspase-9 and caspase-3 were also measured because they were the key caspase proteases in the activation of intrinsic apoptosis pathway [62]. Through the measurement of those apoptotic factors, the efficacy of suppressing muscle cell apoptosis can be verified.
MyoD, MHC-II, MyHC, and myogenin were measured to investigate the level of myogenesis. MyoD, MHC-II, and MyHC are proteins that play a major role in regulating muscle differentiation, and myogenin is a transcription factor involved in coordination of skeletal muscle development or myogenesis and repair.
Mitochondria related proteins like PGC-1 , Cox IV, and NRF-1 were measured to investigate the effect of skeletal muscle metabolism through modulating mitochondrial biogenesis. Also, NCoR1 were measured as a key modulator of the mitochondrial metabolism in muscle, which can reprogram muscle metabolism from a glycolytic to a more oxidative mitochondrial state [63].
In this literature review, we searched for articles on CAM as a treatment modality for muscular atrophy. However, owing to the small number of studies used in the review, only a qualitative analysis of the data could be done. Further studies must be carried out to provide additional evidence to support our findings.

Conclusion
In this study, outcome measures of muscle mass and function were used to evaluate the effects of complementary and alternative medicine on muscular atrophy and, in most of studies, the results have proven it to be significantly effective. Markers of muscle protein synthesis and muscle atrophy inhibitors were measured to find their treatment mechanisms. In those mechanisms, (1) changes in growth hormone/IGF productivity and sensitivity by aging, (2) decrease of muscle protein by proinflammatory cytokines (TNF-, IL-1, IL-6, etc.), (3) acceleration of apoptosis in muscle cells, (4) ubiquitin-proteasome proteolytic pathway, and (5) Akt/mTOR signaling pathway were found to be important factors contributing to treatment effects.
In most of the studies we have investigated, CAM promoted protein synthesis and inhibited degradation by enhancing the PI3K-Akt-mTOR signaling pathway. It also inhibited the ubiquitin-proteasome system which was involved in protein degradation. In several studies, CAM alleviated skeletal muscle metabolism through modulating mitochondrial biogenesis factors, thereby preventing muscle loss. In addition, CAM restored the expression of proteins involved in the cell apoptotic pathway and autophagy pathway in the various models of muscular atrophy.