Amyotrophic lateral sclerosis (ALS) is induced by motor neuron loss through multiple pathological mechanisms, including neuroinflammation, oxidative stress, autophagy, mitochondrial dysfunction, and endoplasmic reticulum stress, leading to muscle atrophy and severely impaired mobility. ALS is caused by mutations in genes, including superoxide dismutase (SOD1), TAR DNA-binding protein, chromosome 9 open reading frame 72 (C9orf72), optineurin (OPTN), and serine–threonine protein kinase-binding kinase 1 (TBK1) [1]. These mutations also contribute to immune dysfunction, affecting microglia, peripheral T lymphocytes, and monocytes. Immune dysfunction in sporadic ALS is associated with disease progression and involves pro-inflammatory macrophages [2]. In an animal study, neuroinflammation induced by activated microglia and astrocytes accelerated disease progression and promoted pathological events in ALS [3]. These anti-neuroinflammatory effects may occur via major histocompatibility complex class I (MHCI) protein upregulation and CD8 T lymphocyte-induced muscle innervation and axonal regrowth enhancement. Furthermore, Song et al. [4] (2016) demonstrated that increased microgliosis and astrocytosis reduce MHCI in the spinal motor neurons of mSOD1 mice and patients with ALS while promoting neurotoxicity.

Inflammation and oxidative stress are intricately connected, with loss of redox balance underlying immune dysfunction in many neurodegenerative diseases. In ALS, mitochondrial dysfunction causes oxidative stress, resulting in motor neuron death, muscle dysfunction, and disease progression. However, whether oxidative stress is a trigger or by-product of other pathological events remains unclear. Indeed, several antioxidants have demonstrated therapeutic effects in patients with ALS. Conversely, while some antioxidants, such as coenzyme Q10 and vitamin E, delay disease progression in animal models of ALS [5], they are reportedly ineffective in humans [6]. Moreover, although edaravone—an antioxidant that mitigates oxidative stress through the free radical peroxynitrate—has been approved by the Food and Drug Administration (FDA) for treating ALS [7], its use is limited to certain patients [8].

Oxidative stress is involved in autophagy, while autophagy dysfunction causes reactive oxygen species (ROS) accumulation. Moreover, insoluble protein degradation is impaired in ALS, hence, aggregation of misfolded proteins (e.g., SOD1, OPTN, VCP, TDP-43, ubiquitin-2, and FUS) is a pathological marker of the disease [9]. In addition, the autophagy-associated gene SQSTM1/p62 is mutated in some ALS cases, accumulating in the lumbar anterior horn of mice with ALS [10]. p62/SQSTM1 is associated with polyubiquitinated proteins and is degraded by autophagy. Similarly, Song et al. [11] revealed that the expression of an autophagy marker (microtubule-associated protein 1 light chain 3 (LC3)-II protein) is increased in the spinal cords of human patients and animal models. Furthermore, Fabbrizio et al. [12] demonstrated that activating purinergic P2 receptors (P2X7) controls autophagic flux by regulating LC3b and p62 proteins in SOD1G93A mice, linking anti-neuroinflammatory events with autophagy and suggesting several targets for ALS treatments.

Herbal medicines are made from plant extracts comprising many compounds and elicit multitarget pharmacological actions [13]. These medicines typically have anti-inflammatory and antioxidant effects and have, thus, been used to treat many diseases for millennia in China and other Asian countries [14]. Indeed, several active compounds in a preparation elicit their effects by targeting different macromolecules, each at low and safe concentrations, making herbal medicines useful for the treatment and prevention of different diseases.

Chaenomeles sinensis Koehne (CS) has been used to treat anaphylaxis, viral infections, and neurodegenerative diseases (e.g., Alzheimer’s disease) and has demonstrated anti-hyperuricemic and nephroprotective effects [15, 16]. Kang et al. [17] demonstrated the antioxidant and anti-inflammatory effects of the CS extract in benign prostatic hyperplasia, and revealed that CS induces the expression of nuclear factor erythroid-2-related factor 2 while reducing that of pro-inflammatory proteins, including cyclooxygenase-2, inducible nitric oxide synthase, tumor necrosis factor-$\alpha$ (TNF-$\alpha$), and interleukin (IL)-2. Collectively, this suggests that CS can help manage chronic diseases through its antioxidant and anti-inflammatory effects [17]. Moreover, Kim et al. [18] revealed that multiple bioactive compounds from CS extracts act on various targets, exhibiting anti-neuroinflammatory (reduction in NO levels), neurotrophic, and cytotoxic effects in vitro.

Although the FDA recently approved riluzole, an anti-glutamatergic agent for treating ALS, it only extends survival by 2–3 months. Therefore, more effective treatments are required for ALS. In this study, we examined the effects of CS extract treatment on hSOD1${}^{\text{G93A}}$ mice, an animal model of ALS. We demonstrated that the CS extract exerted anti-neuroinflammatory activity and reduced autophagic dysfunction while improving the motor functions of the affected animals. Our findings suggest that CS is a potential candidate for the treatment of inflammation in neurodegenerative diseases, including ALS.

ALS is characterized by progressive motor neuron death and muscle atrophy, leading to respiratory failure and death. Although edaravone and riluzole are approved for treating ALS, these medications have limited effects on improving the lifespan, necessitating the development of more effective therapeutic strategies. Therefore, we investigated the effects of CS, an herbal medicine, on the muscles and spinal cords of hSOD1${}^{\text{G93A}}$ mice in a murine model of ALS. ALS is a systemic disease, and herbal medicines contain various compounds that act on multiple targets. We revealed that the anti-inflammatory and antioxidant effects of CS in hSOD1${}^{\text{G93A}}$ mice were associated with improved motor activity and muscle function.

Neuroinflammation is a common pathological mechanism of many neurodegenerative diseases. In ALS, neuroinflammation is characterized by increased activation of microglial and astroglial cells. In addition, pro-inflammatory cytokines, such as IL-6, IL-1$\beta$, IL-6, IL-8, vascular endothelial growth factor, and TNF-$\alpha$, have been detected in the peripheral blood of patients with ALS [23]. Activated microglia and astrocytes induce neuroinflammation, which is aggravated by disease progression in the spinal cords of hSOD1${}^{\text{G93A}}$ mice [24]. However, given that our study did not determine which specific cytokines are related to ALS, additional investigation on the relationship between cytokine levels and disease severity is warranted to potentially identify new diagnostic and prognostic biomarkers.

Beers et al. [24] revealed that CD4+ T lymphocytes augment survival rates by modulating resident microglia and increasing IGF-1 secretion, as T lymphocytes promote protective microglial responses. Activated astrocytes disrupt the clearance of extra glutamate from synaptic clefts due to the loss of the glutamate transporter EAAT2/GLT-1, consequently leading to exacerbated motor neuron degeneration in patients with ALS [25]. Papadeas et al. [25] demonstrated that glial-restricted precursor cells transplanted into hSOD1${}^{\text{G93A}}$ mice differentiated exclusively into astrocytes and facilitated motor neuron death and respiratory dysfunction, a feature of ALS with astrocytic changes. In addition, T cells and CD4+ cells are involved in neuroinflammation in ALS, and abnormal T-cell activation and differentiation in TANK-binding kinase 1 (TBK1) knockout mice augment neuroinflammation via an increase in Foxo 1 phosphorylation [26].

Herbal medicines have been used to prevent diseases worldwide for centuries and are notable in the histories of China, Japan, and Korea. Herbal medicines are extracts and mixtures of multiple bioactive molecules that modulate multiple molecular targets. Some studies have shown that this could contribute to the effectiveness and low toxicity of many herbal medicines. Ginseng is an example of a multitarget herbal medicine. Two disease-modulating compounds with different molecular targets have been isolated from the ginseng plant. Subsequent treatment of an MPTP-based mouse model of Parkinson’s disease with ginsenoside Rb1 ameliorated motor deficits and reduced excitotoxicity. This was likely caused by increased expression of the glutamate transporter GLT-1, promoting glutamate uptake in the midbrains and prefrontal cortices of diseased mice [16]. Meanwhile, administration of ginsenoside Rd increased extracellular glutamate clearance via upregulating an astrocytic glutamate transporter via the PI3K/AKT and ERK1/2 pathways [27]. These studies established that an active compound originating from herbal medicines could modulate glutamate systems in treating neuronal diseases caused by glutamate excitotoxicity, such as ALS. Bioactive compounds have been utilized as starting points in medicinal chemistry efforts, guided by network pharmacology and molecular docking analyses [28]. Similar efforts could be applied to bioactive compounds isolated from the CS extract in future work, potentially leading to the identification of compounds with improved properties.

Disease progression in our ALS model (hSOD1${}^{\text{G93A}}$ mice) was characterized by muscular dysfunction involving impairment of neuromuscular junctions (NMJs) and oxidative stress-induced dysfunction of mitochondria [29]. These dysfunctions are consistent with those observed in other animal models of ALS and human diseases. For example, Xiao et al. [22] reported the emergence of elevated cyclophilin D levels in the muscles of patients with advanced ALS, consistent with mitochondrial dysfunction and ROS production. However, further studies are required to elucidate the signaling pathways involved in this process. Graber et al. [30] demonstrated that activated macrophages in the peripheral sciatic nerve and increased inflammatory events are associated with motor weakness. Moreover, Dupuis et al. [31] reported the involvement of Nogo-A—a member of the reticulon 4 (Rtn4) protein family—in skeletal muscle inflammation and suggested that increased Nogo-A expression could accelerate the progression of motor innervation in an ALS animal model. Furthermore, Bruneteau et al. [32] revealed that Nogo-A is expressed in type I fibers and participates in the degeneration of NMJs in patients with ALS, suggesting that NMJ alterations could be prognostic indicators of motor impairment and neurodegeneration.

In this study, we demonstrated that CS treatment improved motor function and attenuated the expression of inflammatory and oxidative stress-related proteins in the muscles and spinal cords of hSOD1${}^{\text{G93A}}$ mice. The observed changes in body and muscle weights were as expected, with Tg mice weighing less than age-matched nTg mice and consistent with the progression of muscular atrophy in hSOD1${}^{\text{G93A}}$ mice. Notably, weight differences help distinguish between presymptomatic and symptomatic animals. We detected impaired motor function in the footprint test, while CS treatment increased body and muscle weights, although the difference was not statistically significant. The increase in GC muscle weight is partially related to improving muscle function as the GC contributes to the function of other “fast” movements and energy metabolism in hSOD1${}^{\text{G93A}}$ mice. Future experiments are required to understand further the relationship between glucose metabolism and mitochondrial function in the muscle and confirm the relationship between GC muscle weight gain and motor function in CS-treated hSOD1${}^{\text{G93A}}$ mice. Additionally, CS prevented the loss of motor neurons in the SP of the hSOD1${}^{\text{G93A}}$ mice. Our findings suggest that CS treatment augments anti-inflammatory events, improving motor function and the survival of motor neurons in the muscles and spinal cords of hSOD1${}^{\text{G93A}}$ mice. These results are consistent with those of previous studies on the biological activities of CS [17, 18], including antioxidant, antitumor, anti-hepatitis, antimicrobial, immunoregulatory, anti-influenza, and anti-Parkinson effects [33, 34, 35, 36, 37].

ALS is a complex disease involving multiple pathogenic mechanisms and interrelated pathways, including mitochondrial dysfunction, oxidative stress, and autophagic dysfunction. Autophagy regulates skeletal muscle remodeling and maintains muscle function during movement. In addition, neurogenic atrophy is induced by skeletal muscle denervation through the suppression of autophagy. Furthermore, muscle regeneration is involved in autophagic flux, and autophagy contributes to mitochondrial regeneration [38]. The role of dysregulation of autophagy in ALS is well understood. Mutations in the genes encoding SQSTM1/p62, TDP-43, and OPTN [39] contribute to the progression of ALS [40]. p62/SQSTM1 is associated with polyubiquitinated proteins and degraded by autophagy. Simultaneously, TDP-43 aggregation can be eliminated by autophagic flux and autophagosome formation either via mTOR inhibition-dependent or -independent pathways. Indeed, mTOR signaling pathway regulates autophagy and muscle regeneration. Although various drugs have been developed that modulate autophagy, many gaps exist in our understanding of the molecular mechanisms underlying their effects. Nevertheless, mTOR inhibitors are currently being evaluated in ALS clinical trials; however, to our knowledge, no results have been published. Our study shows that CS regulates autophagy in muscles; however, whether CS affects muscle regeneration and neuromuscular connections in hSOD1${}^{\text{G93A}}$ mice remains to be determined. Moreover, future studies are needed to unravel the relationship between muscle metabolism and autophagy regulation by CS in hSOD1${}^{\text{G93A}}$ mice and expand investigations to other animal models of ALS. Furthermore, additional studies investigating the effects of CS in the muscles and SP of other ALS animal models and the survival rate in CS-treated groups are warranted to consider CS treatment in patients with ALS [5].

In this study, we demonstrated that CS improved motor function and enhanced anti-inflammatory events both in the muscles and spinal cord of hSOD1${}^{\text{G93A}}$ mice. Additionally, CS inhibited motor neuron death in the SP and regulated autophagy in the muscles of hSOD1${}^{\text{G93A}}$ mice. The results suggest that CS can delay disease progression and the onset of a systemic disease, such as ALS. However, further studies are needed to investigate the bioactive compounds of CS for the development of drug candidates for ALS treatment.

All data are available within the article and available by reasonable request.

EJY conceptualized the study, performed the animal procedures, and reviewed and edited the manuscript. SHL performed biochemical and molecular studies and analyzed the data. Both authors contributed to editorial changes in the manuscript. Both authors have read and agreed to the published version of the manuscript. Both authors have participated sufficiently in the work and agreed to be accountable for all aspects of the work.

The Institutional Animal Care Committee of the Korea Institute of Oriental Medicine approved the experimental protocol (KIOM protocol # 17-061), and all experiments were performed in accordance with the U.S. National Institutes of Health guidelines and guidelines of the Animal Care and Use Committee at the KIOM.

Not applicable.

This work was supported by the Basic Science Research Program through the National Research Foundation of Korea, funded by the Ministry of Science, ICT, and Future Planning, South Korea, under grant NRF-2020R1A2C2006703 and KIOM, KSN2212010 and C18040.

The authors declare no conflict of interest.