Anti-Inflammatory Effects of Chaenomeles sinensis Extract in an ALS Animal Model

Background : Amyotrophic lateral sclerosis (ALS) is a systemic disease with multiple pathological effects, including neuroinflammation, oxidative stress, autophagy, mitochondrial dysfunction, and endoplasmic reticulum stress. Despite many studies seeking to identify and develop effective therapies, effective ALS treatment has yet to be approved. Hence, patients with ALS ultimately experience muscle atrophy and loss of motor neurons. Herbal medicines have been used to treat numerous diseases by modulating multiple biological processes and exerting pharmacological effects, including anti-inflammatory and antioxidant properties. In particular, Chaenomeles sinensis Koehne (CS) exhibits anti-hyperuricemic and nephroprotective effects and is used to treat anaphylaxis, viral infections, and neurodegenerative diseases, such as Alzheimer’s disease. This study monitored the effects of CS supplementation on muscle function and motor neurons in hSOD1 G93A mice, an established ALS animal model. Methods : Body weight measurements and behavioral tests were performed; additionally, western blotting and immunohistochemistry analyses were conducted using the mice gastrocnemius, tibialis anterior, and spinal cord. Results : CS augmented anti-inflammatory and antioxidant effects in the muscle and spinal cord of hSOD1 G93A mice. Furthermore, CS improved motor function and regulated autophagy in the muscles of the hSOD1 G93A mice. Conclusions : CS might represent a promising supplement for improving motor function and delaying ALS progression. However, its development for clinical use warrants further investigation.


Introduction
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 kinasebinding 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 antineuroinflammatory 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 antineuroinflammatory 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 antiinflammatory 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-α (TNF-α), 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 antiglutamatergic 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 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.

Animals
Male hemizygous B6SJ/hSOD1 G93A mice (8 weeks old) were purchased from Jackson Laboratory (Bar Harbor, ME, USA) and maintained in an air-conditioned room at 20-23 °C under a 12 h light/dark cycle and relative humid-ity of 50 ± 10%.Male hSOD1 G93A mice were randomly divided into the following groups: non-transgenic mice (nTg) (n = 8), hSOD1 G93A transgenic mice (Tg) (n = 8), and CS extract-treated hSOD1 G93A transgenic mice (Tg+CS) (n = 8).The CS extract (1 mg/g) was orally administered to hSOD1 G93A transgenic mice once daily for six weeks, starting at eight weeks of age.The nTg and Tg mice were administered distilled water as controls.

Preparation of Chaenomeles sinensis Koehne Extract
CS was purchased from Kwang Myung Dang Medical Herbs Co. Ltd. (Ulsan, Republic of Korea).CS extract was prepared as previously described [19].CS was extracted using distilled water.The CS extracts were dried and stored in powder form.Before administration, powdered CS extract was dissolved in distilled water.Human-equivalent doses of CS in mice were calculated based on results from adult human subjects (5 g/60 kg body weight/day) [20].

Measurement of Muscle Weight and Footprint Test
Mice were euthanized by intraperitoneal injection of avertin (250 mg/kg).Gastrocnemius (GC) muscle weight was measured using a previously described method [21].The footprint test was performed one day before the mice were sacrificed to prepare tissues for biochemical analysis.The footprint test was conducted as previously described [19].Stride length was measured by staining mouse hind paws and allowing them to walk on an alley floor (70 cm × 6 cm × 16 cm) covered with white paper.Each mouse performed the footprint test thrice; the average stride length was determined for each group.

Immunohistochemistry
Spinal cord tissues obtained after sacrificing mice were fixed in 4% paraformaldehyde, embedded in paraffin (Merck), and sectioned.The sections were deparaffinized and incubated with 3% hydrogen peroxide (H 2 O 2 ) and 5% bovine serum albumin in 0.01% PBS-Triton X-100 (Sigma-Aldrich, Oakville, ON, Canada).Immunohistochemical staining of SP tissue sections was performed using anti-ChAT (Thermo Scientific, Wilmington, DE, USA), anti-Iba1 (Fujifilm Wako Chemical, Richmond, VA, USA), and anti-GFAP (Agilent Technologies, Santa Clara, CA, USA).Anti-horseradish peroxidase-conjugated mouse or rabbit IgG (GenDEPOT, Katy, TX, USA) secondary antibodies were the added.For visualization, the slides were incubated with diaminobenzidine (DAB) (Vector Laboratories, Newark, CA, USA) before images were captured using an Olympus BX51 microscope (Olympus, Tokyo, Japan), and the intensity of the primary antibodies was measured using the ImageJ software.
Nissl staining was performed using 0.1% cresyl violet.The stained slides were dehydrated using an alcohol gradient and mounted with histomounting media (Sigma-Aldrich, Oakville, ON, Canada).

Ethics Approval
The Institutional Animal Care Committee of the Korea Institute of Oriental Medicine approved the experimental protocol (korea institute of oriental medicine (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.

Chaenomeles sinensis Koehne Improves Motor Activity of hSOD1 G93A Mice
To investigate the effects of CS administration in an ALS murine model, we measured the body and muscle weights and performed a footprint test.The body and  GC muscle weights of Tg mice were lower than those of nTg mice; however, CS treatment did not alter the body weight of Tg mice (Fig. 1A).CS treatment increased the weight of the GC muscle compared to that in Tg mice; however, these changes were not significant.Furthermore, CS treatment increased stride length by 1.2-fold in Tg mice (Fig. 1B).These results suggest that CS supplementation improves motor activity and muscle function in an ALS murine model.

Chaenomeles sinensis Koehne Attenuates Inflammation in Muscle and Spinal Cords of hSOD1 G93A Mice
To examine the molecular mechanisms underlying the effects of CS on muscle function, we studied its antiinflammatory effects in the muscles and spinal cords of hSOD1 G93A mice.The levels of the inflammation-related proteins CD11b and GFAP were reduced by 1.2-and 2.1fold, respectively, in the TA of Tg+CS mice compared to the TA of Tg mice (Fig. 2A).In addition, the expression of CD11b was decreased by 1.4-fold in the GC of CS-treated Tg mice compared to the GC of Tg mice (Fig. 2B).CS treatment reduced the expression levels of TLR4 and IL-1β by 2.0-and 4.2-fold, respectively, in the SP of CS-treated Tg mice compared to the SP of Tg mice (Fig. 2C).We confirmed the anti-neuroinflammatory effects of anti-GFAP and anti-Iba1 in the SP via immunohistochemical analysis in each group (nTg, Tg, and Tg+CS groups).GFAP-and Iba1-positive intensities were significantly decreased by 2.3-and 3.0-fold, respectively, in the spinal cords of the Tg+CS group compared with those of the Tg group.Furthermore, the abundance of Nissl-and ChAT-positive motor neurons had increased by 2.0-and 5.3-fold, respectively, in the Tg+CS group compared with the Tg group (Fig. 2D).These findings suggest that CS supplementation prevents motor neuron cell death by inhibiting neuroinflammatory signaling in hSOD1 G93A mice.

Chaenomeles sinensis Koehne Shows Antioxidant Effects in the Muscles of hSOD1 G93A Mice
Considering the primary role of oxidative stress in inflammatory events, the antioxidant effect of CS was investigated in the muscles and spinal cords of hSOD1 G93A mice.In the TA, the levels of the oxidative stress-related proteins HO1, Bax, and ferritin were increased by 2.9-, 4.2-, and 2.7-fold, respectively, in Tg mice compared with nTg mice.However, CS treatment significantly reduced the expression of HO1, Bax, and ferritin by 1.3-, 1.3-, and 1.4fold, respectively, in the TA of hSOD1 G93A mice (Fig. 3A).Similarly, CS treatment reduced the levels of oxidative stress-related proteins (transferrin) by 1.9-fold in the SP of hSOD1 G93A mice (Fig. 3B).These results are consistent with the antioxidant effects of CS, which induced improvements in motor function and motor neuron survival in an ALS murine model.

Chaenomeles sinensis Koehne Regulates Autophagy in the Muscles of hSOD1 G93A Mice
Impaired autophagy can induce muscle dysfunction [22].Autophagy-related proteins, including p62 and LC3b, were significantly reduced by 1.4-and 1.3-fold, respectively, in the TA of CS-treated hSOD1 G93A mice compared with that in hSOD1 G93A mice (Fig. 4A).In addition, CS treatment decreased the levels of the muscle atrophy-related proteins p-mTOR and SMAD2 by 1.3-and 2.8-fold, respectively, in the TA of hSOD1 G93A mice (Fig. 4A).The p62 and LC3b levels were also reduced by 1.5-and 1.5-fold, respectively, in the GC of CS-treated hSOD1 G93A mice compared to those of hSOD1 G93A mice (Fig. 4B).These findings suggest that CS prevents muscle atrophy by regulating autophagic dysfunction in hSOD1 G93A mice.

Discussion
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 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 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, proinflammatory cytokines, such as IL-6, IL-1β, IL-6, IL-8, vascular endothelial growth factor, and TNF-α, 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 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 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 MPTPbased 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 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 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 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 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 G93A mice.Additionally, CS prevented the loss of motor neurons in the SP of the hSOD1 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 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 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 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].

Conclusions
In this study, we demonstrated that CS improved motor function and enhanced anti-inflammatory events both in the muscles and spinal cord of hSOD1 G93A mice.Additionally, CS inhibited motor neuron death in the SP and regulated autophagy in the muscles of hSOD1 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.

Fig. 1 .
Fig. 1.Chaenomeles sinensis Koehne (CS) improves muscle function in hSOD1 G93A mice.(A) After administration of CS (1 mg/g) for six weeks to hSOD1 G93A mice, the body and muscle weight of the gastrocnemius in the posterior leg were measured in the non-Tg, Tg, and Tg+CS groups (n = 6/group).The weight of the gastrocnemius muscle was measured after the skin had been peeled off.(B) To examine motor function, a footprint test was performed in each group of mice (n = 7/group).Bars represent the mean ± tandard error of the mean (SEM).*p < 0.05, ***p < 0.001, ****p < 0.0001.ns, not significant; non-Tg, D.W.-treated non-transgenic mice; Tg, D.W.-treated transgenic mice; Tg+CS, C. sinensis Koehne-treated transgenic mice.

Fig. 2 .
Fig. 2. Chaenomeles sinensis Koehne (CS) attenuates inflammatory events in the muscle and spinal cord of hSOD1 G93A mice.(A) Mice were sacrificed under avertin (250 mg/kg) anesthesia, and the tibialis anterior (TA) in the anterior leg, gastrocnemius (GC) in the posterior leg, and spinal cord (SP) of each animal were collected for western blotting.Western blotting was performed using anti-CD11b and anti-GFAP antibodies in the TA of the non-Tg, Tg, and Tg+CS groups.(B) Immunoblotting with anti-CD11b in GC was performed for the non-Tg, Tg, and Tg+CS groups.Actin was used for the normalization of the immunoblots.(C) Anti-TLR4 and anti-IL-1β antibodies were used for western blotting of the spinal cords of the non-Tg, Tg, and Tg+CS groups.Tubulin was used as the loading control.(n = 3, 4/group).(D) Spinal cord tissues obtained after sacrificing the mice were fixed and sectioned for immunohistochemistry. Immunohistochemistry was performed using anti-GFAP and anti-Iba1, along with anti-ChAT and Nissl staining in the spinal cord of the non-Tg, Tg, and Tg+CS groups.Quantification of GFAP and Iba1 intensity was performed using ImageJ software.Scale bar = 100 mm; quantification bars represent the mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.ns, not significant; non-Tg, D.W.-treated non-transgenic mice; Tg, D.W.-treated transgenic mice; Tg+CS, C. sinensis Koehne-treated transgenic mice.