The Role of Inflammation Mediators in Neurological DiseasesView this Special Issue
Treatment with Herbal Formula Extract in the hSOD1G93A Mouse Model Attenuates Muscle and Spinal Cord Dysfunction via Anti-Inflammation
Amyotrophic lateral sclerosis (ALS), a multicomplex neurodegenerative disease, has multiple underlying pathological factors and can induce other neuromuscular diseases, leading to muscle atrophy and respiratory failure. Currently, there is no effective drug for treating patients with ALS. Herbal medicine, used to treat various diseases, has multitarget effects and does not usually induce side effects. Each bioactive component in such herbal combinations can exert a mechanism of action to increase therapeutic efficacy. Herein, we investigated the efficacy of an herbal formula, comprising Achyranthes bidentata Blume, Eucommia ulmoides Oliver, and Paeonia lactiflora Pallas, in suppressing the pathological mechanism of ALS in male hSOD1G93A mice. Herbal formula extract (HFE) (1 mg/g) were orally administered once daily for six weeks, starting at eight weeks of age, in hSOD1G93A transgenic mice. To evaluate the effects of HFE, we performed footprint behavioral tests, western blotting, and immunohistochemistry to detect protein expression and quantitative PCR to detect mRNA levels in the muscles and spinal cord of hSOD1G93A mice. HFE-treated hSOD1G93A mice showed increased anti-inflammation, antioxidation, and regulation of autophagy in the muscles and spinal cord. Thus, HEF can be therapeutic candidates for inhibiting disease progression in patients with ALS. This study has some limitations. Although this experiment was performed only in male hSOD1G93A mice, studies that investigate the efficacy of HEF in various ALS models including female mice, such as mice modeling TAR DNA-binding protein 43 (TDP43) and ORF 72 on chromosome 9 (C9orf72) ALS, are required before it can be established that HEF are therapeutic candidates for patients with ALS.
Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease with various underlying pathological factors, including genetic and environmental factors that induce other neuromuscular diseases, leading to muscle atrophy and respiratory failure. The known pathological mechanisms of ALS are mitochondrial dysfunction, oxidative stress, and neuroinflammation in the glial cells . Activated astrocytes and microglia release proinflammatory cytokines and toxic factors that contribute to neurotoxicity , and energy metabolism plays a key role in driving the onset and progression of ALS . In preclinical studies, drugs have targeted these mechanisms for treating ALS; however, most drugs targeting a single process have been unsuccessful in clinical trials . Therefore, there is an urgent need for an effective cure for ALS. Patients with ALS and their families have a poor quality of life. Moreover, only a few drugs, such as riluzole and edaravone, can delay death by 3–4 months. ALS results from several pathological dysfunctions, including metabolic dysfunction in the spinal cord and muscle, and therefore, new therapies and drug discoveries should be aimed at multiple targets in the muscles and spinal cord [5, 6].
Herbal medicines have been used to treat various diseases because they rarely induce side effects and have multitarget effects . In ALS, conventional drugs, such as riluzole and edaravone, which are mostly ineffective and cause side effects, do not greatly prolong the survival of patients; therefore, herbal medicines have been evaluated for use in patients with ALS . Each component of this herbal combination may use a different mechanism of action, thus increasing therapeutic efficacy. According to our previous study, treatment with Bojungikgi-tang showed neuroprotective effects and delayed disease progression in an ALS animal model . Achyranthes bidentata Blume strengthens bones and muscles and protects against N-methyl-D-aspartate-induced excitotoxicity in hippocampal neurons  and nerve crush injury in mice . Eucommia ulmoides Oliver improves metabolic functions by decreasing ATP levels and increasing the use of ketone bodies and glucose in the skeletal muscle . Paeonia lactiflora Pallas exerts protective, analgesic, anti-inflammatory, and immunomodulatory effects in vitro and in vivo . Since ALS is a complex disease caused by muscle dysfunction and loss of motor neurons, we examined the effects of an herbal medicine formula containing A. bidentata Blume, E. ulmoides Oliver, and P. lactiflora Pallas to harness the multitarget advantage of herbal medicine on tibialis anterior (TA), gastrocnemius (GC), and spinal cord (SP) of hSOD1G93A mice. In this study, we demonstrated that HFE treatment exhibited anti-inflammatory and antioxidative effects and enhanced autophagy dysfunction in the TA, GC, and SP of hSOD1G93A mice, suggesting that multitargeted treatment with HFE regulates the ALS-inducing pathological mechanism.
2. Materials and Methods
Male B6SJL-hSOD1G93A and female B6SJL mice were purchased from Jackson Laboratory (Bar Harbor, ME, USA) and mated to obtain hemizygous male hSOD1G93A mice. Hemizygous hSOD1G93A mice were selected as described previously . Two to three mice were placed in each cage, habituated to the specific pathogen-free cages, and maintained in the specific pathogen-free animal facility at a temperature of 20 ± 2°C and humidity of 50 ± 10%, with a 12-h : 12-h light : dark cycle. Food and tap water were provided ad libitum. Behavioral tests were performed by experimenters who were blinded to the experimental groups. All mouse experiments were approved by the Institutional Animal Care and Use Committee of the Korea Institute of Oriental Medicine (KIOM protocol# 17-061). Male hSOD1G93A mice were randomly divided into the following groups: nontransgenic mice (nTg), =8; hSOD1G93A transgenic mice (Tg), =8; and herbal formula extract (HFE)-treated hSOD1G93A transgenic mice (Tg + HFE), =8. HFE (1 mg/g) was orally administered once daily for six weeks, starting at eight weeks of age in hSOD1G93A transgenic mice. The nTg and hSOD1G93A transgenic mice were used as controls and were administered distilled water (Figure 1).
2.2. Treatment with Herbal Extract
The herbal formula composed of A. bidentata Blume, E. ulmoides Oliver, and P. lactiflora Pallas (1 : 1 : 1) was purchased from Kwangmyungdang Medicinal Herbs Co. (Ulsan, Republic of Korea). The HFE was prepared as previously described .
2.3. Footprint Test
A footprint test was conducted on the day before the mice were sacrificed to measure motor activity. The footprint test was performed as previously described .
2.4. Tissue Preparation and Western Blotting
After anesthetizing the mice with avertin (250 mg/kg), the tibialis anterior (TA), gastrocnemius (GC), and spinal cord (SP) were collected and homogenized in RIPA buffer (50 mM Tris-Cl [pH 7.4], 1% NP-40, 0.1% sodium dodecyl sulfate [SDS], and 150 mM NaCl) containing a protease and phosphatase inhibitor cocktail (Thermo Fisher Scientific, Waltham, MA, USA). Homogenized tissues were centrifuged at 13,000 rpm for 15 min at 4°C. The total protein was quantified using a bicinchoninic acid assay kit (Pierce, Rockford, IL, USA).
Protein samples for western blotting were heated with SDS sample buffer. Equal amounts of total protein were separated on a 4–12% SDS-PAGE precast gel (Thermo Fisher Scientific) and transferred onto polyvinylidene fluoride membranes. The membranes were incubated with the specific primary antibodies for 12 h at 4°C. The primary antibodies used were anti-β catenin (1 : 1,000; Cell Signaling Technology, Danvers, MA, USA), anti-P62 (1 : 1,000; Cell Signaling Technology), anti-LC3b (1 : 1,000; Cell Signaling Technology), anti-GFAP (1 : 5,000; Agilent Technologies, Santa Clara, CA, USA), anti-CD11b (1 : 1,000; Abcam, Cambridge, MA, USA), anti-BAX (1 : 1,000; Santa Cruz Biotechnology, Dallas, TX, USA), anti-HO1 (1 : 1,000; Abcam), anti-ferritin (1 : 1,000; Abcam), anti-transferrin (1 : 1,000; Santa Cruz Biotechnology), anti-TGF-β (1 : 1,000; Cell Signaling Technology), anti-SMAD2 (1 : 1,000; Cell Signaling Technology), anti-β-actin (1 : 1,000; Santa Cruz Biotechnology), and Tubulin (1 : 1,000; Abcam). The membranes were then incubated with horseradish peroxidase-conjugated secondary antibodies (anti-rabbit or anti-mouse; Santa Cruz Biotechnology) and washed with TBST. The membranes were probed with enhanced chemiluminescence reagents (Thermo Fisher Scientific) and visualized using an imaging system (Bio-Rad, Hercules, CA, USA) for chemiluminescence detection. All immunoblots were quantified using the ImageJ software (National Institutes of Health, Bethesda, MD, USA).
The spinal cord was fixed in 4% paraformaldehyde and rinsed in 0.01 M PBS. Sectioned paraffin tissues were deparaffinized, and immunohistochemistry was performed as described previously . The primary antibodies used were anti-CHAT (1 : 500; Thermo Scientific, Wilmington, DE, USA), anti-Iba1 (1 : 2,000; Fujifilm Wako chemical, Richmond, VA, USA), and anti-GFAP (1 : 5,000; Agilent Technologies), and the secondary antibody was an anti-horseradish peroxidase-conjugated mouse or rabbit IgG (GenDEPOT, Katy, TX, USA). The spinal cord slides were incubated with anti-mouse or rabbit IgG biotinylated secondary antibodies (Vector Laboratories, Burlingame, CA, USA) using diaminobenzidine (Vector Laboratories). Diaminobenzidine-stained slides were dehydrated with serial ethanol and xylene solutions and mounted with a coverslip and mounting solution. Images were captured using an Olympus BX51 microscope, and the intensity of the primary antibodies was quantified using ImageJ software.
2.6. RNA Extraction and Real-Time Reverse Transcription PCR
Total RNA was extracted from the muscle tissues using the RNA extraction kit (Intron Biotechnology, Seongnam-Si, Korea). RNA extraction and the RT-PCR were performed as previously described . The primer sequences used in this study are shown in Table 1. The relative mRNA levels of the target genes were presented as fold values.
2.7. Statistical Analysis
All data are reported as the mean ± standard error (SEM). Statistical analyses were performed using GraphPad Prism 9 software (GraphPad, Inc., La Jolla, CA, USA). The results were analyzed with a one-way analysis of variance followed by Tukey’s test for multiple comparisons. Statistical significance was set at .
3.1. HFE Treatment Improved Motor Activity and Reduced Inflammation-Related Protein Levels in the TA and GC of hSOD1G93A Mice
To investigate the effect of HFE on motor activity, we measured the stride length of the mice in a footprint test. As shown in Figure 2, HFE improved motor function by 1.3-fold compared with the Tg group ().
Next, to examine the biological mechanism involved in improving motor function following HFE treatment, we evaluated the effect of HFE on inflammation and oxidative stress in the TA and GC of hSOD1G93A mice. Levels of inflammation-related proteins such as β-catenin, CD11b, and GFAP were increased by 3.4-, 6.6-, and 2.0-fold, respectively, compared with those in nTg mice (, , ). However, HFE treatment significantly reduced these levels by 1.9-. 2.4-, and 1.6-fold, respectively, compared with those in Tg mice (, , Figures 3(a) and 3(c)).
In addition, HFE treatment reduced the mRNA level of IL18 by 1.5-fold compared with that in the TA of Tg mice (, Figure 3(b)). Oxidative stress is involved in inflammation . Therefore, we also determined the effect of HFE on the expression levels of oxidative stress-related proteins in the TA of the nTg, Tg, and Tg-HFE groups. The levels of oxidative stress-related proteins, including HO1, ferritin, transferrin, and Bax, were increased by 4.1-, 6-, 1.5-, and 5-fold, respectively, in the TA of the Tg groups compared with those in the nTg group (, , , , Figure 3(d)). Furthermore, HFE treatment dramatically reduced HO1, ferritin, transferrin, and Bax levels in the TA of the Tg group by 3-, 2.7-, 1.6-, and 1.5-fold, respectively (, , , , Figure 3(d)). Furthermore, the mRNA level of COX IV1a was significantly reduced by 1.2-fold in the TA of HFE-treated mice compared with that in Tg mice (, Figure 3(e)). These findings suggest that HFE treatment improved motor activity by regulating inflammatory reactions in the skeletal muscle of hSOD1G93A mice.
3.2. HFE Treatment Regulates Autophagy Function in TA and GC of hSOD1G93A Mice
Autophagy dysfunction is a pathological feature of the muscles and spinal cord in ALS. In addition, because muscle atrophy is caused by autophagy dysfunction, we investigated the expression of autophagy-related proteins, such as p62 and LC3b, and atrophy-related proteins, including TGFβ and SMAD2, in the TA and GC of hSOD1G93A mice. As shown in Figure 4, the levels of p62 and LC3b proteins were reduced by 2.0- and 1.5-fold (, ) in the HFE-treated TA and 1.5- and 2.0-fold in the HFE-treated GC, respectively, compared with those in Tg mice (, , Figures 4(a) and 4(c)).
In addition, the expression of TGFβ and SMAD2 was decreased by 2.3- and 1.7-fold (, ) in the HFE-treated TA and 1.8- and 1.5-fold in the HFE-treated GC (, ), respectively, compared with those in Tg mice (Figures 4(b) and 4(e)). Furthermore, HFE treatment significantly attenuated the mRNA levels of muscle denervation-related genes, myogenin (Myog). and cholinergic receptor nicotinic gamma subunit (Chrng), by 1.5- and 1.3-fold (, ), respectively, in the TA compared with those in the Tg group (Figure 4(d)).
3.3. HFE Treatment Attenuates Motor Neuronal Cell Death in the Spinal Cord of hSOD1G93A Mice
To examine the effect of HFE on motor neuron death, we investigated the immunoreactivity of CHAT, Iba1, and GFAP in the spinal cord of hSOD1G93A mice. The number of motor neuron cells positive for CHAT increased by 6.3-fold in the ventral horn of the spinal cord in HFE-treated mice than in hSOD1G93A mice (, Figure 5(a)). In addition, the expression of neuroinflammatory proteins, Iba1 and GFAP, was significantly reduced by 1.8- and 2.1-fold (, ), respectively, in the spinal cord of HFE-treated hSOD1G93A mice compared with that in hSOD1G93A mice (Figure 5(a)). Furthermore, SMAD2, p62, and ferritin levels were dramatically decreased by 1.7-, 1.6-, and 3.2-fold (, , ), respectively, in the spinal cord of hSOD1G93A mice following HFE treatment compared with those in Tg mice (Figure 5(b)).
Thus far, an effective drug that can improve the quantity of life of patients with ALS is yet to be developed. Therefore, we examined the effects of herbal medicines against the multiple pathological mechanisms of ALS, including metabolic and muscle dysfunction, inflammation, autophagy dysfunction, and oxidative stress [17–19] in the muscle and spinal cord of an ALS animal model. We found that the HFE (A. bidentata Blume, E. ulmoides Oliver, and P. lactiflora Pallas) improved muscle function by reducing inflammation and autophagy dysfunction in the muscle and spinal cord of hSOD1G93A mice.
Neuroinflammation or inflammation is critical in neurodegenerative diseases, including Alzheimer’s disease, Parkinson’s disease, and ALS. In ALS, microglia activation and activated astrocytes are increased in the spinal cord, inducing motor neuron death . In addition, neuroinflammation in hSOD1G93A mice is involved in the upregulation of NLRP3-inflammasome-related proteins . However, based on these findings, most drugs target a single mechanism, such as those with antiglutamatergic (ceftriaxone, memantine, and talampanel), anti-inflammation (celecoxib, erythropoietin, and NP001), antioxidative stress, and neurotrophic effects, have failed in clinical trials . TAR DNA-binding protein 43 (TARDBP, TDP-43) and ORF 72 on chromosome 9 (C9orf72) mutation were detected in sporadic and familiar ALS cases and involved in neuroinflammation [22, 23].
ALS is associated with muscular disease, including muscle weakness, inflammation, and denervated atrophy . Specifically, inflammation and oxidative stress affect muscle homeostasis and myogenesis following FOXO activation, leading to muscle atrophy. Lawler et al. showed that muscle atrophy is induced by Foxo3 activation under oxidative stress and inflammation conditions. Huang et al. also demonstrated that oxidative stress and inflammation suppression attenuated denervation-induced muscle atrophy .
Autophagy is important for maintaining muscle functions; however, dysfunction in autophagy induces muscle degeneration, inflammation, oxidative stress, and mitochondrial dysfunction in ALS [26–29]. In ALS, inflammatory events in the skeletal muscle induce motor weakness, neuromuscular junction impairment, and motor neuron death. Autophagy dysfunction induces oxidative stress  and antioxidants through redox signaling, and the Nrf2-Keap1 pathway regulates autophagy function . However, it is unclear whether autophagy activation or inactivation causes ALS. Zhang et al. reported that autophagy activation decreased the accumulation of misfolded proteins in the motor neurons of hSOD1G93A mice . However, Bhattacharya et al. showed that autophagy activation augmented motor neuron degeneration and did not extend the life span of hSOD1G93A mice . These results suggest that other mechanisms such as oxidative stress and inflammation should be considered therapeutic targets, as ALS is a complex disease. In this study, we demonstrated that HFE treatment exhibited anti-inflammatory and antioxidative effects and attenuated autophagy dysfunction in the TA, GC, and SP of hSOD1G93A mice, suggesting that multitargeted treatment with HFE regulates the ALS-inducing pathological mechanism.
In the skeletal muscle, denervation-induced muscle atrophy and neuromuscular junction impairments occur with motor neuron loss, and mitochondria are involved in inducing apoptosis for denervation . TGF-β, which causes muscle fibrosis, Smad, and histone deacetylase 4 (HDAC4), is related to ALS progression and is upregulated in ALS [35, 36]. Furthermore, HDAC4 activates the synaptic acetylcholine receptors MuSK to promote muscle reinnervation, and HDAC4 deletion contributes to neurogenic atrophy . Consistently, we found that the expression of TGF-β and Smad proteins was increased in the muscle of hSOD1G93A mice; however, treatment with HFE significantly reduced the levels of these proteins. Furthermore, denervation-related genes (Myog and Chrng) were reduced by HFE in the TA of hSOD1G93A mice. Thus, treatment with HFE may prevent muscle atrophy and delay motor neuron loss in hSOD1G93A mice.
Muscle metabolism is involved in body energy homeostasis, and denervation of muscle and atrophy is related to muscle metabolism. Therefore, metabolic dysfunction is a key factor in ALS. Dobrowonly et al. reported a relationship between metabolic changes and disease progression in a hSOD1G93A mouse model  and suggested that the muscle is a critical therapeutic target in ALS. Muscle metabolism is related to mitochondrial function. Mitochondrial abnormalities with dysregulation of respiratory complexes, such as complexes I and IV, were observed in the muscles of patients with ALS and an hSOD1G93A mouse model [39, 40]. We previously observed that herbal medicine improved motor activity and inhibited mitochondria cristae disruption in hSOD1G93A mice . Therefore, herbal medicines may improve motor function by regulating muscle metabolism via inhibition of mitochondria dysfunction in hSOD1G93A mice.
The HFE treatment improved motor function through anti-inflammation, antioxidative, and autophagy regulation effects in the TA, GC, and SP of hSOD1G93A mice. These findings suggest that HFE are therapeutic candidates for treating patients with ALS or inhibiting disease progression. However, this study has some limitations. The effects of HFE treatment on neuromuscular junction impairment in skeletal muscles and motor neurons should be further investigated in an ALS animal model. In addition, muscle metabolism and mitochondria dysfunction in hSOD1G93A mice should be examined, as HFEs improve motor function. Chang et al. had reported that TDP43-induced aggregation was reduced by berberine, herbal medicine, by the regulation of mTOR-autophagy signaling pathway . Therefore, studies are also needed to investigate the efficacy of HFE in various ALS models, such as mice modeling TDP43 and C9orf72 ALS, as this experiment was performed only in hSOD1G93A mice before it can be established that HFE are therapeutic candidates for patients with ALS.
All the data are available within the article.
The study was conducted according to the guidelines of the Korea Institute of Oriental Medicine, and the study was approved by the Institutional Animal Care Committee of the Korea Institute of Oriental Medicine (protocol number: 17-061).
Conflicts of Interest
The authors declare no conflict of interest.
EJY contributed to the study conception and design. Material preparation, data collection, and analysis were performed by EJY, SHL, and MC. The first draft of the manuscript was written by EJY and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript. Eun Jin Yang and Sun Hwa Lee contributed equally to this work.
This work was supported by the Basic Science Research Program through the National Research Foundation of Korea, funded by the Ministry of Science, ICT & Future Planning, South Korea, under Grant NRF-2020R1A2C2006703 and Korea Institute of Oriental Medicine grant C18040.
R. Bonafede and R. Mariotti, “ALS pathogenesis and therapeutic approaches: the role of mesenchymal stem cells and extracellular vesicles,” Frontiers in cellular neuroscience, vol. 11, p. 80, 2017.View at: Google Scholar
T. W. Tefera and K. Borges, “Metabolic dysfunctions in amyotrophic lateral sclerosis pathogenesis and potential metabolic treatments,” Frontiers in neuroscience, vol. 10, p. 611, 2017.View at: Google Scholar
D. Petrov, C. Mansfield, A. Moussy, and O. Hermine, “ALS clinical trials review: 20 years of failure. Are we any closer to registering a new treatment?” Frontiers in aging neuroscience, vol. 9, p. 68, 2017.View at: Google Scholar
C. Cook and L. Petrucelli, “Genetic convergence brings clarity to the enigmatic red line in ALS,” Neuron, vol. 101, no. 6, pp. 1057–1069, 2019.View at: Google Scholar
R. Chia, A. Chiò, and B. J. Traynor, “Novel genes associated with amyotrophic lateral sclerosis: diagnostic and clinical implications,” The Lancet Neurology, vol. 17, no. 1, pp. 94–102, 2018.View at: Google Scholar
M. S. A. Khan, I. Ahmad, and D. Chattopadhyay, New Look to Phytomedicine : Advancements in Herbal Products as Novel Drug Leads, Academic Press, London, 2019.
M. Cai, S. H. Lee, and E. J. Yang, “Bojungikgi-tang improves muscle and spinal cord function in an amyotrophic lateral sclerosis model,” Molecular Neurobiology, vol. 56, no. 4, pp. 2394–2407, 2019.View at: Google Scholar
H. Shen, Y. Yuan, F. Ding, J. Liu, and X. Gu, “The protective effects of Achyranthes bidentata polypeptides against NMDA-induced cell apoptosis in cultured hippocampal neurons through differential modulation of NR2A-and NR2B-containing NMDA receptors,” Brain research bulletin, vol. 77, no. 5, pp. 274–281, 2008.View at: Google Scholar
Y. Yuan, H. Shen, J. Yao, N. Hu, F. Ding, and X. Gu, “The protective effects of Achyranthes bidentata polypeptides in an experimental model of mouse sciatic nerve crush injury,” Brain Research Bulletin, vol. 81, no. 1, pp. 25–32, 2010.View at: Google Scholar
T. Fujikawa, T. Hirata, A. Wada et al., “Chronic administration of Eucommia leaf stimulates metabolic function of rats across several organs,” British Journal of Nutrition, vol. 104, no. 12, pp. 1868–1877, 2010.View at: Google Scholar
L. Zhang and W. Wei, “Anti-inflammatory and immunoregulatory effects of paeoniflorin and total glucosides of paeony,” Pharmacology & Therapeutics, vol. 207, p. 107452, 2020.View at: Google Scholar
S. L. Lee, M. Cai, and E. J. Yang, “Anti-inflammatory Effects of a Novel Herbal Extract in the Muscle and Spinal Cord of an Amyotrophic Lateral Sclerosis Animal Model,” Front Neurosci, vol. 15, p. 743705, 2020.View at: Google Scholar
M. Cai and E. J. Yang, “Combined treatment with Bojungikgi-tang and riluzole regulates muscle metabolism and dysfunction in the hSOD1(G93A) mouse model,” Antioxidants (Basel), vol. 11, no. 3.View at: Google Scholar
L. Xiong, M. McCoy, H. Komuro et al., “Inflammation-dependent oxidative stress metabolites as a hallmark of amyotrophic lateral sclerosis,” Free Radical Biology and Medicine, vol. 178, pp. 125–133, 2022.View at: Google Scholar
M. C. Evans, Y. Couch, N. Sibson, and M. R. Turner, “Inflammation and neurovascular changes in amyotrophic lateral sclerosis,” Molecular and Cellular Neuroscience, vol. 53, pp. 34–41, 2013.View at: Google Scholar
S. Boillée, C. V. Velde, and D. W. Cleveland, “ALS: a disease of motor neurons and their nonneuronal neighbors,” Neuron, vol. 52, no. 1, pp. 39–59, 2006.View at: Google Scholar
M. R. Turner, R. Bowser, L. Bruijn et al., “Mechanisms, models and biomarkers in amyotrophic lateral sclerosis,” Amyotrophic Lateral Sclerosis and Frontotemporal Degeneration, vol. 14, no. sup1, pp. 19–32, 2013.View at: Google Scholar
J. E. Burda and M. V. Sofroniew, “Reactive gliosis and the multicellular response to CNS damage and disease,” Neuron, vol. 81, no. 2, pp. 229–248, 2014.View at: Google Scholar
B. Debye, L. Schmülling, L. Zhou, G. Rune, C. Beyer, and S. Johann, “Neurodegeneration and NLRP3 inflammasome expression in the anterior thalamus of SOD1 (G93A) ALS mice,” Brain Pathology, vol. 28, no. 1, pp. 14–27, 2018.View at: Google Scholar
J. H. Jara, M. Gautam, N. Kocak et al., “MCP1-CCR2 and neuroinflammation in the ALS motor cortex with TDP-43 pathology,” Journal of neuroinflammation, vol. 16, no. 1, pp. 1–16, 2019.View at: Google Scholar
D. Lall and R. H. Baloh, “Microglia and C9orf72 in neuroinflammation and ALS and frontotemporal dementia,” The Journal of clinical investigation, vol. 127, no. 9, pp. 3250–3258, 2017.View at: Google Scholar
Y. Iwasaki, H. Sugimoto, K. Ikeda, K. Takamiya, T. Shiojima, and M. Kinoshita, “Muscle morphometry in amyotrophic lateral sclerosis,” International journal of neuroscience, vol. 58, no. 3-4, pp. 165–170, 1991.View at: Google Scholar
Z. Huang, Q. Fang, W. Ma et al., “Skeletal muscle atrophy was alleviated by salidroside through suppressing oxidative stress and inflammation during denervation,” Frontiers in pharmacology, vol. 10, p. 997, 2019.View at: Google Scholar
Z. Fan and Q. Xiao, “Impaired autophagic flux contributes to muscle atrophy in obesity by affecting muscle degradation and regeneration,” Biochemical and Biophysical Research Communications, vol. 525, no. 2, pp. 462–468, 2020.View at: Google Scholar
Y. Xiao, C. Ma, J. Yi et al., “Suppressed autophagy flux in skeletal muscle of an amyotrophic lateral sclerosis mouse model during disease progression,” Physiological reports, vol. 3, no. 1, p. e12271, 2015.View at: Google Scholar
A. Sadeghi, M. Shabani, S. Alizadeh, and R. Meshkani, “Interplay between oxidative stress and autophagy function and its role in inflammatory cytokine expression induced by palmitate in skeletal muscle cells,” Cytokine, vol. 125, p. 154835, 2020.View at: Google Scholar
F. Ko, P. Abadir, R. Marx et al., “Impaired mitochondrial degradation by autophagy in the skeletal muscle of the aged female interleukin 10 null mouse,” Experimental gerontology, vol. 73, pp. 23–27, 2016.View at: Google Scholar
J. Lee, S. Giordano, and J. Zhang, “Autophagy, mitochondria and oxidative stress: cross-talk and redox signalling,” Biochemical Journal, vol. 441, no. 2, pp. 523–540, 2012.View at: Google Scholar
A. L. Levonen, B. G. Hill, E. Kansanen, J. Zhang, and V. M. Darley-Usmar, “Redox regulation of antioxidants, autophagy, and the response to stress: implications for electrophile therapeutics,” Free Radical Biology and Medicine, vol. 71, pp. 196–207, 2014.View at: Google Scholar
X. Zhang, L. Li, S. Chen et al., “Rapamycin treatment augments motor neuron degeneration in SOD1G93A mouse model of amyotrophic lateral sclerosis,” Autophagy, vol. 7, no. 4, pp. 412–425, 2011.View at: Google Scholar
A. Bhattacharya, A. Bokov, F. L. Muller et al., “Dietary restriction but not rapamycin extends disease onset and survival of the H46R/H48Q mouse model of ALS,” Neurobiology of aging, vol. 33, no. 8, pp. 1829–1832, 2012.View at: Google Scholar
P. M. Siu and S. E. Alway, “Mitochondria‐associated apoptotic signalling in denervated rat skeletal muscle,” The Journal of physiology, vol. 565, no. 1, pp. 309–323, 2005.View at: Google Scholar
G. Bruneteau, T. Simonet, S. Bauché et al., “Muscle histone deacetylase 4 upregulation in amyotrophic lateral sclerosis: potential role in reinnervation ability and disease progression,” Brain, vol. 136, no. 8, pp. 2359–2368, 2013.View at: Google Scholar
Y. Si, S. Kim, X. Cui et al., “Transforming growth factor beta (TGF-β) is a muscle biomarker of disease progression in ALS and correlates with Smad expression,” PLoS One, vol. 10, no. 9, p. e0138425, 2015.View at: Google Scholar
T. J. Cohen, D. S. Waddell, T. Barrientos et al., “The histone deacetylase HDAC4 connects neural activity to muscle transcriptional reprogramming,” Journal of Biological Chemistry, vol. 282, no. 46, pp. 33752–33759, 2007.View at: Google Scholar
G. Dobrowolny, E. Lepore, M. Martini et al., “Metabolic changes associated with muscle expression of SOD1G93A,” Frontiers in physiology, vol. 9, p. 831, 2018.View at: Google Scholar
V. Crugnola, C. Lamperti, V. Lucchini et al., “Mitochondrial respiratory chain dysfunction in muscle from patients with amyotrophic lateral sclerosis,” Archives of neurology, vol. 67, no. 7, pp. 849–854, 2010.View at: Google Scholar
D. Capitanio, M. Vasso, A. Ratti et al., “Molecular signatures of amyotrophic lateral sclerosis disease progression in hind and forelimb muscles of an SOD1G93A mouse model,” Antioxidants & redox signaling, vol. 17, no. 10, pp. 1333–1350, 2012.View at: Google Scholar
C. F. Chang, Y. C. Lee, K. H. Lee et al., “Therapeutic effect of berberine on TDP-43-related pathogenesis in FTLD and ALS,” Journal of biomedical science, vol. 23, no. 1, pp. 1–12, 2016.View at: Google Scholar