Amyotrophic lateral sclerosis (ALS) is the most frequently diagnosed adult-onset motor neuron disease. ALS involves the progressive loss of upper and lower motor neurons. Within a few years of developing ALS, patients die from progressive respiratory muscle paralysis; unfortunately there are no currently available therapies that can alter the disease course effectively (Duffy et al., 2011; Brown and Al-Chalabi, 2017; Aizawa et al., 2021). Whether it is sporadic ALS or familial ALS, because of its involvement of progressive muscle weakness, and even atrophy, in Traditional Chinese medicine (TCM), ALS should be classified as an “amyotrophy and weakness disease” (Gao et al., 2017). According to its pathogenesis, TCM doctors think that the disease is mainly caused by deficiency of some related organs, which deficiencies are mainly concerned with the yang of the spleen and kidney, mixed with deficiency of the stomach, liver, and lung, and weakness or deficiency of qi (one the circulating energy materials in TCM theory). Therefore, treatment methods for ALS involving tonifying the yang of the kidney and spleen, and invigorating qi in China suggested a certain clinical efficacy (Wang et al., 2022). According to the theory of TCM differentiation and TCM constitution theory, we followed-up of nearly 500 patients with ALS and found that in the early stage of ALS, deficiency the yang of the spleen and kidney is the main syndrome type, together with deficiency of the stomach, liver, and lung; and in the middle and late stage, in addition to deficiency the yang of the spleen and kidney, syndromes of deficiency and excess are mostly intermingled, accompanied by blood stasis and phlegm (Grimm and Eckert, 2017). According to the classic text of traditional Chinese medicine Huang Di Nei Jing, the “spleen controls the main muscle function of limbs,” “qi is flowing energy” and the “kidney fills and manages circulation of energy to the muscle as the officer” (Hosaka et al., 2019). The long-term clinical experience of the Shanghai University of TCM led to the use of pure TCM herbs, named the Wen-Shen-Jian-Pi (WSJP) prescription, to delay the progression of ALS and improve the clinical symptoms of patient with ALS (Kwak et al., 2010; Jin et al., 2019); however, the mechanism of action of WSJP is not clear.

A study of ALS indicated that the toxicity mechanism is mediated by GluR2 [Glutamate Ionotropic Receptor α -amino-3-hydroxy-5-methyl-4-isooxazole-propionic acid (AMPA) Type Subunit 2], which plays an important role in both the pathogenesis of sporadic ALS and animal models of superoxide dismutase 1 (SOD1)-related familial ALS (Liu et al., 2018). Based on research by Professor Shin, we successfully made an ALS model comprising mice knocked out for Adar2 (encoding the RNA editing, enzyme adenosine deaminase acting on RNA 2), referred to as AR2 mice, which can reflect the occurrence and progression of ALS (Mehta et al., 2021). The AR2 model was treated with the WSJP prescription to observe its effect on the motor function of AR2 mice, and the mechanism of action of the observed effect was investigated.

The results of the present study revealed that the WSJP prescription administered orally to AR2 mice for 12 weeks prevented ALS progression significantly, and ameliorated mitochondria and autophagosome pathology-associated motor neuron cell death. The successful rescue of the ALS phenotype of the AR2 mice, which are a mechanistic model of sporadic ALS, suggested that the WSJP prescription could be used to treat ALS.

We found WSJP-treated AR2 mice gradually gained body weight (Figures 1A,A′). WSJP could significantly reduce ALS-associated weight loss, improve the activity of mouse limbs, the activity persistence of muscles, and the physical flexibility and muscle strength of the mice. The result suggested that oral WSJP treatment might have the efficacy to prevent the development of atrophy in ALS, which is consistent with clinical studies of ALS using other TCM materials (Petrov et al., 2017; Qu et al., 2020). From 8 to 12 weeks, WSJP-treated AR2 mice spent a longer time turning on the pole in rotarod test and had more powerful grip strength than the mice in the vehicle group, whereas the mice in the vehicle group showed a decreasing trend of motor function (Figures 1B,B′,C,C′). These results indicated that the WSJP prescription might have prevented the loss of motor function and even improved the motor function for AR2 mice compared with that of the vehicle group.

Cell survival and metabolism critically rely on mitochondria, which are especially important in neurons (Shigematsu et al., 2021). In neurons, mitochondria have important functions in apoptosis, phospholipid synthesis, and calcium homeostasis, besides their well-known functions in oxidative phosphorylation-driven ATP production. The metabolic demands of neurons are high; consequently, 20% of the ATP produced by the body is consumed by the brain, despite representing only 2% of the body mass (Smith et al., 2019). Mitochondrial maintenance of energy production and calcium homeostasis are particularly important in the maintenance of neuronal function because of neurons’ high metabolic activity and energy requirements. Many proteins associated with sporadic and familial ALS, including SOD1, TAR DNA-binding protein 43 (TDP-43), FUS RNA binding protein (FUS), C9orf72-SMCR8 complex subunit (C9orf72), and glycine/arginine (GR) dipeptide repeat proteins (DPR) associated with repeat amplification of C9ORF72GGGCC, have been shown to interact with mitochondria (Qu et al., 2020). Decreased ATP production and cellular respiration have been demonstrated clearly in ALS, and after the death of patients with sporadic ALS, decreased electron transport complexes I, II, III, and IV activities were observed in the spinal cord (Song and Pan, 2014; Song et al., 2022). In SOD1^G93Atransgenic mice, before the onset of motor symptoms, a reduced mitochondrial respiration rate and impaired ATP synthesis were observed in the spinal cord and brain cord, which persisted throughout the disease process (Sun et al., 2020), accompanied by decreased activities of complex I + III, II + II, I, and IV. As expected, WSJP-treated AR2 mice had an almost normal number of normal mitochondria (Figure 2C) and decreased numbers of abnormal mitochondria and autophagosomes (Figures 2D,E). The abnormal mitochondria and autophagosome were seen almost exclusively in the nuclei of motor neurons in ALS (Wang Y. et al., 2021).

The differentiation syndrome in TCM theory for ALS might be divided into seven syndrome types; however, the syndrome type with the largest proportion is deficiency of the yang of the spleen and kidney, at more than 60% (Grimm and Eckert, 2017; see Supplementary Material 1). The TCM, WSJP prescription, is often used in the clinic to treat ALS, and is clinically proven to be effective (Kwak et al., 2010; Petrov et al., 2017; Jin et al., 2019; Qu et al., 2020). To explore the mechanism by which the WSJP prescription treats ALS, we investigated the molecular mechanism and material basis of WSJP via network pharmacology. We used the disease database and the TCM database to acquire the potential targets of the prescription in ALS via Kyoto Encyclopedia of Genes and genomes (KEGG) and Gene Ontology (GO) using the DAVID tool, established a protein-protein interaction (PPI) network via STRING and a Targets network using Cytoscape. The results showed that WSJP might inhibit neuronal death and neuro inflammation via multiple pathways to treat ALS (see Supplementary Material 2).

Although traditional Chinese medicine cannot replace western medicine in the treatment of neuro degenerative diseases such as Parkinson’s disease, Alzheimer’s disease; nonetheless, TCM dialectical treatment has achieved certain clinical effects according to the TCM pathogenesis of these degenerative diseases (Wong et al., 2013; Yamashita et al., 2017; Wang Z. Y. et al., 2021). The WSJP prescription, which can tonify the yang of the spleen and kidney and increase the power and energy of qi circulation in the TCM theory, is based on the traditional formula, Si-Jun-Zi decoction (Yamashita and Kwak, 2014), but with increased Radix Astragali, Radix herba Cistanche deserticola, and Herba Epimedii. The prescription also demonstrated powerful effects for muscle weakness and atrophy in many clinical studies (Yu et al., 2014, 2020; Ye and Dong, 2017; Yamashita and Kwak, 2019).

Tonifying the yang of the kidney and spleen, and strengthening and warming qi can delay the progression of ALS in mice and locally improve motor function. Whether the long-term use of this method can delay the progression of ALS also requires further study. In addition, recent studies have suggested that the early onset of neurodegenerative diseases, including Parkinson’s disease and Alzheimer’s disease, is related to the imbalance of intestinal flora (Zhang et al., 2014, 2021). The adjustment of intestinal flora in TCM is mainly carried out by the spleen and coordinated by the liver. Treating neurodegenerative diseases from the spleen might become a new therapeutic target of TCM. Aizawa et al. (2021) found that perampanel, an AMPA receptor antagonists, restored the size of anterior horn cells to that found in age-matched wide-type mice, which indicated that the physiological functions disturbed by increased Ca²⁺ influx via abnormal AMPA receptors had been restored (Zhu et al., 2017). Perampanel was effective in asymptomatic AR2 mice and in AR2 mice during disease progression, as reported in a study using adeno-associated virus-mediated ADAR2 gene delivery. However, the AR2 mice treated with perampanel did not gain body weight, which was recognized an adverse effect of perampanel in clinical trials. TDP-43-related pathology, in which TDP-43 is mislocalized from the nucleus to abnormal cytoplasmic inclusion bodies in motor neurons, is found in the motor neurons of the majority of patients with ALS. This represents a pathological hallmark of ALS and is considered to be closely involved in the pathogenesis of ALS (Zhu et al., 2017). In the present study, we did not measure the increased number of TDP-43-positive neurons, especially those neurons with nuclear and nucleocytoplasmic patterns, nor the subcellular localization of TDP-43 in the AR2 mouse motor neurons. In addition, we did not confirm whether the WSJP-treated AR2 mice showed amelioration of the increased Ca²⁺ influx in ADAR2-depleted motor neurons.

WSJP prescription, which according to TCM, can tonify the yang of the spleen and kidney and improve the power and circulation of qi, can improve clinical symptoms of ALS, slow disease progression, and reduce the loss of motor activity in AR2 mice, and is thus worthy of clinical application.

The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.

The animal study was reviewed and approved by the Committee of Animal Handling of the Shanghai University of Traditional Chinese Medicine.

WP and TL contributed to conception and design of the study. WP organized the database and wrote the first draft of the manuscript. MW performed the statistical analysis. XZ, CW, and MW wrote sections of the manuscript. All authors contributed to manuscript revision, read, and approved the submitted version.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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