**1. Introduction**

Amyotrophic lateral sclerosis (ALS), also known as Lou Gehrig's disease, is characterized by a loss of motor neurons, muscle weakness, and spasticity [1]. ALS can be divided into familiar ALS (fALS), which is caused by autosomal dominant mutations in genes such as superoxide dismutase (SOD)1, and sporadic ALS (sALS). However, some gene mutations have been found to be involved in both fALS and sALS, including mutations of TAR DNA-binding protein (TDP) 43, fused in sarcoma (FUS), valosin-containing protein (VCP), and TATA-binding protein-associated factor 15 (TAF15) [2].

Several pathological mechanisms underlying ALS have been reported, including proteasome and autophagy dysfunction, ER stress, oxidative stress, and mitochondrial disorders [3]. Most notably, a dysregulated immune response plays a critical role in disease progression, as revealed by both ALS animal model and clinical studies [4–6].

In the central nervous system (CNS), neuroinflammation that is mediated by microglia is involved in the pathogenesis of neurodegenerative diseases such as Alzheimer's Disease (AD), Parkinson's Disease (PD), and ALS. In ALS, specific gene mutations in the CNS have been found to contribute to immune dysfunction, including mutations of SOD1, TARDBP, and C9orf72 [7–9]. A mutant SOD1 overexpressed animal model was found to exhibit motor neuron dysfunction that was induced by an increase in activated microglia in the peripheral nervous system and CNS [10]. In addition, the expression of IL-6 has been reported to increase via activation of microglia and macrophages in both an animal model of and patients with ALS [11,12]. In the muscles, alternation of neuromuscular junction (NMJ) and muscle denervation that involves a loss of presynaptic terminals, Schwann cells, and axonal degeneration, has been found to lead to clinical weakness and an increased disease severity in patients with ALS [13]. Furthermore, activated macrophages reportedly surround NMJs in symptomatic and end-stage mouse models of ALS [14], and complement factors are upregulated to recruit macrophages in the denervated muscle of a SOD1G93A mouse model [15]. Therefore, immune enhancers could be a candidate for attenuating disease progression and enhancing homeostasis of the body in patients with ALS.

Herbal medicine has been widely used in Asian countries for thousands of years because of antinociceptive, analgesic, and anti-inflammatory effects, both centrally and peripherally [16,17]. Simply put, herbal medicine can stimulate the immune system and maintain the internal balance of the body. In the case of AD, bioactive components from herbal medicines such as Radix Polygalae, *Panax ginseng*, and *Ginko biloba* have been shown to effectively improve AD symptoms by targeting autophagy [18]. In ALS, many experimental studies have demonstrated that Chinese prescriptions have anti-inflammatory and anti-oxidant effects. In patients with ALS, Chinese prescriptions, including Jiawei Sijunzi, and Dihuang Yinzi, have been found to improve phenotype symptoms and functional rating scales [19,20]. However, further evidence for the efficacy, mechanisms of action, and safety of herbal medicines in the treatment of ALS is required.

Hochu-ekki-to (HET) in Japanese herbal (Kampo) medicine is similar to Bojungikgi-Tang (BJIGT) in Korea and Bu-Zhong-Yi-Qi Tang in Chinese medicine. HET has ten component herbs, as follows: Astragali radix (16.7%, *A. membranaceus Bunge*), Atractylodes lancea Rhizome (16.7%, rhizomes of *A*. *lancea* DC.), Ginseng radix (16.7%, *P. ginseng* C.A. Meyer), Angelica Radix (12.5%, *Angelica acutiloba* Kitagawa), Bupleuri radix (8.3%, *Bupleurum falcatum* L.), Zizyphi fructus (8.3%, *Zizyphus jujuba* Miller var. inermis Rehder), Aurantii nobilis pericarpium (8.3%, *Citrus unshu* Markovich), Glycyrrhizae radix (6.3%, *Glycyrrhiza uralensis* Fisch et DC.), Cimicifugae Rhizoma (4.2%, *Cimicifuga simplex* Worms kjord), and Zingiberis Rhizoma (2%, *Zingiber o*ffi*cinale* Roscoe) and it was provided by Tsumura pharmaceutical company [21,22]. In addition, Dan et al., and Yae et al., had already reported chemical profile of HET by 3-dimensional HPLC.

HET has been used to enhance the immune system in respiratory disorders [23,24] and to improve the nutritional status associated with chronic diseases [25]. Thus, many studies have investigated the immunopharmacological activities of HET [26–28]. In addition, Kiyohara et al. reported that HET enhanced the mucosal immune system [29]. Shih et al. found that HET improved learning and memory, and had an anti-aging effects in a senescence-accelerated mouse model [29]. Furthermore, the authors suggested that HET can penetrate the blood–brain barrier by increasing dopamine and noradrenaline levels in the brain.

ALS causes both motor neuron death and skeletal muscle paralysis. A suitable therapy with optimal treatment effects for patients with ALS would involve a motor neuron target combined with a skeletal muscle target. In this sense, herbal medicine is effective for complex disease because herbal medicine consists of multiple components. Therefore, we investigated the effect of HET on neuroinflammation, motor function, and muscle weakness in a hSOD1G93A animal model.

#### **2. Materials and Methods**
