## 4 Discussion  
ALS is an incurable neurodegenerative disease that has no effective treatment. ALS has various neuropathological signatures, such as oxidative stress, mitochondrial dysfunction, defective axonal transport, glutamate excitotoxicity, and neuroinflammation. Defects occur that influence free radical accumulation in motor neurons, including the loss of anti-oxidant function of enzymes (e.g., SOD1) and alterations in DNA repair. Herbal medicines are composed of multiple compounds and have multiple effects, such as anti-inflammatory, anti-oxidant, and anticancer effects [39]. In this study, we investigated the effects of a CHM composed of _G. elata_, COM, and _O. Koreanum_ in hSOD1G93A mice to determine whether a multi-targeting herbal medicine can be effective for the treatment of ALS. To investigate whether CHM affects ALS progression, 8-week-old hSOD1G93A mice were treated with CHM for 6 weeks. CHM administration increased the stride length of movement in symptomatic hSOD1G93A mice. Furthermore, the loss of motor neurons was substantially attenuated by CHM treatment via anti-inflammatory effects as well as anti-oxidant and autophagy-regulating effects in the muscles and SC of hSOD1G93A mice. Inflammation can be acute or chronic. Chronic inflammation induces cancer, autoimmune disorders, metabolic disorders, and neurological diseases, such as AD, PD, and ALS. In the brain, inflammation induces the reactions of glial cells (astrocyte and microglial acti  
Figure 4: CHM treatment regulates autophagy function in the tibialis anterior (TA) and gastrocnemius (GC) muscles and spinal cord (SC) of hSOD1G93A mice. (**a**) The expression of the muscle atrophy-related protein SMAD2 was decreased in the Tg-CHM group (\(n\) = 3–4/group). CHM treatment increased the expression of autophagy-related proteins (LC3b and p62) in the (**b**) GC muscles and (**c**) SC of hSOD1G93A mice. CHM treatment reduced the Prox1 and SMAD2 levels in the (**b**) GC muscles and (**c**) SC of hSOD1G93A mice. Actin or tubulin was used as a loading control to normalize the levels of the target proteins for the western blot analyses. nTg (\(n\) = 4), Tg (\(n\) = 4), and Tg-CHM (\(n\) = 3). Data are normalized relative to those of the control groups and presented as mean \(\pm\) SEM. * \(p\) \(<\) 0.05, **_p_ \(<\) 0.01, and ***_p_ \(<\) 0.001 compared with the control group. DW-treated nTg mice (nTg), hSOD1G93A Tg mice (Tg), CHM-treated Tg mice (Tg-CHM). vation) and an increase in the production of proinflammatory cytokines, such as IL-2 and IL-13 [40], by circulating immune cells, including monocytes, neutrophils, and lymphocytes. In addition, neurodegenerative diseases are characterized by the impairment of inflammatory events. In ALS, the loss of T regulatory cells is involved in neuroinflammation, and the transfer of T regulatory cells extends lifespan [41]. Specifically, activation of microglia and astrocytes induces toxic reactions and motor neuronal cell death in ALS. Inflammation can be caused by ROS produced via the mitochondrial respiratory chain [42]. In NMJs, ROS produced by impaired mitochondria activate intracellular Ca\({}^{2+}\) to induce presynaptic decline in an ALS model [43]. In addition, neuroinflammation is accompanied by oxidative stress, which is a fundamental feature of the progression of neurodegenerative diseases. Anti-oxidants from plants or herbal medicines, as preventive medicines, include flavonoids and phenolic compounds, which have no adverse effects and are inexpensive [44]. In addition, flavonoids and phenolic compounds exhibit anti-inflammatory activities by inhibiting signaling pathways, such as NF-\(\kappa\)B and MAPK, which induce the production of proinflammatory cytokines [45]. Furthermore, [PERSON] et al. demonstrated that natural anti-oxidants obtained from consumption of natural foods prevent disease development [46]. In this study, we found that CHM treatment reduced the levels of oxidation-related or inflammatory proteins in the muscles (TA and GC muscles) and SC of hSODIG93A mice.