## 3 Results  
### CHM Treatment Improves Movement in hSODIG93A Mice  
To investigate the effect of CHM on the movement of hSOD1G93A mice, we performed the footprint behavioral test to analyze movement. The stride length of movement of hSOD1G93A mice was reduced by 1.4-fold compared with that of nTg mice (Figure 1a). However, CHM administration to hSOD1G93A mice improved the stride length by 1.3-fold compared with that of the distilled water-treated hSOD1G93A mice, according to the footprint test. Furthermore, the motor neuron survival of CHM-treated hSOD1G93A mice increased compared with that of hSOD1G93A mice, as determined using immunohistochemistry analysis (Figure 1b). In addition, the intensity of positive-CD11b immunostaining was also significantly reduced by the CHM treatment in hSOD1G93A mice (Figure 1b). Figure 1: Treatment with combined herbal medicine (CHM) comprising _Gastrodia elata_, _Cnidium officinal Makino_ (COM), and _Ostericum forearm_ improves motor function and prevents motor neuron death in hSOD1G93A mice. (**) Footprint test (\(n=8\)). (**) Representative image of anti-ChAT-positive immunohistochemistry analysis. nTg (\(n=4\)), Tg (\(n=4\)), and Tg-CHM (\(n=3\)). Data are presented as mean \(\pm\) SEM. \({}^{*}p<0.05\), \({}^{**}p<0.01\), and \({}^{***}p<0.001\) compared with the control group. Scale bar = 2 \(\upmu\)m. DW-treated nTg mice (nTg), hSOD1G93A Tg mice (Tg), CHM-treated Tg mice (Tg-CHM). CHM Treatment Suppresses the Occurrence of Inflammation-Related Events in the Muscles and SC of hSOD1G93A Mice  
To determine the molecular mechanism underlying the effects of CHM in hSOD1G93A mice, TA and GC muscles and SC tissues were analyzed using western blotting. Abnormal GFAP expression in muscle indicates the degeneration of neuromuscular junctions (NMJs) and motor neurons in ALS [37]. As shown in Figure 2a,b, the expression of GFAP protein increased by 3.7- and 4.5-fold in the TA and GC muscles of hSOD1G93A mice, respectively, compared with that in the TA and GC muscles of nTg mice. However, CHM treatment reduced the GFAP level by 2.4- and 3.4-fold in the TA and GC muscles of hSOD1G93A mice, respectively, compared with that in the TA and GC muscles of hSOD1G93A mice (Figure 2a,b). In the SC, the expression of the neuroinflammation-related proteins CD11b and GFAP was downregulated by 1.5- and 2.2-fold, respectively, in CHM-treated hSOD1G93A mice compared with that in hSOD1G93A mice. Inflammation is involved in oxidative stress-related events. Therefore, we examined the effect of CHM on the expression of oxidative stress-related proteins in the muscles (TA and GC muscles) and SC of hSOD1G93A mice. The expression of ferritin was significantly reduced, by 2.2- and 2.8-fold, in the TA and GC muscles of CHM-treated hSOD1G93A  
Figure 2: CHM treatment reduces the levels of inflammatory proteins in the tibialis anterior (TA) and gastrocnemius (GC) muscles and spinal cord (SC) of hSOD1G93A mice. (**a,b**) Western blots showing the changes in GFAP protein level in the (**a**) TA and (**b**) GC muscles of mice from the nTg, Tg, and Tg-CHM groups. (**c**) Representative immunoblots of CD11b and GFAP proteins in the SC of nTg, Tg, and Tg-CHM mice. Quantification of immunoblots showed the expression ratio of GFAP and CD11 in the TA and GC muscles and SC of each group compared with that in the nTg group. 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 group 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). mice, respectively, compared with that in the TA and GC muscles of hSOD1G93A mice (Figure 3a,b).