**3. Results**

#### *3.1. Hochu-Ekki-(HET) Extended Survival and Improved Motor Function*

To examine the effects of HET on physical function, we measured the body and muscle weight of symptomatic HET-treated hSOD1G93A mice (Tg-HET). As shown in Figure 1A, body weight of hSOD1G93A mice (Tg) was lower than that of age-matched non-Tg (nTg); however, there was no significant difference in body weight between the Tg and Tg-HET groups. HET treatment resulted in a 1.6-fold significant increase in the weight of the gastrocnemius muscle compared to that of Tg mice

(Figure 1B). Furthermore, we found that HET treatment resulted in a 2.8-fold improvement in motor function in symptomatic hSOD1G93A mice, as revealed in the rota-rod test (Figure 1C). Motor activity was assessed by measuring stride length through the foot print test. The stride length of Tg-HET mice was 1.5-fold greater than age-matched Tg mice (Figure 1D). Furthermore, HET treatment extended the survival rate compared to that of Tg mice (Figure 1E). These findings sugges<sup>t</sup> that HET treatment can prevent motor neuron death and skeletal muscle paralysis in hSOD1G93A mice.

**Figure 1.** Hochu-ekki-to (HET) treatment ameliorates motor activity and prolongs the life span of a mouse model of amyotrophic lateral sclerosis (ALS). HET (1 mg/g) was orally administered once a day for 6 weeks from the age of 2 months. (**A**) Comparison of body weight between the nTg, Tg, and HET-treated Tg groups. (**B**) Comparison of gastrocnemius weight between the nTg, Tg, and HET-treated Tg groups. (**C**) Motor function was measured by the rota-rod test in all groups. (**D**) The representative average of stride length (*n* = 7/group) of each group, measured using the foot print test. (**E**) Survival rate was calculated by Kaplan-Meyer analysis in Tg and HET-treated Tg (*n* = 8/group). Data are shown as the mean ± SEM. \* *p* < 0.05, \*\* *p* < 0.01, \*\*\* *p* < 0.001. nTg: non-transgenic mice, Tg: hSOD1G93A, Tg-HET:HET-treated hSOD1G93A.

#### *3.2. Hochu-Ekki-To (HET) Reduces Neuroinflammation and Oxidative Stress in the Spinal Cord of hSOD1G93A Mice*

In our previous study, we found that hSOD1G93A transgenic mice had increased neuroinflammation, indicated by an increase in CD11b, GFAP, Iba-1, and TLR4 (inflammatory proteins in spinal cord) [33,34]. To investigate the effect of HET on neuroinflammation of the spinal cord in hSOD1G93A mice, we investigated the expression of neuroinflammation-related proteins (Iba-1, GFAP, and TLR4) using immunoblotting. As shown in Figure 2A,B, the expression levels of Iba-1, GFAP, and TLR4 in the spinal cord were significantly greater by 18-, 2.1-, and 2.8-fold in symptomatic Tg mice compared

to those of nTg mice. However, HET treatment dramatically reduced the levels of Iba-1, GFAP, and TLR4 proteins by 2.3-, 2.7-, and 1.7-fold compared to that of Tg mice. In addition, proinflammatory cytokine, IL-1β immunoreactivity was increased in anterior horn of spinal cord of symptomatic Tg mice, but it was reduced by treatment with HET (Figure 2C). Furthermore, we found evidence for anti-neuroinflammatory effects of HET, and observed a reduction of oxidative stress in the spinal cord of Tg mice. Oxidative stress-related proteins HO1, transferrin, and BAX were significantly lower by 7-, 2.6-, and 1.6-fold in the spinal cord of Tg-HET mice compared to that of age-matched Tg mice (Figure 2D,E). Taken together, HET treatment seems to enhance neuroimmune systems to maintain motor neuron survival and consequently improve motor function in the ALS animal model.

**Figure 2.** Hochu-ekki-to (HET) increases anti-inflammation and anti-oxidative stress effects in the spinal cord of an ALS mouse model. (**A**) Representative Western blots on inflammatory protein levels of Iba-1 (a marker of microglia), GFAP (a marker of astrocytes), and TLR4 in the spinal cord of each group (nTg, Tg, and Tg- HET). Tubulin was used as a loading control. (**B**) Quantification of the expression level of Iba-1/Tubulin, GFAP/Tubulin, and TLR4/Tubulin in each immunoblot. (**C**) Representative images of IL-1β immunoreatcivity in the anterior horn of the spinal cord in each group. Scale bars = 100 μm (**D**) Representative images of oxidative stress-related proteins (BAX, HO1, and Transferrin) in the spinal cord of each group mice. (**E**) Quantification of the expression levels of BAX/Tubulin, HO1/Tubulin, and transferrin/Tubulin. Data are presented as the mean ± SEM (*n* = 3/group). \* *p* < 0.05, \*\* *p* < 0.01, \*\*\* *p* < 0.001. nTg: non-transgenic mice, Tg: hSOD1G93A, Tg-HET:HET-treated hSOD1G93A.

#### *3.3. Hochu-Ekki-To (HET) Attenuates Muscle Dysfunction*

In our previous study, we found that HO1, Transferrin, BAX, and Ferritin (as oxidative stress-related proteins) were increased in the spinal cord of hSOD1G93A mice [35,36]. To examine the effect of HET on the weakness of skeletal muscle during ALS progression, we investigated the expression level of inflammatory and oxidative stress-related proteins in the gastrocnemius muscle of symptomatic hSOD1G93A mice. The smaller myocytes with abnormal nuclei that had moved to the center of the cells in the gastrocnemius of hSOD1G93A mice. As shown in Figure 3A,B, we found that the percentage of central nuclei was increased by 7.8-fold in the gastrocnemius muscle of symptomatic hSOD1G93A mice compared to nTg mice (Figure 3B). In addition, the average CSA of myocytes was reduced by 2.2-fold in symptomatic hSOD1G93A mice compared to nTg mice (Figure 3B). However, HET treatment led to decrease 4.8-fold in the percentage of central nuclei and increase 2.4-fold the average CSA of myocytes in the gastrocnemius of hSOD1G93A mice.

In addition, myocyte was small in the gastrocnemius muscle of symptomatic hSOD1G93A mice. However, HET treatment inhibited the muscle atrophy seen in the gastrocnemius by H&E staining (Figure 3). This suggests that HET treatment can reduce muscle damage and inflammation in the gastrocnemius of symptomatic hSOD1G93A mice. To address this hypothesis, we investigated the expression level of inflammatory proteins including CD11b and GFAP and oxidative stress-related proteins such as Ferritin, HO1, and BAX in gastrocnemius of symptomatic hSOD1G93A mice. As expected, HET treatment significantly reduced the expression levels of GFAP and CD11b by 1.7- and 2.5-fold, respectively, in the gastrocnemius of hSOD1G93A mice (Figure 4A,B). In addition, proinflammatory cytokine, IL-1β immunoreactivity was increased in the gastrocnemius of symptomatic Tg mice, but it was reduced by treatment with HET (Figure 4C). Furthermore, HET treatment significantly reduced the levels of Ferritin, HO1, and BAX by 1.9-, 1.6-, and 2.2-fold, respectively, in the gastrocnemius of hSOD1G93A mice (Figure 4D,E). These findings sugges<sup>t</sup> that HET treatment may boost the immune system to protect from muscle loss and damage in this model of ALS.

**Figure 3.** Hochu-ekki-to (HET) treatment has a protective e ffect against muscle atrophy in the gastrocnemius of an ALS mouse model. ( **A**) Representative images of H&E staining showing the muscle atrophy condition, such as smaller myocytes and abnormal nuclei in gastrocnemius of hSOD1G93A mice. Arrowheads indicate abnormal nuclei (central nucleation) in myocytes. (**B**) Abnormal nuclei were expressed as a percentage of abnormal nuclei (left panel). Quantified average myocyte cross-sectional area (CSA) (right panel). Scale bar = 50 μm Data are presented as the mean ± SEM (*n* = 3/group). \* *p* < 0.05. nTg: non-transgenic mice, Tg: hSOD1G93A, Tg-HET:HET-treated hSOD1G93A.

**Figure 4.** Hochu-ekki-to (HET) treatment enhances anti-inflammation, anti-oxidative stress effects, and regulates autophagy in the gastrocnemius of an ALS mouse model. (**A**) Representative Western blots of inflammatory related proteins, CD11b and GFAP, in the gastrocnemius of each group. Actin was used as a loading control. (**B**) Quantification of the expression levels of CD11b/Actin and GFAP/Actin. (**C**) Representative images of IL-1β immunostaining in the gastrocnemius of each group. Scale bars = 100 μm (**D**) Representative images of oxidative stress-related proteins (Ferratin, HO1, and BAX) in the gastrocnemius of each group. (**E**) Quantification of the expression levels of Ferritin/Actin, HO1/Actin, and BAX/Actin. Data are presented as the mean ± SEM (*n* = 3/group). \* *p* < 0.05, \*\* *p* < 0.01. nTg: non-transgenic mice, Tg: hSOD1G93A, Tg-HET:HET-treated hSOD1G93A.
