**4. Discussion**

ALS is a disease with complex pathological mechanisms and no effective drug treatment. Herbal medicine is composed of multiple components and is used for multi-targets. In addition, herbal medicine focuses on boosting the immune system and maintaining an internal balance of the body. To investigate the possibility of using herbal medicine as treatment for ALS, we investigated the effect of HET treatment on the spinal cord and skeletal muscle in an animal model of ALS.

Neuroinflammation in the brain occurred via microglial proliferation and astrocytic hypertrophy. Microglia are immune cells in the CNS that play a role in clearing pathogens through phagocytosis and play a critical role in homeostasis [37]. Microglial cell activation increases the expression of inflammatory cytokines such as IL-6 and IL1β and leads to oxidative stress and neuroinflammation, which results in augmented microglial NADPH-derived ROS accumulation [38,39]. In ALS, microglial activation is correlated with neuroinflammation and disease progression [40]. Correspondingly, minocycline treatment has been found to reduce neuroinflammation and microglial activation in clinical trials with patients with ALS [41]. However, it is not effective in patients with ALS who have other neurological disorders. Hence, herbal medicine may be a good, more effective candidate for protecting neurons and skeletal muscle from degeneration, primarily because herbal medicine contains multiple compounds and targets. In this study, HET treatment reduced the expression levels of CD11b and GFAP in the spinal cord and gastrocnemius of symptomatic hSOD1G93A mice. This suggests

that HET treatment can enhance the body's immune system and extend the survival rate of these mice. As expected, we found that HET treatment increases gastrocnemius weight and survival rate of hSOD1G93A mice.

Oxidative stress and inflammation are significant factors in ALS pathogenesis, and lead to motor neuron death and severe muscle degeneration. While motor neurons control muscle function, retrograde signals can pass from the muscle back to motor neurons via the NMJ [42]. In addition, previous work has found that oxidative stress leading to muscle atrophy was increased in the pre-symptomatic stages in hSOD1G86R mice [43]. In our study, we found that oxidative stress-related proteins such as Ferritin, HO1, Transferrin, and BAX were dramatically increased in the gastrocnemius and the spinal cord of symptomatic hSOD1G93A mice. Furthermore, HET treatment significantly attenuated the expression level of oxidative stress-related proteins in the muscle and spinal cord of hSOD1G93A mice. Patients with ALS have defective energy homeostasis, and skeletal muscle degeneration is a critical factor in the pathogenesis of ALS and its symptoms. Some studies have provided consistent evidence by demonstrating that atrophy occurred before motor neuron loss and neurodegeneration [44,45]. Furthermore, studies with patients with ALS (fALS and sALS) and animal models of ALS (hSOD1G93A and G86R models) have reported increased energy expenditure and a defective energy balance due to increased oxidative stress, mitochondrial dysfunction, and inflammation [46–48].
