## 4 Discussion
Whether the cause is liver fat accumulation or portal circulation inflammation, inflammatory cytokines such as IL-6 and TNF-\(\alpha\) are overproduced in a damaged liver. Furthermore, produced inflammatory substances such as IL-6, TNF-\(\alpha\), and IL-1\(\beta\) can enhance osteoclastogenesis [44]. Our results suggested that the expression of IL-6, TNF-\(\alpha\), IL-1\(\beta\), and iNOS increased in the OVX group, indicating liver damage (Figure 3), and GAM decreased the expression of these inflammatory biomarkers compared with the OVX group (Figure 3). These results showed that GAM had anti-inflammatory effects that protected the liver from lipid accumulation and alleviated osteoporosis. Bone remodeling requires a balance between bone formation (osteoblasts) and bone resorption (osteoclasts). BMD and trabecular bone volume were assessed using a \(\mu\)-CT system to evaluate the extent of osteoporosis. BMD indicates the amount of calcium and other types of minerals in analyzed areas of bone. A deficiency of bone replacement during menopause can cause an increase in bone mass loss because bone resorption outpaces bone formation, leading to a decrease in BMD [45]. The \(\mu\)-CT system is highly accurate for evaluating the trabecular bone volume. We investigated bone quality in our osteoporosis model by measuring BMD, percent bone volume ratio, and trabecular bone number, and we confirmed OVX-induced bone loss in the \(\mu\)-CT images of the femur. Our histological visualization of the \(\mu\)-CT results shows that GAM treatment filled the trabecular bone. Additionally, our finding that the bone morphometric markers (BMD, BV/TV, Tb.N) increased (Figure 4b) indicates that GAM treatment for 4 weeks prevented trabecular bone loss in estrogen deficiency-induced osteoporosis (Figure 4b). Therefore, GAM could have anti-osteoporosis effects in postmenopausal osteoporosis. ALP activity is an important biochemical marker for bone formation during osteoblast differentiation [46]. After menopause, bone resorption markers increase due to the activation of bone resorption by estrogen deficiency, so bone formation must also increase to fill the higher number of resorption cavities, causing an increase in the serum levels of both ALP and OC [43; 44]. That is why high levels of ALP in elderly people suggest the presence of osteoporosis [44]. We investigated bone formation activity by measuring ALP and OC levels in our OVX-induced model rats. We found that the serum ALP and OC levels were higher in the OVX group than in the Sham group (Figure 5), suggesting that OVX increases bone formation markers to maintain the balance of bone turnover. The serum concentrations of both ALP and OC decreased following GAM treatment for 4 weeks, which suggests that GAM prevents OVX-induced bone loss by decreasing bone resorption. Furthermore, in our in vitro experiment, we found that increasing ALP levels indicate increasing bone mineralization. Because ALP activity can indicate the degree of differentiation of osteoblasts, it is used to evaluate the mineralization ability of osteoblasts [47]. We measured ALP using alizarin red staining in osteoblasts differentiated from MC3T3-E1 cells (Figure 7) and found that GAM treatment increased ALP levels compared with the control group (Figure 7b). The histological visualization of alizarin red staining showed that GAM treatment increased mineralization in osteoblasts differentiated from MC3T3-E1 cells (Figure 7d). Increased ALP activity and alizarin red staining both reflect osteogenic mineralization. Therefore, GAM could prevent postmenopausal osteoporosis by regulating the ALP concentration. The inhibition of osteoclastogenic signals could be an effective method for treating bone loss-related diseases. CTX1 and NTX1 are released by osteoclasts and are therefore markers of bone resorption [48]. Bone resorption activity was investigated by measuring CTX1 and NTX1 in our OVX-induced rat model.