## 4 Discussion  
Perimenopause is a medical condition that develops in middle-aged females and is characterized by irregular or lack of estradiol and follicular-stimulating hormone levels. During the menopausal transition, imbalances in levels of ovarian hormones such as E\({}_{2}\) and P4 can lead to health problems, including hot flashes, night sweats, sleeping difficulty, osteoporosis, and heart disease [46; 47]. In the present study, we exposed ovariectomized rats to hind-limb ischemic stress in order to induce conditions similar to those of PVD during the menopausal transition. We then examined blood perfusion and capillary density in the hind-limb and evaluated the angiogenic effects of TTK extract based on changes in endothelial cell migration, tube formation activities, and angiogenic factors. Figure 6: Angiogenic effects of TTK in ischemic limb. Western blotting assay to determine the expression of phosphor-ERK(1/2), ERK(1/2), phosphor-Akt, Akt, phosphor-FAK, FAK, phosphor-PLCy1 and PLCy1 contained in the ischemic hind limb on day 28 after surgery. * \(p<0.05\), ** \(p<0.01\) vs. OH. In the case of PVD, risk factors indicating inadequate blood perfusion, perimenopause, postmenopausalopause, or lipid metabolism disorders contribute to peripheral ischemic symptoms such as intermittent claudication [15; 20]. In other study, in vivo models of hind-limb ischemia in ovariectomized rats were developed, including ones for examining blood perfusion, capillary density, and angiogenic factors in the presence or absence of estradiol [5; 24; 48]. In these studies, estrogen deficiency exacerbated blood perfusion by attenuating angiogenesis or neovascularization in a mouse model of hind-limb ischemia. In our previous study, treatment with TTK extract improved risk factors for CVD or PVD, including high-fat mass, overweight status, and lipid profiles [35]. Thus, we hypothesized that TTK extract could have a beneficial effect on blood circulation by decreasing risk of PVD in a murine hind-limb ischemic model. We focused on improvement of blood perfusion by evaluating the blood flow ratio between ischemic (left hind-limb) and non-ischemic (sham-operated; right hind-limb) lesions. TTK treatment resulted in marked increases in blood perfusion, and a significant difference was first observed at 7d after ischemic surgery in the high TTK group. Additionally, blood perfusion improvements were observed at 14 to 28d after surgery in all treatment groups (positive control, low TTK, and high TTK groups). Notable sexual dimorphism was previously reported in a rodent model of liver ischemic injury [49]. This result suggests that the protective mechanisms in hind-limb ischemic mice are dependent upon the up-regulation and activation of angiogenic signaling mediated by estradiol [49; 50; 51], and growth factors for angiogenesis are mediated via active-kinase signal pathways, including PI3K/Akt, ERK1/2, FAK, and p38/MAPK. Kinases regulate endothelial cell functions such as cell migration, survival, and vascular cell permeability [52; 53]. Therefore, in our model, expression of angiogenic factors might have initially been stimulated in ischemic tissues of thigh and calf muscles by excision of femoral vessels. Expression levels of pERK(1/2), pAkt, pFAK, and pPLCy1 were elevated by TTK treatment in a dose-dependent manner. Mitogen-activated protein kinases ERK1 and ERK2 (ERK1/2) are critical for angiogenesis and endothelial cell function [54; 55]. FAK, also known as cytoplasmic tyrosine kinase, is important for cell migration and integrin-related signal production [56]. The serine-threonine kinase Akt is activated downstream of PI3K and mediates survival of endothelial cells [57], phospholipase Cy 1 (PLC-y1) activity at Ser1248, and enhancement of cellular motility [58]. However, the current in vivo studies had limitations lacking in inhibitor studies, such as siRNA and kinase inhibitors, in pERK(1/2), pAkt, pFAK, and pPLCy1 expression.