## 4 Discussion  
The increased prevalence of OA over the past decade is attributed to the expansion of both an aging society and the consumption of a \"western-type\" high energy diet leading to increased obesity [30]. In addition, menopause is considered one of the most significant causes of OA in women [3]. This study's experimental design was intended to assess which of these factors (aging, diet, and menopause) independently or synergistically cause OA and how they determine the disorder's signature pathological pattern. This study successfully established the multiple phenotype of OA in a female rat model by using the mixed factors of aging, obesity, and ovariectomy. Interestingly, GSM consumption directly alleviated cartilage destruction without modulation of the predisposing factors including obesity, hyperglycemia, and bone loss due to estrogen deficiency. We speculate that the biological activity of GSM can be mainly attributed to omega-3 fatty acids, docosahexaenoic acid (DHA), and eicsapentaenoic acid (EPA), which are abundant in GSM powder [31] and well known to have anti-inflammatory properties [32; 33; 34; 35; 36]. However, GSM also contains other nutrient compositions such as a serious of unusual lipids (non-methylene interrupted FAs and plasmalogens), proteins, carbohydrates, and insoluble fiber, which are not well elucidated in their biological activity [37; 38], but these may contribute partially to some effects in this study. Therefore, the flash-dried GSM used in the current study may produce an anti-arthritis effect as effectively as GSM oil has been shown to do in previous studies [22; 24] It was observed in the current study that in the early cohort rats, whole GSM powder significantly reduced CTX-II [31], an early prognostic biomarker for OA. Similarly, in the late cohort rats, whole GSM powder significantly reduced OA in rats with a high Mankin score, which was the only definitive diagnostic marker for OA. Diet-induced obesity models have been used in rats for many decades. Feeding a diet with high proportions of fat and sugar accelerates bodyweight gain. The main bodyweight gain is attributed to an increase in body fat, the characteristic of obesity. Interestingly, inclusion of GSM in the diet of sham rats resulted in increased bodyweight but it was opposite in the OVX rats. It is possible that some components in GSM might be able to facilitate the digestion or absorption of macronutrients in the gastrointestinal tract resulting in an increase of nutrient bioavailability [39]. However, the mechanism of GSM on weight reduction in the OVX rats due to less fat mass gain cannot be explained by the current available information. Obese rats have a substantially increased proportion of body fat, mainly in the abdominal cavity (visceral fat) and under the skin (subcutaneous fat) [40]. Visceral fat is composed of white adipocytes which are responsible to adipokine production [41]. Leptin, one of the adipokines highly correlated with body fat mass, is known to be a critical mediator that initiates low-grade chronic inflammation by activating Toll-like receptors on macrophages [42]. Subsequently, activated macrophages release various types of proinflammatory cytokines into the circulatory system, manifesting inflammatory responses [43]. In this study, plasma leptin was dramatically elevated in the HFHS fed rats proportional to bodyweight gain. Estrogen deficiency had an explicit influence on increased bodyweight and plasma leptin [44]. To confirm the source of leptin production, the perigonadal fat pad, one portion of visceral fat, was assessed for leptin mRNA expression. A similar incremental pattern between plasma leptin and gene expression was revealed in all groups, indicating that perigonadal fat is one of the main sources of circulating leptin production in obesity which corroborates with previous rat studies [45; 46]. Plasma adiponectin levels in ND sham rats were high (-7 ug/mL) and similar to those found in humans under matching physiological conditions (5-10 ug/mL) [47].