## 3 Results
### Pathological Changes in Knee joints  
Total Mankin score of knee joints examined histologically was compared in rats of different ages fed a similar diet (ND) (Figure 5). Younger rats (26 weeks old) showed lower scores than the aged rats (48 weeks old), demonstrating that age alone in the absence of HFHS diet or OVX resulted in significant knee joint damage. Figure 4: Analysis of cartilage and glucose metabolism in systemic circulation. Plasma analysis was measured in the rats at 48 weeks of age. Bar graphs and error bars show means and SE respectively (\(n=11\)–\(12\), per group). Two-way ANOVA was used to evaluate the influence of effects: diet, surgical procedure, and GSM. If the effect was statistically significant, the symbol * is indicated. One-way ANOVA was used to identify significant differences across all groups. In case data reached the criteria, a multiple comparison test is performed using Turkey method, and then letters are indicated on the bar. Groups showing different letters are significant differences. The table shows \(p\) values calculated from two-way ANOVA analysis. \(p<0.05\) was used to determine statistical significance in all methods. Figure 5: Comparison of knee joint scores between young rats and aged rats. Rats on normal control diet at age of 26 weeks (young rats, \(n=12\)) and 48 weeks (aged rats, \(n=10\)) were sacrificed and knee joints were processed to pathological slide preparation stained with Safranin-O. The criteria of Mankin score were used to evaluate articular cartilage and then summed into mean \(\pm\) SE presented in the bar chart. Mann–Whitney U test was used to analyzed statistical differences between young rats and aged rats as which asterisk *** shows significance at \(p<0.001\). Figure 6 demonstrates the pathological findings of knee joints in the 48-week-old rats from each of the test groups, with images selected to visually represent the various criteria used to inform the Mankin score. Severity of cartilage erosion was graded from 0 to 14 with the higher scores corresponding to more severe deterioration. As can be seen, all rat groups had only modest severity (4-5) except the OVX rats fed HFHS; this group expressed a Mankin score of approximately 7, which is considered being moderate severe disease. Rats on the ND developed only modest OA with no significant differences due to other interventions. Rats on the HFHS diet that did not undergo OVX likewise had only moderate scores, although the score was decreased slightly but not significantly with GSM addition. Thus, neither OVX nor HFHS diet alone induced additional joint damage, but the combination resulted in a synergistic effect causing moderate damage. Of greatest interest was the observation that inclusion of GSM in the OVX rats fed HFHS resulted in complete mitigation of the induced pathological lesions (Figure 6). Figure 6: Pathological changes in rat’s knee joints at 48 weeks of age. Knee joints from rats at the end of the study were dissected, formalin-fixed, and decalcified prior to processing tissue slides. Slides were stained with Safranin-O showing in panels A–F. Panel (**A**) shows normal features of rat tibia bone as which articular cartilage is stained red (CA) and subchondral and trabecular bone are pink (SB). The cartilage can be divided by tide mark (dotted line) into two zones; non-calcified cartilage (from the surface area to tide mark junction) and calcified cartilage (under tidemark to subchondral bone plates). The abnormality of articular cartilage can be marked as surface irregularity (black arrows in panel (**B**)), cloning and clusters of chondrocytes (**C**), pale staining of cartilage matrix (**D**), degeneration and necrosis of chondrocytes (black arrow in panel (**E**)), and duplicated tide mark (**F**). Mankin score criteria were used to grade the severity of cartilage lesion in each group and mean \(\pm\)SE is showed in the table (**G**). Finally, all criteria were summed and presented as a mean \(\pm\)SE of Mankin score in the bar chart (**H**).