**4. Discussion**

In the current study, we found that both daily oral SNO supplementation and 6 weekly doses of IAHA alone are sufficient to attenuate post-traumatic OA-induced cartilage deterioration. In terms of the knee joint swelling and pain assessment, we found a marked difference between the two modalities of treatment, in which the long-term daily oral SNO supplement resolved better reduction in inflammatory signs/symptoms of the knee as well as the synovial reaction in the joint cavity than IAHA alone. The combination of both treatments demonstrated an additive effect, the SNO + IAHA OA rats showed the best histological scores, and at 10 weeks of oral SNO supplement provided additional anti-inflammatory and antinociceptive effects on visco-lubricative IAHA chondro-protection. Moreover, long-term oral SNO supplementation caused no alteration in metabolic profiles, such as serological uric acid, total cholesterol, and HDL levels, compared to those of control OA rats. Surprisingly, there was a minor reduction in fasting blood glucose and significantly reduced TG levels in the SNO-treated rats.

It is worth noting, a reduction of body weight was also observed in SNO and SNOHA treated rats. This similar finding was reported recently in obese rats [29]. The anti-obesity effect of SNO could be the result of reduction of OA pain, or vice versa. Given the fact that the clinical and pathogenic correlation between OA and metabolic disorder has been extensively reviewed [34], thus the managemen<sup>t</sup> of body weight is strongly recommended by OARSI, American Academy of Orthopaedic Surgeons (AAOS), and the American College of Rheumatology [35–37]. Clinical studies on weight loss and preclinical studies targeting metabolic abnormalities in OA are an area of research interest and have achieved important improvement in OA progression [38]. In rat model of type 2 diabetes mellitus, Onur et al. demonstrated the metabolic disease itself contributes to the onset and progression of knee osteoarthritis [39]. Mooney et al. showed that surgically-induced OA mice fed a high-fat diet presented not only higher fasting glucose levels and body weights compared to those of lean OA mice, but also had worse OARSI histological scores and less cartilage thickness [40]. Moreover, a recent report also demonstrated that cartilage deterioration was sustained even after the high-fat diet was withdrawn from the OA mice, and the blood glucose and body weight were restored to the levels in normal diet mice [41]. These findings suggested that an increased weight load is not the sole cause of the severity of OA progression; instead, the lipid/glucose metabolic pathways could also jeopardize cartilage integrity and synthesis.

Moreover, an emerging concept of gut–joint axis has associated the gu<sup>t</sup> dysbiosis (perturbation of gu<sup>t</sup> microbiota (GM) biodiversity and function), and the leaky gu<sup>t</sup> syndrome with the joint disease progression [42,43]. In both human and rodent model, an increase in serum level of the pro-inflammatory marker and bacterial metabolites were associated with OA severity [44,45]. This chronic low-grade inflammation as result of dysbiosis explains a new OA phenotype, indicated as the metabolic OA [46]. In fact, long-term diet or prebiotic supplements have shown to shift GM colony with the improvement of cartilage integrity [47]. The diary ingestion of high triterpenes SNO could have potential modulation on the GM diversity, which may associate with both metabolic change and cartilage protection. Future evaluation of GM colonies under long-term oral SNO remains to be explored.

On the other hand, in vitro evidence shows that a mixture of triterpenes ( α, β-amyrin) significantly reduced lipid droplet formation via suppression of PPARγ and C/EBP α expression, while enhancing the translocation of glucose transporter GLUT4 onto the plasma membrane of 3T3-L1 cells [48]. Furthermore, reduction in blood glucose, total cholesterol, and TG levels were also observed in streptozotocin-induced diabetic mice treated with an α, β-amyrin mixture [49]. Moreover, triterpene as lupeol was also found to have a hypolipidemic e ffect (decreased total cholesterol, TG, and phospholipids) in rats fed a high cholesterol diet [50]. The e ffect of SNO (with a high concentrate of triterpenes) on lipid metabolism may be connected to the molecular mechanism of its chondro-protective e ffect.

As previously shown, the predominant fatty acids in femoral head cartilage are palmitic (16:0), oleic (18:1), and linoleic (18:2) acids [51]. In the animal model of OA, those fatty acids were significantly reduced in mice after destabilization of the medial meniscus [52]. Nonetheless, oleic acid exposure downregulates the expression of MMP-1 and COX-2 in TNFα stimulated human chondrocytes culture while linoleic acid increased PGE2 production [53]. These results sugges<sup>t</sup> that local fatty acid concentrations could be results of OA and also contribute to OA progression. The high proportion of oleic acid of SNO could be one of the chondro-protection factors.

The interval and multiple doses of IAHA showed a significant chondro-protective e ffect in the treated group compared with the OA-control in our ACLT + MMx injured OA rats. Surprisingly, we observed neither an antinociceptive e ffect nor a reduction in inflammatory signs, as demonstrated by the weight-bearing asymmetry and knee swelling tests. In fact, several animal studies on OA pain reported similar findings on the HA e ffect. Ikeuchi et al. employed a monoiodoacetate (MIA)-induced OA pain model and found no significant di fference in weight-bearing asymmetry in HA-treated rats [54]. Boettger et al. also demonstrated in a rat bradykinin/PGE2 pain model that HA lost its antinociceptive e fficacy (shown as weight-bearing asymmetry) from day 7 after injection [55]. Recently, IAHA was found unable to reduce ankle swelling in MIA-induced ankle OA [56], which is similar to the knee width exam in our ACLT + MMx OA model. However, the limited time of follow-up and the small sample size are the two major limitations of this study. An evaluation with an extended follow-up observation will further elucidate the long-term effect of IAHA alone or in combination with oral SNO for the treatment of chronic osteoarthritis.
