## 4 Discussion
In the current study, MIA exposure alone did not lower the SOX9 level in the chondrocytes, supporting the speculation that chondrocytes might be damaged, but their self-repair mechanism was not overwhelmed. Co-treatment of AnTT and MIA stimulated the expression of SOX9, which coincides with the effects of this treatment on COL2\(\alpha\)1 and aggrecan level. Taken together, these observations suggest that AnTT enhanced chondrocytes' self-repair mechanism when challenged by MIA. ADAMTS4 is one of the metalloproteinases responsible for the degradation of type II collagen and aggrecan in the cartilage [60]. ADAMTS4 levels in the serum and synovial fluid increases in patients with early-stage OA compared to intermittent and late-stage OA [61; 62]. Intra-articular MIA administration has been shown to increase ADAMTS4 expression in rat cartilage samples [63; 64]. In this study, MIA exposure led to a significant decrease in ADAMTS4 levels in chondrocytes, probably due to activation of the self-repair mechanism. PT3 (25 \(\upmu\)g/mL) restored ADAMTS4 levels in chondrocytes exposed to MIA. It is postulated that PT3 negated the effects of MIA, and the cellular self-repair mechanism was not triggered to reduce ADAMTS4 levels. Despite the potential anabolic effects on MIA-induced chondrocytes, as discussed above, AnTT did not normalise ADAMTS4 levels. Furthermore, we cannot rule out the possibility that the direct chemical interaction between MIA andtoctirenols may partially contribute to the protective effects oftoctirenols during concurrent treatment. Iodoacetate has been demonstrated to bind with nucleophiles such as glutathione or glyceraldehyde-3-phosphate dehydrogenase via electrophilic addition [37]. Furthermore,tocopherol is also reported to bind with mutagenic electrophiles like nitrogen dioxide and peroxynitrite via its electron-rich chromatol ring [65; 66; 67]. Tocotrienol, with its similar chromatol ring and a tri-unsaturated aliphatic tail, may serve as a nucleophile in scavenging MIA. Nevertheless, the interaction betweentoctirenols and MIA may be minimal, given that concurrent treatment withtoctirenols did not completely abrogate the MIA-mediated cytotoxicity and alteration in metabolism. Therefore, further study is required to confirm the postulated chemical interaction between MIA andtoctirenols. Comparison between PT3 and AnTT revealed that only AnTT significantly improved the anabolic activities of chondrocytes by increasing the COL2\(\alpha\)1/COL1\(\alpha\) ratio, SOX9, COL2\(\alpha\)1 and aggrecan levels. AnTT is postulated to enhance the self-repair mechanism of chondrocytes independently of their antioxidant activities. On the other hand, PT3 lowered the MIA-mediated oxidative stress but did not activate the self-repair mechanism, unlike AnTT. Based on the vitamin E isomer constitution, the AnTT-mediated self-repair mechanism may be contributed to by \(\delta\)-tocotrienol, given that 20 ug/mL AnTT contains a 2.8-fold higher amount of \(\delta\)-tocotrienol than PT3 (50 ug/mL). The other isomers in PT3, such as \(\alpha\)-tocopherol and \(\alpha\)-tocotrienol, may contribute to its antioxidant activities but they did not promote anabolic activities in the chondrocytes. The difference in potency of biological actions for each occotrienol isomer has been discussed previously [46]. For instance, \(\delta\)-tocotrienol has been shown to be the most potent isomer in suppressing 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGCR) [68], activating peroxisome proliferating-activated receptor-\(\delta\) or -\(\gamma\)[69] and improving insulin synthesis [70]. Additionally, the current study demonstrates the absence of anabolic activities for PT3 at 50 ug/mL, despite containing 6 ug/mL \(\delta\)-tocotrienol, compared to AnTT at 10 ug/mL (8.4 ug/mL \(\delta\)-tocotrienol). A potential antagonism interaction is postulated between vitamin E isomers. Tocopherol has been shown to antagonise the action oftoctirenols in lowering lipoprotein and inhibiting HMGCR [71; 72].