## 4 Discussion
The increased oxidative stress was probably a sequela of mitochondrial dysfunction induced by MIA, consistent with previous reports [34; 35]. Additionally, MIA also depleted the intracellular glutathione via non-enzymatic binding, which may explain the increase in the endogenous ROS upon induction [37]. Both AnTT and PT3 were shown to suppress MIA-induced lipid peroxidation completely in this study. The function of toctorienol as a free radical scavenger has been widely reported [46]. The chromatol ring of toctorienols readily scavenges ROS byexchanging electrons with free radicals [47; 48], thereby terminating the lipid peroxidation chain reaction. The reduction of oxidative stress by AnTT and PT3 could explain the reversal of MIA-induced viability suppression in chondrocytes. Collagens and non-collogenous proteins such as aggrecan are the major constituents of the extracellular matrix synthesised by chondrocytes [49]. Type II collagen is a marker of well-differentiated chondrocytes, while type I collagen is a marker of dedifferentiated chondrocytes [50]. Thus, COL2\(\alpha\)1/COL1\(\alpha\)1 ratio could be used as a marker of chondrocyte differentiation. In this study, MIA caused a significant reduction in COL1\(\alpha\)1 levels but did not alter COL2\(\alpha\)1 and aggrecan levels. In contrast, previous studies have reported that MIA suppressed type II collagen expression [51; 52], while increasing type I collagen expression in articular cartilage samples in rats [53]. Other studies have reported a concurrent reduction of type I and type II collagen and aggrecan protein or mRNA expression in articular cartilage samples of rats induced with MIA [54; 55]. It is speculated that MIA at the current dose did not overwhelm the repair capacity of chondrocytes, thus the production of type II collagen and aggrecan was preserved at an extent similar to unstimulated chondrocytes. In this study, AnTT and PT3 alone or PT3 in the presence of MIA did not significantly alter the COL1\(\alpha\)1 and COL2\(\alpha\)1 expressions of the chondrocytes. However, concurrent treatment with AnTT or PT3 with MIA increased the COL2\(\alpha\)1/COL1\(\alpha\)1 indices, and it was found that AnTT treatment was more potent despite being at a lower concentration. Unexpectedly, co-treatment of AnTT and MIA stimulated the COL2\(\alpha\)1 and aggrecan expression of the chondrocytes. It is hypothesised that AnTT enhanced the repair response of chondrocytes exposed to MIA. In line with this speculation, it can be suggested that tocotrienols alone did not exert anabolic effects in unstimulated chondrocytes in the current study because the repair response was not activated. By way of comparison, palm vitamin E mixture (Palmyitee) alone was ineffective in preventing the diminished type II collagen expression and the upregulation of type II collagen expression in a monolayer culture of primary chondrocytes from the human nasal septum [56]. However, palm vitamin E mixture promoted type II collagen expression in reconstructed cartilage embedded in vivo [56]. Parallel with our findings, [PERSON] et al. demonstrated that pre-treatment of \(\alpha\)-tocopherol upregulated the expression of gene coding for COL2\(\alpha\)1 and aggrecan, and downregulated gene coding for COL1\(\alpha\)1 in primary chondrocytes exposed to hydrogen peroxide for 3 h. This resulted in an increased COL2\(\alpha\)1/COL1\(\alpha\)1 ratio in the treated cells [40]. The observations above are supported by the results for SOX9 levels in the chondrocytes treated with both AnTT and MIA. SOX9 is a member of the SRY-related high-mobility group (HMG) box (SOX) family of transcription factors. It plays an important role in chondrogenesis, cell survival and activation of transcription for cartilage components, such as type II collagen and aggrecan [57; 58]. MIA administration was reported to suppress SOX9, type II collagen and aggrecan expression in the articular cartilage sample of rats [54; 59].