*3.3. Oxidative Damage Protection of Hyt@tgel*

Several studies have concluded that OA progression is significantly related to an imbalance between the production of reactive oxygen species (ROS) and their clearance by an antioxidant defense system [52,53]. During OA pathogenesis, chondrocytes become both source and target of elevated amounts of reactive chemical species, particularly oxygen and nitrogen species triggering a vicious circle that leads to further damage of cartilage cells and matrix [54]. A wide body of evidence suggested that Hyt has antioxidant activity by inhibition and/or scavenging of reactive oxygen species (ROS) [54,55]. Moreover, a gene expression profiling study has suggested that Hyt affect the expression of genes involved in oxidative stress, inflammation, cell proliferation, or differentiation, suggesting that the beneficial effects of this molecule may be multifactorial and context-dependent [56]. The efficiency of Hyt@tgel10 to reduce the intracellular ROS generation was assessed in C20A4 cells in the presence of hydrogen peroxide (H2O2) (Figure 3A,B). The stimulation of chondrocytes with H2O2 mimics the in vivo condition observed in OA cartilage inducing the production of cellular and mitochondrial ROS and producing proinflammatory and procatabolic responses [57,58]. A significant increase (*p* < 0.001) in chondrocyte intracellular oxidants by approximately 2.8 times was obtained with respect to untreated cells (Control) after H2O2 24 h treatment (Figure 3A). A short-time pre-incubation (24 h) with Hyt@tgel10 considerably reduced (*p* < 0.01) H2O2-induced ROS production of about 1.4-fold with respect to the H2O2 group. Moreover, the protective effect of released Hyt was greatly enhanced ((*p* < 0.001) by a longer pre-treatment (96 h) resulting in slight fluorescence increase with respect to control cells. MDA, a lipid peroxidation end product, is abundant in synoviocytes from patients with OA. Under oxidative stress, polyunsaturated fatty acids of cellular membrane lipids represent the prime targets of ROS attack. The lipid peroxidation leads to the formation of chemically reactive lipid aldehydes, such as MDA, capable of causing severe damage to nucleic acids and proteins, altering their functions and leading to the loss of both structural and metabolic function of cells [59]. As reported in Figure 3B, treatment of cells with H2O2 increased intracellular lipid peroxidation to 2-fold relative to control (*p* < 0.001). Conversely, the presence of Hyt@tgel10 for 24 h markedly diminished (*p* < 0.01) the MDA level (1.1-fold) compared with H2O2 treated cells, with a marked decrease (*p* < 0.001) after 96 h leading the MDA formation to levels almost similar to control.

**Figure 3.** Antioxidant capacity of Hyt@tgel in H2O2-treated chondrocytes. C20A4 cells were incubated in the presence of hydrogel for 24 and 96 h and then treated with hydrogen peroxide for 24 h. (**A**) ROS release was determined by oxidized H2DCFDA (DCF). (**B**) Malondialdehyde quantity was used as a marker of lipid peroxidation. (**C**) Superoxide dismutase (SOD2) activity measured by assay kit. (**D**) SOD2 mRNA transcription level. Results are expressed as the mean of three independent experiments ± S.D (*n* = 3). \*\* *p* < 0.01, \*\*\* *p* < 0.001 versus untreated cells (control). ### *p* < 0.001 versus H2O2 group.

To prevent an accumulation of ROS-mediated damage, chondrocytes produce a number of antioxidant enzymes including the superoxide dismutases (SOD), catalase, and glutathione peroxidase [60]. The three SOD family members SOD1, SOD2, and SOD3 transform O2 − into hydrogen peroxide (H2O2), limiting the formation of highly aggressive compounds such as ONOO− and OH−. All SOD are expressed at lower levels in OA cartilage compared to normal control cartilage, at both the messenger RNA (mRNA) and protein level. In particular, Ruiz-Romero et al. demonstrated through a proteomics approach, a significant decrease in the major mitochondrial antioxidant protein manganese-superoxide dismutase (SOD2) in the superficial layer of OA cartilage. This SOD2 reduction makes cartilage more susceptible to ROS damage suggesting a central role of mitochondrial redox imbalance in OA pathogenesis [61]. To verify if the antioxidant actions of Hyt have been related not only to its free radical scavenging activity, but also to the ability to enhance the endogenous defense system by inducing antioxidant/detoxifying enzymes activity, SOD2 activity was assayed. As shown in Figure 3C, treatment with H2O2 leads to decrease in antioxidant enzyme activity of about 54% with respect to untreated cells. When chondrocytes were pre-treated with Hyt@tgel10 for 24 and 96 h, SOD2 activity was 18% and 42%, respectively, higher than that in H2O2-treated cells, demonstrating a good ability to protect mitochondria from oxidative damage. Moreover, Hyt@tgel10 pretreatment restored the SOD2 transcript to above their control levels, by significantly increasing its expression by 2.4-fold for 24 h and 5.5-fold after 96 h over the H2O2-depressed level (Figure 3D).

Taken together, the results reported herein confirm a key role of Hyt@tgel10 pretreatment to effectively suppress the production of intracellular ROS and lipid peroxidation and also elevated the activity of antioxidant enzymes such as SOD, limiting oxidative stress-induced damage in the OA in vitro model.

## *3.4. Hyt@tgel Suppresses Inflammatory Response in Chondrocytes*

Increases in the levels of the cytokines in joints plays a central role in the pathogenesis of OA by modulating oxidative stress, cartilage ECM turnover, and chondrocytes apoptosis [62,63]. The current drugs for treating OA are developed primarily to relieve pain and control symptoms, failing to cure the disease [63]. Epidemiologic studies demonstrated the lower incidence of inflammatory chronic disease, such as OA in people of the Mediterranean basin. One of the possible reasons is that Mediterranean people have a high intake of olive and olive oil rich in polyphenolic compounds with antioxidant and anti-inflammatory properties [14,64,65]. During the pathophysiological processes of OA, cytokines, hormone-like proteins, are responsible for the loss of metabolic homeostasis of tissues forming joints by promoting catabolic and destructive processes. Olive-oil-rich extracts inhibit the production of proinflammatory cytokines, including IL-1β, TNF-α, IL-6, and prostaglandin E2 in arthritic joints [66,67]. Richard and colleagues demonstrated a pivotal role of Hyt extracted from olive vegetation water in diminished secretion of cytokines (IL-1 α, IL-1 β, IL-6, IL-12, TNF-α), and chemokines (CXCL10/IP-10, CCL2/MCP-1) in murine macrophages (RAW264.7 cells) stimulated with lipopolysaccharide (LPS) [68]. Another study showed a decrease in the severity of the disease and an overall anti-IL-1β effect after treatment with olive and grape seed extract in animal models of post-traumatic OA [69]. In the present study, secreted IL-6, IL-8, and TNF-α were detected in the supernatant of chondrocytes cell line C20A4 by enzyme-linked immunosorbent assays (ELISA). As expected, incubation of cells for 24 h with Hyt@tgel10 significantly reduces the amount of released cytokines with respect to the control in a time-dependent manner (Figure 4A–C). Consistently, the protective effects of Hyt were confirmed also by RT-qPCR analysis. As reported in Figure 4D–F, the mRNA levels of all tested cytokines (relative to the housekeeping gene) were significantly upregulated (*p* < 0.01) in H2O2-treated cells, compared with the control group. As expected, the H2O2-driven release of IL-6, IL-8, and TNF-α was decreased by Hyt@tgel10 pre-treatment with a 50% reduction in interleukin expression levels with respect to H2O2-treated cells.

**Figure 4.** Hyt@tgel10 inhibits H2O2-induced inflammatory response in chondrocytes. The effect of Hyt on the production of IL-6 (**A**,**D**), IL-8 (**B**,**E**), and TNF-α (**C**,**F**) was measured by ELISA assay (**A**–**C**) and qRT-PCR (**D–F**). C20A4 cells were pre-treated with Hyt@tgel10 for 24 h, then stimulated with H2O2 for 24 h (ELISA assay) or 4 h (qRT-PCR). Results are expressed as the mean of three independent experiments ± S.D (*n* = 3). ### *p* < 0.001 H2O2 vs. CTL, \* *p* < 0.05, \*\* *p* < 0.01, and \*\*\* *p* < 0.005 Hyt@tgel10 vs. H2O2.
