Theaflavin-3,3′-Digallate Inhibits Erastin-Induced Chondrocytes Ferroptosis via the Nrf2/GPX4 Signaling Pathway in Osteoarthritis

There is evidence that osteoarthritis (OA) is associated with ferroptosis which is a kind of lipid peroxidation-related cell death. Theaflavin-3,3′-digallate(TF3), a polyphenol compound extracted from black tea, possesses antioxidative and anti-inflammatory properties, but its effects on chondrocyte ferroptosis in osteoarthritis (OA) remain unclear. Our present study aims at exploring the protective role and underlying mechanisms of TF3 against erastin-induced chondrocyte ferroptosis in OA. In human primary chondrocytes treated with erastin alone or combined with different doses of TF3, cell viability was assessed by MTS. Ferroptosis-related proteins, including Gpx4, HO-1, and FTH1, were detected by western blot. The levels of lipid peroxidation and Fe2+ were determined by fluorescence staining. Meanwhile, the change of related proteins in the Nrf2/Gpx4 signaling pathway was determined by western blot. siRNA-mediated Nrf2 knockdown and the Gpx4 inhibitor RSL3 were used to explore molecular mechanisms for TF3-induced ferroptosis in OA chondrocyte. The magnetic resonance imaging (MRI), HE staining, Masson's staining, and immunohistochemistry were used to evaluate articular cartilage damages in the rat OA model. The results showed that Gpx4 expression was markedly downregulated in the chondrocytes of OA patients. TF3 reversed erastin-induced ferroptosis of human cultured chondrocytes, lipid ROS, and Fe2+ production in mitochondria. Moreover, the expression of Gpx4, HO-1, FTH1, and Nrf2 was markedly induced by TF3 in the erastin-treated chondrocytes. The antiferroptotic effect of TF3 was related to enhance Nrf2/Gpx4 signaling pathway. Finally, TF3 inhibited OA progression by alleviating in vivo cartilage damage related to chondrocyte ferroptosis. Thus, TF3 significantly inhibits chondrocyte ferroptosis by activating the Nrf2/Gpx4 signaling pathway, suggesting that TF3 serves as a potential therapeutic supplement for OA treatment.


Introduction
Osteoarthritis (OA), a chronic degenerative disease, has become a notable cause of disability in elderly patients. The incidence of OA among people over 75 years old is approximately 70% to 90%, and more than 100 million people worldwide suffer from OA [1]. The clinical manifestations of OA are joint pain, swelling, joint deformities, and restricted mobility [2]. Cartilage degeneration, destruction, and osteophyte formation are the main pathological features of OA [2]. Many physiological processes contribute to the development of OA, such as pyroptosis, apoptosis, and autophagy of chondrocytes. Chondrocytes undergo ferroptosis during OA, and that ferroptosis contributes to the progression of the disease [3]. Thus, inhibition of chondrocyte ferroptosis promises a potential OA therapy.
Ferroptosis is a type of iron-dependent lipid peroxidation-related cell death process that requires the accumulation of iron-dependent cellular active oxidation products [4]. Ferroptosis can lead to the progression of various diseases, including ischemia-reperfusion injury [5], neurodegenerative diseases [6], and tumors [7]. Iron is essential for cell physiological and biochemical processes, including participation in oxygen transport, DNA and ATP synthesis, and the tricarboxylic acid (TCA) cycle [8,9]. In addition, Fe 2+ promotes the lipid peroxidation of saturated fatty acids and the oxidative phosphorylation of mitochondria [10,11]. The oxidative reaction induced by iron deposition leads to oxidative stress, causing protein, nucleic acid, and liposome damage directly or indirectly, which gives rise to cell damage or ferroptosis [12]. Firstly, liposome peroxidation causes damage to cell membrane composition and structure [13,14]. In addition, liposome peroxidation produces a large amount of lethal reactive oxygen species (ROS), malondialdehyde (MDA), and peroxide dismutase (SOD), and these products can react with DNA or protein, further exerting toxic effects [15]. Glutathione peroxidase 4 (Gpx4), a lipid repair enzyme, is regulated by glutathione (GSH), which has been illustrated to block ferroptosis by reducing lipid peroxidation products and lethal reactive oxygen species accumulation [16,17]. Activation of the antioxidant enzyme Gpx4 may have a potential to suppress ferroptosis.
Theaflavin-3,3′-digallate (TF3), a polyphenol compound extracted from black tea [18], has attracted medical attention because of its extensive pharmacological effects, such as anticancer, antiviral, antioxidant, anti-inflammatory [19], and immunomodulatory [20] effects. A previous study indicates that TF3 regulates the expression of copper transporter 1 and GSH through the wnt/β-catenin pathway to inhibit ovarian cancer [21,22]. TF3 inhibited RANKL-induced osteoclast formation in bone marrow-derived macrophages (BMMs) in animal models [23]. In addition, TF3 could activate plasma kallikrein expression, reduce the deposition of fat and antagonize the oxidative damage induced by H 2 O 2 in hepatocytes [24]. In human chondrocytes, theaflavins (TFs) reduced oxidative stress-induced apoptosis and modulated AKT activation [25]. Moreover, ferroptosis is affected by multiple signaling pathways in cells [26]. The Keap1/ Nrf2 pathway is involved in glioma cell proliferation and ferroptosis [27]. MAPK/ERK/p38 [28,29], Stat3/Nrf2/Gpx4 [30,31], and ALK4/5 [32] signaling pathways are critical for ferroptosis induction. Despite these exciting discoveries, it remains that the role of TF3 in OA chondrocyte ferroptosis and related signaling pathway is far from elucidated. In this study, we aimed to further explore the role of TF3 in OA chondrocyte ferroptosis and elucidate its underlying mechanisms.  Table S1. Rabbit polyclonal antibodies were used to detect human proteins related to the Nrf2/Gpx4 signaling pathways and ferroptosis-related proteins Slc7a11, FTH1, and HO-1. The human β-actin antibody was used as an internal control for protein loading, and relative expression levels were quantified using Quantity One software.

Propidium Iodide Stain.
After different treatments, 2 μl of PI solution reagent (Vazyme Biotech Co., Ltd., Nanjing, China) was pipetted into each well plate. Then, the plate was incubated at room temperature for 5 minutes without light and was observed under a fluorescence microscope (Nikon Eclipse Ti, Japan).
2.8. Nuclear Protein Extraction. Nuclear protein was extracted using a nuclear protein extraction kit (Beyotime, China, P0028) according to the manufacturer's protocol. The human lamin B and β-actin antibodies were used as the internal controls for protein loading, and relative expression levels were quantified using Quantity One software. 2.12. Animal Experiments. In total, 25 male SD rats, weighing 150 to 200 g, were purchased from Cavens Experimental Animal Co. Ltd. (Changzhou, China). All rats were randomly divided into five groups: control, OA, OA + Erastin, OA + Erastin+TF3, and OA + Erastin+DFO. DFO, an effective iron chelator, has been used to inhibit ferroptosis in various degenerative disease models [33]. In an OA animal model, DFO delayed the progression of primary OA. Therefore, DFO was selected as a positive control for animal experimental treatment [34]. The OA model was successfully established by medial meniscus destabilization (DMM) surgery. TF3 (1 mg/kg), erastin (1 mg/kg), or DFO (1 mg/kg) were injected into the articular cavity twice a week, and the rats were sacrificed six weeks later. The progression of OA was evaluated using the Osteoarthritis Research Society International (OARSI) scored by two blinded investigators. The effects of TF3 and DFO 3 Oxidative Medicine and Cellular Longevity treatments were measured by morphological analysis and magnetic resonance imaging (MRI) examination. The animal study proposal was approved by the Animal Care Committee of Nanjing Medical University. All animal experimental procedures were performed in accordance with the Regulations for the Administration of Affairs Concerning Experimental Animals approved by the State Council of the People's Republic of China.

MRI Examination.
Six weeks after the model operation, cartilage damage was examined by MRI.

Immunohistochemical Staining and Histomorphometric
Measurements. Tibial and femoral tissues were separated and fixed in 10% formalin, decalcified in 10% ethylenediaminetetraacetic acid (EDTA) for 3 weeks after washing with water, and then dehydrated in graded alcohols. Specimens were embedded in paraffin and cut into 5 μm serial sections. 3 sections per rat were analyzed, and all sections were from medial femoral condyles. H 2 O 2 (3%) and BSA (5%) were used to block endogenous peroxidase activity and nonspecific binding sites, respectively. The Gpx4 primary antibodies were incubated overnight at 4°C. Next, the appropriate HRP-conjugated secondary antibody was added to the sections for incubation and counterstaining with haematoxylin at room temperature. Each glass slide was stained with HE and Masson's staining according to the manufacturer's instructions.
2.15. Statistical Analysis. Statistical analyses were performed using Prism8 (GraphPad Software, San Diego, CA, US). Unpaired Student's t -test was used for two groups; oneway ANOVA was used for more than two groups, and the Mann-Whitney U test was used for ranked data analysis. All quoted p values were 2-tailed, and those less than 0.05 were considered statistically significant.

TF3 Shows No Significant Cytotoxicity to Chondrocytes
at Appropriate Concentrations. The molecular structure of TF3 was shown in Figure 2(a). To evaluate the dose effect of the ferroptosis activator erastin, cultured OA chondrocytes were incubated with five different concentrations of erastin (1, 2, 5, 10, and 20 μM) and the solvent DMSO as a control. As demonstrated in Figure 2(b), chondrocyte viability was decreased in a dose-dependent manner. Notably, when chondrocytes were treated with 5 μM erastin, chondrocyte ferroptosis was successfully induced with a reduced viability after 12 hrs. Therefore, a 5 μM concentration of erastin was used to induce ferroptosis in the subsequent experiments. Next, we used different concentrations of TF3 (5, 10, 15, 30, and 60 μM) to treat OA chondrocytes for 12 hrs or 24 hrs. The MTS results showed that TF3 did not significantly affect chondrocyte viability at different concentrations (Figures 2(c) and 2(d)), suggesting that TF3 had no significant cytotoxicity to culture chondrocytes at given concentrations.   Oxidative Medicine and Cellular Longevity Figure 3(h)). Together, these observations indicated that TF3 inhibits chondrocyte ferroptosis by improving the Gpx4 expression suppressed by erastin.

TF3 Promotes Iron Metabolism in Cultured OA
Chondrocytes. Abnormal iron metabolism is another contributor to ferroptosis, and iron deposits are found in OA [35]. Ferrogreen was used to detect the Fe 2+ level. The results showed that Fe 2+ was accumulated in chondrocytes treated with erastin. In contrast, TF3 reduced the accumulation as reflected by the intensity of green fluorescence (Figures 3(i)  and 3(j)). In addition, Fe 2+ was stored in FTH1, and FTH1 participated in iron metabolism progression. Western blot results showed that TF3 upregulated the expression of FTH1 in the erastin-treated chondrocytes (Figure 3(g)).
Taken together, TF3 improves iron metabolism in OA chondrocytes.
3.6. TF3 Protects Chondrocytes from Ferroptosis via the Nrf2/ Gpx4 Signaling Pathway. To further investigate molecular mechanisms for TF3 in protecting chondrocytes from ferroptosis, we detected change of the signaling pathwayrelated proteins. TF3 significantly increased Nrf2, Keap1, p-MEK1/2, and p-Erk1/2 expressions in the total cell lysate (Figures 4(a) and 4(b)). Next, we separated the nucleus component from cytoplasm and carried out western blot analysis. A significantly increased expression of Nrf2 was observed in the nucleus and the cytoplasm upon TF3 treatment (Figures 4(c) and 4(d)). Then, we knockdown Nrf2 expression by using small interfering RNA (si-RNA) and  Figure 5(a). We observed that the combination treatment of erasin with TF3 significantly increased the PI-positive cell percentage, levels of lipid ROS, Fe 2+ in mitochondria in the si-Nrf2 chondrocytes compared with the si-NC chondrocytes ( Figures 5(b)-5(g)). On the contrary, the expression of ferroptosis-related protein (Slc7a11, Gpx4, FTH1, and HO-1) was markly decreased in erastin+TF3 + si-Nrf2 group in comparison with the erastin+TF3 + si-NC group (Figures 5(h) and 5(i)). Moreover, we further evaluated the TF3 protective role of Gpx4 by applying the Gpx4 inhibitor RSL3 to cultured OA chondrocytes for 2 hrs. The MTS results showed that TF3 failed to improve chondrocyte viability in the presence of RSL3 (10 μM) (Figure 6(a)). Meanwhile, TF3 did not decrease PI-positive chondrocytes number (Figures 6(b) and 6(c)), lipid ROS (Figures 6(d) and 6(e)), and Fe 2+ level in mitochondria (Figures 6(f) and 6(g)). However, TF3 partly reversed the expression of FTH1, Gpx4, HO-1, and Slc7a11 (Figures 6(h) and 6(i)). Collectively, these data suggested that the Nrf2/Gpx4 signaling pathway is involved in TF3-regulated chondrocyte ferroptosis.

TF3 Attenuates Cartilage Degradation and Increased
Proteoglycans and Gpx4 Expression in a Rat OA Model. To further explore the role of TF3-regulated ferroptosis in invo, we established a rat OA model. The gross morphological images of the rat's knee were shown in Figure 7(a). In the OA group, the articular surface was rough and ulcerated. The degree of cartilage joint injuries was more serious in OA + erastin group, while the cartilage joint injuries were repaired in the OA + erastin+TF3 group to some extent.  The MRI results illustrated that the degree of articular surface cartilage destruction in OA + erastin group was also more serious than that of in the OA group, while the degree of articular cartilage destruction was significantly alleviated in the TF3 treatment groups (Figure 7(b)). Furthermore, we also observed that cartilage damage was reversed after TF3 treatment, as showed by HE staining (Figure 7(c)) and OARSI score (Figure 7(d)). These observations suggested that articular cartilage damage might be repressed by TF3.
The proteoglycans in the cartilage were gradually lost as OA progresses. We used Masson's staining to assess the changes of proteoglycans in cartilage. Intra-articular injection of 1 mg/kg TF3 significantly reversed the proteoglycans levels in the cartilage, as assessed in Figure 8(a). Immunohistochemistry staining showed that the number of Gpx4positive chondrocytes was reduced in the OA + erastin group, but significantly increased in OA + erastin+TF3 group (Figures 8(b) and 8(c)). In summary, TF3 alleviates OA progression by Gpx4-mediated inhibition of chondrocyte ferroptosis in a rat OA model.

Discussion
In this study, we found that TF3 delayed the progression of OA and protected chondrocytes from ferroptosis via modulation of the Nrf2/Gpx4 signaling pathway. Currently, nonsteroid anti-inflammatory drugs (NSAIDS) is prescribed for treatment of OA. While it affords some protection to ferroptossis, it has noticeable side effects. Researchers have reported drug-drug interactions resulting in liver damages [36]. In erastin-induced chondrocyte ferroptosis, TF3, at nontoxic concentrations, reversed cell viability in a dosedependent manner, suggesting that TF3 may have the fewer The levels of Slc7a11, Gpx4, FTH-1, HO-1, and Keap1 in chondrocytes after stimulation were examined by western blotting; representative images are shown. (i) Quantitative analysis for the expression of Slc7a11, Gpx4, FTH-1, HO-1, and Keap1 in chondrocytes. Chondrocytes were pretreated with TF3 for 2 hours and then transfected with Nrf2 si-RNA and stimulated with erastin (5 μM) for 24 hours. The data are presented as the mean ± SD of three independent experiments, and statistical significance was determined by one-way ANOVA. * p < 0:05, * * p < 0:01, * * * p < 0:001, #p < 0:0001; n.s.: no significant difference. 10 Oxidative Medicine and Cellular Longevity Erastin, a ferroptosis inducer, can reduce glutathione levels by directly inhibiting cystine/glutamate antiporter system Xc -activity and activating the ferroptotic response [37], increasing ROS and iron accumulation further inducing ferroptosis [38]. Thus, we chose erastin to induce chondrocyte ferroptosis. Lipid peroxidation and iron accumulation were key factors in ferroptosis. Gpx4, an antioxidant agent, was negatively correlated with lipid peroxidation and showed protective effect on ferroptosis. Studies have indicated that the expression of Gpx4 was decreased in synovial fluid from the patients with OA and rheumatoid arthritis (RA) [39,40], which is consistent with our findings. Its inhibition blocks intracellular iron metabolism, resulting in lipid peroxidation products and ROS accumulation and thus accelerating ferroptosis progress [41]. Additionally, ROS is related to cartilage damage in OA [42]. Consistent with previous studies, the level of Gpx4 was decreased in the erastin-treated chondrocytes. In the presence of TF3, the decreased level of Gpx4 and lethal ROS accumulation were reversed in a concentration-dependent manner, suggesting the antiliposome peroxidation ability of TF3.
Abnormal iron metabolism contributes to ferroptosis induced by the production of ROS from the Fenton reaction [35,43]. Divalent metal transporter 1 (DMT1, also named Slc11a2) mediates the release of Fe2 + from the endosome into a labile iron pool in the cytoplasm [44]. Excess iron is stored in an iron storage protein complex including ferritin light chain (FTL) and ferritin heavy chain1 (FTH1) [45]. Increased iron uptake and reduced iron storage may contribute to iron overload during ferroptosis [46]. Consistently, our results showed that the level of Fe 2+ was increased in the erastin-treated chondrocytes and reversed by addition of TF3. In addition, TF3 promoted FTH1 expression. These observations suggested that TF3 inhibits ferroptosis by promoting iron metabolism in OA chondrocytes.
Recently, growing attention has been given to the role of the Nrf2 transcription factor in cartilage homeostasis [54]. Nrf2 is capable of regulating the basal and inducible expression of a plethora of antioxidant and detoxification enzymes, including CAT, SOD, Gpxs, heme-oxygenase1 (HO-1), NADPH, and quinone oxidoreductase1 (NQO1) [55,56]. Pharmacological activation of Nrf2 or overexpression of Nrf2 [57] has been shown to limit IL-1β-induced reactive oxygen species generation and reduce the absorption of iron in chondrocytes, demonstrating the importance of Nrf2 activity on the antioxidant response in cartilage. We found that the MEK1/2, ERK1/2, and keap1/Nrf2/Gpx4 signaling pathways were activated in erastin-treated chondrocytes in the presence of TF3. Under normal conditions, the DGR region of Keap1 binds to the DLG and ETGE sequences of Nrf2, which stabilizes Nrf2 in the cytoplasm and induces Nrf2 ubiquitination and proteasome degradation [58]. Keap1 transforms its connection with Nrf2 by sensing changes in ROS, promoting nucleus Nrf2 accumulation binding with the promoter antioxidant response element (ARE) to induce downstream antioxidant proteases transcription and translation [59]. The increased ROS level was associated with the elevated Nrf2 expression, by which cells maintain oxidative stress balance [60]. These studies were   13 Oxidative Medicine and Cellular Longevity the Nrf2/Gpx4-related pathway is a mediator of the protective effects of TF3 in OA chondrocytes.
Delaying articular cartilage degeneration was the key intervention method for OA treatment [62]. Systemic administration of DFO reduces cartilage lesion development in the OA model [34]. Our animal experiments results showed that TF3 could alleviate the degeneration of OA cartilage, which was manifested by a lower OARSI score and lower degree of articular cartilage destruction. Meanwhile, TF3 notably upregulated the expression of proteoglycans 14 Oxidative Medicine and Cellular Longevity and antiferroptosis protein Gpx4. These in vivo results indicated the protective effect of TF3 on OA was similar to that of DFO.

Conclusions
In summary, we found that TF3 protects chondrocytes against erastin-induced ferroptosis via the Nrf2/Gpx4 signaling pathway activation, suggesting that TF3 might be a novel and promising therapeutic option for OA (graphical abstracts). Oxidative Medicine and Cellular Longevity