Four-Octyl Itaconate Protects Chondrocytes against H2O2-Induced Oxidative Injury and Attenuates Osteoarthritis Progression by Activating Nrf2 Signaling

Nrf2 is a critical regulator of the antioxidant defense systems in cellular protection. Emerging evidence has shown that four-octyl itaconate (OI) activates Nrf2 cascade. In this study, the chondroprotective effects of OI on H2O2-stimulated chondrocytes and DMM-induced osteoarthritis (OA) progression were investigated. In primary murine chondrocytes, OI interrupted the binding of Keap1 and Nrf2, leading to accumulation and nuclear translocation of Nrf2 protein, as well as transcription and expression of Nrf2-dependent genes, such as HO-1, NQO1, and GCLC. Furthermore, OI inhibited cell death and apoptosis, as well as H2O2-stimulated ROS generation, lipid peroxidation, superoxide accumulation, and mitochondrial depolarization in chondrocytes, which were abolished by the silence or depletion of Nrf2. In addition, in vivo experiments revealed the therapeutic effects of OI on OA progression in a DMM mouse model. Collectively, these results suggested that OI might serve as a potential treatment for OA progression.


Introduction
Osteoarthritis (OA), featured by progressive cartilage degradation, is one of the most prevailing degenerative joint disorders among the elderly, leading to severe pain and joint dysfunction [1,2]. Studies have shown that oxidative stress caused by sustained reactive oxygen species (ROS) production is the major reason for chondrocyte apoptosis, eventually leading to OA pathogenesis [3]. In contrast, clearance of ROS offers significant chondrocyte protection against oxidative injury.
Previous researches have confirmed that itaconate could activate Nrf2 pathway [13,14]. Specifically, itaconate can modify Keap1, leading to separation of Nrf2 from Keap1, followed by Nrf2 stabilization, accumulation, and activation. Additionally, several studies have shown that the itaconate derivative four-octyl itaconate (OI) potently exerts cytoprotection against oxidative injury via activating Nrf2 signaling [13,[15][16][17]. For example, Tang et al. revealed OI stimulated Keap1-Nrf2 pathway to efficiently downregulate the generation of proinflammatory cytokines in macrophages [16]. Another study revealed that OI induced Keap1-Nrf2 cascade to protect neurons against hydrogen peroxide (H 2 O 2 ) [17]. Moreover, a recent study revealed that OI can protect osteoblasts against H 2 O 2 via activating Nrf2 pathway [18]. However, the underlying effect of OI on chondrocytes remains to be fully elucidated. The purpose of this research was to test whether OI could exert chondrocyte protection against oxidative injury and explore the possible mechanism. In addition, the therapeutic value of OI in OA mouse model was also evaluated in vivo.

Western
Blotting. Western blotting assays were carried out as previously reported [19,20]. In brief, total proteins were extracted from chondrocytes and isolated in RIPA lysis buffer. Then, 40 μg of total proteins were loaded into well on sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a polyvinylidene difluoride (PVDF) membrane (Bio-Rad, USA). Next, the membranes were blocked with 5% skim milk, followed by incubation with primary antibodies against GAPDH, Tubulin, CypD, p53, ANT-1, Lamin B1, Keap1, Nrf2, HO1, NQO1, and GCLC. Antibodies were all used at 1 : 1,000 dilution unless otherwise indicated. Subsequently, the membranes were incubated with the relevant secondary antibody, and the protein bands were visualized by an enhanced chemiluminescence kit, with ImageJ software used to quantify the band intensity.
2.3. Real-Time PCR. After treatment, total RNA was isolated from chondrocytes with TRIzol reagents (Invitrogen). Next, cDNA was prepared with 1 mg of total RNA (MBI Fermantas, Germany). The thermocycler settings were 95°C for 10 min, then 95°C for 15 s, and 60°C for 1 min, conducted for 40 cycles. The process was performed via the CFX96 Real-Time PCR system (Bio-Rad, USA). The cycle threshold (CT) value was obtained and standardized to GAPDH levels. The relative mRNA levels to each target gene were measured via the 2 −ΔΔCt approach. The mRNA primers for the listed genes were obtained from Dr. Di [17].
2.5. CRISPR-Cas9-Mediated Gene Knockout (KO). Primary murine chondrocytes were transfected with a CRISPR/ Cas9-Nrf2-KO-GFP-puro construct or a CRISPR/Cas9-Keap1-KO-GFP-puro construct, purchased from Santa Cruz Biotechnology. A FACS-mediated selection of GFP-positive cells was performed. And single cells were cultured in a 96-well plate to generate monoclonal stable cells. Nrf2 KO or Keap1 KO were verified by western blotting and qPCR analyses.

2.7.
Co-Immunoprecipitation (co-IP). The detailed Co-IP protocol has been reported elsewhere [23]. In brief, total cell lysates were precleared with protein A/G beads (Sigma), followed by Keap1 antibody incubation overnight. The Keap1-immunoprecipitated proteins were captured by protein A/G beads and measured by western blotting analyses.
2.9. ROS Detection. ROS content was determined following an earlier described method [25]. In brief, chondrocytes were incubated with 1 μM of carboxy-H2DCFDA, followed by detection of the DCF fluorescence signal at a wavelength of 550 nm with a fluorescence spectrofluorometer (Thermo Scientific, China).
2.10. Lipid Peroxidation. As reported previously [25], the level of cellular lipid peroxidation was determined by thiobarbituric acid reactive substances (TBAR) activity.
2.11. Superoxide Detection. The level of cellular superoxide was detected by the superoxide colorimetric assay kit (Beyotime, Wuhan, China). Briefly, treated chondrocytes were incubated with superoxide detection reagent, followed by test of the superoxide absorbance at a wavelength of 450 nm.
2.12. Single Strand DNA (ssDNA). The level of cellular ssDNA was determined following the previously reported method [22].
2.13. Primary Cultivation of Mouse Chondrocytes. Immature C57BL/6 mice were provided from the Animal Center of Chinese Academy of Sciences (Shanghai, China) and euthanized with excess pentobarbital sodium. First, articular cartilage tissues from the knee joints were isolated. Next, the collected cartilage was washed with PBS 2 to 3 times, shredded, digested by 0.1% collagenase II for 4 h at 37°C, and centrifuged. After resuspension in PBS, the cartilage was incubated in DMEM/F12 added with 10% fetal bovine serum and 1% penicillin/streptomycin at 37°C with 5% . As mentioned previously [26], a mouse model of OA was established by surgical destabilization of the medial meniscus (DMM). Then, the mice (n = 15) were randomly classified into three groups of five mice each: sham group, DMM + saline group, and DMM + OI group, respectively. Mice in the DMM + OI group were intraperitoneally administered OI at doses of 25 mg/kg/day in (2-hydroxypropyl)-β-cyclodextrin diluted in PBS or vehicle control for 8 weeks. The control mice received an equivalent volume of saline instead. After eight weeks, the cartilage tissue samples were collected.
2.17. Statistical Analysis. All experiments were conducted three times. The values are represented as mean ± standard deviation (SD). SPSS 20.0 was utilized for statistical analyses. Data were processed by one-way analysis of variance (ANOVA), and the Tukey test was used for comparisons between groups. Nonparametric data (OARSI scores) were analyzed by the Kruskal-Wallis H test. A P value of less than 0.05 is considered to be significant.

OI Activated Nrf2 Signaling in Primary Murine
Chondrocytes. The potential effect of OI on the Nrf2 cascade in cultured primary murine chondrocytes was examined.
The results of coimmunoprecipitation (co-IP) assays (Figures 1(b) and 1(c)) revealed that Keap1 immunoprecipitated with Nrf2, and the connection between Keap1 and Nrf2 was disrupted by OI, with the concentration of 25 μM based on previous researches [17,18]. Then, Nrf2 protein was stabilized and accumulated in chondrocyte cytosol with OI treatment, while the Keap1 protein remained unchanged.
In addition, the results demonstrated that Nrf2 translocated into the nucleus, evidenced by the potently elevated Nrf2 protein levels in the nuclei determined by western blotting analysis (Figure 1(d)). Notably, the protein (Figure 1(e)) and mRNA (Figure 1(f)) levels of Nrf2-ARE target genes (HO1, NQO1, and GCLC), as well as those of the ARE luciferase activity (Figure 1(g)), were robustly increased in OI-treated chondrocytes, with unchanged Nrf2 mRNA levels ( Figure 1(f)) and Keap1 expression (Figure 1(d)). More importantly, the Nrf2 protein level was not significantly upregulated by OI in chondrocytes treated with MG-132, an established cell-permeable proteasome inhibitor (Figure 1(h)). Furthermore, the Nrf2 protein level was not distinctly affected by OI in chondrocytes after treatment with cycloheximide (CHX), a well-known protein synthesis inhibitor (Figure 1(i)). These results suggested that OI-induced Nrf2 protein augmentation was not the result of protein synthesis but protein stabilization. Taken together, the results suggested that the Nrf2 cascade was activated by OI in primary murine chondrocytes.   Figures 3(a) and 3(b)), superoxide accumulation (Figure 3(c)), GSH/GSSG ratio decrease (Figure 3(d)), and lipid peroxidation (Figure 3(e)). These actions were largely attenuated with OI treatment.

Oxidative Medicine and Cellular Longevity
Additionally, our results revealed that H 2 O 2 treatment activated programmed necrosis in primary murine chondrocytes. Mitochondrial immunoprecipitation (Mito-IP) assay results demonstrated that H 2 O 2 induced immunoprecipitation of p53 with CypD and ANT-1, two major components of the mitochondrial permeability transition pore (mPTP) [27], while the expression of p53, CypD and ANT-1 remained unchanged (Figure 3(f), "Input"). In addition, cytosolic cytochrome C (Cyto-C) release (Figure 3(g)) and mitochondrial depolarization (Figures 3(h) and 3(i)    Oxidative Medicine and Cellular Longevity or depleted. Two lentiviral Nrf2 shRNAs with nonoverlapping sequences were individually transfected into proliferating primary murine chondrocytes, and two stable cells were employed after puromycin selection (sh-Nrf2-a and sh-Nrf2-b). In addition, stable chondrocytes with the CRISPR/ Cas9-Nrf2-KO-GFP construct were established (ko-Nrf2). And results of Figures 4(a) and 4(b) showed that OIinduced Nrf2 protein stabilization as well as the mRNA and protein levels of HO1, NQO1, and GCLC were almost completely blocked with Nrf2 silenced or depleted in chondrocytes.
Functionally, sh-Nrf2 and ko-Nrf2 chondrocytes were more vulnerable to H 2 O 2 stimulation, showing reduced viability (Figure 4(c)) and enhanced cell death (Figure 4(d)). However, OI was ineffective in ameliorating the reduced viability and cell death stimulated by H 2 O 2 , indicating that the cytoprotective effect of OI was abrogated by Nrf2 silenced or depleted. These findings proved that Nrf2 cascade was involved in the cellular protection of OI in H 2 O 2 -stimulated chondrocytes.

Keap1 Knockout Abolished OI-Induced Cytoprotection from H 2 O 2 .
It has been demonstrated that OI modifies Keap1, leading to separation of Nrf2 from Keap1 and activation of Nrf2 signaling [13]. Based on this, we predicted that the cytoprotective effect of OI on chondrocytes would be nullified by Keap1 depletion. To examine this prediction, a CRISPR/Cas9-KO-Keap1 construct was used to transfect primary murine chondrocytes, and stable chondrocytes were established (ko-Keap1). As illustrated in Figures 5(a) and 5(b), ko-Keap1 led to depletion of Keap1 protein and stabilization of Nrf2 protein, as well as potently upregulated levels of HO1, NQO1, and GCLC. Furthermore, viability decrease ( Figure 5(c)) and apoptosis ( Figure 5(d) (Figure 6(a)), the OA group revealed more severe cartilage erosion, more proteoglycan loss, and fewer cells than the control and OI groups. Quantitative analysis using Osteoarthritis Research Society International (OARSI) scores ( Figure 6(b)) was in line with the findings of HE and safranin O staining. The OARSI scores of the OI group was distinctly lower than that of the OA group. Furthermore, immunohistochemical staining of the cartilage tissues for Nrf2 and MMP13 was performed. As shown in Figures 6(c)-6(e), higher level of MMP13 was observed in the OA group than in the control group, whereas the level of MMP13 was decreased in the OI group. Notably, the OI group showed a significantly higher number of Nrf2 nuclear-positive chondrocytes than the control and OA groups. In addition, to investigate whether Nrf2 was involved in OI-induced protection of OA progression, Nrf2 in the cartilage were measured by immunofluorescence staining. Results of Figure 6(f) showed that Nrf2 was mostly concentrated in cytosol in the sham and DMM groups, whereas the DMM + OI group showed significantly increased Nrf2 nuclear-positive chondrocytes. Quantitative analysis (Figure 6(g)) also confirmed that. Collectively, these data indicated that OI ameliorated OA progression and that the Nrf2 cascade was involved in this process.

Discussion
It has been widely shown that the Nrf2-associated signaling pathway suppresses ROS generation and subsequently offers vital protection against oxidative damage. Once activated, Nrf2 separates from Keap1, enters the nucleus, and associates with AREs to initiate increase of some antioxidant    Oxidative Medicine and Cellular Longevity enzymes and detoxifying genes, thereby exerting ROS scavenging and antioxidant functions [6,10,28]. Accumulating evidence has indicated that oxidative stress contributes greatly to the progression of OA. In contrast, radical scavengers and antioxidant agents are considered therapeutic strategies to protect chondrocytes against oxidative injury to attenuate OA progression [29]. Therefore, the Nrf2 pathway may be a promising target to treat OA.  [17]. Furthermore, OI activated Keap1-Nrf2 pathway to protect human umbilical vein endothelial cells against high glucose [15]. Therefore, we hypothesized that OI could protect chondrocytes against oxidative injury via activating Nrf2 pathway. In the current study, our findings indicated that OI boosted Nrf2 pathway, resulting in stabilization and nuclear translocation of Nrf2 protein, as well as upregulation of the levels of Nrf2-target genes (HO-1, NQO1, and GCLC) in chondrocytes. Importantly, as shown in the subsequent studies, treatment of OI potently reduced H 2 O 2 -stimulated ROS generation, oxidative stress, superoxide, lipid peroxidation, and DNA damage, revealing that treatment with OI potently inhibited H 2 O 2 -induced cell death and apoptosis in primary murine chondrocytes.
However, even several studies revealed that OI could protect different cells against oxidative stress, few reports focused on in vivo experiments. To investigate whether OI could protect against OA progression, the DMM mouse models were established in the current study. As expected in the DMM mice, the treatment of OI effectively attenuated ECM degradation and reduced the OARSI scores. Notably, a significantly higher number of Nrf2 nuclear-positive chondrocytes was observed in the OI-treated DMM groups, showing that OI administration ameliorated the OA progression via activation of Nrf2 in the DMM model, which was consistent with the in vitro results. These data offered evidence that OI could protect chondrocytes and inhibit OA progression via the Nrf2 signaling pathway. Besides, matrix metalloproteinases (MMPs) mainly consist of aggrecan and collagen II and are closely associated with the cartilage ECM degradation [30]. MMP-13, the main components of MMPs, plays an essential role in OA progression through preferentially decreasing collagen II. In our study, OI reduced production of MMP-13 via activation of Nrf2 path-way, so as to exert the anti-inflammatory effects on OA progression. Furthermore, it has been reported that the mPTP consists of at least three main proteins (VDAC, ANT-1, and CyPD) and plays a pivotal role in cell apoptosis and programmed necrosis [27,31,32]. Once stimulated by H 2 O 2 , p53 translocates to mitochondria and binds to CypD and ANT-1. Then, the Cyp-D-p53-ANT-1 complex results in mitochondrial depolarization, mPTP opening, and cytochrome C release, subsequently inducing programmed necrosis. In this study, we demonstrated that OI dramatically suppressed H 2 O 2 -induced programmed necrosis in primary murine chondrocytes. Programmed necrosis, together with apoptosis, might explain the potential cellular protective effect of OI against H 2 O 2 in chondrocytes.
Importantly, our results showed that OI-induced cytoprotection in H 2 O 2 -treated chondrocytes was almost blocked by Nrf2 silencing or depletion, indicating that   Figure 7: Schematic of the chondroprotective effect of OI via the Nrf2 pathway. OI protects chondrocytes against H 2 O 2 -induced oxidative stress by activating Nrf2, which translocates into the nucleus to increase the transcription and expression of Nrf2-dependent antioxidant proteins (HO1, NQO1, and GCLC), and then attenuates OA progression.