Luteolin Protects Chondrocytes from H2O2-Induced Oxidative Injury and Attenuates Osteoarthritis Progression by Activating AMPK-Nrf2 Signaling

Osteoarthritis (OA) is a chronic degenerative disease featured by cartilage erosion and inflammation. Luteolin, a member of the flavonoid family, has been shown to exert anti-inflammatory and antioxidative activities. However, the potential biological effects and underlying mechanism of luteolin on chondrocytes and OA progression remain largely elusive. In this study, the potential effect and mechanism of luteolin on OA were investigated in vitro and in vivo. Our data revealed that luteolin inhibited H2O2-induced cell death, apoptosis, oxidative stress, programmed necrosis, and inflammatory mediator production in primary murine chondrocytes. In addition, luteolin could activate the AMPK and Nrf2 pathways, and AMPK serves as a positive upstream regulator of Nrf2. In vivo results demonstrated the therapeutic effects of luteolin on OA in the DMM mouse model. Collectively, our findings showed that luteolin might serve as a novel and effective treatment for OA and provided a new research direction for clinical OA therapies.


Introduction
Osteoarthritis (OA) is a chronic joint degenerative disease with no cure currently [1][2][3]. The progressive chondrocyte apoptosis and extracellular matrix (ECM) degradation are the major pathogenic features of OA. Accumulating evidence has revealed that some oxidative stimuli will induce chondrocytes to generate reactive oxygen species (ROS) and inflammatory cytokines [4]. Inflammatory mediators, such as IL-1β and TNF-α, could damage the imbalance of cartilage homeostasis and activate inflammation response, resulting in chondrocyte apoptosis. As a major type of ROS, hydrogen peroxide (H 2 O 2 ) induces mitochondrial damage, lipid peroxidation, and DNA damage, leading to ECM degradation and chondrocyte apoptosis [5]. Therefore, research on the discovery of novel and effective antioxidants to protect chondrocytes from oxidative injury has received increasing attention regarding OA treatment.
AMP-activated kinase (AMPK), a highly conserved serine/threonine kinase, serves as a master central hub that regulates cellular energy and metabolism homeostasis by suppressing inflammation and oxidative injury [14,15]. Notably, emerging evidence has revealed that boost of the AMPK signaling pathway could protect cells against oxidative injury by targeting Nrf2 signaling [16,17], indicating crosstalk between the AMPK and Nrf2 signaling pathways. However, the underlying mechanism by which the AMPK/ Nrf2 pathway protects chondrocytes against oxidative stress remains largely unknown.
In the past few decades, most studies have focused on the therapeutic potential of natural products for OA treatment. Among those natural compounds, luteolin has attracted growing attention. As an important member of the flavonoid family, luteolin is widely found in many plants, including green pepper, celery, carrots, cauliflower, and spinach [18,19]. Studies have shown that luteolin exhibits a series of therapeutic properties, such as antiinflammatory, antioxidant, and antitumor effects [20,21]. For example, Xiao et al. demonstrated that luteolin could inhibit cardiac ischaemia/reperfusion injury by activating the Nrf2 signaling pathway [18]. Furthermore, another study revealed that luteolin could activate the AMPK cascade to protect human umbilical vein endothelial cells from oxidative injury [19]. In addition, Wang et al. reported that luteolin alleviated allergic asthma by activating PI3K/Akt/mTOR signaling [22]. However, the potential biological effects and underlying mechanism of luteolin on chondrocytes and OA models remain largely elusive. Here, we explored the chondroprotective effects and the potential mechanism of luteolin in H 2 O 2 -induced chondrocytes and explored the in vivo protective effect of luteolin in the mouse OA model. The aim of the present study was to examine whether luteolin could protect primary murine chondrocytes from H 2 O 2 -induced injury and explore the potential mechanism. Additionally, the effect of luteolin on OA progression was assessed via the DMM mouse model.

Primary Murine Chondrocyte
Culture. Ten C57BL/6 mice (less than 2 weeks of age) were euthanized by pentobarbital sodium, followed by harvest of articular cartilage pieces from the knee joints under sterile conditions. The cartilage was dissected, rinsed with phosphate-buffered saline PBS, and treated with 0.2% collagenase II at 37°C for 4 to 6 h. The digested cartilage tissue was centrifuged, and the cartilage tissue suspension was seeded in culture medium containing 10% FBS, DMEM/F12, and 1% penicillin/streptomycin under 5% CO 2 at 37°C. The cells were harvested with 0.25% trypsin-ethylenediaminetetraacetic acid (EDTA) when they reached 80%~90% confluence, and only cells at passages 1 to 3 were used in our experiments.

Cell Viability.
Primary murine chondrocytes (3000 cells per well) were seeded in 96-well plates. After the indicated treatments, cell viability was examined by the Cell Counting Kit-8 (CCK-8, Dojindo Co., Kumamoto, Japan) assay, and CCK-8 OD values at 450 nm were recorded.

Western
Blotting. The detailed western blotting procedures were described elsewhere [23]. Antibodies were all used at 1 : 1,000 dilution unless otherwise indicated.

Quantitative Real-Time Polymerase Chain Reaction
(qPCR). After the indicated treatment, total cellular RNA was extracted with TRIzol reagent (Invitrogen, Shanghai, China), which was reverse transcribed using the ReverTra Ace qPCR RT kit (Toyobo, Tokyo, Japan) on an ABI Prism 7600H fast Real-Time PCR system (Applied Biosystems, Foster City, CA). The mRNA primers of the target genes were provided by Dr. Di [24]. Melt curve analysis was performed to calculate the product melting temperatures. The 2 −ΔΔCt method was used to quantify the targeted mRNAs, using GAPDH as the reference gene.
2.7. Nrf2 shRNA and AMPKα1 shRNA. Nrf2 shRNA and AMPKα1 shRNA lentiviral particles (Santa Cruz Biotech, Santa Cruz, CA) were individually added to cultured primary murine chondrocytes for 24 h, followed by puromycin selection for 12 days. In stable cells, over 95% knockdown of target proteins (Nrf2 and AMPKα1) was detected by western Trypan blue (%)      Single cells were further cultured in 96-well plates to generate monoclonal cells, followed by puromycin selection of stable cells. Nrf2 KO and AMPKα1 KO were verified by western blotting and qPCR analyses.

Mitochondrial Depolarization.
In the presence of mitochondrial depolarization, JC-1 fluorescent dye aggregates in mitochondria to form green monomers [25]. The method for the JC-1 assay was reported previously [26].
2.10. Coimmunoprecipitation (Co-IP). The methods for Co-IP were reported previously [27]. In brief, 800 μg of total cell lysates were precleared with protein A/G Sepharose ("Beads", Sigma-Aldrich). Then, the anti-Keap1 antibody was added to the lysates and incubated overnight, followed by the protein A/G Sepharose adding to the lysates. The Keap1-immunoprecipitated proteins were then captured by the beads and examined by western blotting.
2.11. Intracellular ROS Measurement. ROS levels were measured by the carboxy-H2DCFDA dye assay according to the previous report [28].
2.12. Lipid Peroxidation. As reported [28], the level of cellular lipid peroxidation was quantified by thiobarbituric acid reactive substance (TBAR) activities.
2.13. Superoxide Detection. A superoxide colorimetric assay kit (BioVision, San Francisco, CA) was used to measure cellular superoxide levels based on the kit protocol.
2.14. TUNEL Assay. Cells were initially seeded in six-well tissue culture plates (30,000 cells/cm 2 ). Following the treat-ments, the cells were stained with TUNEL (5 mM) for 30 min at room temperature in the dark. Cells with positive nuclear TUNEL staining were labelled apoptotic cells. The TUNEL ratio (TUNEL/DAPI × 100%) was calculated by counting 500 cells in five random fields of views (1 × 200 magnification) for each treatment.
2.15. Glutathione Levels. Primary murine chondrocytes were seeded in six-well tissue plates. Following H 2 O 2 stimulation, the levels of GSH and GSSG were tested, and the GSH/GSSG ratio was determined using a previously described protocol [29].
2.16. Caspase-3 Activity. Primary murine chondrocytes were seeded in six-well tissue plates. Following H 2 O 2 stimulation, 30 μg of total cell lysates were incubated with AFC-bound caspase-3 substrate (Invitrogen, Shanghai, China). The AFC absorbance, which indicates the relative caspase-3 activity, was measured by a Fluoroskan Ascent FL instrument at excitation and emission wavelengths of 355 nm and 525 nm, respectively.
2.17. Annexin V FACS. After being treated, the cells were incubated with Annexin V and propidium iodide (PI) (both 5 mg/mL). Then, FACS was performed on a FACSCalibur (BD Biosciences, Shanghai, China) to analyze the cells. The Annexin V ratio was recorded.
2.18. Single-Stranded DNA (ssDNA). The cellular ssDNA level was tested following the previously reported method [26].
2.19. ARE Promoter Activity. Chondrocytes were seeded in six-well plates and transfected with pGL4.37 and pGL4.74 plasmids in accordance with the manufacturer's protocol. After the indicated treatments, a dual-luciferase reporter assay system (Dual-Glo® Luciferase Assay System) was used to analyze ARE-driven promoter activity.   The NO concentration in culture medium was measured by Griess reagent as previously described [30]. The concentrations of PGE 2 , TNF-α, IL-6, collagen II, aggrecan, ADAMTS-5, and MMP13 in cell culture supernatants were measured by ELISA kits (R&D Systems, Minneapolis, MN) based on the manufacturer's instructions.
2.21. Animal Model. Male C57BL/6 mice (20-25 g; 8 weeks old) were obtained from Shanghai Animal Centre of the Chinese Academy of Sciences and housed under specific pathogen-free (SPF) conditions in our facility. All animal procedures were conducted in accordance with the National Institutes of Health guide for the care and use of Laboratory animals (NIH Publications No. 8023, revised 1978) and approved by the Institutional Animal Care and Use Committee of Soochow University. The mouse OA model was established by surgical destabilization of the medial meniscus (DMM) of the right knee as described previously [31]. Then, the mice were randomly assigned to three groups: sham group (n = 10), DMM group (OA group, n = 10), and DMM+luteolin group (n = 10).

Histopathological Analysis.
Freshly dissected mouse knee joints were fixed in 4% paraformaldehyde (PFA) for 24 h and decalcified in a 10% EDTA solution for 4 weeks. The knee samples were embedded in paraffin blocks and sliced (5 μm). After a series of processing steps, the joint sections were stained with hematoxylin and eosin (HE) and safranin O/fast green (Sigma-Aldrich, Oakville, Ontario, Canada) in accordance with the manufacturer's recommendations. The slides were assessed by two independent histology researchers in a blinded manner. The Osteoarthritis Research Society International (OARSI) scoring system was used to quantify the degeneration of cartilage as previously described [32].

Statistical Analysis.
Each experiment was repeated in triplicate. The data are presented as the mean ± standard deviation (SD). Statistical analysis was conducted through SPSS 20.0. Statistical analyses were performed by one-way analysis of variance (ANOVA), and the Tukey test was used for comparisons between groups. OARSI scores were analyzed by the Kruskal-Wallis H test. P < 0:05 was considered statistically significant.  (Figures 1(g) and 1(h)), indicating apoptosis activation. Conversely, these effects were dramatically ameliorated by luteolin treatment (Figures 1(d)-1(h)). Notably, treatment with luteolin alone failed to affect chondrocyte viability or cell death (Figures 1(d)-1(h)). These results suggested that luteolin treatment effectively protected chondrocytes from H 2 O 2 -elicited cell death and apoptosis.

Luteolin Inhibited H 2 O 2 -Stimulated Oxidative Injury and Programmed Necrosis in Primary Murine
Chondrocytes. The effect of luteolin on H 2 O 2 -stimulated ROS generation and oxidative injury in chondrocytes was evaluated. Our results showed that ROS production (DCF-DA intensity increase, Figures 2(a) and 2(b)) was potently increased in response to H 2 O 2 stimulation, and oxidative injury was significantly induced, which was supported by superoxide accumulation (Figure 2(c)), a reduction in the  (Figure 2(d)), and the presence of lipid peroxidation (TBAR intensity increase, Figure 2(e)). Notably, luteolin treatment significantly reversed these effects (Figures 2(a)-2(e)).
Additionally, programmed necrosis was also activated in H 2 O 2 -stimulated primary murine chondrocytes, as indicated by the association of mitochondrial CypD-p53-ANT-1 (Figure 2(f)), cytosolic cytochrome C (Cyto-C) release (Figure 2(g)), and mitochondrial depolarization (Figures 2(h) and 2(i)). Again, these effects were largely attenuated by luteolin treatment. Collectively, these data suggested that luteolin reversed H 2 O 2 -stimulated oxidative injury and programmed necrosis in murine chondrocytes.   (Figures 3(h)-3(k)). Furthermore, H 2 O 2 markedly mitigated the levels of collagen II and aggrecan, which contributed to chondrocyte survival, and significantly enhanced the expression of MMP-13 and ADAMTS-5, which were the major ECM degradation enzymes, and all these H 2 O 2 -induced alterations were reversed by luteolin treatment (Figures 3(l)-3(p)). Taken together, these data suggested that luteolin inhibited H 2 O 2 -induced inflammatory mediators and protects chondrocytes against ECM degradation.

Luteolin Activated AMPK and Nrf2 Signaling in Primary
Murine Chondrocytes. To further explore the protective mechanism of luteolin treatment on H 2 O 2 -induced oxidative injury in chondrocytes, the AMPK and Nrf2 pathways were analyzed by western blotting. As demonstrated in Figures 4(a) and 4(b), the coimmunoprecipitation assay results revealed that Keap1 immunoprecipitated with Nrf2 and that treatment with luteolin damaged the connection between Keap1 and Nrf2, resulting in the stabilization and accumulation of the Nrf2 protein in the cytoplasm, while Keap1 expression remained unchanged. In addition, elevated Nrf2 protein levels were detected in the nuclear fractions of luteolin-treated chondrocytes (Figure 4(c)), indicating the translocation of stabilized Nrf2 from the cytosol to the nucleus. Importantly, luteolin robustly strengthened ARE luciferase activity in primary murine chondrocytes (Figure 4(f)), leading to enhanced transcription of Nrf2dependent genes (HO-1, NQO1, and GCLC; Figures 4(d) and 4(e)). Notably, the Nrf2 protein level was robustly increased in luteolin-treated chondrocytes, whereas the Nrf2 mRNA level remained unchanged (Figure 4(e)). The results indicated that luteolin treatment resulted in the stabilization, nuclear translocation, and activation of Nrf2 in primary murine chondrocytes.
Next, we examined AMPK signaling. As shown in Figure 4(g), luteolin upregulated the phosphorylation levels of AMPKα1 in primary murine chondrocytes, while the total levels of AMPKα1 remained unchanged. Additionally, AMPK activity was potently augmented by luteolin treatment (Figure 4(h)). Thus, luteolin activated AMPK signaling in primary murine chondrocytes. Collectively, these data showed that luteolin-induced protection against H 2 O 2 in chondrocytes might occur through the AMPK/Nrf2 pathway.

Nrf2 Signaling Activation Mediated Luteolin-Induced
Chondroprotection from H 2 O 2 . Next, the relationship between the Nrf2 cascade and luteolin-induced cellular protection in H 2 O 2 -stimulated chondrocytes was further examined. Chondrocytes were transfected with lentiviral Nrf2 shRNA, and stable cells were selected by puromycin. Besides, a lentiCRISPR/Cas9-Nrf2-GFP-knockout (KO)  11 Oxidative Medicine and Cellular Longevity construct was used to knock out Nrf2 in primary murine chondrocytes, and stable cells were established. Our findings demonstrated that Nrf2 protein expression was dramatically suppressed in Nrf2 shRNA and Nrf2-KO chondrocytes treated with luteolin ( Figure 5(a)). Furthermore, luteolinstimulated Nrf2 protein stabilization, as well as enhancement of HO-1, NQO1, and GCLC, was abolished in Nrf2 shRNA and KO cells (Figures 5(a) and 5(b)). More importantly, H 2 O 2 -induced cell viability decrease ( Figure 5(c)), cell death ( Figure 5(d)), and mitochondrial depolarization (Figures 5(e) and 5(f)) were intensified in Nrf2-silenced cells, and luteolin treatment did not eliminate these effects (Figures 5(c) and 5(d)). Therefore, Nrf2 silencing or depletion affects luteolin-induced effects on primary murine chondrocytes. Taken together, our data showed that Nrf2 cascade activation mediated the protective effect of luteolin against H 2 O 2 in chondrocytes.

AMPK Activation Mediated Luteolin-Induced
Chondroprotection against H 2 O 2 . To explore whether AMPK signaling activation mediates the luteolin-induced protection against H 2 O 2 , genetic methods were employed to silence AMPK activation. First, AMPKα1 shRNA lentivirus was transfected into primary murine chondrocytes, followed by the establishment of stable cells via puromycin selection (sh-AMPKα1). In addition, a lentiCRISPR/Cas9-AMPKα1-GFP-KO construct was used to knock out AMPKα1 in primary murine chondrocytes, and stable cells were established (ko-AMPKα1). In sh-AMPKα1 and ko-AMPKα1 cells, reduced AMPKα1 expression levels were confirmed by western blotting (Figure 6(a)). More importantly, cell viability reductions (Figure 6(b)), cell death (Figure 6(c)), and mitochondrial depolarization (JC-1 intensity, Figures 6(d) and 6(f)) induced by H 2 O 2 were not affected by luteolin treatment after AMPKα1 silencing or depletion. These data indicated that AMPK activation mediated luteolin-induced protection against H 2 O 2 in chondrocytes.

AMPK of Downstream Nrf2 Signaling Activation Was
Required for Luteolin-Induced Chondroprotection from H 2 O 2 . Based on the widely reported interaction between the Nrf2 and AMPK cascades, the association of luteolinmediated AMPK and Nrf2 activation was further investigated in primary murine chondrocytes. Genetic strategies (shRNA and CRISPR/Cas9-KO) were used to block AMPK and Nrf2. The results showed that luteolin-mediated Nrf2 activation was effectively blocked by AMPK shRNA or AMPK KO (Figures 7(a) and 7(b)). In contrast, Nrf2 silencing or depletion had no significant effect on luteolin-induced AMPK activation (Figure 7(c)). These data suggested that luteolin-induced AMPK activation acted as an upstream signal for Nrf2 signaling activation.

Luteolin Ameliorated OA Progression in the DMM Mouse Model.
To assess the protective effect of luteolin on OA progress in vivo, DMM was established in mice, followed by intragastric administration of 10 mg/kg/day luteolin in 0.5% carboxymethylcellulose (CMC) or vehicle alone (0.5% CMC) for 8 consecutive weeks until the mice were sacrificed. The protective effect of luteolin on cartilage morphological structure was examined. As presented by HE and safranin O staining (Figure 8(a)), the DMM group exhibited robust cartilage damage, chondrocyte decreases, and notable proteoglycan decreases compared to those in the sham control group. Moreover, these effects were mitigated in the luteolin-treated DMM group (DMM+luteolin), which revealed a smoother cartilage surface than that of the DMM group. OARSI scores were also determined for quantitative analysis (Figure 8(b)). The DMM group had significantly higher OARSI scores than the sham control group. Contrarily, the DMM+luteolin group showed markedly lower  13 Oxidative Medicine and Cellular Longevity OARSI scores than the DMM group. Furthermore, to investigate the effect of luteolin on ECM in vivo, immunohistochemical staining of Nrf2 and MMP13 in cartilage samples was carried out. The immunohistochemistry results (Figures 8(c)-8(e)) demonstrated higher expression of MMP13 in the DMM group than in the sham group, and Nrf2 expression showed no significant difference between the sham and DMM groups; however, the level of MMP13 was decreased and Nrf2 was upregulated in the DMM+luteolin group. Collectively, these results indicated that luteolin alleviated OA progression in vivo.

Discussion
In the current study, we investigated the potential protective effect of luteolin on H 2 O 2 -induced chondrocytes and DMMinduced OA in mice. The results showed that luteolin distinctly mitigated H 2 O 2 -induced cell death and apoptosis in primary murine chondrocytes and markedly inhibited OA progression in the mouse model. Besides, our study revealed that the AMPK/Nrf2 signaling pathway was involved in luteolin-induced cytoprotection.
Growing evidence reveals that inflammation and oxidative stress are the major pathological processes responsible for OA progression [4,5]. The excessive ROS could damage biomolecules or modify proteins and genes to activate signaling cascades [33]. As a result, some transcription factors and proinflammatory genes stimulated by ROS lead to the onset and development of inflammation [34]. Reflexively, an enhanced ROS generation due to the inflammatory response induces oxidative stress and tissue injury, such as ECM degradation. Thus, inflammation and oxidative stress are inextricably interrelated, creating a vicious cycle to pro-voke the occurrence and progression of a series of diseases, including OA.
Oxidative stress resulting from excessive ROS generation stimulates lipid peroxidation, protein damage, and DNA breaks, ultimately leading to chondrocyte apoptosis and articular cartilage degradation, ultimately contributing to OA pathogenesis [5]. In contrast, inhibiting ROS production and oxidative stress can effectively protect chondrocytes. In the current study, treatment with luteolin dramatically reversed the apoptosis-related effects of H 2 O 2 , as evidenced by ROS production, an increase in caspase-3 activity, and the accumulation of ssDNA. These results indicated that luteolin potently ameliorated H 2 O 2 -induced oxidative injury and apoptosis in chondrocytes.
Further, inflammatory factors have been shown to play a principal role in OA development. iNOS can synthesize NO, which not only stimulates MMP production but also inhibits collagen II and proteoglycan generation, ultimately leading to ECM degradation [35]. PGE2, another core inflammatory mediator, is produced by COX-2 and facilitates ECM degradation by increasing the levels of MMP-13 and ADAMTS5 [36]. Eventually, the accumulation of NO and PGE2 results in chondrocyte apoptosis and contributes to OA progression [37]. Collagen II and aggrecan, the main components of the ECM, are synthesized and secreted by chondrocytes and are usually degraded under inflammatory conditions by MMP-13 and ADAMTS5, respectively [38]. Thus, articular cartilage degeneration could be delayed by inhibiting the expression of inflammatory mediators. Our study showed that the H 2 O 2 -induced generation of PGE 2 , NO, TNF-α, IL-6, COX-2, and iNOS was reversed by luteolin in OA chondrocytes. Furthermore, luteolin suppressed the generation of MMP-13 and ADAMTS5, as well as the degradation of collagen II and aggrecan. These data suggested that luteolin could effectively inhibit the production of inflammatory cytokines and protect chondrocytes from H 2 O 2 .
Then, the underlying mechanism of luteolin-induced cytoprotection was investigated. It has been generally recognized that both the AMPK and Nrf2 signaling pathways play vital roles in regulating cellular energy and metabolism homeostasis by inhibiting inflammation and ROS generation, thus protecting cells under stress conditions. Therefore, the potential connection between luteolin-mediated protective effects and AMPK or Nrf2 signaling was examined.
AMPK is a phylogenetically conserved fuel-sensing enzyme and plays a vital role in regulating cellular energy homeostasis [14,15]. It exists as a heterotrimer composed of a catalytic α subunit and regulatory β and γ subunits. The kinase is activated by stresses that inhibit ATP generation or promote its consumption, such as glucose deprivation, hypoxia, and ischemia. Recent studies have demonstrated that

16
Oxidative Medicine and Cellular Longevity AMPK serves as a therapeutic target for various metabolic diseases. For instance, AMPK is reported to be involved in the regulation of nonalcoholic fatty liver disease [39], ischemic stroke-induced brain injury [40,41], acute lung injury [42], and OA progression [43]. In addition, it is well-established that Keap1/Nrf2 system forms the cellular defense against oxidative stress and inflammation. The modification of Keap1 leads to the separation of Nrf2 from Keap1 and Nrf2 accumulation, nuclear translocation, and binding to AREs, followed by the activation of several cytoprotective genes [44,45]. Several [47]. In this study, the results indicated that luteolin led to the stabilization, nuclear translocation, and boost of Nrf2 in primary murine chondrocytes, followed by upregulated expression of Nrf2-ARE-dependent detoxifying enzymes and antioxidant genes, including HO-1, NQO1, and GCLC. Furthermore, luteolin-induced activation of Nrf2 signaling was dose-dependent. These results indicated that luteolin could potently activate the Nrf2 signaling pathway in chondrocytes. Besides, AMPK was activated by luteolin treatment in primary murine chondrocytes, as evidenced by increased AMPKα1 phosphorylation. Then, we confirmed that luteolin-induced activation of both the AMPK and Nrf2 pathways protected chondrocytes from H 2 O 2 . Conversely, silencing AMPK or Nrf2 with shRNA or KO reversed luteolin-mediated effects on cell death and apoptosis. Notably, luteolin-induced protection against H 2 O 2 was almost completely abolished by Nrf2 or AMPK silencing via shRNA or KO in chondrocytes. Taken together, the findings demonstrated that both AMPK and Nrf2 cascade activation mediated luteolin-induced protection against H 2 O 2 in chondrocytes.
Interestingly, emerging evidence has revealed that AMPK could serve as a key upstream target of the Nrf2 cascade. For example, a recent study showed that AMPK activation improved inflammation and redox imbalance by mediating Nrf2 signaling [16]. Another study revealed that the neuroprotective effect of Nrf2 activation was dependent on the AMPK pathway [17]. Therefore, the interplay between the AMPK and Nrf2 signaling networks in chondrocytes was further clarified. In this study, AMPK silencing or depletion with shRNA or KO completely abolished luteolin-mediated Nrf2 activation. Intriguingly, luteolin-induced AMPK activation, as indicated by increased AMPKα1 phosphorylation, was not affected by Nrf2 silencing or depletion. These results indicated that luteolin showed the potential to protect chondrocytes against oxidative injury caused by H 2 O 2 by activating the AMPK pathway, subsequently resulting in an increase in the downstream Nrf2 cascade.
Finally, we further explored the effects of luteolin in a DMM-induced mouse model, which is widely used for in vivo analysis of OA [31]. Histological analysis by HE and safranin O staining, as well as quantitative analysis by OARSI scores, indicated that treatment with luteolin inhibited OA progression, as by less cartilage erosion, chondrocyte loss, and smaller OARSI scores than in DMM mice. Notably, the in vivo results also confirmed that luteolin could improve ECM homeostasis, as indicated by decreased MMP13 and increased Nrf2 expression, which was consistent with our in vitro results.
There are several limitations that should be put forward. First, the effect of luteolin on OA was based on mouse models instead of human beings. And its effect on mouse kidney and liver was not explored. Besides, the ideal dose, timing, duration of management, and long-term effects by future studies would provide a better understanding of its therapeutic value.

Conclusion
We demonstrated that luteolin exerted cytoprotective effects against oxidative injury, the inflammatory response, and ECM degradation in primary murine chondrocytes via activating the AMPK/Nrf2 signaling pathway and that AMPK served as a positive upstream regulator of Nrf2. Furthermore, oral administration of luteolin attenuated OA progression in a DMM-induced mouse OA model ( Figure 9). Therefore, our findings suggested that luteolin might serve as a novel and effective treatment for OA and provided a new research direction for clinical OA therapies.

Data Availability
The data used to support the findings of this study are included within the article.

Conflicts of Interest
The authors declare that they have no conflict of interest. Reviews Figure 9: Schematic of the chondroprotective effect of luteolin via the AMPK/Nrf2 pathway. Luteolin protected chondrocytes against H 2 O 2induced oxidative stress and inflammation by increasing levels of phosphorylated AMPK and activating Nrf2, which translocates into the nucleus to increase the transcription and expression of Nrf2-target genes, such as HO-1, NQO1, and GCLC. 18 Oxidative Medicine and Cellular Longevity