Hydroxysafflor Yellow A Regulates Inflammation and Oxidative Stress by Suppressing the HIF-1α/JAK/STAT3 Signaling Pathway to Attenuate Osteoarthritis

Hydroxysafflor yellow A is extracted from Carthamus tinctorius L., Asteraceae, and has extensive pharmacological properties. In this study, interleukin-1 beta was used to establish the osteoarthritis model in vitro, and the impacts of hydroxysafflor yellow A on the cell model were analyzed. CCK8 was used to measure cell viability, and flow cytometry was used to evaluate apoptosis and reactive oxygen species. An enzyme-linked immunosorbent assay was performed to calculate the release of inflammatory cytokines and oxidative stress index. Western blotting was performed to measure the expression of collagen-related proteins. The protein levels in the HIF-1α/JAK/STAT3 signaling pathway were also measured. The results showed that hydroxysafflor yellow A promoted cell viability and inhibited apoptosis and oxidative stress. In addition, quinochalcone C-glycoside upregulated the expression of collagen II and Sry-related HMG box-9, while downregulating the expression of matrix metalloproteinase-13. Interleukin-1 beta induced high levels of interleukin-6 and tumor necrosis factor-α that were inhibited by hydroxysafflor yellow A. Meanwhile, hydroxysafflor yellow A inhibited the interleukin-1 beta–induced high levels of reactive oxygen species and malondialdehyde and enhanced the interleukin-1 beta–induced low levels of superoxide dismutase and glutathione peroxidase. Furthermore, hydroxysafflor yellow A downregulated the mRNA expression of HIF-1α, JAK, STAT3, and interleukin-6 as well as the protein expression of HIF-1α, p-JAK, and p-STAT3. The results suggest that hydroxysafflor yellow A inhibited the JAK/STAT3 signaling pathways by inhibiting HIF-1α. Therefore, hydroxysafflor yellow A regulates the inflammatory response and oxidative stress in vitro.


Introduction
Osteoarthritis is a common disease that often causes pain, stiffness, and disability among older adults (O'Neill et al. 2018), imposing a considerable economic burden on families and society. The main pathological features of osteoarthritis are articular cartilage degeneration and inflammation (Thomas et al. 2017). Osteoarthritis produces synovia, menisci, and ligaments in the bone. Briefly, osteoproliferation leads to joint space narrowing, subchondral bone sclerosis leads to meniscal loss of function, and inflammation leads to synovitis and ligament stiffness (O'Neill et al. 2018). As a complex disease, the risk factors for osteoarthritis include age, gender, body mass index, physical activity, genetics, and joint-level factors (Palazzo et al. 2016).
Multiple studies have demonstrated that oxidative stress is a risk factor for osteoarthritis (Marchev et al. 2017;Feng et al. 2019), which is closely associated with cartilage inflammation, fibrosis, and apoptosis (Xu et al. 2022). Moreover, inflammatory processes have been found in the tissues of the synovium, chondrocytes, and synovial fluid in osteoarthritis (Pelletier et al. 2001;O'Neill et al. 2018). Inflammation in osteoarthritis is chronic, low-grade, and mediated by the innate immune system (Robinson et al. 2016). Studies have suggested that the inflammatory response regulates chondrocyte catabolism and causes low-grade synovitis (Scanzello 2017). However, inflammatory cytokines can activate the Janus kinase (JAK)/signal transducer and activator of transcription (STAT) signaling pathway, thereby regulating oxidative stress, apoptosis, and inflammation during osteoarthritis (Malemud 2017;Zhou et al. 2022). Additionally, the JAK/ STAT signaling pathway is involved in cartilage remodeling (Huang et al. 2011). Thus, the JAK/STAT signaling pathway may be a therapeutic target for managing osteoarthritis.
Hydroxysafflor yellow A (1), which has a quinochalcone C-glycosidic structure, is one of the main components extracted from Carthamus tinctorius L., Asteraceae (Asgarpanah and Kazemivash 2013). It has various pharmacological properties including anti-inflammatory, antioxidative, antitumor, immunosuppressive, analgesic, and antidiabetic (Asgarpanah and Kazemivash 2013;Delshad et al. 2018). Yang et al. (2016) reported that hydroxysafflor yellow A modulates inflammatory cytokine release via the TLR4-Myd88 and MAPK-NF-κB signaling pathways. Compound 1 was also found to regulate cholesterol metabolism and prevent fatty liver disease in hyperlipidemic mice (Bao et al. 2015). However, whether hydroxysafflor yellow A regulates the JAK/STAT3 signaling pathway to alleviate osteoarthritis has not yet been studied. Given the high costs of joint replacement surgery, pharmacological treatments became increasingly important in the treatment of osteoarthritis. Paracetamol, NSAIDs, corticosteroid injections, and tramadol are recommended by the guidelines for the treatment of osteoarthritis. However, these drugs often have adverse effects (Kloppenburg and Berenbaum 2020). For example, NSAIDs can cause gastrointestinal discomfort and corticosteroid injections can cause osteoporosis (Wang et al. 2022). Currently, the inhibition of osteoarthritis-relevant signaling pathways is a new treatment strategy. A recent study reported that inhibiting the JAK/STAT3 signaling pathway can attenuate osteoarthritis (Sun and Xue 2022). Therefore, this study explored whether hydroxysafflor yellow A could act as an inhibitor of the HIF-1α/JAK/STAT3 signaling pathway and alleviate the symptoms of osteoarthritis by inhibiting inflammation and oxidative stress.

Cell Culture and Treatment
SW1353 cells (Chinese Academy of Sciences Cell Bank, Shanghai, China) were cultured in Dulbecco's modified Eagle medium (Gibco, USA) supplemented with 10% FBS (Gibco, Australia), 100 units/ml penicillin, and 100 μg/ml streptomycin and incubated at 37 °C with 5% CO 2 . To assess the effects of hydroxysafflower yellow A on cell viability, SW1353 cells were cultured in 96 wells overnight and then treated with increasing amounts of hydroxysafflor yellow A (0, 20, 40, 60, 80, 100, and 200 μM) for 24 h. Next, 10 μl of CCK-8 reagent was added to each well for further incubation of 2 h. IL-1β was used to induce the osteoarthritis cell model in vitro. After the cells were cultured in 96 wells overnight, they were treated with different doses of IL-1β (0, 2, 4, 6, 8, and 10 μM) to determine the optimum concentration. For the protective effect of hydroxysafflor yellow A, the SW1353 cells were treated with 6 μM IL-1β for 24 h, before the treatment with or without 40 μM of hydroxysafflor yellow A for 2 h. Then, they were treated with a CCK-8 reagent and incubated for 2 h. The absorbance was assessed by a microplate reader (BioTek Epoch, USA) at 450 nm. The test was repeated thrice.

Flow Cytometry
We measured the effects of hydroxysafflower yellow A (the test sample) on cell apoptosis using flow cytometry. The cells were divided into control, IL-1β, test sample (40 μmol/l), and IL-1β 1 3 + test sample groups. The cells were pretreated as part of the protective effect of the test sample and then digested with trypsin. After centrifugation and resuspension in the buffer, the cells were stained with the Annexin V-FITC/PI kit (Invitrogen, USA) for apoptosis detection and with the reactive oxygen species detection kit (Beyotime, China) for ROS detection. The data were collected using a flow cytometer (Beckman, USA).

Co-immunoprecipitation
Co-immunoprecipitation experiments on SW1353 cell lysates were performed using a Pierce co-immunoprecipitation kit (Thermo Scientific). Antibodies HIF-1α or STAT3 were used for column binding, and IgG antibody (A7016, Beyotime, China) as a control. After incubation with the antibody/ lysate mixture for 1 h at room temperature, the columns were washed for 40 min. The retained proteins were eluted using 40 μl of buffer (Thermo Fisher) at a pH value of 3. Finally, the protein complexes were detected by western blotting.

Western Blot Analysis
RIPA buffer containing 0.1% protease inhibitor was used to homogenize the cell samples, and the lysates from cells were centrifuged at 8496 × g for 15 min at 4 °C; then, the supernatants were collected for protein detection. The total protein concentrations were measured using a BCA kit (Thermo Scientific, Rockford, IL, USA). Equal amounts of protein (20 μg per lane for cell samples) were separated by sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE) and then transferred onto PVDF membranes. The membranes were blocked in TBS-T (20 mM Tris-HCl pH 7.6, 150 mM NaCl, and 0.1% Tween-20) at room temperature for 2 h with 5% skim milk or 5% BSA. After being washed in TBS-T three times, the immunoblots were incubated (3% BSA dilution) with the Col2, SOX9, MMP13, HIF-1α, JAK, p-JAK, STAT3, and p-STAT3 primary antibodies overnight at 4 °C. The membranes were incubated with secondary antibodies (1% BSA dilution) for 2 h at room temperature. The membranes were washed again with TBST and visualized using ECL. The band sizes were quantified using Scion Image 4.0 software (Scion Corporation, Frederick, MD, USA). The sample loading was normalized relative to β-actin as a reference standard. The results are shown as "fold changes" in comparison with the control group.

Real-Time Quantitative PCR
TRIzol reagent (Invitrogen, USA) was used to extract the total RNA from SW1353 cells. To quantify the mRNA expression of HIF-1α, JAK, IL-6, and STAT3, a SYBR Green assay (Vazyme, China) was performed on samples based on the manufacturer's protocol. β-Actin acted as a control and the 2 −ΔΔCt method was used to quantify the relative gene expression level. The primers used are listed in Table 1.

Statistical Analyses
GraphPad Prism 9.1.2 (GraphPad Software, Inc.) was used to analyze the data and generate the charts in this experiment. All data are presented as the mean ± standard deviation, and all tests were performed three times. Unpaired, two-tailed Student's t-test was used to statistically analyze the differences between the two groups. Differences among the three groups were analyzed by one-way analysis of variance followed by Dunnett's post hoc test. For all experiments, p < 0.05 was considered statistically significant.

Results and Discussion
Cell Viability SW1353 cells were treated with increasing concentrations of compound 1 to test its effects on cell viability. As shown in  Fig. 1A (F = 24.61), compound 1 had no cytotoxicity when the dose was below 40 μM, while it significantly inhibited cell viability when the dose was over 40 μM. IL-1β was used to induce osteoarthritis cell models in vitro (Fig. 1). The results showed that the inhibition ratio of SW1353 increased with increasing levels of IL-1β (F = 455.5). When the concentration of IL-1β was 6 μM, the inhibition ratio was approximately 50%. Therefore, we used 6 μM IL-1β to establish the osteoarthritis cell model.

Apoptosis
Chondrocyte degradation is a classical characteristic of osteoarthritis. To discover whether compound 1 can inhibit this progress, the 6 μM IL-1β induced cells were treated with 40 μM of this test sample. As shown in Fig. 1C, it is apparent that 6 μM IL-1β suppressed cell viability (F = 142.4). However, this effect was attenuated with compound 1 treatment. The results showed that 40 μM remarkably upregulated the cell viability of SW1353. Flow cytometry was performed to measure the effects of compound 1 on apoptosis (Fig. 2D, E) (F = 553.8). The results showed that compound 1 markedly downregulated the IL-1β induced apoptosis rate.

Levels of Collagen-Relevant Protein
To detect the effects of compound 1 on the levels of collagenrelated protein in the osteoarthritis cell model, the protein expression levels of Col2 (F = 17.39), SOX9 (F = 89.74), and MMP13 (F = 178.4) were analyzed ( Fig. 2A-D). Compared with the model group (IL-1β group), compound 1 increased the levels of Col2 and SOX9 and decreased the expression of MMP13, which was induced by IL-1β. However, Col2, SOX9, and MMP13 showed no changes when treated with 1 separately in SW1353 cells. These results indicate that compound 1 promoted cell viability and inhibited apoptosis.

Release of Inflammatory Cytokines
Cytokine release is the primary cause of osteoarthritis development. A vital therapy for controlling this disease is inhibition of the release of inflammatory cytokines. Thus, we measured the expression of IL-6 (F = 303.0), TNFα (F = 27.39), and CRP (F = 218.1) with the compound 1 treatment (Fig. 3A-C). Compound 1 successfully inhibited the expression of IL-6, TNFα, and CRP when compared with the IL-1β-induced cell model group.

Oxidative Stress
Abnormal oxidative stress levels have also been observed in osteoarthritis. The ROS (Fig. 3F, G) (F = 35.23), MDA (F = 58.96), SOD (F = 25.23), and GSH-Px (F = 48.56) levels were also measured (Fig. 3D, E). In comparison with the IL-1β group, the ROS, SOD, and GSH-Px levels were before treatment with or without 40μM HSYA for 2 h, and the con group with no treatment. Measured by CCK-8. D-E Analysis of cell apoptosis by flow cytometry assay. All experiments performed three times at last, and data are presented as means ± SD. **p < 0.01, ***p < 0.001 compared with the con group; ### p < 0.001 compared with the IL-1β group significantly elevated. The MDA levels were inhibited by the compound 1 treatment. These results demonstrate the effects of compound 1 on the regulation of oxidative stress.
As a chronic disease, osteoarthritis is one of the main causes of physical disability and reduces the quality of life among adults worldwide (Nelson 2018). The mechanism underlying the development of osteoarthritis is complex, and many factors contribute to its progression. Therefore, further understanding of its pathogenesis and identification of effective and low-sideeffect medicines are urgently needed. Hydroxysafflower yellow A (1), a natural product extracted from the Chinese C. tinctorius, has various pharmacological properties. In this study, we evaluated its pharmacological functions in osteoarthritis cell models to identify an alternative therapy.
To maintain the integrity and physiology of the joints, it is essential to maintain an intact structure of the articular cartilage and subchondral bone. However, chondrocyte death and matrix loss have been observed during osteoarthritis development (Hwang and Kim 2015). In addition, the decreased expression of collagen II and SOX9 and increased expression of MMP13 in osteoarthritis have been reported (Ouyang et al. 2019). Interestingly, several recent studies have shown that flavonoids, such as fisetin and scutellarin, modulate this process. The elevated expression of MMP13 and decreased levels of collagen II and SOX9 are inhibited by these plant polyphenols (Zheng et al. 2017;Wang et al. 2019). In this study, hydroxysafflower yellow A (1) had a similar function that inhibited the IL-1β-induced high expression of MMP13 and increased the expression of collagen II and SOX9. In addition, compound 1 significantly increased cell viability and decreased the apoptosis ratio of SW1353 cells. Therefore, these results indicate that compound 1 may have a protective function by deterring the degradation of cartilage cells; however, this extrapolation needs to be further studied in vivo.
In affected joints and other tissues damaged by osteoarthritis, inflammatory cytokines are released, promoting the inflammatory response in chondrocytes (Lieberthal et al. 2015). Several cytokines such as IL-1β, TNFα, IL-6, IL-15, IL-17, and many other cytokines are associated with the pathogenesis of osteoarthritis. Research showed that compound 1 reduced the levels of hs-CRP, IL-1β, and IL-6 in the serum of acute myocardial infarction (Zhou 2013). These results agree with the findings of Jin et al. (2013), who showed that the administration of compound 1 significantly inhibited the expression of TNFα, IL-1β, and IL-6 in mice with acute lung injury (Jin et al. 2013). Furthermore, the CRP levels are Fig. 4 Effects of hydroxysafflor yellow A on HIF-1α/JAK/STAT3 signaling pathway. SW1353 cells were treated with 6μM IL-1β for 48 h, before treatment with or without 40 μM HSYA for 2 h, and the con group with no treatment. qRT-PCR was performed to measure the mRNA expression levels of HIF-1α (A), IL-6 (B), JAK (C), and STAT3 (D). WB (E) was performed to measure the levels of HIF-1α (F), p-JAK/JAK(G), and p-STAT3/STAT3 (H). All experiments performed three times at last, and data are presented as means ± SD. **p < 0.01, ***p < 0.001 compared with the con group; # p < 0.05, ## p < 0.01. ### p < 0.001 compared with the IL-1β group significantly associated with the progression of osteoarthritis Kondo et al. 2021). In the present study, the inhibitory effects of compound 1 on the release of IL-6, TNFα, and CRP were demonstrated. Therefore, it could be suggested that hydroxysafflower yellow A (1) could alleviate inflammation during osteoarthritis progression.
Numerous studies have reported high levels of oxidative stress in osteoarthritis patients (Devi et al. 2021;Pal et al. 2023), and it has been regarded as a risk factor for initiating this disease (Wang et al. 2020;Peng et al. 2021). The overproduction of ROS is inexorable in osteoarthritis. Antioxidant enzymes such as SOD and GSH-PX usually scavenge ROS (Wu et al. 2012;Shen et al. 2019). Moreover, excess ROS can react with membrane lipids to generate MDA (Wang et al. 2014). Thus, high levels of ROS and MDA and low levels of SOD and GSH-PX mark high levels of oxidative stress. In this study, compound 1 decreased the oxidative stress induced by IL-1β. Importantly, the chemical structure of hydroxysafflower yellow A (1), an active redox agent, is associated with the keto-enol tautomerism of the quinochalcone moiety (Feng et al. 2013).
Many signaling pathways are involved in the development of osteoarthritis, including the NF-κB and PI3K/Akt/ mTOR signaling pathways Woo et al. 2017). Furthermore, previous studies have noted the importance of the JAK/STAT3 signaling pathway in chondrocyteintrinsic inflammatory responses (van Beuningen et al. 2014;Malemud 2017). During the development of osteoarthritis, high levels of IL-6 can stimulate the activation of the JAK/ STAT3 signaling pathway and thus promote cartilage degeneration (Legendre et al. 2003). Moreover, JAK inhibitors are regarded as therapies for osteoarthritis (Chiu et al. 2021).
The results of the present study showed that the levels of HIF-1α, p-JAK, and p-STAT3 decreased with the compound 1 treatment. Additionally, our results suggest that hydroxysafflower yellow A (1) modulates the JAK/STAT3 signaling pathway in osteoarthritis. Furthermore, our results indicate that HIF-1α may be a target of compound 1 during the treatment of osteoarthritis.

Conclusion
This study demonstrated that hydroxysafflower yellow A (1) promoted chondrocyte viability and inhibited chondrocyte apoptosis. This quinochalcone inhibited the release of inflammatory cytokines IL-6 and TNFα and inhibited oxidative stress via the HIF-1α/JAK/STAT3 signaling pathway. This indicates that hydroxysafflower yellow A (1) is a potential drug for the treatment of osteoarthritis. However, in vivo studies are required to determine the exact side effects and therapeutic dosage.

Authors Contribution
The conception and design of the study: SJ and MW; acquisition of data: SJ, PL, LT, and YT; analysis and interpretation of data: XL, BH, and YX; drafting the article or revising it critically for important intellectual content: SJ. All authors gave a final approval of the version to be submitted.
Funding This work was financially supported by the Natural Science Foundation of Sichuan Province (no. 2023NSFSC1803), Innovative topics of Affiliated Sport Hospital of CDSU (no. LCCX22B01), and Innovation team of Chengdu Sport University (No. CXTD201805).
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