Stigmasterol mitigates rheumatoid arthritis progression by decreasing Nrf2/NLRP3-mediated pyroptosis in chondrocyte

doi:10.21203/rs.3.rs-3968785/v1

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Stigmasterol mitigates rheumatoid arthritis progression by decreasing Nrf2/NLRP3-mediated pyroptosis in chondrocyte

https://doi.org/10.21203/rs.3.rs-3968785/v1

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Background

Rheumatoid arthritis (RA) is a long-lasting inflammatory joint disorder characterized by joint swelling, stiffness, pain, and loss of function in joints. Chondrocyte inflammation and apoptosis are closely associated with cartilage destruction. Stigmasterol (Stig) is a phytosterol with anti-inflammatory and anti-oxidant effects. In the study, we aimed to investigate whether Stig mitigates RA progression by decreasing chondrocyte injury.

Methods and Results

A mouse RA model was established by injecting intradermally type Ⅱ collagen into the tail roots of mice. Arthritic score and spleen index were measured in RA mice to assess the effects of Stig on RA progression. Lipopolysaccharide (Lps)-treated chondrocytes were applied as the cell model of RA. The roles of Stig in chondrocyte viability, proliferation, migration, inflammation, and injury were tested using cell counting kit-8 (CCK-8), EdU, transwell, quantitative real-time PCR (qRT-PCR), and western blot assays, respectively. Our results demonstrated that Stig exhibited no significant cytotoxicity against CHON-001 chondrocytes, but Stig effectively inhibited Lps-induced decreases in cell viability, proliferation, and migration. Stig also alleviated Lps-evoked pro-inflammatory responses and CHON-001 cell injury. Mechanistically, Stig inhibited Lps-induced nuclear factor erythroid 2-related factor 2 (Nrf2) activation and subsequent NOD-like receptor protein 3 (NLRP3) inflammasome activation, resulting in CHON-001 pyroptosis. Nigericin, a NLRP3 activator, reversed the effects of Stig on mitigating CHON-001 cell injury. Furthermore, the clinical severity in RA mice was improved after Stig treatment.

Conclusions

In summary, Stig decreases Lps-induced chondrocyte injury and mitigates RA progression by inhibiting Nrf2/NLRP3-mediated pyroptosis, thereby providing the opportunity to treat RA.

rheumatoid arthritis

chondrocyte

stigmasterol

Nrf2

NLRP3

pyroptosis

Globally, the prevalence of rheumatoid arthritis (RA) is about 0.5–1%, affecting 30–60 million people worldwide (1). The commonly used drugs for RA include non-steroidal anti-inflammatory drugs (NSAIDs: hydroxychloroquine, sulfasalazine, methotrexate, leflunomide, etc.), glucocorticoids (prednisone, hydrocortisone, dexamethasone, etc.), and immunosuppressants (anti-CD20 antibodies, IL-6 receptor inhibitors, tumor necrosis factor inhibitors, etc.) (2–5). Despite tremendous progress in RA therapy over the past few decades, a significant number of patients suffer severe side effects or are not sensitive to these drugs.

Chondrocytes are the primary cells that synthesize and sustain the extracellular matrix (ECM) of cartilage (6). Chondrocyte injury caused by pro-inflammatory mediators is closely associated with the occurrence and severity of such disease (7). Inhibition of chondrocyte dysfunction is an effective strategy to treat RA. Nesfatin-1, an energy-regulating peptide, mitigates arthritis symptoms by decreasing chondrocyte injury (8). Emerging studies have demonstrated that medicinal plant-derived natural products play an important role in maintaining chondrocyte function. Li et al. showed that triptolide increases chondrocyte proliferation and secretory function by inhibiting exosomal miR-221 secreted by fibroblast-like synovial (FLS) cells (9). Resveratrol mitigates lymphotoxin α-triggered chondrocyte apoptosis and increases chondrocyte viability and proliferation (10).

Stigmasterol (Stig), a phytosterol found in the fats or oils of many plants (soybeans, calabar beans, and rapeseeds) (11), acts as a significant effect on maintaining the structure and physiology of cell membranes (12, 13). Stig has been shown to exert anti-inflammatory (14), anti-nociceptive (15), and antioxidant effects (16, 17). For example, Stig attenuates inflammatory responses and cognitive deficits in Alzheimer's disease (AD) by inhibiting NLR family pyrin domain containing 3 (NLRP3) signaling (14). Stig mitigates cerebral ischemia/reperfusion injury by increasing nuclear factor erythroid 2-related factor 2 (Nrf2) expression and thus decreasing oxidative stress and inflammation (18). The role of Stig in arthritis has also been revealed. In a rat model of RA, Stig treatment decreases joint destruction and ameliorates histological changes by inhibiting proinflammatory responses (19). In vitro, Stig attenuates IL-1β-induced chondrocyte damage by suppressing ferroptosis (20). Based on the findings that Stig exhibits a potential effect on treating RA, it is necessary to further reveal the mechanism by which Stig alleviates chondrocyte injury.

Pyroptosis is initiated by the activation of inflammasomes and is accompanied by the formation of membrane pores and the release of pro-inflammatory cytokines such as IL-1β and IL-18 (21, 22). To date, the best studied inflammasome is the NLRP3 inflammasome, which is composed of NLRP3, pro-caspase-1, and apoptosis-associated speck-like protein containing a CARD (ASC) (23). Pyroptosis is strongly connected with the inflammatory response, which is one of the leading causes of chondrocyte death (24, 25). Therefore, blocking pyroptosis may be a novel approach to protecting chondrocytes. A group of natural products such as icariin (26), morroniside (27), and loganin (28) have been shown to suppress chondrocyte pyroptosis by inhibiting Nrf2 or NLRP3 signaling (29). Thus, it was inferred that Stig might inhibit chondrocyte injury by suppressing chondrocyte pyroptosis. Considering the active involvement of chondrocytes in RA pathogenesis (30), the present study aimed to explore the effect and mechanism of Stig on RA via Nrf2/NLRP3-mediated chondrocyte pyroptosis, hoping to uncover a potential drug candidate for RA treatments.

Cell culture and treatment

CHON-001 chondrocyte cells were obtained from ATCC (Manassas, VA, USA) and grown in DMEM (Thermo Fisher Scientific, MA, USA) containing 10% FBS (GIBCO, NY, USA) in a humidified 5% CO₂ incubator at 37°C. To measure the cytotoxic effect of Stig, CHON-001 cells were treated with different doses of Stig (0, 1, 5, 10, 20, 30 µg/mL) for 36 h. To investigate the roles of Nrf2 in Stig-regulating activation of the NLRP3 inflammasome, CHON-001 cells were treated with ML385, a specific Nrf2 inhibitor (5 µM), in the presence of Lps and Stig for 36 h. To activate pyroptosis, CHON-001 cells were treated with Nigericin (8 µM) in the presence of Lps and Stig for 36 h.

Cell counting kit-8 (CCK-8)

CHON-001 cells were seeded in 96-well plates (1 × 10⁵ cells per well) overnight. After treatment with the specified reagents, cells were treated with CCK-8 regents (10 µL, Solarbio, Beijing, China) and incubated at 37 ℃ for 1 h. The absorbance was measured with a ReadMax 1200 microplate reader (Shanpu, Shanghai, China).

EdU assay

CHON-001 cells were seeded in 96-well plates (1 × 10⁵ cells per well) overnight and treated with Lps (5 µg/mL) alone or combined with Stig (20 µg/mL) for 36 h. Then cells were fixed with 4% paraformaldehyde (Solarbio) for 15 min, permeabilized with 0.5% Triton-X-100 for 20 min, and incubated in a medium containing 10 µM EdU regents (Beyotime, Shanghai, China) for 2 h. After staining with DAPI, cells were washed with PBS and observed under a fluorescence microscope (XSPY-3201LED, CSOIF, Shanghai, China), and the data was analyzed using Image J (NIH, MD, USA).

Western blot

Total protein was extracted using the RIPA lysis buffer (KeyGEN, Nanjing, China), quantified by the BCA Protein Assay Kit (P0012, Beyotime), separated by 10% SDS-PAGE, and electro-transferred to a PVDF membrane (Thermo Fisher Scientific). After blocking by 5% nonfat milk, the membrane was incubated with primary antibodies against Bcl-2 (ab182858, 1:2000, Abcam), Bax (ab182733, 1:2000, Abcam), MMP13 (ab39012, 1:3000, Abcam), ADAMTS-5 (PA1-1751A, 1µg/ml, Thermo Fisher Scientific), Col II (ab307674, 1:1000, Abcam), NLRP3 (ab263899, 1:1000, Abcam), cleaved caspase 1 (PA5-77886, 1:1000, Thermo Fisher Scientific), pre-GSDMD (ab210070, 1:1000, Abcam), GSDMD-N (ab215203, 1:1000, Abcam), Nrf2 (ab62352, 1:800, Abcam), and β-actin (ab213262, 1µg/ml, Abcam) for 1 h, and the HRP-conjugated secondary antibody (ab6721, 1:2000, Abcam) at room temperature for 1 h. The blots were detected by the Chemidoc system (Bio-Rad, Munich, Germany), and the data were analyzed using Image J (NIH).

Transwell migration assay

Cell migration was assessed using transwell chambers (8 µm, 24-well insert, Corning, NY, USA). After treatment with 5 µg/mL of Lps alone or combined with 20 µg/mL of Stig for 36 h, CHON-001 cells were seeded on the upper surface of a chamber with culture medium containing no FBS. The chamber was then inserted into a bottom well with culture medium containing 10% FBS. After 36 h of migration, cells were fixed with 4% paraformaldehyde. The cells on the lower surface were dyed with 0.1% crystal violet (Beyotime) after wiping the cells on the upper surface. The images were captured using a microscope (Olympus) and analyzed using Image J (NIH).

Quantitative real-time PCR (qRT-PCR)

Total RNA was extracted from CHON-001 cells by the RNeasy Mini Kit (Qiagen, Hilden, Germany), quantified by the NanoDropTM spectrophotometer (Thermo Fisher Scientific), and converted into cDNA using the M-MLV reverse transcriptase (TaKaRa) from 1.5 µg of total RNA. qRT-PCR was conducted by the QuantStudio™ 5 Real-Time PCR System (Thermo Fisher Scientific) with SYBR green qPCR mix (Beyotime). After normalization to β-actin, the mRNA levels were calculated using the 2^−ΔΔCT method, as previously described (31). The primers used in the study were shown in Supplementary Table S1.

Enzyme-linked immunosorbent assay (ELISA)

The IL-6, IL-1β, TNF-α, and LDH levels were assessed with the human IL-6 ELISA kit (ab178013, Abcam), the IL-1β ELISA Kit (KAC1211, Invitrogen, CA, USA), the TNF-α ELISA kit (ab181421, Abcam), and the LDH ELISA kit (ab65393, Abcam) according to the manufacturer’s protocol.

Immunofluorescence (IF)

CHON-001 cells were treated with Lps (5 µg/mL) alone or combined with Stig (20 µg/mL) for 36 h. After fixing with 4% paraformaldehyde for 15 min, cells were permeabilized with 0.4% Triton X-100 and blocked by blocking buffer (5% BSA in PBS) for 1 h. For the collagen II assay, cells were incubated with anti-collagen II primary antibodies (ab34712, 1:100, Abcam) for 1 h and then goat anti-rabbit secondary antibodies (ab150077, 1:200, Abcam) for 1 h. For the Nrf2 assay, cells were incubated with anti-Nrf2 primary antibodies (ab62352, 1:100, Abcam) for 1 h and then goat anti-rabbit secondary antibodies (ab150079, 1:200, Abcam) at room temperature for 1 h. After staining with DAPI, cells were observed under a fluorescence microscope (CSOIF) and analyzed using Image J (NIH).

A mouse model of CIA

The work was approved by the Animal Ethics Committee of Shanghai University of Traditional Chinese Medicine (PZSHUTCM220822024) and performed in compliance with the ARRIVE guidelines. The male DBA/mice were obtained from Vital River (Beijing, China) and cultured in a facility with free access to food and water, 55%± 5% air humidity, 25°C ± 2°C temperature, and a 12 h/12 h light-dark cycle. The CIA model was established by injecting emulsified Type II collagen (C II) at the tail root of mice at a dose of 50 µl/animal. To emulsify the C II, C II powder (Chondrex, Inc., WA, USA) was first dissolved in 10 mM acetic acid to a concentration at 4 mg/ml. Then it was mixed with the equal volume of complete Freund’s adjuvant and emulsified using a homogenizer on ice. Immunization intensification was completed by injecting the same concentration of C II emulsified by incomplete Freund’s adjuvant after 3 weeks.

Arthritic index

To calculate the arthritis index, the lesions of each hind ankle joint were assessed on a 4-point scale, as previously described (19, 32): no significant lesion was scored 0, small toe joint swelling was scored 1, toe joint and plantar swelling was scored 2, paw below the ankle joint swelling was scored 3, and total joint swelling was scored 4. The assessment was conducted every 7 days after the immunization. The rate of swelling was measured with a vernier caliper and calculated by comparing the increase in paw thickness on day x to the original paw thickness after immunization.

Spleen index

On day 28 after immunization, animals were euthanized with 40 mg/kg of pentobarbital sodium salt (Sigma-Aldrich, MO, USA), and the spleens were collected and weighed. The spleen index was calculated in accordance with the ratio of the spleen weight to the body weight (mg/g).

Statistics

Data were displayed as the mean ± SD from three independent replicates. The differences were compared with the student’s t test or one-way ANOVA followed by the Scheffé test. All tests were carried out with GraphPad Prism7.0 software (La Jolla, CA, USA). The difference was regarded as statistically significant when the p value was < 0.05.

The effects of Stig on chondrocyte viability and migration

To investigate the biological roles of Stig in chondrocyte viability, proliferation, and migration, its cytotoxicity was first assessed in CHON-001 cells using CCK-8 assay. Figure 1A revealed that Stig did not exhibit significant cytotoxicity against CHON-001 cells until reaching 20 µg/ml, but 30 µg/ml of Stig significantly decreased CHON-001 cell viability. Subsequently, 20 µg/ml of Stig were applied to treat CHON-001 cells. Functional studies showed that Lps treatment decreased CHON-001 cell viability, whereas these effects were reversed by Stig (Fig. 1B). The results from the EdU assay showed that Stig significantly restored CHON-001 cell proliferation, which was suppressed by Lps (Figs. 1C and D). There was an evident decrease in Bcl-2 protein expression and an increase in Bax protein expression in Lps-treated CHON-001 cells, while Stig treatment reversed the effect (Fig. 1E). Given the correlation of the migrated capacity of chondrocytes with cartilage damage repair (33), the role of Stig in regulating chondrocyte migration was next assessed. As shown in Figs. 1F and G, Lps decreased the migrated capacity of CHON-001 cells, while these effects were reversed by Stig.

Stig alleviated Lps-induced inflammation and chondrocyte injury

To explore the role of Stig in inhibiting Lps-induced inflammation in chondrocytes, CHON-001 cells were treated with Lps and Stig, and pro-inflammatory cytokines were measured using qRT-PCR and ELISA. Figure 2A showed that Stig significantly inhibited Lps-induced mRNA expression of IL-6, IL-1β, and TNF-α. Moreover, Lps promoted pro-inflammatory cytokine secretion in the supernatant of CHON-001 cells, while Stig reversed the effect (Fig. 2B-D). Subsequently, the expression levels of MMP13, ADAMTS-5, and Col II were assessed to evaluate the effect of Stig on regulating chondrocyte injury. Figure 2E showed that Lps increased MMP13 and ADAMTS-5 mRNA levels and decreased Col II mRNA levels, whereas these effects were blocked by Stig. Furthermore, Stig markedly inhibited Lps-induced protein expression of MMP13 and ADAMTS-5 in CHON-001 cells and up-regulated Col II protein expression (Figs. 2F and G). IF assay also displayed that Col II levels were reduced in Lps-treated CHON-001 cells compared with control, while Stig treatment restored Col II expression (Fig. 2H).

Stig inhibited Lps-induced activation of the NLRP3 inflammasome by activating Nrf2 signaling

Given the important roles of Nrf2 signaling and its downstream NLRP3 inflammasome in chondrocyte injury and RA (34–37), we next explored whether Stig mitigates CHON-001 cell injury by regulating the activation of Nrf2 and NLRP3 inflammasome. The nuclear translocation of Nrf2 were measured to evaluate its activation in CHON-001 cells after treatment with Lps and Stig. Figures 3A-C displayed that Lps resulted in a significant increase of the cytoplasmic Nrf2 protein levels, while concurrently decreasing its levels in the nucleus. However, these changes were reversed by Stig treatment, indicating that Stig activated Nrf2 signaling in Lps-exposed CHON-001 cells (Figs. 3A-C). Moreover, IF assay showed that Stig increased the total protein levels of Nrf2 as well as its nuclear translocation in Lps-exposed CHON-001 cells (Fig. 3D).

Subsequently, Nrf2-dependent activation of the NLRP3 inflammasome was explored. As shown in Figs. 3E and F, Lps treatment up-regulated NLRP3 and activated caspase-1 protein levels in CHON-001 cells, while these changes were reversed by Stig. Furthermore, Stig inhibited Lps-evoked IL-18 and IL-1β expression (Fig. 3G). These results suggested that Stig decreased NLRP3 inflammasome activation. Then NLRP3-dependent pyroptosis was measured in CHON-001 cells after treatment with alone or combined with Stig. As shown in Fig. 3H, Lps treatment led to GSDMD cleavage and increased GSDMD-N fragment levels, while these effects were reversed by Stig. As expected, Stig inhibited Lps-triggered LDH production (Fig. 3I). More importantly, the aforementioned effects of Stig were all disrupted by ML385 (Figs. 3E-I). These data demonstrate that Stig inhibits Lps-induced NLRP3 inflammasome activation by activating Nrf2 signaling.

Stig alleviated Lps-induced chondrocyte pyroptosis

To investigate whether Stig alleviates CHON-001 cell injury by inhibiting pyroptosis, CHON-001 cells were treated with a pyroptosis activator (Nigericin) in the presence of Lps and Stig, and cell injury was measured. Figure 4A showed that Nigericin treatment significantly inhibited CHON-001 cell viability and migration in the presence of Lps and Stig (Fig. 4A-C). Nigericin elevated pro-inflammatory cytokine production in CHON-001 cells in the presence of Lps and Stig (Fig. 4D). Nigericin also promoted CHON-001 cell injury, as suggested by an increase in MMP13 and ADAMTS-5 expression and a decrease in Col II expression (Fig. 4E).

Stig treatment ameliorated the clinical severity of RA mice

Finally, the in vivo effects of Stig on regulating RA progression were investigated using a murine RA model by assessing arthritic score and spleen index, as described previously (19, 38). Figure 5A-C showed that CIA mice exhibited a marked increase in arthritic index and foot swelling compared with control mice, while Stig treatment effectively reversed these changes. As an immune organ, the spleen is commonly affected in RA, as evidenced by spleen hyperplasia (39). Figure 5D showed that CIA mice exhibited a significant increase in spleen index compared with control mice, while these effects were reversed by Stig.

Chondrocyte is the only cell type in cartilage tissue, accounting for 1% of articular cartilage (40). They play a critical role in maintaining the integrity and function of joints by secreting ECM. Owing to genetic, epigenetic, and environmental causes, numerous pro-inflammatory mediators in the RA synovial joint lead to chondrocyte dysfunction (7). It is worth noting that chondrocytes are not just victims of joint inflammation, they also experience phenotype alterations to accelerate disease’s progression by increasing pro-inflammatory response and matrix destruction (30, 41, 42). These findings suggest that chondrocytes are a crucial factor in understanding the pathogenesis of RA and developing potential therapeutic strategies.

For thousands of years, plants have been applied as a source of medicine to treat different types of diseases due to their low toxicity and high efficiency (43, 44). A lot of natural products with anti-inflammatory capacities have been extracted from medicinal plants and used in inflammation-related diseases, including RA (45), systemic lupus erythematosus (46), and inflammatory bowel diseases (47). Stig, a phytosterol derived from multiple herbaceous plants (herbs, soybeans, tobacco, etc.), possesses various pharmacological capacities, including anti-inflammation, anti-diabetes, anti-oxidation, and lowering blood cholesterol (12). Especially recent results have revealed the roles of Stig in RA. In a rat model of RA, Stig treatment ameliorates clinical symptoms by inhibiting NF-κB signaling and decreasing the production of pro-inflammatory mediators (19). Stig could also attenuate chondrocyte injury by decreasing oxidative stress and subsequent ferroptosis (20). Mounting studies have demonstrated that oxidative stress-triggered NLRP3 inflammasome activation plays a vital role in chondrocyte dysfunction and RA progression (48–50). Given the antioxidant capacities of Stig and the crucial roles of Nrf2 in inhibiting oxidative stress, we investigated whether Stig mitigates RA progression by activating Nrf2 signaling and thus inhibiting NLRP3 inflammasome activation. The current results demonstrated that, i) Stig increased chondrocyte viability and migrated capacity, ii) Stig alleviated Lps-induced inflammation and chondrocyte injury, iii) Stig inhibited Lps-induced activation of NLRP3 inflammasome by activating Nrf2, iv) Stig alleviated Lps-induced chondrocyte pyroptosis, v) Stig treatment ameliorated the clinical severity of RA mice.

Pyroptosis is a newly identified programmed cell death characterized by the formation of membrane pores, cell swelling and rupture, and pro-inflammatory cytokines (IL-1β, IL-18, etc.) generation (21, 22). Pyroptosis is initiated by the activation of inflammasomes. The NLRP3 is the most widely studied inflammasome (51). NLRP3 inflammasome-dependent pyroptosis has been implicated in the pathogenesis of RA (52). Given the role of pyroptosis in RA pathogenesis, it has been suggested that pyroptosis could be a new target for RA treatment (53). Several natural products derived from medicinal plants have been reported to treat RA through the pyroptosis pathway. Ge et al. demonstrated that punicalagin improves the clinical severity of RA mice by decreasing NLRP3-dependent pyroptosis in macrophages (54). Combination treatment with mangiferin and cinnamic acid mitigates RA by suppressing NF-κB/NLRP3-dependent pyroptosis (55).

Previous studies have demonstrated that Stig treatment contributes to ameliorating the clinical severity by decreasing the production of inflammatory cytokines in RA rats (19) and that Stig inhibits the inflammatory response by repressing NLRP3 inflammasome activation in other diseases (14). We speculated whether Stig improves clinical symptoms in mouse model of RA by decreasing NLRP3 inflammasome-dependent pyroptosis. As expected, Stig mitigates RA progression by inhibiting Nrf2/NLRP3-mediated pyroptosis in chondrocytes. However, although pyroptosis is implicated in this process, the molecular mechanism by which Stig decreases pyroptosis in RA remains poorly understood. Further research is needed to understand how Stig alleviates chondrocyte dysfunction by regulating NLRP3-dependent pyroptosis or other types of cell death (apoptosis, ferroptosis, autophagy, etc.).

Stig decreases Lps-induced chondrocyte injury and mitigates RA progression by activating Nrf2 and thus inhibiting NLRP3-mediated pyroptosis, providing a potential therapeutic agent for RA.

Ethics approval and consent to participate

The work was approved by the Animal Ethics Committee of Shanghai University of Traditional Chinese Medicine (PZSHUTCM220822024)

Consent to Publish

The work described has not been published before.

Authors Contributions

Li Ding, Huijun Lin contributed to the study conception and design. Material preparation, data collection and analysis were performed by Li Ding, Lianbo Xiao, Huijun Lin, Yong He, Sheng Ding, Jiechao Zhang. The first draft of the manuscript was written by Lianbo Xiao. All authors read and approved the final manuscript.

Funding

Sponsored by Shanghai Sailing Program（21YF1438900）

Declaration of competing interest

All authors declare that they have no competing interests.

Data Availability

All data generated or analysed during this study are included in this published article (and its Supplementary Information files).

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No competing interests reported.

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Stigmasterol mitigates rheumatoid arthritis progression by decreasing Nrf2/NLRP3-mediated pyroptosis in chondrocyte

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Abstract

Background

Methods and Results

Conclusions

Figures

Introduction

Materials and methods

Cell culture and treatment

Cell counting kit-8 (CCK-8)

EdU assay

Western blot

Transwell migration assay

Quantitative real-time PCR (qRT-PCR)

Enzyme-linked immunosorbent assay (ELISA)

Immunofluorescence (IF)

A mouse model of CIA

Arthritic index

Spleen index

Statistics

Results

The effects of Stig on chondrocyte viability and migration

Stig alleviated Lps-induced inflammation and chondrocyte injury

Stig inhibited Lps-induced activation of the NLRP3 inflammasome by activating Nrf2 signaling

Stig alleviated Lps-induced chondrocyte pyroptosis

Stig treatment ameliorated the clinical severity of RA mice

Discussion

Conclusions

Declarations

References

Additional Declarations

Supplementary Files

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