NLRC5 attenuates inflammatory response in IL-1β-stimulated human osteoarthritis chondrocytes through the NF-κB signaling pathway

Cell culture of primary human OA chondrocytes and IL-1β treatment

Cartilage samples from 9 OA patients who underwent total knee arthroplasty were collected at Central Hospital Affiliated to Shen Yang Medical Collage (Shenyang, China). Informed consent was obtained from all the patients involved in this study, which was approved by the Ethics Committee of the Central Hospital Affiliated to Shen Yang Medical Collage. The cartilage samples cut with scissors and then digested with 0.25% trypsin-EDTA solution for 30 min, followed by digestion with 0.4% collagenase II (Sigma-Aldrich, St. Louis, MO, USA) for 24 h. Cells were cultured in DMEM/F12 growth medium containing 10% FBS (Hyclone) and 1% penicillin/streptomycin (Sigma). The OA chondrocytes were maintained at 37° C in a humidified atmosphere. Chondrocytes were stimulated by IL-1β (10 ng/ml; Peprotech Asia, Rocky Hill, NJ, USA) for 24 h to induce inflammation. PDTC (5 μM; Sigma) was used to block NF-κB activation.

Quantitative real-time PCR (qRT-PCR)

Total RNA samples were extracted from chondrocytes using Qiazol (Qiagen, Hilden, Germany). RNA was quantified and then applied for the generation of cDNA using a cDNA Synthesis Kit. The mRNA levels of target genes were measured using a SYBR Green qPCR master mix on a 7500 Real-Time PCR System. Results were calculated using the 2^-ΔΔCT method.

Cell transfection

Chondrocytes were inoculated into a six-well plate and incubated for 24 h before transfection. Then the cells were transfected with NLRC5 siRNA (si-NLRC5) or negative control siRNA (si-NC), which were obtained from GenePharma (Shanghai, China). The NLRC5 overexpressing plasmid was constructed by inserting the open reading frame of NLRC5 into the pcDNA3.1 expression vector. Chondrocytes were transfected with siRNAs or vectors using RNAiMAX Reagent or Lipo2000 transfection reagent (Invitrogen).

Western blot

Chondrocytes were lysed in RIPA Lysis Buffer containing PMSF and phosphatase inhibitor (Beyotime), and the lysates were obtained from centrifugation. Proteins were separated on 12% SDS-PAGE gels. Then the proteins on the gels were transferred to PVDF membranes (Thermo) and blocked with 5% skimmed milk powder in TBST buffer for 1 h. The membranes were sequentially incubated with primary antibodies (anti-NLRC5, iNOS, COX-2; were obtained from Abcam, Cambridge, MA, USA; anti-p-p65, p65, IĸBα, or β-actin were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA) at 4° C overnight and HRP-conjugated secondary antibodies (Santa Cruz) for 2 h. The targeted protein bands were developed using ECL reagent (Thermo).

Cell viability assay

Chondrocytes (1 × 10⁴ cells/well) were seeded into 96-well plates and subjected with indicated transfections and treatments. After that, MTT (5 mg/ml) was added to the cells to evaluate the cell viability. After incubation for 4 h, the products were dissolved by incubating with dimethyl sulfoxide (DMSO). Then the OD value at 490 nm was measured using a microplate reader.

Measurement of NO

Twenty-four hours post indicated treatments, the NO production in cell culture samples was measured by a nitrate/nitrite colorimetric assay kit.

ELISA

The secretion of prostaglandin E2 (PGE2), TNF-α, IL-6, MMP-3 and MMP-13 in the supernatants were measured using corresponding ELISA kits purchased from R&D Systems.

OA model in rats and animal treatment

Six-week-old male Sprague-Dawley rats (200 ± 20 g) were purchased from the Animal Center of Chinese Academy of Sciences (Shanghai, China). The OA model was established as described previously [17]. The rats were randomly divided into control group: rats received a sham-operation; model group: rats received an operation; treatment group (NLRC5 group): 1-week after operation, the rats were intraarticularly injected with 50 μl solution (10 ng/ml) every 7 days. After 6 weeks of indicated treatments, the knee joint tissues were collected and stored in 4% paraformaldehyde solution for further histological analysis. The animal experiments were approved by the Animal Care and Use Committee of Central Hospital Affiliated to Shen Yang Medical Collage.

Histological analysis

Knee joint tissues were embedded in paraffin and then cut into sections (5 μm) using a rotary microtome. Then, the sections were stained with hematoxylin-eosin (H&E) and Safranin O. The images were captured using a light microscope. Cartilage destruction was examined by Safranin O staining, finally, the score was evaluated using the Osteoarthritis Research Society International (OARSI) grading system.

Statistical analysis

The data were expressed as the mean ±SD. Two-group comparisons were evaluated by Student’s t test. Multiple-group comparisons were analyzed by one-way analysis of variance (ANOVA). P < 0.05 was considered to indicate a statistically significant difference.

NLRC5 expression was down-regulated in IL-1β-induced chondrocytes

IL-1β signaling plays constructive roles in the pathogenesis of OA [18, 19]. IL-1β is frequently used for inducing inflammation in chondrocytes to simulate OA in vitro [20–22]. Firstly, we investigated NLRC5 expression in IL-1β-induced chondrocytes. Results showed that the mRNA level of NLRC5 was markedly down-regulated in chondrocytes after induction with IL-1β (Figure 1A). Consistently, western blot analysis revealed that NLRC5 is lowly expressed in chondrocytes stimulated with IL-1β (Figure 1B).

Figure 1. NLRC5 expression was down-regulated by IL-1β induction in chondrocytes. Chondrocytes were stimulated by IL-1β (10 ng/ml) for 24 h to induce inflammation. The mRNA and protein levels of NLRC5 were measured using RT-PCR (A) and western blot analysis (B). *p < 0.05.

Overexpression of NLRC5 increased chondrocytes viability and inhibited inflammatory mediators’ production

To further explore the role of NLRC5, pcDNA3.1-NLRC5 was transfected into chondrocytes to overexpress NLRC5. As shown in Figure 2A, a dramatical increase of the protein level of NLRC5 was observed in the pcDNA3.1-NLRC5-transfected chondrocytes. As shown in Figure 2B, overexpression of NLRC5 significantly increased cell viability in OA chondrocytes.

Figure 2. Overexpression of NLRC5 attenuated IL-1β-induced inflammatory injury in human OA chondrocytes. The pcDNA3.1-NLRC5 or pcDNA3.1 vector was transfected into chondrocytes, followed by IL-1β (10 ng/ml) stimulation for 24 h. (A) The expression levels of NLRC5 in chondrocytes were measured using western blot after transfection. (B) Cell viability of chondrocytes was detected using MTT assay. (C–F) The mRNA and protein levels of iNOS and COX-2 were measured using RT-PCR and western blot analysis. (G, H) The production of NO and PGE2 in chondrocytes. *p < 0.05.

The iNOS and COX-2 are two important enzymes, which are responsible for the production of NO and PGE2 [23]. Results indicated that NLRC5-overexpressing cells exhibited markedly reduced expression levels of iNOS and COX-2, as compared to the chondrocytes exposed to IL-1β (Figure 2C–2F). Besides, NLRC5 repressed the IL-1β-induced levels of NO and PGE2 in the culture supernatants of chondrocytes (Figure 2G, 2H).

Knockdown of NLRC5 reduced cell viability and increased inflammatory mediators’ production

Additionally, si-NLRC5 was transfected into chondrocytes to knock down NLRC5. Transfection efficiency assay showed that NLRC5 protein expression was dramatically decreased after transfection with si-NLRC5 (Figure 3A). Knockdown of NLRC5 significantly enhanced the IL-1β-induced reduction in chondrocytes viability (Figure 3B). Transfection with si-NLRC5 induced iNOS and COX-2 expression in IL-1β-stimulated chondrocytes (Figure 3C–3F). Meanwhile, the production levels of NO and PGE2 were also elevated by NLRC5 knockdown in IL-1β-induced chondrocytes (Figure 3G, 3H).

Figure 3. Knockdown of NLRC5 promoted IL-1β-induced inflammatory injury in human OA chondrocytes. The si-NLRC5 or si-NC was transfected into chondrocytes, followed by IL-1β (10 ng/ml) stimulation for 24 h. (A) The expression levels of NLRC5 in chondrocytes were measured using western blot after transfection. (B) Cell viability of chondrocytes was detected using MTT assay. (C–F) The mRNA and protein levels of iNOS and COX-2 were measured using RT-PCR and western blot analysis. (G, H) The production of NO and PGE2 in chondrocytes. *p < 0.05.

Overexpression of NLRC5 suppressed the production of TNF-α, IL-6, and MMP-3/13

TNF-α and IL-6 are majorly increased in the process of OA [24, 25], thus, we examined the effects of NLRC5 on TNF-α and IL-6 production. Results in Figure 4A, 4B revealed that overexpression of NLRC5 suppressed the production of TNF-α and IL-6 in chondrocytes. It has been demonstrated that MMPs expression are upregulated with the increased proinflammatory cytokines levels. MMPs, especially MMP-3 and MMP-13, are implicated in the pathogenesis of OA [26]. Our results showed that overexpression of NLRC5 also inhibited the production of MMP-3 and MMP-13 (Figure 4C, 4D).

Figure 4. Overexpression of NLRC5 suppressed the production of inflammatory cytokines in IL-1β-stimulated human OA chondrocytes. After transfection with pcDNA3.1-NLRC5 or pcDNA3.1 vector and the following incubation with IL-1β, (A–D) the production of TNF-α, IL-6, MMP-3 and MMP-13 were detected using ELISA. *p < 0.05.

Knockdown of NLRC5 increased the production of TNF-α, IL-6, MMP-3/13

In contrast to the effects of NLRC5 overexpression, knockdown of NLRC5 significantly induced the production of TNF-α and IL-6 (Figure 5A, 5B). The levels of MMP-3 and MMP-13 in cell culture of IL-1β-stimulated chondrocytes were increased after transfection with si-NLRC5 (Figure 5C, 5D).

Figure 5. Knockdown of NLRC5 increased the production of inflammatory cytokines in IL-1β-stimulated human OA chondrocytes. After transfection with si-NLRC5/si-NC and the following incubation with IL-1β, (A–D) the production of TNF-α, IL-6, MMP-3 and MMP-13 were detected using ELISA. *p < 0.05.

NLRC5 inhibited IL-1β-induced NF-κB activation in chondrocytes

The levels of p-p65, p65, and IκBα were determined to explore the involvement of NF-κB signaling in inflammatory response. The results implied that the p-p65 expression was decreased, while IκBα expression was increased by NLRC5 overexpression (Figure 6A–6C), indicating that NLRC5 blocked the activation of NF-κB pathway.

Figure 6. NLRC5 inhibited IL-1β-induced NF-κB activation in chondrocytes. After transfection with pcDNA3.1-NLRC5 or pcDNA3.1 vector, the chondrocytes were stimulated by IL-1β (10 ng/ml) for 24 h. (A–C) The expression levels of p-p65, p65, IĸBα were determined using western blot analysis. *p < 0.05.

Inhibition of NF-κB partially reversed the si-NLRC5-mediated inflammatory injury

Subsequently, chondrocytes were treated with pyrrolidinedithiocarbamate (PDTC, an NF-κB pathway inhibitor) to prevent the NF-κB activation. The deceased cell viability caused by si-NLRC5 was mitigated by PDTC (Figure 7A). Additionally, the si-NLRC5-mediated increases in expression levels of iNOS and COX-2, and production of TNF-α, IL-6, MMP-3/13 were attenuated in PDTC-treated cells (Figure 7B–7G).

Figure 7. Inhibition of NF-κB partially reversed the si-NLRC5-mediated promotion of IL-1β-induced inflammatory injury in chondrocytes. Chondrocytes were treated with PDTC (5 ;μM) to prevent the activation of NF-κB signaling pathway. (A) Cell viability of chondrocytes was detected using MTT assay. (B, C) The mRNA levels of iNOS and COX-2 were measured using RT-PCR. (D–G) The production of TNF-α, IL-6, MMP-3/13 in chondrocytes. *p < 0.05.

NLRC5 treatment ameliorated cartilage degeneration in an OA rat model

The role of NLRC5 in OA was further examined in vivo using an OA rat model. Cartilage degeneration in the rats were evaluated by H&E and Safranin O staining. As compared with the sham group, rats in the OA model group presented obvious hypocellularity, extensive proteoglycan loss, cartilage erosion, and superficial cartilage destruction. Compared to the OA model group, rats with intra-articular injection of NLRC5 exhibited remarkable alleviation in cartilage destruction (Figure 8A, 8B).

Figure 8. NLRC5 treatment ameliorated cartilage degeneration in an OA model in rats. (A) HE and Safranin O staining in each group (100×). (B) OARSI scores of each group to assess cartilage degeneration. *p < 0.05 vs sham group, #p < 0.05 vs OA group.