Platelet-rich plasma inhibits inflammation,apoptosis,and the NLRP3/Caspase-1 pathway and induces matrix metalloproteinases and proliferation of IL-1β-induced articular chondrocytes by downregulating T-box transcription factor 3

Abstract

Objectives

Osteoarthritis (OA) is a chronic joint disease characterized by osteoproliferation and the degeneration and destruction of articular cartilage. Platelet-rich plasma (PRP) is rich in various growth factors that have been reported to promote bone defect repair. This study examined the specific role and mechanism of PRP in OA.

Methods

OA model cells were created by treating articular chondrocytes with IL-1β. After treatment of the model cells with PRP or/and a T-box transcription factor 3 (TBX3)-overexpression plasmid, TBX3 expression was monitored via RT-qPCR, western blotting, and immunofluorescence assays. IL-1β, IL-33, and Caspase-3 levels were detected with ELISA kits. Levels of NLRP3, Caspase-1, MMP9, MMP13, and COL2A1 expression were evaluated by western blotting, and cell proliferation was assessed by the CCK-8 assay.

Results

Our results showed that TBX3 expression was upregulated in IL-1β-induced articular chondrocytes. IL-1β stimulation induced inflammation and the production of matrix metalloproteinases, activated Caspase-3 and the NLRP3/Caspase-1 pathway, inhibited the proliferation of articular chondrocytes; however, all those affects mediated by IL-1β could be markedly reversed by PRP. We also found that PRP alleviated IL-1β-induced inflammation, apoptosis, and extracellular matrix degradation in articular chondrocytes by inhibiting TBX3. Our findings suggest that PRP alleviates OA progression in vitro by downregulating TBX3.

Conclusion

PRP suppressed OA progression in vitro by inhibiting TBX3, which may be its mechanism of action in treating OA.

Keywords

Osteoarthritis platelet-rich plasma T-box transcription factor 3 NLRP3/Caspase-1 matrix metalloproteinases

Introduction

Osteoarthritis (OA) is a degenerative disease characterized primarily by pain, stiffness, joint dysfunction, and even disability in the affected joints.^1,2 OA is a primary cause of disability in the elderly, and affects ∼10% of men and 18% of women aged 60 worldwide.³ Due to the limited spontaneous healing and repair of articular cartilage, intervention is necessary for OA treatment.⁴ Clinically, the main treatments for early-stage OA are anti-inflammatory drugs, joint debridement surgery, and joint cavity drug injection^5,6; however, these therapeutic methods do not produce satisfactory clinical results. Therefore, there remains an urgent need to explore the molecular mechanism of OA progression and develop an exact and effective treatment plan.

Platelet-rich plasma (PRP) is mainly derived from peripheral blood, which is a concentrated solution of platelets obtained by centrifugation.⁷ The platelet concentrations in PRP are three to fivefold higher than those in normal whole blood.⁸ PRP contains a mixture of growth factors (GFs), cytokines, and other proteins that play an active role in inducing cell proliferation, migration, differentiation, angiogenesis, and extracellular matrix (ECM) synthesis, and thus facilitate the repair of bone defects.^9–11 In recent years, PRP has been widely used in orthopedics, stomatology, plastic surgery, and other disciplines.^12–14 It has been assumed that OA results from an anabolic-catabolic (a/c) imbalance, and that PRP helps to alleviate OA by modulating critical pro-inflammatory mediators and catabolic enzymes.¹⁵ Thus, PRP has been shown to provide relief from pain and inflammation associated with OA.⁴ Although PRP is highly effective in treating OA, its specific mechanism of action remains unclear.

T-box genes significantly affect the embryonic development of multicellular organisms, as they code for proteins that bind to DNA and regulate embryonic tissue and morphological development.¹⁶ As a member of the T-box protein family, T-box transcription factor 3 (TBX3) has been reported to play roles in embryonic development, hepatocytes, and malignant tumors.¹⁶ Research has shown that TBX3 mainly acts to inhibit cell apoptosis and promote cell proliferation by inhibiting a p53 activator (p14^ARF) or CDK inhibitor (p21^WAF1/CIP1).¹⁷ A recent study suggested that TBX3 could serve as a diagnostic biomarker for rheumatoid arthritis.¹⁸ However, the role played by TBX3 in OA progression and the relationship between PRP and TBX3 have not been fully clarified.

In this study, we established cultures of OA model cells by treating articular chondrocytes with IL-1β, and then verified the influence of IL-1β on various biological functions of the OA model cells. We also explored the effects of PRP on the inflammation, apoptosis, and proliferation of the model cells, as well as its effects on extracellular matrix (ECM) degradation and NLRP3 inflammasomes present in the OA model cells. Furthermore, a rescue experiment was performed to investigate the regulatory effect of PRP on TBX3 during OA progression.

Materials and methods

Cell culture

Articular chondrocytes were acquired from ATCC (Manassas, VA, USA) and cultured in DMEM medium (Gibco, Paisley, UK; Cat. No. 21063-029) containing 10% fetal bovine serum (FBS, HyClone, Logan, UT, USA), 100 U/mL penicillin, and 100 mg/L streptomycin at 37^oC in a 5% CO₂ atmosphere.

Preparations of PRP

100 mL of blood was obtained from five healthy volunteers (3 males and 2 females) with a mean (±SD) age of 33.20 ± 6.20 years without medical history by a licensed phlebotomist. And then, 15 mL of ACD-A was added to the blood as an anticoagulant. 1 mL of the sample was used for platelet count. The rest of the blood was centrifuged with 1,600 rpm for 15 min for separation of erythrocytes and next with 2,800 rpm for 7 min in order to obtain a two-part plasma, with the upper part consisting of platelet-poor plasma and the lower part consisting of leukocyte-poor PRP (LP-PRP).¹⁹ The upper part was discarded to obtain 10 mL LP-PRP. After resuspending the platelets, a 1 ml of LP-PRP was used for a platelet count. The final concentration of PRP was 300,000 platelets/μL. PRP were applied to cell culture within 6 h.

Cell treatment

A TBX3-overexpression plasmid and vector (control) were purchased from Origene (Rockville, MD, USA). Articular chondrocytes were treated with IL-1β, and PRP. Briefly, articular chondrocytes were plated at a cell density of 2 × 10³ cells/well in 96-well plates and cultured in the presence of 5 or 10 ng/mL IL-1β, and with or without 10% PRP for 72 h before RT-qPCR et al. experiments or plasmid transfection were performed. Articular chondrocytes were also transfected with a control vector or TBX3-overexpression plasmid by using Lipofectamine 3000 (Invitrogen, Waltham, MA, USA) according to the manufacturer’s instructions.

RT-qPCR assay

Total mRNA was extracted from the collected articular chondrocytes in each group using TRIzol reagent (Thermo Fisher Scientific, Waltham, MA, USA), and subsequently reverse-transcribed into cDNA using a Bestar qPCR RT kit (DBI Bioscience, China). Gene expression was monitored using SYBR Green PCR Master Mix (Applied Biosystems, Waltham, MA, USA). GAPDH served as an internal reference. The relative levels of TBX3 expression were calculated using the 2^−△△CT method.²⁰

Western blot assay

The total proteins were extracted from articular chondrocytes in each group by using RIPA buffer that contained a protease inhibitor cocktail. The protein concentration in each extract was determined by the BCA method (Beyotime, China). Next, a 40 μg sample of total protein from each extract was separated by 10% SDS-PAGE, and the protein bands were transferred onto PVDF membranes (Roche; Cat. no. 3010040001), which were subsequently blocked with 5% non-fat milk for 2 h. The membranes were then incubated overnight at 4^oC in blocking solution containing the primary antibody, and then incubated with an HRP-conjugated secondary antibody (1:1000, Abcam, Cambridge, UK) for 1 h. Finally, an appropriate amount of ECL solution (Thermo Fisher Scientific) was dropped onto the membrane, and after 1 min, images of the immunostained proteins were captured with a gel imaging system. The primary antibodies used were as follows: TBX3 (1:1000, Abcam; ab249134, NLRP3 (1:1000, Abcam; ab214185), Caspase-1 (1:1000, Abcam), MMP9 (1:1000, Abcam; ab38898), MMP13 (1:1000, Abcam; ab219620), COL2A1 (1:1000, Abcam; ab188570), and GAPDH (1:1000, Abcam; ab125247).

Immunofluorescence assay

Articular chondrocytes in each group were inoculated into a 6-well plate containing glass cover slides and incubated overnight. Next, the cells in each well were fixed with 4% paraformaldehyde (Sigma, Cat. No. P-6148) for 15 min, washed with PBS, and then permeated with 0.5% Triton X-100 for 20 min. Normal goat serum was dropped onto the glass slides, which were then sealed at room temperature for 30 min. Each cover glass was first incubated with a sufficient amount of diluted primary antibody (anti-TBX3) overnight at 4^oC in a humidifying box. Next, each cover glass was incubated with a diluted fluorescent secondary antibody (Abcam) at 37^oC for 1 h in a humidifying box. After washing, the articular chondrocytes were treated with DAPI (Thermo Fisher Scientific, Cat. No. 62249) for 5 min, and then treated with an anti-fluorescence quenching agent. Images of the immunostained cells were observed and photographed under a fluorescence microscope (Olympus, CKX41).

ELISA detection

The levels of IL-1β, IL-33, and Caspase three were examined using the appropriate ELISA kits (eBioscience, San Diego, CA, USA), and read the absorbance at 450 nm with a microtitre plate reader.

CCK-8 assay

Cell Counting Kit-8 (CCK-8; Dojindo, Kumamoto, Japan) was used to evaluate cell proliferation. The articular chondrocytes in each group were inoculated into a 96-well plate at a concentration of 5.0 × 10³ cells/well. Next, 10 μL of CCK-8 solution was added to each well at the following time points: 0, 24, 48, and 72 h. After the cells had been incubated with CCK-8 solution at 37^oC for 3 h, the absorbance of each well was detected with a microplate analyzer.

Statistical analysis

All experiments were repeated three times, and results for quantitative data are presented as a mean value ± standard deviation (SD). All data analyses were performed using IBM SPSS Statistics for Windows, Version 21.0 (IBM Corp., Armonk, NY, USA). Kolmogorov–Smirnov test was used to examine normal distribution of the quantitative data. One-Way ANOVA was used for comparisons of quantitative data. A p < .05 was considered to be statistically significant.

Results

TBX3 was highly expressed in IL-1β-induced articular chondrocytes

To examine changes that occur in TBX3 expression in OA, model OA cells were established by using articular chondrocytes that had been stimulated with 5 or 10 ng/mL IL-1β. RT-qPCR results revealed that relative to the blank group, the levels of TBX3 mRNA expression were increased in the IL-1β treatment groups, and especially in the 10 ng/mL IL-1β treatment group (Figure 1(a)). Similarly, western blotting results (Figure 1(b)) and IF assay results (Figure 1(c)) also showed that IL-1β induction markedly upregulated TBX3 protein expression in articular chondrocytes. These findings suggested that an upregulation of TBX3 is related to OA progression.

Figure 1.

TBX3 levels were upregulated in IL-1β-induced articular chondrocytes. (a) After stimulation with 5 or 10 ng/mL IL-1β, the levels of TBX3 in articular chondrocytes were detected by RT-qPCR analysis. (b) Changes in TBX3 protein expression in IL-1β-stimulated articular chondrocytes were verified by western blotting. (c) The influence of IL-1β on TBX3 expression in articular chondrocytes was also confirmed using an IF assay. Magnification, ×200; scale bar = 20 μm.

IL-1β induced inflammation, MMP production, NLRP3/Caspase-1 pathway activity, and Caspase-3 activity, but suppressed articular chondrocyte proliferation

Subsequently, we further verified the effect of IL-1β induction on the biological function of articular chondrocytes. ELISA data showed that IL-1β and IL-33 levels were increased in the IL-1β treatment groups relative to those in the blank group, and the increase was highest in the 10 ng/mL IL-1β treatment group, suggesting that IL-1β (especially 10 ng/mL) could induce the inflammation of articular chondrocytes (Figure 2(a)). We also found that the levels of NLRP3 and Caspase-1 expression were markedly increased in the IL-1β treatment groups relative to their levels in the blank group, and those increases were highest in the 10 ng/mL IL-1β group (Figure 2(b)). Next, western blotting data showed that when compared to the blank group, the levels of MMP9, MMP13, and COL2A1 expression were notably higher in the IL-1β treated groups, and the upregulation of those three proteins was most obvious in the 10 ng/mL IL-1β group (Figure 2(c)). Moreover, we found that IL-1β stimulation could dramatically decrease the proliferative activity of articular chondrocytes, and the decrease was stronger in the 10 ng/mL IL-1β treatment group (Figure 2(d)). Finally, our data showed that the levels of Caspase-3 in the IL-1β treatment groups were higher than those in the blank group, and Caspase-3 expression was highest in the 10 ng/mL IL-1β treatment group (Figure 2(e)). Thus, we verified that treatment with IL-1β, and especially 10 ng/mL IL-1β, could induce inflammation, NLRP3/Caspase-1 pathway activity, MMP production and Caspase-3 activity, and inhibit the proliferation of articular chondrocytes.

Figure 2.

IL-1β induced inflammation, NLRP3/Caspase-1 pathway activity, MMP production, and Caspase-3, activity and suppressed articular chondrocyte proliferation. (a) After induction with 5 or 10 ng/mL IL-1β, the levels of IL-1β and IL-33 in articular chondrocytes were detected using ELISA kits. (b) The effects of IL-1β induction on NLRP3 and Caspase-1 expression in articular chondrocytes were detected by western blotting. (c) After treatment with 5 or 10 ng/mL IL-1β, changes in MMP9, MMP13, and COL2A1 expression were detected by western blotting. (d) The proliferative activity of IL-1β-induced articular chondrocytes was confirmed using the CCK-8 assay. (e) Changes in Caspase-3 levels in articular chondrocytes after IL-1β treatment were detected by ELISA.

PRP repressed TBX3, inflammation, the NLRP3/Caspase-1 pathway, MMP production, and Caspase-3, but increased the proliferation of IL-1β-induced articular chondrocytes

Because the results of studies described above indicated that IL-1β could induce OA progression in OA model cells (IL-1β-induced articular chondrocytes), we further investigated whether PRP could alleviate the inductive effect of IL-1β on OA cell progression. We treated IL-1β-induced articular chondrocytes with PRP; after which, RT-qPCR results showed that IL-1β significantly increased TBX3 expression in the articular chondrocytes; however, that upregulation of TBX3 could be partially reversed by PRP treatment (Figure 3(a)). Similarly, western blot data also showed that PRP treatment markedly downregulated TBX3 expression in IL-1β-induced articular chondrocytes (Figure 3(b)). ELISA data revealed that IL-1β treatment significantly increased the levels of IL-1β and IL-33 in articular chondrocytes, and those increases could be notably attenuated by PRP treatment (Figure 3(c)). Subsequently, we found that the increases in NLRP3 and Caspase-1 expression that occurred in IL-1β-induced articular chondrocytes could also be dramatically attenuated by PRP treatment (Figure 3(d)). Our findings proved that IL-1β markedly increased the levels of MMP9, MMP13, and COL2A1 expression in articular chondrocytes, and those effects could be reversed by PRP treatment (Figure 3(e)). Moreover, CCK-8 data showed that the proliferative activity of articular chondrocytes could be inhibited by IL-1β, and that inhibitory effect could be attenuated by PRP administration (Figure 3(f)). Similarly, our data showed that PRP markedly reversed the increases in Caspase-3 levels in IL-1β-induced articular chondrocytes (Figure 3(g)). In short, our studies showed that PRP could inhibit OA progression in OA model cells, and that inhibitory effect might be associated with TBX3, the NLRP3/Caspase-1 pathway, and MMPs.

Figure 3.

PRP repressed TBX3, inflammation, the NLRP3/Caspase-1 pathway, MMP production, and Caspase-3, but increased the proliferation of IL-1β-induced articular chondrocytes. After PRP treatment, TBX3 expression in IL-1β-induced articular chondrocytes was detected by RT-qPCR (a) and western blotting (b). (c) After PRP treatment, ELISA assays were performed to examine the IL-1β and IL-33 levels in IL-1β-induced articular chondrocytes. (d) Western blot assays were performed to confirm the effects of PRP on NLRP3 and Caspase-1 expression in IL-1β-induced articular chondrocytes. (e) The effects of TBX3 on IL-1β-induced changes in MMP9, MMP13, and COL2A1 expression in articular chondrocytes were confirmed by western blotting. (f) The CCK-8 assay was used to determine the effect of TBX3 on the proliferation of IL-1β-induced articular chondrocytes. (g) After treatment with IL-1β or/and TBX3, ELISA assays were performed to detect changes in Caspase-3 levels in articular chondrocytes.

Overexpression of TBX3 reversed the inhibition of inflammation, NLRP3/Caspase-1 pathway activity, MMP production, and Caspase-3 activity, but promoted the proliferative activity induced by PRP in IL-1β-induced articular chondrocytes

Because PRP suppressed TBX3 expression in IL-1β-induced articular chondrocytes, we speculated that the inhibitory effect of PRP on OA cell progression might be attributable to TBX3. To test this hypothesis, we treated IL-1β-induced articular chondrocytes with PRP and the TBX3-overexpression plasmid. RT-qPCR data revealed that TBX3 expression in IL-1β-induced articular chondrocytes could be reduced by PRP, while that suppression could also be reversed by TBX3 overexpression (Figure 4(a)). Moreover, western blotting data also indicated that treatment with the TBX3 plasmid upregulated TBX3 expression in IL-1β-induced articular chondrocytes, and the effect could be inhibited by PRP (Figure 4(b)). Additionally, we proved that the downregulation of NLRP3 and Caspase-1 expression mediated by PRP in IL-1β-induced articular chondrocytes could be substantially reversed by TBX3 overexpression (Figure 4(c)). Meanwhile, we found that IL-33, IL-1β, and Caspase-3 levels were significantly increased in the PRP and TBX3 co-treatment group relative to those in the PRP and vector co-treatment group, indicating that overexpression of TBX3 could induce the inflammation and Caspase-3 expression mediated by PRP in IL-1β-induced articular chondrocytes (Figures 4(d)–(f)). CCK-8 assay results showed that PRP could increase the proliferation of IL-1β-induced articular chondrocytes, and that effect of PRP also could be markedly attenuated by TBX3 overexpression (Figure 4(g)). Finally, our data verified that PRP could downregulate MMP9, MMP13 levels, but upregulate COL2A1 level, while treatment with the TBX3 overexpression plasmid could dramatically upregulate and downregulate COL2A1 mediated by PRP in IL-1β-induced articular chondrocytes (Figure 4(h)). These results suggest that TBX3 participates in regulating the effects of PRP on the biological functions of OA cells.

Figure 4.

Overexpression of TBX3 reversed the inhibition of inflammation, NLRP3/Caspase-1 pathway activity, MMP production and Caspase-3 expression, but promoted the proliferative activity induced by PRP in IL-1β-induced articular chondrocytes. After induction with IL-1β, articular chondrocytes were treated with PRP and then transfected with the TBX3-overexpression plasmid. (a) A RT-qPCR analysis of TBX3 expression. (b) A western blotting analysis of TBX3 expression. (c) After treatment with PRP and the TBX3-overexpression plasmid, IL-1β-induced articular chondrocytes were monitored for NLRP3 and Caspase-1 expression by western blotting. The concentrations of IL-33 (d), IL-1β (e), and Caspase-3 (f) were determined by ELISA. (g) The proliferative activity of each group was evaluated with the CCK-8 assay at 0, 24, 48, and 72 h. (h) Western blot analysis of MMP9, MMP13, and COL2A1 expression in each group.

Discussion

Chondrocytes are the key cells in articular cartilage.^21,22 Changes in articular cartilage mainly occur due to an imbalance of tissue remodeling caused by a change in chondrocyte behavior.²³ During the early stage of arthritis, chondrocytes secrete vast amounts of proteoglycans needed to repair damaged articular cartilage under the stimulation of multiple factors.²⁴ As OA develops, chondrocytes begin to secrete chondrocyte-degrading enzymes, such as matrix metalloproteinases (MMPs), which disrupt the anabolic balance.²⁵ MMP13 mainly degrades type II collagen (COL2A) in cartilage.²⁶ MMP-9 can enhance the degradation of extracellular matrix proteins, and thereby cause serious damage to normal tissues and finally induce the formation and development of OA.²⁵ Most studies of OA have focused on chondrocyte autophagy, apoptosis, cartilage matrix degradation, synovitis, and subchondral bone remodeling^27,28; however, the exact pathogenesis of OA remains unclear.

Cellular inflammatory cytokines are relevant to the pathological process of OA.²⁹ IL-1β is an inflammatory cytokine that plays an important role in articular cartilage degeneration.³⁰ IL-1β has been reported to be highly expressed in the synovium and cartilage of patients with OA, and can induce the production of proteases involved in cartilage degradation in joint tissues; it is also the main instigator of OA development.³¹ IL-1β can also cause articular cartilage matrix degradation by inducing MMPs and COL2A. Factors that can prevent the induction of these inflammatory mediators might serve as effective treatments for OA.³² Based on previous literature reports,^33,34 we induced articular chondrocytes with IL-1β to construct an OA cell model.

OA is a non-inflammatory joint disease characterized by destruction of articular cartilage, and an abnormal inflammatory response is a major cause of OA symptoms.³⁵ Inflammatory mediators such as IL-1β, IL-6, monocyte chemotactic protein-1 (MCP-1), and monokines induced by interferon-γ (MIG/CXCL9) are involved in the pathogenesis of OA.^36,37 IL-1β and IL-33 belong to the IL-1 family, and IL-33 can enhance inflammation by promoting the production of cytokines, such as TNF-α.³⁸ Inflammasomes are recently discovered molecular platforms that help to regulate inflammation, and play a crucial role in the pathogenesis of inflammation.³⁹ NLPR3, a vital member of the NLR family, plays a key role in inducing IL-1β secretion.⁴⁰ Activation of NLRP3 causes ASC and caspase-1 to assemble into a NLRP3 inflammasome, which subsequently activates IL-1β precursors to form mature IL-1β that can be secreted into the extracellular matrix and produce inflammatory effects.⁴⁰ Apoptosis also plays an important role in OA.²⁷ While apoptosis is a normal physiological process, excessive apoptosis is pathologic.⁴¹ The apoptosis of chondrocytes is controlled by apoptosis-related proteins, and Caspase-3 is an initiator of cell apoptosis. Caspase-3 becomes activated after receiving an apoptosis signal and subsequently causes programmed cell death.⁴² In the current study, we verified that IL-1β could induce inflammation, Caspase-3 expression, MMP production and NLRP3/Caspase-1 pathway activity, and prevent the proliferation of articular chondrocytes, suggesting the successful establishment of an OA cell model via IL-1β induction.

Currently, small molecule inhibitors of MMP subclasses or inhibitors of the interaction between IL-1 and its receptor are new therapeutic protocols for OA; However, there is no consensus on the scope of the best therapeutic strategy.⁴³

PRP is a platelet concentrate obtained by the centrifugation of autologous whole blood. PRP contains large amounts of growth factors and other bioactive proteins, including vascular endothelial growth factor (VEGF), platelet-derived growth factor (PDGF), and transforming growth factor–beta (TGF-beta).⁴⁴ Studies have proven that PRP can suppress chondrocyte metabolism and induce inflammation.¹⁴ Additionally, PRP administration was shown to reduce MMP levels in ankle OA rats,⁴⁵ and totally or partially inhibit MMP-9 and MMP-2 production. PRP has also been suggested as a safe and effective method for treating OA in dogs.⁴⁶ Moreover, platelet NLRP3 inflammasomes can promote platelet aggregation and pancreatic cancer cell growth.⁴⁷ Therefore, PRP has a certain relieving effect on OA. In our study, we proved that PRP could reduce inflammation, NLRP3/Caspase-1 pathway MMP production, and Caspase-3 activity, and induce the proliferation of IL-1β-induced articular chondrocytes, indicating the therapeutic effect of PRP on OA. We also found that PRP could markedly downregulate TBX3 expression in IL-1β-induced articular chondrocytes, suggesting that TBX3 might be regulated by PRP during OA progression.

TBX3, as a transcription factor in the T-box family, plays crucial roles in early embryonic development and mammogenesis.^48,49 Mutations in the TBX3 gene can result in ulnar mammary syndrome.⁵⁰ Furthermore, studies have verified that TBX3 play roles in congenital heart disease,⁵¹ hepatocellular carcinoma,^52,53 breast cancer,⁵⁴ embryonic skin development,⁵⁵ and rhabdomyosarcoma.⁵⁶ In our study, we found that TBX3 could dramatically reverse the inhibition of inflammation, MMPs and apoptosis, promotion of cell proliferation mediated by PRP in IL-1β-induced articular chondrocytes, indicating the involvement of TBX3 in PRP-treated OA cells. However, the upstream mechanism that regulates TBX3 expression in OA cells requires additional investigation. In the current study, we proved the function of RPR in OA cells; however, we cannot extrapolate our in vitro data to in vivo experiments with animal models of OA. No randomized controlled clinical trials were performed for the PRP. Further animal studies and clinical ramifications are required to elucidate the effects and roles of RPR in vivo and clinical studies. Moreover, it seems that a clear cause–effect relationship is given in the current study, while it is difficult to define the relationship or the concrete mechanism of action, due to the multitude of molecules present in PRP.

Conclusions

Current research suggests that PRP prevents OA progression by suppressing NLRP3 inflammasome activation, inflammation, MMP production and apoptosis, and enhancing cell proliferation. TBX3 expression might be downregulated by PRP during the OA process. TBX3 could possibly serve as a molecular target for treating OA, as evidenced by the effects of PRP in our OA model cells (Figure 5).

Figure 5.

The graphic abstract.

Footnotes

Declaration of conflicting interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

This research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors.

Ethical approval

Written informed consent was obtained from the volunteers. Ethical approval for this study was obtained from Institutional Ethics Committee of Taian city central Hospital (No. 20191013).

Informed consent

Written informed consent was obtained from the volunteers.

ORCID iD

Zong-Yu Tao

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