Aspirin reverses inflammatory suppression of chondrogenesis by stabilizing YAP

Abstract Bone marrow mesenchymal stem cells (BMMSCs) transplantation methods are promising candidates for osteoarthritis (OA) treatment. However, inflammatory factors (such as TNF‐α) that occur at cell transplantation sites are critical factors that impair the effectiveness of the treatment. Previous studies have shown that aspirin (AS) had a regulatory role in stem cell differentiation. However, little is known about the role of AS on the chondrogenesis of BMMSCs. The purpose of this study is to explore the protective role of AS against the negative effects of TNF‐α on BMMSC chondrogenesis. In this study, we investigated the effects of AS and TNF‐α on BMMSCs chondrogenesis by performing the Alcian Blue staining, safranin O‐fast green staining, haematoxylin and eosin staining, and immunohistochemical staining, as well as real‐time RT‐PCR and western blot assays. Our results demonstrated that TNF‐α inhibited chondrogenic differentiation of BMMSCs by disrupting the balance of cartilage metabolism and promoting oxidative stress in BMMSCs, while AS treatment attenuated these effects. Furthermore, a detailed molecular mechanistic analysis indicated that Yes‐associated protein (YAP) played a critical regulatory role in this process. In addition, AS treatment mitigated the progression of cartilage degeneration in a mouse destabilization of the medial meniscus (DMM) model. AS alleviated the inhibitory effect of TNF‐α on chondrogenesis of BMMSCs by stabilizing YAP, which may provide new therapeutic strategies for OA treatment.


| INTRODUCTION
Osteoarthritis (OA), a chronic disease characterized by degenerative changes in articular cartilage accompanied by subchondral bone sclerosis and synovitis, is the cause of both socioeconomic and personal health burdens. 1,2 With an aging global population and increasing obesity, the incidence of OA is increasing year by year. 3 At present, OA is generally treated with conservative drugs in the early stage of the disease, and surgical treatment such as joint replacement in the later stages. 4 However, these treatment strategies are limited by factors such as poor efficacy of drugs, 5 large surgical trauma, and high cost.
Therefore, the development of alternative therapeutic strategies is critical. In recent years, the use of bone marrow mesenchymal stem cells (BMMSCs) that differentiate into cartilage has shown multiple advantages over traditional therapies in the treatment of OA. However, although this treatment strategy has the potential for clinical application, [6][7][8] its use is limited by the low differentiation rate of BMMSCs due to the presence of inflammatory factors, such as Xudong Wang and Hongyi Liao contributed equally to this work. tumour necrosis factor-alpha (TNF-α). 9,10 Thus, the development of strategies to improve the differentiation of BMMSCs into chondrocytes in an inflammatory environment is an important issue that requires further attention.
Aspirin (AS), also known as acetylsalicylic acid, is a commonly used nonsteroidal anti-inflammatory drug, which plays an important role in many physiological processes in the human body, such as anti-inflammation and analgesia, and has been implicated in antitumour responses and prevention of thrombosis. [11][12][13][14] Previous studies have shown that AS can regulate the physiological functions of mesenchymal stem cells (MSCs) in multiple ways, including inhibition of stem cell proliferation, promotion of stem cell apoptosis, promotion of myocardial differentiation and osteogenesis, and inhibition of adipogenic differentiation. [15][16][17][18][19] However, whether AS can reverse the inhibitory effect of TNF-α on chondrogenic differentiation of BMMSCs remains unknown. Therefore, the purpose of this study was to determine whether AS can reverse the TNF-α-induced chondrogenic damage of BMMSCs, in order to provide an experimental basis for the application of AS in the prevention and treatment of OA in the future.

| Isolation and culture of human BMMSCs
The BMMSCs used in this study were isolated and extracted from healthy volunteers as previously described. [20][21][22] Isolated BMMSCs were cultured in low glycemic DMEM mediums containing 10% foetal bovine serum (FBS), 100 U/ml penicillin, and 100 mg/ml streptomycin. The culture medium was changed every 3 days.

| Chondrogenic differentiation of BMMSCs
The high-density hanging drop method was used to culture cartilage pellets as described previously. 20 Pellets were cultured in the Mesenchymal Stem Cell Chondrogenic Differentiation Medium (#7551, ScienCell, USA) with or without TNF-α (10 ng/ml) and AS (100 μM) for 7, 14, and 21 days. Cartilage pellets were collected for follow-up experiments.

| Plasmids and lentivirus construction
Plasmids were obtained from Addgene (Cambridge, MA, USA), and transfection was performed using Lipofectamine 3000 reagent, according to the manufacturer's instructions. The human Yes-associated protein (YAP) coding sequence was cloned into the lentiviral transfer plasmid pSin-puro to construct plasmid pSin-YAP-FLAG. A control vector plasmid was also constructed.
To silence YAP expression, the shRNA sequence was cloned into the lentiviral transfer plasmid pSin-puro to construct pSin-YAP-shRNA. Control scramble-shRNA was also constructed. Lentiviral infection was carried out as described in the previously published article. 23
Finally, samples were visualized and images captured using an Olympus BX63 microscope (Olympus, Japan).

| Statistical analysis
All quantitative data have been analysed using SPSS 23.0 software.
The differences between the 2 and >2 groups were determined by the Student's t-test and analysis of variance methods, respectively.

| AS alleviates TNF-α-induced inhibition of BMMSC cartilage pellet growth
Through YAP overexpression studies ( Figure 6C,D), we found that overexpression of YAP reversed the inhibitory effect of TNF-α on the chondrogenic synthesis markers (COL2A1, ACAN, SOX9),   Figure 7A). We found that treatment with both high and low concentrations of AS did not affect mouse body weight compared to the Sham and PBS groups ( Figure 7B).
Next, we performed H&E staining on heart, liver, spleen, and kidney samples collected 8 and 12 weeks after AS treatment and found that AS treatment was not toxic to these organs ( Figure S2).  differentiation. 25 In addition, AS was found to inhibit osteogenic differentiation and promote adipogenic differentiation of BMMSCs. 26,27 AS was also shown to promote cranial regeneration, as well as reduce bone loss in ovariectomized rats, and could therefore have clinical potential in the treatment of osteoporosis. [28][29][30] Finally, AS was found to improve the immunomodulatory properties of BMMSCs through the 15d-PGJ2/PPARγ/TGF-β1 signalling axis. 31 These studies indicate that AS has the potential to improve the efficacy of stem cell therapy.
AS also plays a critical role in OA prevention and treatment. Our study showed that AS delayed the progression of cartilage degeneration in a DMM mouse model by promoting cartilage synthesis and inhibiting cartilage catabolism. Our findings are consistent with data from a cohort study, which showed that the use of low-dose AS was associated with reduced medial tibial cartilage loss over 2 years in patients with knee OA, suggesting that low-dose AS may be used to slow the progression of knee OA. 16