Vaspin Alleviates Osteoarthritis by Inhibiting NLRP3-mediated Inammation

Background: Osteoarthritis (OA) is a chronic degenerative joint bone disease characterized by cartilage degradation. Visceral adipose tissue-derived serine protease inhibitor (vaspin) is associated with the inammatory and metabolic responses to OA. However, the underlying mechanisms of the pathological process of OA are not clear. The aim of the present study was to examine the protective effects of vaspin both in vitro and in vivo. Methods: Monosodium iodoacetate (MIA)-induced Wistar rat model of OA was used to assess the in vivo effects of vaspin administered for 12 weeks. The characteristics of OA were evaluated by haematoxylin and eosin (H&E) and safranin O/fast green staining. The anti-inammatory effect of vaspin was assessed using immunohistochemical, qRT-PCR, and western blotting analysis. Parallel experiments to detect the molecular mechanism through which vaspin prevents OA were performed using LPS-treated chondrocytes. Our results showed that the degeneration of cartilage and upregulated expression of matrix metalloproteinase (MMP)-1 and MMP-13 were ameliorated by vaspin. Additionally, vaspin suppressed the activation of TXNIP/NLRP3 and secretion of tumor necrosis factor (cid:0) and interleukin-1β in vivo. It was further conrmed that vaspin could also suppress LPS-induced NLRP3 inammasome activation and reduce collagen formation in chondrocytes. Moreover, vaspin inhibited NLRP3 inammasome activation by suppressing the ROS/TXNIP pathway. vivo novel

The past decade has seen a fundamental shift in our understanding of the mechanisms underlying OA. A previous study found that OA is a chronic in ammatory disease [3]. The chondrocytes and synovial cells in OA produce or overproduce various in ammatory mediators characteristic of in ammatory arthritis (such as IL 1β, TNF, and nitric oxide (NO)) [4][5][6]. The in ammasome is a crucial cytoplasmic multiprotein that relies on the maturation and secretion of caspase-1 to promote the release of IL-1β and IL-18, thereby causing in ammation [7,8]. The NLRP3 in ammasome, composed of NLRP3, an apoptosis-related speckle-like protein-containing acupaspase recruitment domain (ASC) and pro-caspase-1, is the most studied member of the NLR family [8]. In a recent study, the expression levels of the NLRP3 protein in the synovial membrane of patients with knee OA increased by 5.4 times compared with that in a control group [9]. This emphasizes the potential role of NLRP3 in OA as well as the possibility of its measurement as a biomarker for OA or its targeted inhibition.
The adipogen vaspin belongs to the family of serine protease inhibitors and is related to insulin resistance and metabolic syndrome [10]. It has been detected in cartilage, synovium, and osteophytes of OA patients undergoing joint surgery. Moreover, it was found that serum levels of vaspin in paired samples with high synovial uid levels [11]. Vaspin is a new type of adipokine with anti-in ammatory properties, and the link between vaspin and arthritis has been demonstrated. Previous study has revealed that vaspin shows anti-in ammatory and anticatabolic effects in chondrocytes by inhibiting the NF-κB signaling pathway [12]. However, the molecular mechanism of vaspin in OA and its relationship with the NLRP3 in ammasome is not yet fully understood. This study aimed to determine the effectiveness of vaspin in mitigating in ammation and the related mechanisms in vitro and in vivo.

Animals
Speci c-pathogen-free Wistar rats (male, 280-320 g, 3-months old) were purchased from Hunan SJA Laboratory Animal Co., Ltd (Changsha, China). The rats were kept under standard laboratory conditions (temperature 24°C, 12 h light-dark cycles). All experimental procedures were conducted according to the Guiding Principles in the Care and Use of Laboratory Animals published by the U.S. National Institutes of Health (NIH Publication No. 8023, revised 1978) and were approved by the Animal Ethics Committee of Qingdao University. After one week of acclimatisation, the OA model was prepared according to a previous study. A single intra-articular injection of 3 mg of monosodium iodoacetate (MIA; Sigma-Aldrich, MO, USA) was administered to the rats to induce OA. An equal volume of saline was injected into the control rats. The rats were divided randomly into three groups (n = 5 per group) as follows: (1) control group; (2) OA group: MIA injection; and (3) OA + vaspin group (vaspin 320 ng/kg in citrate buffer). Vaspin injection was given two weeks after MIA injection and administered for 45 days after MIA injection.
Histopathological and Immunohistochemistry (IHC) Cartilage blocks were immersed in 10% neutral buffered formalin at 4°C for three days, followed by decalci cation for 14 days in 30% formic acid solution and dehydration with ethanol in a conventional gradient. The sample was embedded in para n and cut into 5-µm sections.
The para n sections were dewaxed, hydrated with graded ethanol, stained with haematoxylin solution for 15 min, and counterstained with eosin solution for 5 min. After dehydration, transparency, and sealing with gradient alcohol, the pathological condition of articular cartilage was observed using the Image-Pro image analysis software.
For safranin O/fast green staining, the samples were stained with 0.5% Fast Green for 20 min and 0.5% Safranin O for 5 min, followed by gradient alcohol dehydration, transparent xylene, and sealed with neutral gum. The normal cartilage appeared red, and the background appeared green.
Immunohistochemical staining was performed according to the manufacturer's instructions (Solarbio, Beijing, China) and observed under a microscope (Olympus, Tokyo, Japan).

Isolation and culture of chondrocytes
We shaved the cartilage sections from the joint surfaces of the two knee joints of adult male Wistar rats. Cartilage samples were digested in .25% trypsin at 37°C for 1 h and then transferred to 0.3% collagenase II at 37°C for 6 h until the extracellular matrix was completely digested. The chondrocytes were then ltered through a mesh, and the resulting single-cell suspension was centrifuged at 1500 x g for 10 min. Next, we transferred the cells to a culture ask and incubated them with complete Dulbecco's Modi ed Eagle Medium (DMEM) in a 5% CO 2 incubator at 37°C. Chondrocytes were identi ed by collagen II immunohistochemical staining.

ELISA
According to the manufacturer's instructions, supernatants of the cell culture or rat sera were used to measure IL-1β and tumour necrosis factor (TNF)-(R&D Systems, Minneapolis, MN, USA).

Flow cytometric analysis
A peroxide-sensitive uorescent probe 20.7 0-dichloro uorescein diacetate (DCFH-DA) was used to detect the level of reactive oxygen species (ROS). After treatment, the chondrocytes were washed three times with PBS and incubated with DCFH-DA for 30 min in the dark. Fluorescence was detected with a ow cytometer (BD Biosciences, San Jose, CA, USA).

Western blot analysis
Western blotting was performed as previously described [13]. The primary antibodies used were as follows: NLRP3, ASC, TXNIP, and caspase-1 (1:1000, Abcam, USA), MMP-1, MMP-13, and collagen II (1:2000, Abcam, Cambridge, MA, USA). Antigen-antibody complexes were visualized with the ECL western detection kit (Thermo Fisher Scienti c, Waltham, MA, USA). The protein levels were normalized to the level of β-actin. ImageJ software was used to quantify the density of each band.

Statistical analysis
Data are expressed as the mean ± standard deviation (SD). Statistical signi cance between groups was analysed by Student's t-test between two groups or one-way analysis of variance (ANOVA) followed by Tukey's post-hoc test for multiple comparisons using GraphPad Prism version 6.0. Results with P < 0.05 was considered signi cant.

Results
Vaspin attenuates the histopathological features of OA in an MIA-induced rat model We constructed an MIA-induced rat OA model to investigate the protective effects of vaspin on osteoarthritis in vivo. H&E and safranin O/fast green staining showed normal morphology of joints in the control group, whereas the knee joint in the OA group showed severe cartilage erosion. Treatment with vaspin alleviated the degeneration and erosion of articular cartilage (Fig. 1A-B).
Expression of vaspin in OA rat model Serum vaspin expression was examined by ELISA. As shown in Fig. 2A, serum vaspin was decreased in the OA group compared to the control group. However, the difference was not notable (P > 0.05). The protein expression of vaspin was evaluated by IHC analysis. The results showed that the expression level of vaspin in the OA group was higher than that in the control group, even though the increase in expression level was not signi cant (Fig. 2B).

Vaspin alleviates rat OA by inhibiting NLRP3 activation
To further investigate the effect of vaspin on chondrocyte in ammation in the OA model, IHC staining was performed to detect the expression level of NLRP3. As shown in Fig. 3A, the rat OA model revealed a marked increase in NLRP3 expression, while the vaspin treatment showed a marked reduction in the number of positive cells in the rat cartilage tissue. The expression of NLRP3 in ammasome in joint tissues was analysed by qPCR and western blotting. Our results showed that vaspin downregulated the gene expression of NLRP3, ASC, TXNIP, and caspase-1 in OA rats (Fig. 3B), which was consistent with the results of the protein analysis (Fig. 3C). Western blotting also revealed that the protein levels of MMP-1 and MMP-13 were suppressed, while the protein level of collagen II was increased after vaspin treatment (Fig. 3D). In addition, vaspin inhibited the levels of mature IL-1β and TNF-α in OA rat serum (Fig. 3E-F).
Overall, these results indicate that vaspin exerts a protective effect on OA by regulating the activation of the NLRP3 in ammasome.

Vaspin suppresses LPS-mediated TXNIP/NLRP3 activation in chondrocytes
The expression of proteins involved in collagen formation and NLRP3 in ammasome activation, were analysed by western blotting. Western blot analysis demonstrated that vaspin inhibited the LPS-induced upregulation of MMP-1, MMP13, NLRP3, ASC, and cleaved-caspase-1 while preventing LPS-reduced collagen II expression (Fig. 4A-B). The RT-qPCR results also indicated that vaspin reversed the LPSinduced increase in the expression of NLRP3, ASC, and caspase-1 (Fig. 4C). Moreover, ELISA results showed that vaspin signi cantly reduced the protein levels of IL-1β and TNF-α in the supernatant of chondrocytes (Fig. 4D-E). Next, NLRP3 siRNA was used to further investigate the effect of vaspin on LPSinduced chondrocytes. Western blotting con rmed the successful transfection (Fig. 4F). As shown in Fig. 4G, NLRP3 knockdown signi cantly downregulated the expression of MMP-1 and MMP-13 but reversed the downregulation of collagen II in LPS-induced chondrocytes, which was comparable to the effect induced by vaspin. These ndings demonstrate that vaspin can reverse the LPS-activated NLRP3 in ammasome and its downstream protein expression.

Vaspin suppresses NLRP3 in ammasome activation of rat chondrocytes through inhibition of ROS/TXNIP signalling
To investigate the regulatory mechanism underlying these effects, the ROS release was examined.
chondrocytes were pre-treated with vaspin or NAC and then stimulated with LPS. The results showed that vaspin inhibited the LPS-induced generation of ROS, which was comparable to the effect induced by NAC (a known ROS scavenger) treatment (Fig. 5A). In addition, we found that NAC reduced TXNIP and NLRP3 in ammasome (NLRP3, ASC, and cleaved-caspase-1) expression. The above ndings indicate that vaspin partially inhibits the TXNIP/NLRP3 pathway by reducing ROS production (Fig. 5B).

Discussion
OA is no longer regarded a typical degenerative disease but a multifactorial disease in which chronic in ammation plays a central role [13]. Current treatments for OA can only relieve pain, and no drugs have been approved by the FDA to prevent or slow the progression of the disease. Therefore, we urgently need to develop new drugs that selectively target in amed joints to prevent damage to healthy tissues. Recent studies have found that vaspin improves the in ammation of chondrocytes by inhibiting the NF-κB pathway [12]. Our present research showed that vaspin reduces osteoarthritis in rats by inhibiting TXNIP/NLRP3 signalling and LPS-mediated activation of NLRP3 in ammasome in chondrocytes. Therefore, we discovered a new mechanism by which vaspin can prevent OA, which further proves the protective effect of vaspin against OA.
The chondrocytes in OA cannot maintain the balance between anabolic and catabolic activities in the tissue, which leads to disturbances in the synthesis and degradation of the extracellular matrix [14]. Various molecular components and mechanisms may turn joint trauma, chronic injury, or overuse into an in ammatory process [15,16]. The pathogenesis of OA involves several cytokines such as TNF, IL 1β, IL -15, and leukemia inhibitory factor. Among them, IL 1β and TNF are involved in inducing cartilage catabolism and inhibiting anabolic processes that are essential for cartilage homeostasis [17,18]. Due to the postulated role of IL-1β in the pathology of OA and the main role of in ammasomes in the maturation of IL-1β, the NLRP3 in ammasome has recently received extensive attention. Curcumin or estradiol inhibits the NLRP3 in ammasome and may downregulate in ammatory cytokines and prevent OA progression [19]. The ways to activate NLRP3 have been extensively explored, such as K + e ux and ROS production [20]. Thioredoxin-interacting protein (TXNIP) inhibits cell apoptosis and in ammation by inhibiting thioredoxin (TRX), thereby promoting cell growth and reducing in ammation caused by oxidative stress [21][22][23]. The results of a study by Seong et al. showed that the ROS-TXNIP-NLRP3 in ammasome axis is crucial in the pathogenesis of in ammation [24]. In the present study, activation of the NLRP3 in ammasome was found in both OA models and LPS-induced chondrocytes, and the activation mechanism may be related to ROS/TXNIP. Activated NLRP3 is responsible for the pathogenic effects of OA, driving cartilage degeneration, and synovitis through the production of IL-1β, IL-18, and matrix-degrading enzymes [25]. They can change the differentiation and function of chondrocytes and stimulate chondrocytes to release cartilage-degrading enzymes, such as metalloproteases (MMPs) and aggrecanases. These enzymes degrade type II collagen and aggrecan from the extracellular matrix, resulting in cartilage loss [26]. Therefore, regulating the NLRP3 in ammasome is crucial to maintaining adequate immune protection while preventing tissue damage caused by the overproduction of cytokines.
Many studies have proposed a link between vaspin and cardiovascular disease [27]. Moreover, vaspin expression in arthritis and its valuable role have been discovered in recent studies [28,29]. Bao et al. found that the serum vaspin level in OA was higher than that in healthy individuals, and all joint tissues of OA patients, including the cartilage, synovium, meniscus, fat pad, and osteophytes, expressed the vaspin gene [11]. Our study also found that the expression level of vaspin in the serum of OA rats was signi cantly lower than that in the control group. Although at a low level, vaspin was expressed in the joint tissues, and the expression level in the joint tissues was higher for the OA group than for the control group. Recent studies indicate that Vaspin inhibits the expression of MMP-2 and MMP-9 induced by leptin and that it inhibits the production of NO and TNF-α induced by leptin, indicating that vaspin has anti-in ammatory and anticatabolic effects on chondrocytes [12]. Li et al. found that vaspin prevents diabetic cardiomyopathy (DCM) by inhibiting NLRP3 in ammasome activation [27]. Based on these results, we hypothesized that vaspin would improve the in ammatory response of OA models by inhibiting the activation of the NLRP3 in ammasome. As expected, vaspin inhibited the activation of TXNIP/NLRP3 and the release of in ammatory factors such as IL-1β both in vitro and in vivo. In addition, the knockdown of NLRP3 mimicked the negative regulatory effect of vaspin on the inhibition of NLRP3 in ammasome activation and IL-1β and TNF-α secretion. In vitro experiments showed that vaspin and NAC (ROS inhibitors) signi cantly reduced ROS production and inhibited NLRP3 in ammasome activation.

Conclusion
In conclusion, the NLRP3 in ammasome promotes the pathogenesis of OA. Vaspin can inhibit the TXNIP/NLRP3 pathway, thereby reducing the damage to rat chondrocytes and the occurrence of OA. Vaspin has the potential to become a target drug for the treatment of OA. However, further research is needed to better understand its other effects. The present study was approved by the Animal Ethics Committee of Qingdao University.

Consent for publication
Not applicable.

Availability of data and materials
The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.