Imperatorin alleviated NLRP3 inammasome cascade-induced synovial brosis and synovitis in rats with knee osteoarthritis

Background: To clarify the therapeutic potential of imperatorin (IMP) in knee osteoarthritis (KOA). Methods: Thirty 3-month-old SD male rats were randomly divided into Normal group, monosodium iodoacetate (MIA) group and MIA+IMP group. Their synovial tissues were subjected to histopathological analysis. Primary synovial broblasts obtained from additional normal rats were treated by lipopolysaccharide (LPS) and then IMP. The mRNA and protein expressions of factors related to synovitis and synovial brosis were detected by qRT-PCR and Western blotting, respectively. The levels of inammatory factors IL-1β and IL-18 were measured by ELISA. Results: IMP reduced HIF-1α, NLRP3 inammasome expression and IL-1β, IL-18 production in synovial broblasts induced by LPS. IMP also down-regulated synovial brosis markers. In vitro study revealed that MIA-induced synovitis and synovial brosis were relieved by IMP. Conclusion: IMP exerts anti-inammatory effects associated with synovitis and synovial brosis. It reduces the production of pro-inammatory mediators and cytokines and inhibits TGF-β1, TIMP-1 and VEGF expressions that promote synovial brosis. growth


Background
Knee osteoarthritis (KOA) is a common and disabling condition that represents a substantial and increasing health burden with notable implications for the individuals affected, health-care systems, and wider socioeconomic costs [1,2]. Synovial brosis is important pathological processes characterized by abnormal deposition of extracellular matrix (ECM), as well as cell migration and proliferation in the occurrence and development of knee osteoarthritis. Synovial brosis is very common in KOA, which are the main causes of pain and joint stiffness. Up-regulation of brogenic factors, such as TGF-β1 [3], are signs of the development of synovial brosis. TGF-β1 is the most widely known brosis factor, and plays a key role in many pro brotic processes, including promoting tissue matrix metalloproteinase (TIMP) expressions [4]. TIMPs are found to be elevated in some diseases related to brosis, for example liver brosis, as well as in the synovium of mice with OA and human end-stage OA patients. In addition, vascular endothelial growth factor (VEGF) is an effective stimulator of angiogenesis and may also promote synovial brosis through extravasation [5]. These three are con rmed to be brogenic factors [6].
KOA is a complex disease, the pathogenesis of it involving not only mechanical but in ammatory and metabolic factors, leading to destruction and failure of the knee joints [7]. Each common osteoarthritis risk factor can lead to different mechanisms of knee osteoarthritis, such as increased in ammatory components [8] and oxidative stress. In addition, the extended metabolic activity characteristics of knee synovium contribute to imbalance of oxygen homeostasis and enhance hypoxia in the microenvironment [9]. Hypoxia-induced factor (HIF)-1α is recognized as a major regulator of hypoxia signaling, which mediates the adaptive response of cells to hypoxia by activating the transcription of genes encoding proteins. HIF-1α expression can be triggered in an in ammatory microenvironment even under normoxic conditions [10]. Clinical studies have shown that HIF-1α levels in serum, synovial uid, and articular cartilage in patients with KOA are associated with progressive joint damage. It can be used as a biomarker for KOA progression and prognosis. The previous studies have already shown that HIF-1α is associated with upregulation of genes encoding pro-in ammatory cytokines and growth factors, thereby activating broblasts and mediating brosis. Besides, the NLRP3 in ammasome can be activated by HIF-1α [11]. The NLRP3 in ammasome has been implicated in the pathogenesis of a number of arthritic disorders, producing proin ammatory cytokines and degradative enzymes, such as interleukin-1 beta (IL-1β) which drive cartilage degeneration and synovial in ammation [12]. Inhibiting NLRP3 in ammasome can alleviate many types of brosis, especially synovial brosis [13,14]. Imperatorin (IMP) is a secondary metabolite of plants. It is one of furanocoumarin derivatives and is widely used in many traditional Chinese herbal medicines (e.g. Angelica dahurica) which are used for treatment of KOA. It has the effects of antitumor [15], antibacterial [16], anti-in ammatory [17], but little is known about its action in the suppression of synovial in ammatory and brosis. Also, IMP is one of the active ingredients in "Yiceng", a layer used to treat KOA [18]. Therefore, in this study, we examined the effects of IMP on synovial brosis provoked by monosodium iodoacetate (MIA) in vivo, and in ammatory model in primary synovial broblasts induced by lipopolysaccharide (LPS) in vitro. The therapeutic effect of IMP on synovitis and synovial brosis reveals it as a potential candidate for drug development. TransStart Green qPCR SuperMix was obtained from Takara (Dalian, China). The primers and rat GAPDH Endogenous Reference were supplied by Sangon Biotech (Shanghai, China). Enzyme linked immunosorbent assays (ELISA) kits for IL-1β and IL-18 were supplied by Invitrogen (Life Technologies Corp., California, USA). All other chemicals were of reagent grade.

Animals
Thirty 3-month-old SD male rats, weight ranging from 250-290 g, 10 for each group (provided by Beijing Vital River Laboratory Animal Technology Co., Ltd.), were used for experimental KOA studies. Animals were maintained in a speci c pathogen-free laminar-ow housing apparatus under controlled temperature, humidity, and 12 h light/dark regimen. All animal protocols were approved by the Animal Care and Use Committee of the Nanjing University of Chinese Medicine. All experiments were conducted in accordance with the National Institutes of Health Guidelines for the Care and Use of Laboratory Animals.

Induction of KOA and Drug Administration
The rat osteoarthritis model was made according to a previous literature [6]. Thirty rats were randomly divided into three groups (Normal group, MIA group, MIA+IMP group). For Normal group, injection of 0.9% saline into articular joint was performed; for MIA group, 2 mg MIA dissolved in 50 µl 0.9% saline; for MIA +IMP group, we chose 5 mg/kg/day as oral administration concentration from 2 weeks after injection as previously described [19]. IMP was dissolved in 0.5% carboxymethylcellulose sodium (CMC-Na), and 0.5% CMC-Na was given by intragastric administration alone in sham group and MIA group every day for 6 weeks until the rats were sacri ced. All rats were sacri ced after eight weeks' post-injection and the synovial tissues were processed for histological analysis and further experiments.

Histopathological analysis
For hematoxylin and eosin (H&E) staining, synovial tissues were frozen and xed in 4% paraformaldehyde, soaked in 0.5 M EDTA, embedded in para n for routine H&E staining and the para n blocks were sectioned at a thickness of 5 μm.
Isolation and primary culture of synovial broblasts Primary rat synovial broblasts were obtained from additional normal rats. In brief, synovial tissues were washed 2-3 times with phosphate-buffered saline and then minced into pieces of 2-3 mm 2 and digested in 0.1% collagenase type II (Sigma Aldrich, St. Louis, MO, USA) for 30 min. Following cell dissociation, the samples were ltered through a cell strainer. After dissociation, broblasts were pelleted by centrifugation at 1500 rpm for 4 min and cultured in DMEM supplemented with 10% FBS (Gibco, Thermo Fisher Scienti c, Waltham, MA, USA) and antibiotics (100 U/ml penicillin, 100μg/ml streptomycin). Cells were cultured at 37°C in a humidi ed 95% air and 5% CO 2 atmosphere. Passages 3-6 of the synovial broblasts were used for the experiments.
Fibroblasts were stimulated with LPS (5 μg/ml) in DMEM for 6 h to stimulate the in ammatory response and activate the NLRP3 in ammasome. The broblasts exposed to DMEM with same volume of saline served as control. Before administration of LPS, IMP were used for 24 h or 48 h for continued experiments.

Synovial extraction and preservation in rats
After 14 days of modeling or 14 days of treatment, the rats were sacri ced by CO 2 asphyxia method, and the rat knee joint hair was removed. The ligament was incised on both sides of the patellofemoral ligament. The upper edge of the humerus was transversely cut to the distal end of the quadriceps muscle until the femur. The ophthalmologist picked up the free tibia and its surrounding tissue and opened it to the distal end. The pale yellow translucent synovial membrane was seen. The synovial tissue was carefully cut with a surgical blade. Paraformaldehyde was preserved for pathological sectioning, and the rest was placed in a cryotube at -70°C.

Real-time PCR
RNA was isolated from synovial tissues and broblasts with Trizol (Invitrogen, CA, USA), respectively. The reverse transcription was performed by using a rst strand cDNA synthesis kit (Takara, Otsu, Japan) according to manufacturer's instructions. qPCR was performed using Premix Ex Taq SYBR-Green PCR (Takara) according to manufacturer's instructions on an ABI PRISM 7300 (Applied Biosystems, Foster City, CA, USA).
Primer was designed and synthesized by Shanghai Biotechnology Service Co. Ltd. in accordance with the gene sequence in GenBank gene sequence design, together with Oligo v6.6. Sequences for primers shown in Table 1. The mRNA level of individual genes was normalized to GAPDH and calculated by the 2 −ΔΔCt method.

Western blotting
Dissect the synovial tissue, weigh and mix with RIPA lysate. The samples were centrifuged at 15,000 r/min for 15 minutes at 4°C. The cultured broblasts were washed and lysed. Protein levels were then quanti ed using the BCA protein assay kit. Protein samples were electrophoresed in SDS-PAGE to separate protein bands. The protein was transferred from the gel to a PVDF membrane and blocked with 5% skimmed milk for 2 hours. PVDF membranes were incubated with polyclonal rabbit antibodies speci c for NLRP3, caspase-1, ASC, TGF-β1, VEGF and TIMP-1 overnight at 4°C. The next day, the membrane was incubated with the secondary antibody for 2 hours. The bands were visualized using the ECL method, and ImageJ software was used to quantify the total gray value (average gray value × gray value area) of protein band to calculate the relative value of target protein.

ELISA
Peripheral serum of rats and culture supernatant of cells were respectively collected and centrifuged at 10,000 rpm for 20 min at 4°C, after which the levels of IL-1β and IL-18 were measured by ELISA kits. All steps were performed according to the manufacturer's instructions.

Statistical analysis
Statistical analysis was performed using GraphPad Prism 8.0 Software (San Diego, CA, USA). Data were represented as mean ± standard deviation. Group comparisons were assessed with the one-way ANOVA or student's t-test for comparison of multiple columns. P < 0.05 (two-tailed) was considered statistically signi cant.

Results
Synovial brosis and hypoxia were found in MIA-induced KOA model rats The chemical structure of IMP is shown in (Fig. 1A). To elucidate the role of Synovial brosis and hypoxia and associated regulatory genes in synovial brosis, we examined mRNA expression levels of HIF-1α and brogenic factors, TGF-β1, TIMP1 and VEGF by real-time PCR. Protein expression levels were measured by Western blotting. The upregulation of HIF-1α in pathological conditions suggested that the synovial membrane was under hypoxia conditions with synovial brosis induced by MIA in rats. HIF-1α protein and mRNA levels were markedly elevated in KOA synovial membranes (Fig. 1B). Then we investigated the mRNA and protein expression level of TGF-β1, TIMP1 and VEGF in brotic synovium were all upregulated in pathological conditions induced by MIA in rats (Fig. 1C). Representative synovium tissues of each group stained with collagen ber staining by Sirius red staining or H&E staining (Fig. 1D).
Activation of NLRP3 in ammasome in synovial membranes resulted in synovitis in rats As synovial brosis usually followed by synovitis. The activation of NLRP3 in ammasome is a classic pathway which can promote the expressions of in ammatory factors IL-1β and IL-18. Activation of NLRP3 in ammasome partly contribute to synovitis. Key in ammatory factors of NLRP3 in ammasomedependent cytokines, IL-1β and IL-18, were measured by ELISA, which were signi cantly upregulated in the MIA group compared with the NORMAL group (Fig. 2C). Caspase-1 recruited by NLRP3 in ammasome cleaved pro-IL-1β and pro-IL-18 that promoted the maturation of in ammatory cytokines. We then evaluated the mRNA and protein expression of NLRP3 in ammasome in the MIA group. Both mRNA and protein levels were upregulated compared to the NORMAL group ( Fig. 2A and B).

IMP inhibited experimental synovitis and synovial brosis in rats
IMP, one of the representative components in furanocoumarin, is extracted from Angelica dahurica. In MIA+IMP group, less resident cell hyperplasia, formation of lining cell layers and in ammatory in ltration existed in H&E staining compared with MIA group. In sirius red staining, the collagen bers were red, the nucleus was green, and other components were yellow. The MIA group showed more collagen bers than the Control group and IMP ameliorate synovitis and synovial brosis (Fig. 1D). After the administration of IMP, the expression of NLRP3, ASC, caspase-1 and caspase-1 p10 were determined by qRT-PCR and Western blotting. All these components related to NLRP3 in ammasome were signi cantly downregulated in MIA+IMP group when compared with the MIA group both gene and protein level. The production of caspase-1 (p10) was also signi cantly reduced by IMP compared with MIA group, as well as IL-1β and IL-18 ( Fig. 2A-C). Besides, brogenic factors TGF-β1, TIMP-1 and VEGF were downregulated after the administration of IMP both in gene and protein level (Fig. 1C). Collectively, the administration of IMP inhibited synovitis, synovial brosis and hypoxia pathogenesis in rats.

IMP inhibited LPS-induced upregulation of brogenic factors in broblasts
We next examined the in vitro role of IMP in LPS-stimulated primary cultured broblast-like synoviocytes. The upregulated expression of brogenic factors suggested their possible involvement in synovial brosis. Treatment of broblast-like synoviocytes with LPS (5 μg/mL) signi cantly upregulated TGF-β1, TIMP-1, and VEGF both in gene and protein level. These brogenic factors contribute to synovial brosis in vivo. After the administration of IMP, all those factors were downregulated signi cantly (Fig. 3A and B).

IMP inhibited activation of NLRP3 in ammasome in LPS-stimulated broblasts
Then we observed signi cantly upregulated NLRP3, ASC and caspase-1 expression in the LPS group compared with the NORMAL group both in gene and protein level. IMP signi cantly inhibited the gene and protein expression of NLRP3 in ammasome compared with the LPS group. Production of cleaved caspase-1 and caspase-1 p10 was also con rmed by Western blotting and qRT-PCR. It showed signi cantly upregulation in the LPS group compared with the NORMAL group and this trend was suppressed by IMP ( Fig. 4A and B). The downstream production IL-1α and IL-18 were upregulated after LPS stimulation, and these in ammatory cytokines secreted by broblasts were downregulated by IMP (Fig. 4C).

Discussion
Hypoxia, in ammation and brosis persistently exist in the pathological progress of KOA [20]. Herein, we demonstrated the therapeutic effects of IMP on KOA. IMP signi cantly inhibited the expression of HIF-1α gene and protein, activation of NLRP3 in ammasome and downregulated the level of brogenic factors in KOA. KOA is characterized by the progressive destruction of articular cartilage and surrounding tissues, especially synovial tissue [21]. In this study, we show for the rst time that the IMP intervened with the pathological processes of KOA. The therapeutic effects of IMP may be related to the suppression of in ammation and synovial brosis.
Increased HIF-1α is highly involved in the progression of diseases, as well as synovial brosis and in ammation. It is well established that low oxygen tension exists in the synovium when KOA happens because of synovial angiogenesis and in ammatory cell in ltration [22]. Hypoxia in microenvironment is mainly marked by the HIF-1α expression. Compared with normal group rats, the expression of HIF-1α and its target genes VEGF and TIMP-1 in synovial tissue were signi cantly increased in KOA group [23]. We inferred that inhibition of HIF-1α expression could inhibit synovial brosis to some extent. In addition, HIF-1α can regulate NLRP3 expression. Under the condition of hypoxia, the expression of hypoxia-inducible factor-1α increased, the expression of NLRP3, Caspase-1and IL-1β also increased [24]. HIF-1α is known to regulate a plethora of human diseases. By regulating NLRP3 (transcript) expression under these conditions, it becomes a key node in linking hypoxia response to pro-in ammatory status. Increased expression of NLRP3 and enzymatic activation of caspase-1 are one of the conditions for the upregulation of IL-1β and IL-18. And these increased in ammatory factors were driven by the NLRP3 in ammasome by caspase-1. We interestedly found that IMP can not only inhibit the expression of HIF-1α in broblasts, but also the activation of NLRP3 in ammasome and the expression of downstream IL-1β and IL-18. Our previous research shows that inhibition of HIF-1α can effectively lead to synovial brosis in KOA rats, so we observed three brogenic factors (TGF-β1, VEGF and TIMP-1) at the same time.
Synovial brosis is a pathological process observed in several musculoskeletal diseases. HIF-1α regulates the expression of genes and proteins related to angiogenic growth factors, such as the expression of VEGF and TGF-β1 in RA [25]. TGF-β1 is one of the major indicators of synovial brosis that activate myoblasts, promote ECM gene expression, and inhibit ECM degradation [26]. However, the triggering mechanism of synovial brosis in the knee joint is not completely clear. Ko et al. found that TIMP1 was up-regulated both in OA broblasts stimulated with TGF-β1 and in mice with TGF-β-induced brosis [27]. TIMP-1 is an inhibitor of matrix metalloproteinases. We found that TIMP-1 is also elevated in the synovium of human end-stage OA patients [5]. TIMP-1 is induced by TGF-β, but is usually considered as an accelerator of brosis development not itself involved in inducing brosis [28]. A recent study showed that the articular capsule of the xed knee joint was in a state of hypoxia, VEGF was up-regulated at the mRNA and protein levels after immobilization. The decoy ODN transfected with HIF-1 successfully suppressed the transcriptional activation of HIF-1. Expression of VEGF was subsequently suppressed [29]. In RA, hypoxia is caused by increasing metabolic requirements for white blood cells to enter RA joints, which can cause HIF-1α to accumulate in the cytoplasm and induce expression of RA synovial tissue broblast and secrete VEGF. Positive feedback regulation of the HIF-1α and VEGF pathways can trigger angiogenesis during hypoxia. In addition, the levels of VEGF and HIF-1α in synovial tissue were positively correlated with microvascular density [30]. In this study, IMP decreased TGF-β1, TIMP1 and mRNA and protein levels in vitro and in vivo. Therefore, IMP may inhibit synovial brosis by inhibiting the expression of brogenic factors.
IMP is one of the furanocoumarin derivatives and exists in many Chinese herbal medicines with antitumor, antibacterial, cardiovascular, anti-in ammatory activities. In our previous studies, we found that synovial brosis is highly correlated with the activation of HIF-1α and NLRP3 in ammasome. These results indicate that IMP could improve synovitis and synovial brosis by inhibiting HIF-1α/NLRP3 in ammasome signaling.

Conclusions
In summary, IMP can improve synovial hypoxia and synovitis, and thus improved MIA-induced synovial brosis in KOA rats. It can reduce the release of in ammatory mediators and up-regulate brosis markers induced by MIA or LPS by inhibiting the activation of NLRP3 in ammasome. Taken together, IMP may be a potentially effective therapy for KOA, especially for synovial brosis. Further studies are required to determine the signal pathway involved in HIF-1α/NLRP3 in ammasome activation/ brosis. Its mechanism provides new ideas and means for understanding and treating KOA-related synovitis and synovial brosis.