Imperatorin inhibits proliferation, migration and inflammation via blocking NF-κB and MAPK pathways in rheumatoid fibroblast-like synoviocytes

doi:10.21203/rs.3.rs-1560945/v1

PDF

Research Article

Imperatorin inhibits proliferation, migration and inflammation via blocking NF-κB and MAPK pathways in rheumatoid fibroblast-like synoviocytes

https://doi.org/10.21203/rs.3.rs-1560945/v1

This work is licensed under a CC BY 4.0 License

Version 1

posted 20 Apr, 2022

You are reading this latest preprint version

Background: Rheumatoid arthritis (RA) is a chronic joint inflammatory disease that is associated with the aberrant activation of fibroblast‐like synoviocytes (FLSs). Searching for natural compounds that may suppress the activation of FLSs has become a complementary approach for RA treatment. Here, we investigated the effects and mechanisms of imperatorin (IPT) on proliferation, migration, and inflammation in primary cultured arthritic FLSs.

Methods: Cell viability was evaluated by an MTS assay. The effects of IPT on FLS proliferation, apoptosis and migration were determined by the EdU incorporation assay, flow cytometry and wound healing assay, respectively. Inflammation was assessed by detecting the expression of multiple pro-inflammatory cytokines using quantitative real-time PCR. Western blot and immunofluorescent staining were used to analyze the potential underlying molecular mechanisms. Statistical comparisons were performed using one-way ANOVA, followed by Tukey’s post hoc analysis.

Results: we found that IPT significantly suppressed TNFα-induced proliferation and migration of arthritic FLSs, but showed little effects on survival and apoptosis. In addition, IPT treatment significantly reduced TNFα-induced transcripts of proinflammatory cytokines (IL-1β, TNFα, IL-6 and IL-8) in arthritic FLSs. Further mechanism studies suggested that IPT inhibited the activations of p38 and ERK. Also, IPT blocked NF-κB activation via suppressing degradation of IκBα, and preventing the translocation of p65.

Conclusion: Our results demonstrated that IPT could inhibit the over-activated phenotypes of arthritic FLSs via MAPK (p38 and ERK) and NF-KB pathways. These findings suggested IPT has the potential to be developed as a novel agent for RA treatment.

Imperatorin

fibroblast-like synoviocyte

proliferation

migration

inflammation

NF-κB

MAPK

Rheumatoid arthritis (RA) is a chronic autoimmune disease, accompanied by long-term synovitis and destruction of cartilage and bone[1]. Although multiple cell types are involved in the pathological process of RA, fibroblast-like synoviocytes (FLSs) have been considered to play crucial roles in both joint damage and the propagation of inflammation [2, 3]. FLSs in RA are over-activated and exhibit a unique aggressive and transformed phenotype, which is characterized by increased proliferation and migration, evasion of apoptosis and overproduction of inflammatory cytokines and catabolic enzymes [3–6]. These destructive properties of FLSs have been demonstrated to tightly correlate with histological and radiographic damage in RA and its rodent models [7–9]. Thus, targeting FLSs has been considered to improve clinical outcomes in inflammatory arthritis without suppressing systemic immunity.

It is well known that the goal of treatment for RA is to reduce joint inflammation, inhibit the development of lesions and irreversible bone destruction, protect joint function as much as possible, and ultimately achieve disease relief or low disease activity[10, 11]. Nonsteroidal anti-inflammatory drugs (NSAIDs) can effectively reduce inflammation response and control the symptoms, however, these drugs can’t block the progression of RA[12]. Disease-modifying anti-rheumatic drugs (DMARDs) and biologics can prevent the process of bone destruction, but long-term use may cause obvious side effects. Furthermore, expensive prices prevent most patients from purchasing biologics. Therefore, the development of new drugs with better curative effect and less side effects is becoming an area of active research of RA drug treatment.

Imperatorin (IPT), a naturally occurring furanocoumarin, which can be found in citrus fruits, umbelliferous vegetables, and some herbs such as Angelica dahurica, Angelica archangelica and Glehnia littoralis. This compound has been demonstrated to possess various pharmacological activities such as anticancer[13], analgesic[14], antioxidant, anti-inflammation[15], and diastolic blood vessels[16]. Zhai et al. (2014) has found that IPT is one of the major active ingredients in the Fengshiding capsule which is a widely used traditional Chinese medicine for the treatment of RA. In addition, IPT has been reported to protect against collagen induced arthritis in rats [17]. However, the mechanism of its anti-arthritic action is yet not fully known. Given the therapeutical role of IPT in RA and the importance of FLSs in RA development, we investigated the effect of IPT on pathogenic behaviors of arthritic FLS and further explore its underlying molecular mechanisms

2.1. Reagents

IPT (C₁₆H₁₄O₄, Purity ≥ 98%) was obtained from Mansite Bio-Technology Co., Ltd. (Chengdu, Sichuan, China). Recombinant tumor necrosis factor alpha (TNFα) was purchased from Peprotech (PeproTech, Inc., Rocky Hill, NJ, USA). Fetal bovine serum (FBS) and Dulbecco’s modified Eagle’s medium (DMEM) were obtained from Gibco (Gibco BRL, Grand Island, NY, USA). MTS reagents, 5-Ethynyl-2ʹ-deoxyuridine (EdU), lyophilized native chicken type II collagen (CII), Freund’s complete adjuvant (CFA) and DAPI solution were purchased from Sigma-Aldrich (St. Louis, Missouri, USA). Annexin V-FITC apoptosis detection kit was obtained from KeyGen Biotech, Co., Ltd. (Nanjing, Jiangsu, China). TRIzol reagent was from Invitrogen (Carlsbad, CA, USA). Antibodies against extracellular signal-regulated kinase (ERK), phosphorylated ERK (p-ERK), p38, phosphorylated p38 (p-p38), c-Jun N-terminal kinase (JNK), phosphorylated JNK (p-JNK) and p65 were purchased from Cell Signaling Technology (Beverly, MA, USA). Antibodies against IκBα and GAPDH were obtained from Santa Cruz Biotechnology (Dallas, CA, USA). Enhanced Chemiluminescence (ECL) solution was purchased from Amersham Pharmacia Biotechnology (Piscataway, NJ, USA)

2.2. Isolation, culture and identification of arthritic FLS cells

Four female Wistar rats (160–180 g) were purchased from Shanghai Slac Laboratory Animal Co. Ltd. (Shanghai, China). A collagen-induced arthritis (CIA) rat model was established as our previous descriptions [18–20]. In brief, the rats were intradermally immunized with 1.5 mg CⅡ emulsified with an equal volume of CFA. Seven days later, a subcutaneous booster was given with half the amount of CⅡ emulsified with Freund’s incomplete adjuvant (IFA). The animal experiment was approval by the Experimental Committee of Nanjing Normal University (SYXK 2020-0047). The knee synovium of rats with CIA that had significant signs of disease was dissected and digested by 0.4% type II collagenase and 0.25% trypsin. The primary synovial cells were cultured in H-DMEM supplemented with 10% FBS (v/v), 100 U∙mL^− 1 penicillin and 100 µg∙mL^− 1 streptomycin at 37℃ in an atmosphere of 5% CO₂. The FLS cells were identified by staining for vascular cell adhesion molecule-1 (VCAM-1), as our previous reports [18, 21]. Cells obtained from the 3 ~ 10th passages were used in the subsequent experiments.

2.3. Cell viability assay

To determine the effect of IPT on the viability of arthritic FLSs, the cells were seeded into 96well plates and subsequently treated with different concentrations of IPT (0, 5, 10, 20, 40, 80 and 160 µM) for 48 h. MTS/PMS complex was then added to each well and incubated for another 4 hours. The absorbance of each well was measured at a wavelength of 490 nm using a microplate reader (Model 680, BioRAD, Hercules, CA, USA).

2.4. Wound healing assay

Arthritic FLSs were cultured into 12-well plates and serum starved overnight. A linear scratch on the cell monolayer was formed using a sterile 200 ul pipette tip. After washing the suspended cell debris with PBS, the cells were pretreated with different concentrations of IPT (0, 1, 2.5, 5 and 10 µM) for 1 h, followed by stimulation with TNFα (50 ng∙mL^-1) for 24 h. The effect of IPT on cell migration ability was measured by comparing the remaining cell-free area in the identical fields using ImageJ software.

2.5. Cell proliferation assay

Arthritic FLSs were cultured into 24-well plates, pre-treated with 2.5 µM IPT for 6 h, and then stimulated with or without 50 ng∙mL^-1 TNFα for another 24 h. According to the manufacturer’s instructions, the cells were incubated with 10 µM EdU for 6 h and then fixed with methanol. The cell nuclei were stained with Hoechst 33342. The numbers of the proliferating cells and total nucleated cells were counted by Image Plus Pro software. The proliferation rate was calculated according to the follow formula: the proliferation rate = (number of proliferating cells / number of total nucleated cells) × 100%.

2.6. Apoptosis detection by flow cytometry

Apoptosis assay was performed using an Annexin V-FITC apoptosis detection kit according to the manufacturer's instructions. Briefly, arthritic FLSs were treated with different concentrations of IPT (0, 2.5, 5 and 10 µM) for 24 h and then suspended in binding buffer. The cells were stained with Annexin V and propidium iodide (PI) solution. Flow cytometric analysis was performed with FACScan (Becton Dickinson) with the CellQuest program.

2.7. RNA extraction and quantitative real-time PCR

Arthritic FLSs were treated with different doses of IPT (0, 1, 2.5, 5 and 10 µM) for 1 h and then stimulated with TNFα (50 ng∙mL^-1) for 24 h. Total RNA was extracted using TRIzol reagent and cDNA was synthesized using random primers and oligdT primers. Quantitative real-time PCR amplification was performed using the following primer sets: IL-1β, 5′-ATGATGGCTTATTACAGTGGCAA-3′ (forward), 5′-GTCGGAGATTCGTAGCTGGA-3′ (reverse); IL-6, 5'-AACCTGAACCTTCCAAAGATGG-3' (forward), 5'-TCTGGCTTGTTCCTCACTACT-3' (reverse); IL-8, 5'-CATACTCCAAACCTTTCCACCCC-3' (forward), 5'- TCAGCCCTCTTCAAAAACTTCTCCA-3' (reverse); TNFα, 5′-ATACACTGGCCCGAGGCAAC-3′ (forward), 5′-CCACATCTCGGATCATGCTTTC-3′ (reverse); β-actin, 5’-CCACACTGTGCCCATCTACG-3' (forward), 5'-AGGATCTTCATGAGGTAGTCAGTCAG-3' (reverse). The PCR reaction conditions were as follows: 95°C denature for 30 s, followed by 95°C 10 s, and 60°C 30 s for 40 cycles. PCR was performed on Mastercycler ep realplex 2 systems (Eppendorf, Hamburg, Germany). The relative expression of each target gene compared with β-actin was calculated using the 2^−ΔΔCt method.

2.8. Western blot analysis

Arthritic FLSs were treated with different concentrations of IPT (0, 1, 2.5, 5 and 10 µM ) and then stimulated with or without TNFα (50 ng∙mL^-1) for 15 minutes. The cells were lysed with radioimmunoprecipitation assay (RIPA) lysis buffer and the lysate was collected by centrifugation. Protein was separated by SDS-PAGE electrophoresis and transferred to polyvinylidene fluoride (PVDF) membranes. The membranes were incubated with primary antibodies against p-ERK, p-JNK, p-p38, ERK1/2, p38, JNK, IκBα and GAPDH. Immunological responses were detected by ECL solution. Each independent experiment was repeated three times and grayscale was analyzed by ImageJ software.

2.9 Immunofluorescent staining for p65 localization

Arthritic FLS cells were cultured into 24-well plates containing sterile cover slips and treated with IPT (5 µM) for 4 h. After stimulation with TNFa (50 ng·mL^− 1) for 30 minutes, the cells on cover slips were washed, fixed and permeabilized with 0.5% Triton-X 100. After blocking with 10% goat serum, the cells were incubated with NF-κB p65 antibody overnight. DAPI solution was used to stain nuclei. The nuclear translocation of p65 was imaged using a Nikon A1R resonance scanning confocal microscope with spectral detector (Nikon, Tokyo, Japan).

2.10. Statistical analysis

All data were expressed as the mean ± SD of results obtained from three or more experiments. Statistical comparisons were performed using one-way ANOVA, followed by Tukey’s post hoc analysis. P < 0.05 was considered statistically significant.

3.1. Effect of IPT on arthritic FLSs viability

To explore whether IPT has an inhibitory action on the pathogenic behaviors of

FLSs, we firstly isolated, cultured and identified arthritic FLSs from the knee synovium of CIA rats. As shown in Fig. 1, the isolated cells displayed a spindle shape which was in consistence with FLS morphological feature. Immunofluorescence staining showed that there were more than 98% cells expressing VCAM-1, suggesting that these cultured cells corresponded most likely to the intimal subpopulation of FLSs (Fig. 1). We used these primary cultured arthritic FLS for the subsequent experiments.

To evaluate the possible cytotoxic effect of IPT on arthritic FLSs, an MTX assay was performed. As shown in Fig. 2B, IPT had no significant effect on the viability of FLS cells, even at concentration as high as 160 µM. In the subsequent experiments, concentrations of IPT used were less than 10 µM.

3.2. Effect of IPT on proliferation and apoptosis of arthritic FLSs

The synovial tissues of RA patients are well known to be abnormally hyperplastic due to the enhanced proliferative property of FLSs in an inflammatory environment. Thus, suppression of FLS’s proliferation has been proposed as a potential therapeutic strategy for RA. In this study, EdU incorporation assay showed that TNFα stimulation dramatically increased the proliferative potential of arthritic FLSs, however, this increase was significantly inhibited by 2.5 µM IPT (Fig. 2C and 2D). In addition, we also detected the effect of IPT on synovial cell apoptosis by flow cytometry. As shown in Fig. 2E, IPT at the given doses had little effect on apoptosis of arthritic FLSs.

3.3. Effect of IPT on migrative ability of arthritic FLSs

The migration of RA FLSs is a critical process in cartilage and bone destruction and functionally differs from proliferation. In view of this, we analyzed the effect of IPT on TNF-induced migration by a wound healing assay. As shown in Fig. 2F and 2G, the migration area of arthritic FLSs was significantly increased upon TNFα induction, while IPT significantly suppressed this increased migration ability at doses greater than 1 µM.

3.4. Effect of IPT on the expression of pro-inflammatory mediators in arthritic FLSs

In the pathogenesis of RA, synovial cells can produce a large number of cytokines which promote the occurrence and development of RA by acting on a variety of cells and regulating each other to form a complex network. To assess the inhibitory effect of IPT on pro-inflammatory cytokine production, we examined the mRNA expression of TNFα, IL-1β, IL-6 and IL-8 in TNFα-induced arthritic FLSs. As expected, the transcripts of these pro-inflammatory cytokines were markedly induced after TNFα stimulation. However, the induction was significantly suppressed by IPT treatment (Figs. 3A-D).

3.5. Effect of IPT on TNFα-induced activations of nuclear factor of kappaB (NF-κB) and mitogen-activated protein kinases (MAPK)

To reveal the mechanisms through which IPT suppressed the pathologic phenotypes of arthritic FLSs, TNFα-induced MAPKs signaling pathways were investigated. As shown in the Fig. 4A and 4B, the phosphorylations of ERK, p38, JNK were clearly up-regulated after stimulation with TNFα. However, IPT treatment robustly suppressed TNFα-induced activations of ERK and p38. In contrast, IPT had little effect on TNFα-induced JNK phosphorylation. In addition to MAPK signaling pathway, we also tested the activation of NF-κB which has been demonstrated to play a crucial role in the development and progression of RA [22, 23]. As shown in Fig. 4C and 4D, IκBα was effectively degraded upon TNFα stimulation. However, this degradation was significantly suppressed by IPT treatment. It is well known that IκB binds to NF-κB in the cytoplasm of normal cells and inhibits NF-κB entry into the nucleus. TNFα-stimulated IκBα degradation can release NF-κB protein (such as p65) into the nucleus and triggers the expression of a subset of NF-κB target genes. In our study, NF-κB p65 was clearly observed to translocate into the nuclei upon TNFα stimulation for 30 minutes (Fig. 4E). And IPT substantially inhibited p65 nuclear translocation, as evidenced by the retention in the cytoplasm of the p65 proteins (Fig. 4E).

Synovitis, The predominant pathological change of RA, is characterized by the abnormal proliferation and migration of FLSs which lead to hyperplasia of synovial tissues and the formation of pannus, with subsequent bone and cartilage destruction [24]. Under normal physiological condition, FLSs are located in the lining of synovium, which is involved in regulating the function of leukocytes, nourishing the joint environment and remodeling the matrix in tissue injury [25, 26]. In RA patients with inflamed synovium, FLSs are activated and showed a lot of tumor-like biologic behaviors, such as excessive proliferation, apoptosis resistance, escape of growth inhibition, and enhancement of migration rate. Moreover, RA FLSs can also produce and secrete inflammatory mediators that lead to recruitment and activation of immune and no-immune cells along with angiogenesis induction resulting in joint damage [27]. Thus, inhibiting RA FLSs proliferation, migration, and over-production of inflammatory cytokine may be a promising strategy for RA treatment. It is well known that TNFα is a central cytokine in the inflammatory cascade and its expression level is positively correlated with the severity of RA [28, 29]. FLS with TNFα stimulation was widely used as an in vitro model to explore RA pathologenesis and screen potential therapeutic drugs. In this study, we evaluated the effects of IPT on proliferation, apoptosis, migration and inflammatory cytokine synthesis of TNFα-stimulated FLSs, and revealed the underlying molecular mechanisms.

It should be noted that although primary human FLSs from RA patients have commonly been used to study the effects of a variety of drugs and phytochemicals, they present certain inconveniences. These RA-derived FLSs produce a broad range of results due to the individual responses of each patient sample [30]. And it is routinely difficult for many labs to acquire synovial tissue samples from RA patient. In our study, we established a CIA rat model and isolated FLSs from knee synovium of rats with CIA that had significant signs of RA, as our previous reports [18, 20]. Cell morphology and VCAM-1 immunofluorescence staining both demonstrated that these primary cultured cells belonged to the intimal subpopulation of FLSs. Using these arthritic FLSs, we found IPT significantly suppressed TNFα-induced proliferation and migration. This suppression of IPT was not due to its cytotoxicity and apoptosis-inducing effect, which could be demonstrated by the MTT assay and flow cytometry. IPT has been reported to adequately suppress synovial hyperplasia and pannus formation of collagen induced arthritis in rats [31]. As we known, the over-proliferation and increased migration of RA FLSs is the main cause of synovial hyperplasia and invasive pannus formation. Therefore, our result could, at least in part, interpret the therapeutic action of IPT in CIA rats reported by the previous study [31].

The previous studies have demonstrated that IPT has an anti-inflammatory property in multiple cell types and animal models [32–34]. Huang et al. reported that IPT could suppress the protein expression of iNOS and COX2 in LPS-stimulated RAW264.7 cells and thereby inhibited carrageenan-induced paw edema in mice [33]. Zhang et al. demonstrated that oral administration of IPT significantly inhibited inflammatory reactions in three different animal models (dimethylbenzene‑induced ear edema, acetic acid‑induced vascular permeability and cotton pellet‑induced granuloma) and reduced release of TNFα, IL-1β and IL-6 through blocking NF-κB pathway [34]. In consistence with these previous reports, our results showed that IPT effectively decreased the transcripts of TNFα, IL-1β, IL-6 and IL-8 in TNFα-induced arthritic FLSs. Compared with the current single mediator therapy such as biological agents, this capacity of IPT to down-regulate a wide spectrum of inflammatory cytokines might have a therapeutic advantage.

Having demonstrated that IPT could suppress the pathogenic behaviors of TNFα-induced arthritic FLSs, we further explore the underlying molecular mechanisms. Increasing evidence demonstrates that MAPKs, including p38, ERK, and JNK, have been abnormally up-regulated in RA synovial tissues and RA-derived FLSs [35]. The reversion of these changes usually is deemed as a therapeutic aim due to their important roles in the pathogenesis of RA. In this study, we found that IPT significantly suppressed phosphorylation of p38 and ERK, but without any effect on p-JNK. These results suggested that the inhibitory effect IPT on FLS’s destructive phenotype could occur through suppressing ERK and p38 pathways. NF-κB is a central regulator of inflammatory signaling in several tissues and cells [36, 37]. Moreover, NF-κB plays a crucial role in maintaining the proliferative and aggressive phenotypes of RA FLS [38]. Inhibition of NF-κB pathway demonstrates protective effect against RA-FLSs. In our study, IPT was demonstrated to suppress IκBα degradation and thereby block the nuclear translocation of the p65 subunit of NF-κB. This was further confirmed by our qPCR results that IPT could significantly decrease the mRNA expression of a subset of NF-κB downstream target genes. Collectively, our data revealed that IPT played an inhibitory effect on over-activated arthritic FLSs via multiple targets (Fig. 5). The limitation of this research was not unveil IPT’s direct binding sites, which would be conducted in future.

In conclusion, we demonstrated, for the first time, that IPT could inhibit proliferation, migration and inflammation via MAPKs (p38 and ERK) and NF-KB pathways in TNFα-induced arthritic FLSs. These findings suggested IPT has the potential to be developed as a novel agent for RA treatments.

IPT, Imperatorin; FLSs, fibroblast-like synoviocytes; RA, Rheumatoid arthritis; TNFα, tumor necrosis factor alpha; NF-κB, nuclear factor of kappaB; IκBα, inhibitor of nuclear factor κB alpha; MAPK, mitogen-activated protein kinases; ERK, extracellular signal-regulated kinase; JNK, c-Jun Nterminal kinase; CIA, collagen-induced arthritis; CⅡ, chicken type II collagen; EdU, 5-Ethynyl-2ʹ-deoxyuridine

Ethics approval and consent to participate

The animal experiment was approval by the Experimental Committee of Nanjing Normal University (SYXK 2020-0047).

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.

Competing interests

The authors declare that they have no competing interests.

Funding

This study was supported by National Natural Science Foundation of China (Grant No. 31870895) and the Priority Academic Program Development of Jiangsu Higher Education Institutions.

Authors’ contributions

ML and CW conceived and designed the experiments. WL, GC, YM, XM, JZ and XY performed the experiments. ML and CW analyzed the data. ML wrote the manuscript. All authors read and approved the final manuscript.

Acknowledgements

Not applicable.

Bernard MA, Bénichou J, Blin P, Weill A, Bégaud B, Abouelfath A, Moore N, Fourrier-Réglat A, Team C, Use of health insurance claim patterns to identify patients using nonsteroidal anti-inflammatory drugs for rheumatoid arthritis. Pharmacoepidemiology & Drug Safety 2012; 21: 573-83.
Wu Z, Ma D, Yang H, Gao J, Zhang G, Xu K, Zhang L, Fibroblast-like synoviocytes in rheumatoid arthritis: Surface markers and phenotypes. Int Immunopharmacol 2021; 93: 107392. 10.1016/j.intimp.2021.107392
Bartok B, Firestein GS, Fibroblast-like synoviocytes: key effector cells in rheumatoid arthritis. Immunol Rev 2010; 233: 233-55. 10.1111/j.0105-2896.2009.00859.x
Ma JD, Jing J, Wang JW, Yan T, Li QH, Mo YQ, Zheng DH, Gao JL, Nguyen KA, Dai L, A novel function of artesunate on inhibiting migration and invasion of fibroblast-like synoviocytes from rheumatoid arthritis patients. Arthritis Res Ther 2019; 21: 153. 10.1186/s13075-019-1935-6
Zhang Q, Liu J, Zhang M, Wei S, Li R, Gao Y, Peng W, Wu C, Apoptosis Induction of Fibroblast-Like Synoviocytes Is an Important Molecular-Mechanism for Herbal Medicine along with its Active Components in Treating Rheumatoid Arthritis. Biomolecules 2019; 9. 10.3390/biom9120795
Falconer J, Murphy AN, Young SP, Clark AR, Tiziani S, Guma M, Buckley CD, Review: Synovial Cell Metabolism and Chronic Inflammation in Rheumatoid Arthritis. Arthritis Rheumatol 2018; 70: 984-99. 10.1002/art.40504
Brand DD, Latham KA, Rosloniec EF, Collagen-induced arthritis. Nat Protoc 2007; 2: 1269-75. 10.1038/nprot.2007.173
Oliver SJ, Banquerigo ML, Brahn E, Suppression of collagen-induced arthritis using an angiogenesis inhibitor, AGM-1470, and a microtubule stabilizer, taxol. Cell Immunol 1994; 157: 291-9. 10.1006/cimm.1994.1223
Masoumi M, Bashiri H, Khorramdelazad H, Barzaman K, Hashemi N, Sereshki HA, Sahebkar A, Karami J, Destructive Roles of Fibroblast-like Synoviocytes in Chronic Inflammation and Joint Damage in Rheumatoid Arthritis. Inflammation 2021; 44: 466-79. 10.1007/s10753-020-01371-1
Mor A, Abramson SB, Pillinger MH, The fibroblast-like synovial cell in rheumatoid arthritis: a key player in inflammation and joint destruction. Clinical Immunology 2005; 115: 118-28.
Noss EH, Brenner MB, The role and therapeutic implications of fibroblast-like synoviocytes in inflammation and cartilage erosion in rheumatoid arthritis. Immunological Reviews 2008; 223: 252-70.
Paula FS, Alves JD, Non-tumor necrosis factor-based biologic therapies for rheumatoid arthritis: present, future, and insights into pathogenesis. Biologics Targets & Therapy 2014; 8: 1-12.
Zheng YM, Lu AX, Shen JZ, Kwok AHY, Ho WS, Imperatorin exhibits anticancer activities in human colon cancer cells via the caspase cascade. Oncology Reports 2016; 35: 1995-2002.
Li-xin W, Ying C, Yi-ming K, Hui-jie Y, Qian Z, Yun Y, Dong Z, Yan T, Feng-xian M, Jin-yu W, Effect of Single and Mixed Decoction of Angelicae Dahuricae Radix and Myrrha on Content of Imperatorin and Their Analgesic Effect. Chinese Journal of Experimental Traditional Medical Formulae 2018.
Huang GJ, Deng JS, Liao JC, Hou WC, Wang SY, Pingjyun S, Yuehhsiung K, Inducible Nitric Oxide Synthase and Cyclooxygenase-2 Participate in Anti-inflammatory Activity of Imperatorin from Glehnia littoralis. Journal of Agricultural & Food Chemistry 2012; 60: 1673.
Zhou N, Zhu Y, Zhang P, Zhang Y, Zhou M, Wang T, He L, Imperatorin derivative OW1 inhibits the upregulation of TGF-β and MMP-2 in renovascular hypertension-induced cardiac remodeling. Experimental & Therapeutic Medicine 2016; 11: 1748-54.
Zhai K, Gao G, Cao W, Zhao L, Fang X, Duan H, Simultaneous HPLC determination of four active compounds in fengshiding capsules, a chinese medicine. Indian J Pharm Sci 2014; 76: 445-9.
Liu Z, Zhou L, Ma X, Sun S, Qiu H, Li H, Xu J, Liu M, Inhibitory effects of tubeimoside I on synoviocytes and collagen-induced arthritis in rats. J Cell Physiol 2018; 233: 8740-53. 10.1002/jcp.26754
Yu X, Zhou J, Zhao F, Liu X, Mao Y, Diao L, Wen C, Liu M, Tomatidine Suppresses the Destructive Behaviors of Fibroblast-Like Synoviocytes and Ameliorates Type II Collagen-Induced Arthritis in Rats. Front Pharmacol 2021; 12: 670707. 10.3389/fphar.2021.670707
Qiu H, Sun S, Ma X, Cui C, Chen G, Liu Z, Li H, Liu M, Jatrorrhizine Hydrochloride Suppresses Proliferation, Migration, and Secretion of Synoviocytes In Vitro and Ameliorates Rat Models of Rheumatoid Arthritis In Vivo. Int J Mol Sci 2018; 19. 10.3390/ijms19051514
Qiu H, Hui L, Liu Z, Sun S, Ma X, Mei L, Isolation,Culture and Identification of Fibroblast-Like Synoviocytes from Collagen-Induced Arthritis(CIA) Rats. Journal of Nanjing Normal University 2017.
Ilchovska DD, Barrow DM, An Overview of the NF-kB mechanism of pathophysiology in rheumatoid arthritis, investigation of the NF-kB ligand RANKL and related nutritional interventions. Autoimmun Rev 2021; 20: 102741. 10.1016/j.autrev.2020.102741
van Loo G, Beyaert R, Negative regulation of NF-κB and its involvement in rheumatoid arthritis. Arthritis Res Ther 2011; 13: 221. 10.1186/ar3324
Huber LC, Distler O, Tarner I, Gay RE, Gay S, Pap T, Synovial fibroblasts: key players in rheumatoid arthritis. Rheumatology 2006; 45: 669-75. 10.1093/rheumatology/kel065 %J Rheumatology
Tykocinski LO, Lauffer AM, Bohnen A, Kaul NC, Krienke S, Tretter T, Adam I, Mohapatra SR, Saikali P, Löhning M, Neidhart M, Gay S, Oezen I, Platten M, Opitz CA, Lorenz HM, Synovial Fibroblasts Selectively Suppress Th1 Cell Responses through IDO1-Mediated Tryptophan Catabolism. J Immunol 2017; 198: 3109-17. 10.4049/jimmunol.1600600
Del Rey MJ, Valín Á, Usategui A, Ergueta S, Martín E, Municio C, Cañete JD, Blanco FJ, Criado G, Pablos JL, Senescent synovial fibroblasts accumulate prematurely in rheumatoid arthritis tissues and display an enhanced inflammatory phenotype. Immun Ageing 2019; 16: 29. 10.1186/s12979-019-0169-4
Elshabrawy HA, Chen Z, Volin MV, Ravella S, Virupannavar S, Shahrara S, The pathogenic role of angiogenesis in rheumatoid arthritis. Angiogenesis 2015; 18: 433-48. 10.1007/s10456-015-9477-2
Brennan FM, McInnes IB, Evidence that cytokines play a role in rheumatoid arthritis. J Clin Invest 2008; 118: 3537-45. 10.1172/jci36389
Moelants EA, Mortier A, Van Damme J, Proost P, Regulation of TNF-α with a focus on rheumatoid arthritis. Immunol Cell Biol 2013; 91: 393-401. 10.1038/icb.2013.15
Chang JH, Lee KJ, Kim SK, Yoo DH, Kang TY, Validity of SW982 synovial cell line for studying the drugs against rheumatoid arthritis in fluvastatin-induced apoptosis signaling model. Indian J Med Res 2014; 139: 117-24.
Zhai KF, Duan H, Chen Y, Khan GJ, Cao WG, Gao GZ, Shan LL, Wei ZJ, Apoptosis effects of imperatorin on synoviocytes in rheumatoid arthritis through mitochondrial/caspase-mediated pathways. Food Funct 2018; 9: 2070-79. 10.1039/c7fo01748k
Hu R, Li Z-l, Yang L, Yang J-r, Anti-inflammatory Effect of Imperatorin from Radix Angelicae Dahurica on Rats with Acute Pleurisy. Journal of Emergency in Traditional Chinese Medicine 2012; 21: 576-77.
Huang GJ, Deng JS, Liao JC, Hou WC, Wang SY, Sung PJ, Kuo YH, Inducible nitric oxide synthase and cyclooxygenase-2 participate in anti-inflammatory activity of imperatorin from Glehnia littoralis. J Agric Food Chem 2012; 60: 1673-81. 10.1021/jf204297e
Zhang X, Li W, Abudureheman A, Cheng T, Peng P, Imperatorin possesses notable anti‑inflammatory activity in vitro and in vivo through inhibition of the NF‑κB pathway. Mol Med Rep 2017; 16: 8619-26. 10.3892/mmr.2017.7706
Choe JY, Lee SJ, Park SH, Kim SK, Tacrolimus (FK506) inhibits interleukin-1β-induced angiopoietin-1, Tie-2 receptor, and vascular endothelial growth factor through down-regulation of JNK and p38 pathway in human rheumatoid fibroblast-like synoviocytes. Joint Bone Spine 2012; 79: 137-43. 10.1016/j.jbspin.2011.03.018
Lawrence T, The nuclear factor NF-kappaB pathway in inflammation. Cold Spring Harb Perspect Biol 2009; 1: a001651. 10.1101/cshperspect.a001651
Baker RG, Hayden MS, Ghosh S, NF-κB, inflammation, and metabolic disease. Cell Metab 2011; 13: 11-22. 10.1016/j.cmet.2010.12.008
Simmonds RE, Foxwell BM, Signalling, inflammation and arthritis: NF-kappaB and its relevance to arthritis and inflammation. Rheumatology (Oxford) 2008; 47: 584-90. 10.1093/rheumatology/kem298

No competing interests reported.

PDF

Version 1

posted 20 Apr, 2022

You are reading this latest preprint version

Imperatorin inhibits proliferation, migration and inflammation via blocking NF-κB and MAPK pathways in rheumatoid fibroblast-like synoviocytes

Status:

Version 1

Abstract

Figures

1. Introduction

2. Materials And Methods

2.1. Reagents

2.2. Isolation, culture and identification of arthritic FLS cells

2.3. Cell viability assay

2.4. Wound healing assay

2.5. Cell proliferation assay

2.6. Apoptosis detection by flow cytometry

2.7. RNA extraction and quantitative real-time PCR

2.8. Western blot analysis

2.9 Immunofluorescent staining for p65 localization

2.10. Statistical analysis

3. Results

3.1. Effect of IPT on arthritic FLSs viability

3.2. Effect of IPT on proliferation and apoptosis of arthritic FLSs

3.3. Effect of IPT on migrative ability of arthritic FLSs

3.4. Effect of IPT on the expression of pro-inflammatory mediators in arthritic FLSs

3.5. Effect of IPT on TNFα-induced activations of nuclear factor of kappaB (NF-κB) and mitogen-activated protein kinases (MAPK)

4. Discussion

Conclusions

Abbreviations

Declarations

Ethics approval and consent to participate

Consent for publication

Availability of data and materials

Competing interests

Funding

Authors’ contributions

Acknowledgements

References

Competing Interests

Status:

Version 1