Imperatorin suppresses IL-1β-induced iNOS expression via inhibiting ERK-MAPK/AP1 signaling in primary human OA chondrocytes

doi:10.1016/j.intimp.2020.106612

International Immunopharmacology

Volume 85, August 2020, 106612

https://doi.org/10.1016/j.intimp.2020.106612 Get rights and content

Highlights

•
Imperatorin inhibits NO production in primary human OA chondrocytes.
•
Imperatorin suppresses iNOS expression in primary human OA chondrocytes.
•
Imperatorin binds to iNOS and inhibits its activity.
•
Imperatorin inhibited the IL-1β induced activation of ERK-MAPK/AP1 pathway.

Abstract

Joint inflammation is a key player in the pathogenesis of osteoarthritis (OA). Imperatorin, a plant-derived small molecule has been reported to have anti-inflammatory properties; however, its effect on chondrocytes is not known. Here, we investigated the effects of Imperatorin on interleukin-1β (IL-1β) induced expression of inducible nitric oxide synthase (iNOS) and nitric oxide production in primary human OA chondrocytes and cartilage explants culture under pathological conditions and explored the associated signaling pathways. We pretreated chondrocytes or explants with Imperatorin (50 μM) followed by IL-1β (1 ng/ml), and the culture supernatant was used to determine the levels of nitrite production by Griess assay and chondrocytes were harvested to prepare cell lysate or RNA for gene expression analysis of iNOS by Western blot or qPCR and in explants by immunohistochemistry (IHC). Pretreatment of primary chondrocytes and cartilage explants with Imperatorin suppressed IL-1β induced expression of iNOS and NO production. Imperatorin blocked the IL-1β-induced phosphorylation of ERK-MAPK/AP1 signaling pathway to suppress iNOS expression. The role of ERK in the regulation of iNOS expression was verified by using ERK inhibitor. Interestingly, we also found that Imperatorin binds to iNOS protein and inhibits its activity in vitro. Our data demonstrated that Imperatorin possess strong anti-inflammatory activity and may be developed as a therapeutic agent for the management of OA.

Graphical abstract

Introduction

Nitric oxide (NO) is an important inflammatory mediator which is involved in the pathogenesis of osteoarthritis (OA) [1], [2], [3]. The excessive production of NO in cartilage inhibits matrix synthesis and enhances its degradation [4]. Additionally, NO reacts with oxidants such as superoxide anion, thereby promoting cellular injury. NO is produced by conversion of L-arginine to L-citrulline and is catalyzed by nitric oxide synthase (NOS) enzyme. NO is a gaseous free radical with a very short half-life of a few seconds, so its level in biological fluids is measured indirectly by estimating the level of nitrite (NO2)^-, which is the more stable metabolite of NO by using Griess assay [5]. NO production in OA cartilage has been illustrated by immunostaining with anti-nitrotyrosine antibodies [6]. The expression of inducible NOS (iNOS) is upregulated in OA chondrocytes, resulting in excessive NO production and eventual release of other catabolic and inflammatory cytokines [7]. iNOS expression is preferentially upregulated in the superficial zone of OA cartilage, suggesting active production of NO in that area [8]. The importance of iNOS in OA pathogenesis can be emphasized by the observation that iNOS knock out mice are resistant to developing experimental OA [9]. These experiments suggest that iNOS is a potential therapeutic target for the management of OA.

Imperatorin, known as 9-[(3-methyl-2-buten-1-yl)oxy]-7H-furo[3,2-g]chromen-7-one or 8-(1,1-dimethylallyloxy)-psoralen, is a plant secondary metabolite belonging to the family of furanocoumarins [10]. Imperatorin is mainly found among plants belonging to the Apiaceae and Rutaceae families, particularly in Angelica dahurica, A. archangelica, Notopterygium, Peucedani, and Radix Glehniae [11] and is known for many traditional applications. It is a noted constituent in many traditional medications, especially in traditional Chinese medicine system [12]. It has been reported that Imperatorin has a wide range of pharmacological properties, that include anti-inflammatory, anti-bacterial, anti-convulsant and anti-tumor activities [13], [14]. However, its anti-inflammatory effect has not been explored in OA and the chondrocytes, the only cell type directly affected in OA.

In this study, we used an in vitro and ex vivo model of OA pathogenesis to investigate the effects of Imperatorin on IL-1β induced expression of iNOS, production of NO and associated signaling pathways. We have demonstrated that Imperatorin treatment significantly suppressed the expression of iNOS in primary human OA chondrocytes and cartilage explants and the levels of nitrite in the culture supernatant. Interestingly, in addition to suppressing the expression of iNOS, we also found that Imperatorin can bind to iNOS protein and inhibit its activity. Our data also showed that Imperatorin suppressed iNOS by inhibiting the IL-1β induced activation of ERK-MAPK/AP1 signaling pathway, but was independent of the NF-κB pathway.

Section snippets

Reagents

Imperatorin (99% pure by HPLC, #0519S) was purchased from Alkemist labs. Gene-specific TaqMan assays were purchased from Integrated DNA Technologies. DMEM/F12 Media (#11320082) and FBS used for cell culture were purchased from HyClone Laboratories. Pronase (# 10165921001) and collagenase (# 10103578001) enzymes were purchased from Sigma-Aldrich. Recombinant human IL-1β (#201-LB) was purchased from R&D Systems. Antibody against iNOS (#ab3523) was from Abcam and antibodies against p38 (#sc-7972),

iNOS expression was upregulated in human OA cartilage and chondrocytes under pathological conditions

The human OA cartilage sections were stained with Safranin O/Fast Green to determine the extent of ECM degradation. Compared to the deep zone, the surface area of OA cartilage exposed to synovial cavity showed higher level of proteoglycan degradation (Fig. 1A). Investigation of iNOS expression by IHC in human OA cartilage (Mankin score >3) showed high level of iNOS in the damaged area compared to the undamaged area of cartilage from the same patient (Fig. 1B&C). We quantified the expression of

Discussion

This study reports the inhibitory effects of Imperatorin on NO production and expression of iNOS in OA chondrocytes and cartilage explants. To our knowledge, this is the first study reporting the inhibition of IL-1β induced expression of iNOS by Imperatorin and demonstrating that this was achieved by inhibiting the activation of ERK-MAPK/AP1 signaling pathway in IL-1β stimulated OA chondrocytes. In the present study, we showed that pretreatment of OA chondrocytes with Imperatorin significantly

Conclusion

In conclusion, these results revealed that Imperatorin treatment inhibited the expression of iNOS and effectively suppressed the production of NO in OA chondrocytes under pathological conditions. These results have potential clinical significance considering the role of NO in joint metabolism and cartilage homeostasis. Although further studies are required to analyze the observed inhibitory response in mouse OA model, these results for the first time demonstrate that Imperatorin can counteract

CRediT authorship contribution statement

Nashrah Ahmad: Conceptualization, Data curation, Methodology, Visualization, Investigation, Validation, Writing - original draft, Formal analysis. Mohammad Y. Ansari: Conceptualization, Methodology, Data curation, Supervision, Formal analysis, Writing - review & editing, Visualization, Investigation. Shabana Bano: Software, Data curation, Visualization, Investigation, Formal analysis. Tariq M Haqqi: Conceptualization, Supervision, Data curation, Writing - review & editing, Resources, Funding

Funding

This work was supported by the NIH/National Institute of Arthritis and Musculoskeletal and Skin Diseases and the NIH/National Center for Complementary and Integrative Health of the National Institutes of Health under Award Numbers R01AR067056 and AT007373 respectively.

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

References (36)

D. Jang et al.
Nitric oxide in arthritis
Free Radic. Biol. Med.
(1998)
S.B. Abramson
Osteoarthritis and nitric oxide
Osteoarthritis Cartilage
(2008)
W.Q. Yang et al.
Dimeric furanocoumarins from the roots of Angelica dahurica
Nat. Prod. Res.
(2017)
J. Sun et al.
Preventive effect of Imperatorin on acute lung injury induced by lipopolysaccharide in mice
Int. Immunopharmacol.
(2012)
N.M. Khan et al.
Wogonin, a natural flavonoid, intercalates with genomic DNA and exhibits protective effects in IL-1beta stimulated osteoarthritis chondrocytes
Chem. Biol. Interact.
(2017)
N. Akhtar et al.
Inhibition of cartilage degradation and suppression of PGE2 and MMPs expression by pomegranate fruit extract in a model of posttraumatic osteoarthritis
Nutrition
(2017)
M.Y. Ansari et al.
Parkin clearance of dysfunctional mitochondria regulates ROS levels and increases survival of human chondrocytes
Osteoarthritis Cartilage
(2018)
N.M. Khan et al.
Wogonin, a plant derived small molecule, exerts potent anti-inflammatory and chondroprotective effects through the activation of ROS/ERK/Nrf2 signaling pathways in human Osteoarthritis chondrocytes
Free Radic. Biol. Med.
(2017)
N. Schmidt et al.
Transcriptional and post-transcriptional regulation of iNOS expression in human chondrocytes
Biochem. Pharmacol.
(2010)
M. Ameyar et al.
A role for AP-1 in apoptosis: the case for and against
Biochimie
(2003)

K. Vuolteenaho et al.

The role of nitric oxide in osteoarthritis

Scand. J. Rheumatol.

(2007)

A.R. Amin et al.

The role of nitric oxide in articular cartilage breakdown in osteoarthritis

Curr. Opin. Rheumatol.

(1998)

S.B. Abramson et al.

Nitric oxide and inflammatory mediators in the perpetuation of osteoarthritis

Curr. Rheumatol. Rep.

(2001)

D. Tsikas

Methods of quantitative analysis of the nitric oxide metabolites nitrite and nitrate in human biological fluids

Free Radic. Res.

(2005)

R.F. Loeser et al.

Detection of nitrotyrosine in aging and osteoarthritic cartilage: Correlation of oxidative damage with the presence of interleukin-1beta and with chondrocyte resistance to insulin-like growth factor 1

Arthritis Rheum.

(2002)

A. Leonidou et al.

Inducible nitric oxide synthase as a target for osteoarthritis treatment

Expert. Opin Ther. Targets

(2018)

L. Salerno et al.

Progress in the development of selective nitric oxide synthase (NOS) inhibitors

Curr. Pharm. Des.

(2002)

K.S. Wang et al.

Imperatorin efficiently blocks TNF-alpha-mediated activation of ROS/PI3K/Akt/NF-kappaB pathway

Oncol. Rep.

(2017)

Cited by (32)

Effects and potential mechanisms of Saposhnikovia divaricata (Turcz.) Schischk. On type I allergy and pseudoallergic reactions in vitro and in vivo
2024, Journal of Ethnopharmacology
The incidence of allergic disease is constantly increasing, but its pathogenesis is not fully understood. Saposhnikovia divaricata (SD), called ‘Fangfeng’ in China, not only can be used for antipyretic, analgesic and anti-inflammatory as a traditional Chinese medicine, but also as an active ingredient in about 8% prescriptions. However, its effects on type I allergy and pseudoallergy have not been clarified.
To explore the treatment and potential mechanisms of SD and its major bioactive component Prim-O-glucosylcimifugin (POG) on type I allergy and pseudoallergy in vitro and in vivo.
The inhibitory effect of SD decoction and POG on type I allergy and its possible mechanism were evaluated by using RBL-2H3 cells model in vitro and the passive cutaneous anaphylaxis (PCA) mouse model in vivo. The cell degranulation of RBL-2H3 cells induced by DNP-IgE/DNP-BSA and Compound 48/80 (C48/80) was investigated, and the molecules of degranulation related signaling pathway was further detected by qRT-PCR and Western Blot analysis. Meanwhile, therapeutic effect of SD Decoction and POG were evaluated using PCA models in vivo. The molecular docking technology was conducted to explore the potential mechanisms.
In cells model induced by DNP-IgE/DNP-BSA, the release rate of β-Hex in high dose of SD and POG groups were 43.79% and 57.01%, and the release amount of HA in high dose of SD and POG groups were 26.19 ng/mL and 24.20 ng/mL. They were significantly lower than that in the model group. Besides, SD decoction and POG could significantly inhibit intracellular Ca²⁺ increasing and cell apoptosis. But there is no obvious effect on cells degranulation induced by C48/80. The molecular docking results showed that 5-O-Methylvisamioside and POG could bind with FcεRI α with stronger binding ability, but weak binding ability to Mrgprx2. Moreover, qPCR and Western blot analyses indicated that SD could down-regulate Lyn/Syk/PLCγ, MAPK and PI3K/AKT/NF-κB signal pathway to inhibit IgE-dependent cell degranulation. In mice PCA model, both SD and POG could dose-dependently attenuate the Evans Blue extravasation, paw and ear swelling induced by DNP-IgE/DNP-BSA, but no significant inhibition under the PCA models induced by C48/80.
In conclusion, SD is effective for the therapeutic of type I allergies, suggesting that SD is a potential candidate for the treatment of type I allergy, and the underlying mechanism of these effects needs to be further studied.
Nitric oxide inhibitory phenolic constituents isolated from the roots and rhizomes of Notopterygium incisum
2022, Bioorganic Chemistry
Citation Excerpt :
As shown in Fig. 5, these compounds were anchored tightly inside the active tunnel of iNOS with free binding energies (FBEs) ranging from −9.9 to −8.5 kcal/mol (Table 5). The docking poses of 6a/6b, 7a, 6′a and 7′a displayed that their aromatic motifs (eg. phenyl rings at C-2, styrene fragments, and benzyl groups) were embedded in the hydrophobic pocket consisting of Phe369, Pro350, Val352, and Ala351 [38–40], stacking against the heme group with a slight tilt angle (6a and 6′a) or orientating perpendicularly to it (6b, 7a, and 7′a). Additionally, they extended their rest moieties toward the entrance of the active cavity, forming hydrogen bonds with the catalytic residues, including Arg388, Asn354, Arg266, Gln263, and so on [41–43] (Fig. 5 and Table 5).
Fourteen phenolic constituents, notopheninetols A−E (1–5), notoflavinols A and B (6 and 7), and (2R)-5,4′-dihydroxy-7-O-[(E)-3,7-dimethyl-2,6-octadienyl]flavanone (8a), along with 12 known analogues (8b and 9–19) were isolated from the roots and rhizomes of Notopterygium incisum. Compounds 1–4 and 6–8 were seven pairs of enantiomers, and they were separated by chiral HPLC to obtain the optically pure compounds. The structures of the new compounds were elucidated based on detailed analyses of 1D and 2D NMR and HRESIMS data, and the absolute configurations were determined by quantum chemical calculations of the electronic circular dichroism (ECD) spectra, comparison of the experimental ECD data with those reported, and chemical methods. Compounds 1 and 2 possessed a 1-benzyl-2-methyl-indane skeleton, which was unprecedented in natural source. All of the isolated compounds were evaluated for their nitric oxide (NO) inhibitory effects on RAW264.7 cells induced by LPS, and compounds 6a/6b, 7a, 8a/8b, and the hydrogenated products 6′a and 7′a showed moderate inhibitory activities with IC₅₀ values in the range of 6.2–20.6 μM. Moreover, the interactions of these bioactive compounds with inducible nitric oxide synthase (iNOS) were explored by employing molecular docking simulation.
ERK1/2-mediated activation of DRP1 regulates mitochondrial dynamics and apoptosis in chondrocytes
2022, Osteoarthritis and Cartilage
To determine the Dynamin-related protein 1 (DRP1) regulation of mitochondrial fission in chondrocytes under pathological conditions, an area which is underexplored in osteoarthritis pathogenesis.
DRP1 protein expression was determined by immunohistochemistry (IHC) or immunofluorescence (IF) staining of cartilage sections. IL-1β-induced DRP1 mRNA expression in chondrocytes was quantified by qPCR and protein expression by immunoblotting. Mitochondrial fragmentation in chondrocytes was visualized by MitoTracker staining or IF staining of mitochondrial marker proteins or by transient expression of mitoDsRed. Mitochondrial reactive oxygen species (ROS) levels were determined by MitoSOX staining. Apoptosis was determined by lactate dehydrogenase (LDH) release assay, Caspase 3/7 activity assay, propidium iodide (PI), and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining and IF staining of cleaved caspase 3. Cytochrome c release was determined by confocal microscopy. Surgical destabilization of the medial meniscus (DMM) was used to induce osteoarthritis (OA) in mice.
Expression of DRP1 and mitochondrial damage was high in human OA cartilage and in the joints of mice subjected to DMM surgery which also showed increased chondrocytes apoptosis. IL-1β-induced mitochondrial network fragmentation and chondrocyte apoptosis via modulation of DRP1 expression and activity and induce apoptosis via Bax-mediated release of Cytochrome c. Pharmacological inhibition of DRP1 activity by Mdivi-1 blocked IL-1β-induced mitochondrial damage and apoptosis in chondrocytes. Additionally, IL-1β-induced activation of extracellular signal-regulated kinase 1/2 (ERK1/2) is crucial for DRP1 activation and induction of mitochondrial network fragmentation in chondrocytes as these were blocked by inhibiting ERK1/2 activation.
These findings demonstrate that ERK1/2 is a critical player in DRP1-mediated induction of mitochondrial fission and apoptosis in IL-1β-stimulated chondrocytes.
Nitric oxide mediates disruption of human placental trophoblast invasion induced by perfluorobutane sulfonate
2021, Environmental Pollution
Perfluorobutane sulfonate (PFBS), an emerging pollutant, is associated with disruption of placental functions and adverse birth outcomes. However, the precise mechanism of this disruption remains unclear. Extravillous trophoblasts make up the majority of cells in the placenta, and have invasive abilities, which plays a critical role in a successful pregnancy. It has been reported that inducible nitric oxide (iNOS) and nitric oxide (NO) signaling is associated with trophoblast migration and invasion. In this study, PFBS exposure was found to enhance trophoblast invasion and increase matrix metalloproteinase 9 (MMP-9) levels. Additionally, PFBS upregulated iNOS levels and stimulated NO generation. iNOS inhibitor treatment attenuated the increased invasion of trophoblasts and MMP-9 expression induced by PFBS. Extracellular signal-regulated kinase (ERK) phosphorylation was also enhanced by PFBS exposure. In the presence of an ERK pathway inhibitor, however, the increases in trophoblast invasion, the induction of NO production, iNOS expression and MMP-9 expression induced by PFBS were attenuated. Taken together, these results suggest that iNOS/NO signaling is triggered by activation of the ERK signaling pathway, and that iNOS/NO signaling mediates PFBS-induced stimulation of trophoblast invasion.
Cartilage-targeting peptide-modified dual-drug delivery nanoplatform with NIR laser response for osteoarthritis therapy
2021, Bioactive Materials
Citation Excerpt :
During the early stage of OA, chondrocytes reactively secreted some proinflammatory cytokines and chemokines [53]. Consequently, cumulative inflammatory factors, such as TNF-α and IL-6, activated inflammatory-related signaling pathways in chondrocytes [54]. Generally speaking, the cartilage extracellular matrix (ECM) mainly comprises type II collagen and aggrecan and can be degraded by proteases such as MMP9 and ADAMTS5 during the progression stage of OA [55].
Cartilage-targeting delivery of therapeutic agents is still an effective strategy for osteoarthritis (OA) therapy. Recently, scavenging for reactive oxygen species (ROS) and activating autophagy have been increasingly reported to treat OA effectively. In this study, we designed, for the first time, a dual-drug delivery system based on metal organic framework (MOF)-decorated mesoporous polydopamine (MPDA) which composed of rapamycin (Rap) loaded into the mesopores and bilirubin (Br) loaded onto the shell of MOF. The collagen II-targeting peptide (WYRGRL) was then conjugated on the surface of above nanocarrier to develop a cartilage-targeting dual-drug delivery nanoplatform (RB@MPMW). Our results indicated the sequential release of two agents from RB@MPMW could be achieved via near-infrared (NIR) laser irritation. Briefly, the rapid release of Br from the MOF shell exhibited excellent ROS scavenging ability and anti-apoptosis effects, however responsively reduced autophagy activity, to a certain extent. Meanwhile, following the NIR irradiation, Rap was rapidly released from MPDA core and further enhanced autophagy activation and chondrocyte protection. RB@MPMW continuously phosphorylated AMPK and further rescued mitochondrial energy metabolism of chondrocytes following IL-1β stimulation via activating SIRT1-PGC-1α signaling pathway. Additionally, the cartilage-targeting property of peptide-modified nanocarrier could be monitored via Magnetic Resonance (MR) and IVIS imaging. More significantly, RB@MPMW effectively delayed cartilage degeneration in ACLT rat model. Overall, our findings indicated that the as-prepared dual-drug delivery nanoplatform exerted potent anti-inflammation and anti-apoptotic effects, rescued energy metabolism of chondrocytes in vitro and prevented cartilage degeneration in vivo, which thereby showed positive performance for OA therapy.
High glucose activates ERK1/2 to stabilize AP1 and increase MMP9 expression in diabetic foot ulcers
2021, Experimental Cell Research
Citation Excerpt :
We wondered how AP1 was activated in diabetic skin. Because C-Jun and C-FOS are downstream substrate of ERK1/2 signaling, we detected phosphorylated ERK1/2 (p-ERK1/2) in high glucose-stimulated cells [13,14]. As shown in Fig. 5A, p-ERK1/2 expression was increased in HaCat cells after treating with high glucose.
Increased matrix metalloproteinase 9 (MMP9) expression is involved in delayed wound healing in diabetic foot ulcers. We created skin wounds in normal SD rats and STZ-induced diabetic SD rats, then we found protein levels of activator protein-1 (AP1), a crucial transcription factor to increase MMP9 transcription, as well as MMP9 was up-regulated in epithelium of diabetic skin tissues. Then, we evaluated the mRNA and protein stability of AP1 subunits C-FOS/C-Jun in HaCaT cells after high glucose treatment. Results showed that high glucose could increase protein stability of C-FOS and C-Jun. Additionally, high glucose also activated extracellular signaling-related kinase 1/2 (ERK1/2). ERK1/2 inhibitor could rescue phosphorylation of C-FOS and C-Jun, increased protein stability of C-Jun, and increased MMP9 expressions. Thus, our study demonstrated that high glucose could activate ERK1/2 to stabilize AP1 and increase MMP9 expression in diabetic skin and HaCaT cells.

View all citing articles on Scopus

View full text