Genkwanin suppresses mitochondrial dysfunction to alleviate IL-1β-elicited inflammation, apoptosis, and degradation of extracellular matrix in chondrocytes through upregulating DUSP1

Kanna Xu; Haoran Wang; Zhongqing Wu

doi:10.4103/cjop.CJOP-D-23-00031

ORIGINAL ARTICLE

Year : 2023 | Volume : 66 | Issue : 4 | Page : 284-293

Genkwanin suppresses mitochondrial dysfunction to alleviate IL-1β-elicited inflammation, apoptosis, and degradation of extracellular matrix in chondrocytes through upregulating DUSP1

Kanna Xu¹, Haoran Wang², Zhongqing Wu³
¹ Emergency Department, The First People's Hospital of Huzhou, Huzhou, Zhejiang, China
² Department of Orthopedics, Hangzhou Children's Hospital, Hangzhou, Zhejiang, China
³ Department of Orthopedics, The First People's Hospital of Huzhou, Huzhou, Zhejiang, China

Date of Submission	03-Mar-2023
Date of Decision	22-Apr-2023
Date of Acceptance	27-Apr-2023
Date of Web Publication	22-Aug-2023

Correspondence Address:
Dr. Zhongqing Wu
Department of Orthopedics, The First People's Hospital of Huzhou, No. 158, Guangchang Hou Road, Huzhou, Zhejiang 313000
China

Source of Support: None, Conflict of Interest: None

DOI: 10.4103/cjop.CJOP-D-23-00031

Abstract

Osteoarthritis (OA) is a form of chronic degenerative disease contributing to elevated disability rate among the elderly. Genkwanin is an active component extracted from Daphne genkwa possessing pharmacologic effects. Here, this study is designed to expound the specific role of genkwanin in OA and elaborate the probable downstream mechanism. First, the viability of chondrocytes in the presence or absence of interleukin-1 beta (IL-1β) treatment was detected by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling assay was used to assess cell apoptosis. Inflammatory response was estimated through enzyme-linked immunosorbent assay and Western blot. In addition, immunofluorescence staining and Western blot were utilized to measure the expression of extracellular matrix (ECM)-associated proteins. Dual-specificity protein phosphatase-1 (DUSP1) expression was tested by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and Western blot. Following DUSP1 elevation in genkwanin-treated chondrocytes exposed to IL-1β, inflammatory response and ECM-associated factors were evaluated as forementioned. In addition, 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolocarbocyanine iodide staining was to assess the mitochondrial membrane potential. Adenosine triphosphate (ATP) level was examined with ATP assay kit, and RT-qPCR was used to test mitochondrial DNA expression. Results indicated that genkwanin administration enhanced the viability while ameliorated the apoptosis, inflammatory response, and ECM degradation in IL-1β-induced chondrocytes. Besides, genkwanin treatment fortified DUSP1 expression in IL-1β-exposed chondrocytes. DUSP1 interference further offsets the impacts of genkwanin on the inflammation, ECM degradation, and mitochondrial dysfunction in IL-1β-challenged chondrocytes. In short, genkwanin enhanced DUSP1 expression to mitigate mitochondrial dysfunction, thus ameliorating IL-1β-elicited inflammation, apoptosis, and degradation of ECM in chondrocytes.

Keywords: Dual-specificity protein phosphatase-1, extracellular matrix degradation, genkwanin, inflammatory response, osteoarthritis

How to cite this article:
Xu K, Wang H, Wu Z. Genkwanin suppresses mitochondrial dysfunction to alleviate IL-1β-elicited inflammation, apoptosis, and degradation of extracellular matrix in chondrocytes through upregulating DUSP1. Chin J Physiol 2023;66:284-93

How to cite this URL:
Xu K, Wang H, Wu Z. Genkwanin suppresses mitochondrial dysfunction to alleviate IL-1β-elicited inflammation, apoptosis, and degradation of extracellular matrix in chondrocytes through upregulating DUSP1. Chin J Physiol [serial online] 2023 [cited 2023 Sep 26];66:284-93. Available from: https://www.cjphysiology.org/text.asp?2023/66/4/284/384127

Introduction

Osteoarthritis (OA) is a chronic degenerative joint disease accompanying joint stiffness, swelling, pain, and loss of functionality.^[1] Relevant evidence has put forward that the elderly and obese people are susceptible to OA considering that age and obesity belong to the most dominant risk factors.^[2],[3] It is worth noting that, along with the gradual development of population aging and diet structure, the prevalence of OA follows an escalating trend.^[4] Based on statistical data, around 37% of people aged over 60 years suffer from OA.^[5] The current therapy for OA is chiefly devoted to relieving symptoms applying drugs represented by acetaminophen, nonsteroidal anti-inflammatory drugs, and opioids.^[6] Nevertheless, the existing side effects limit the drug efficacy to a large extent and even endanger the cardiovascular system and gastrointestinal tract.^[7] Thereafter, seeking for both effective and safe drugs is the foremost strategy to treat OA. It adopts the mode of multidisciplinary division of labor and cooperation between surgeons, rehabilitation teams, nurses, and managers, among which the accelerated rehabilitation nursing measures including preoperative education, preoperative prerehabilitation and postoperative rehabilitation, pain management, nutritional support, psychological counseling, and behavioral therapy.^[8] Accelerated rehabilitation nursing combines evidence-based nursing with clinical practice, improves the traditional nursing and rehabilitation mode, and provides patients with the best nursing and rehabilitation treatment in each stage of the perioperative period, so as to promote the recovery of patients, reduce the occurrence of adverse events, and shorten the treatment cycle.^[9]

In recent years, Chinese herbal medicine has been strongly supported to elicit protective efficacy against chondrocyte injury to treat OA.^[10],[11] Daphne genkwa is a traditional Chinese medicine possessing diverse pharmacologic effects to defend against tumors and inflammatory response and modulate immune function.^[12] As an active component isolated from Daphne genkwa, genkwanin [Figure 1]a has been proposed to exert functions on relieving pain, immunoregulation, protecting against tumors as well as inflammatory response. For instance, Wang et al. have highlighted the antitumor and immunomodulatory activity of genkwanin on colorectal cancer in the APC^Min/+ mice.^[13] Genkwanin plays a suppressive role in inflammatory response in lipopolysaccharide (LPS)-activated macrophages through mediating miR-101/mitogen-activated protein kinase phosphatase 1 (MKP-1)/mitogen-activated protein kinase (MAPK) signaling.^[14] Genkwanin inactivates janus kinase/signal transducer of activators of transcription and nuclear factor kappa B signaling to reduce the inflammation and bone destruction in joint tissues.^[15] Nonetheless, its impacts on OA demand clarification.

Figure 1: Genkwanin treatment exacerbates the viability and reduces the apoptosis of IL-1β-challenged ATDC5 cells. (a) Chemical structure of genkwanin. (b) MTT assay judged the impacts of genkwanin treatment on cell viability. (c) MTT assay judged the viability of IL-1β-treated ATDC5 cells. (d) TUNEL assay appraised the apoptosis of IL-1β-treated ATDC5 cells (×200). Data from three independent replicates were presented as mean ± SD. *P < 0.05, **P < 0.01, and ***P < 0.001. IL-1β: Interleukin-1 beta, MTT: 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, TUNEL: Terminal deoxynucleotidyl-transferase-mediated dUTP nick end labeling, SD: Standard deviation.

Click here to view

Intriguingly, STITCH database (http://stitch.embl.de/) predicted that dual-specificity protein phosphatase-1 (DUSP1) is a possible target of genkwanin. DUSP1, also called MKP1, is the first discovered member of MKPs family.^[16] As reported, abundant evidence has covered the involvement of DUSP1 in diverse biological events, such as metabolism, skeletal muscle function, inflammatory response, together with cancer development, and so on.^{[17],[18],[19]} Meanwhile, according to Gene Expression Omnibus (GEO) database (GSE169077), DUSP1 has been discovered to exhibit declined expression in cartilage tissues of OA patients. Further, DUSP1 alleviates OA fibroblast-like synoviocytes through blocking MAPK signaling.^[20] However, the specific role of DUSP1 and the relationship between genkwanin and DUSP1 in OA also need exploration.

The main function of chondrocytes is to maintain the integrity of cartilage and ensure the function of cartilage. Interleukin-1 beta (IL-1β) is an important cytokine involved in OA progression, which will lead to apoptosis and proliferation inhibition of chondrocytes.^[21],[22] Therefore, a large number of in vitro experiments have used IL-1β as the inducer of OA cell model.^[23],[24] The crucial point of the current study is to determine the significance of genkwanin and determine the possible relationship between genkwanin and DUSP1 in IL-1β-induced chondrocytes.

Materials and Methods

Cell treatment

The culture medium for murine chondrogenic ATDC5 cells supplied by BeNa Culture Collection was Dulbecco's modified Eagle's medium (DMEM; Hyclone, Rockford, IL, USA) containing glutamine and sodium pyruvate (Beijing Solarbio Science and Technology Co., Ltd) decorated by 10% fetal bovine serum (FBS; Gibco, Waltham, MA, USA) and routinely maintained under the circumstance of 5% CO₂ at 37°C. ATDC5 cells were challenged with 10 ng/ml of IL-1β R&D System, Minneapolis, MN, USA) for 24 h to stimulate OA model in vitro.^[25] For genkwanin-treated groups, ATDC5 cells were pretreated with different concentrations (12.5, 25, and 50 μM) of genkwanin (Alfa Biotechnology Co., Ltd., Chengdu, China) for 2 h before treatment with IL-1β.^[14] For other groups, chondrocytes were transfected with small interfering RNA targeting DUSP1 (si-DUSP1) or the negative control (si-NC) and then stimulated with genkwanin or IL-1β. Then, ATDC5 cells pretreated with or without IL-1β were administrated by elevated concentrations (12.5, 25, and 50 μM) of genkwanin (Alfa Biotechnology Co., Ltd., Chengdu, China) for 2 h.^[14]

Cell transduction

The transduction of si-DUSP1 (GeneCopoeia, Inc.) and the negative control si-NC (GeneCopoeia, Inc.) into treated ATDC5 cells was executed adopting Lipofectamine 2000 (Invitrogen Corporation). Cells were harvested 48 h later for the ensuing experiments.

3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay

In short words, the supplementation of MTT solution (5 mg/ml; Biosharp Life Sciences) to the treated cells plated into 96-well plates lasted for extra 4 h at 37°C after 24, 48, and 72 h of cultivation. Dimethyl sulfoxide was used to solubilize the formazan following the abandonment of the medium. Optical density (OD) value at 490 nm was examined with a microplate reader (BMG LABTECH, Offenburg, Germany).

Terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling assay

Treated ATDC5 cells received immobilization and permeabilization, respectively, adopting 4% paraformaldehyde and 0.1% Triton X-100. TUNEL assay kit (Enzo Life Sciences, Farmingdale, NY, USA) was supplemented for 60 min of cultivation in the light of the manufacturer's instructions before cultivation in the mounting medium containing 10 mg/ml 4',6-diamidino2-phenylindole (DAPI) for 10 min. Finally, a fluorescence microscope (Nikon Eclipse 80i) was employed to capture images.

Enzyme-linked immunosorbent assay

Tumor necrosis factor-alpha (TNF-α; Cat. No. OKBB00263) and interleukin-6 (IL-6; Cat. No. OKRC01229) levels in the cell supernatant were confirmed by corresponding enzyme-linked immunosorbent assay (ELISA) kits procured from Aviva Systems Biology in compliance with the manufacturer's guidance. A microplate reader (BMG LABTECH, Offenburg, Germany) was to examine OD450 nm value.

Western blot

Utilizing radioimmunoprecipitation assay lysis buffer (Nanjing Jiancheng Bioengineering Institute), the acquired total protein extracts from ATDC5 cells resolved on 10% sodium dodecyl sulfate-polyacrylamide gel were shifted to polyvinylidene difluoride membranes whose nonspecific interaction was determined by 5% nonfat milk. Following, the membranes were successively immunoblotted with primary antibodies overnight at 4°C and goat anti-rabbit horseradish peroxidase antibody (Cat. No. ab205718; 1/2000; Abcam) for 1 h. The blots were visualized by the super-sensitive enhanced chemiluminescence reagent (Dalian Meilun Biology Technology Co., Ltd.) and analyzed by Quantity One (version 4.0; Bio-Rad Laboratories). Cyclooxygenase-2 (Cox-2; Cat. No. ab179800; 1/1000; Abcam), inducible nitric oxide synthase (iNOS; Cat. No. ab178945; 1/1000; Abcam), phosphorylated nuclear factor kappa B (NFκB)-p65 (p-NFκB p65; Cat. No. ab76302; 1/1000; Abcam), NFκB p65 (Cat. No. ab32536; 1/1000; Abcam), matrix metallopeptidase 13 (MMP13; Cat. No. ab39012; 1/3000; Abcam), a disintegrin-like and metalloproteinase with thrombospondin type-1 motifs-5 (ADAMTS-5; Cat. No. ab41037; 1/250; Abcam), aggrecan (Cat. No. NB100-74350; 1/1000; Novus Biologicals), DUSP1 (Cat. No. ab138265; 1/500; Abcam), and GAPDH (Cat. No. ab9485; 1/2500; Abcam); primary antibodies were all provided by Abcam.

Reverse transcription-quantitative polymerase chain reaction

With the aid of Omniscript® RT Kit (Qiagen), complementary DNA (cDNA) was produced from isolated RNA by means of easy-BLUE™ Total RNA Extraction Kit (iNtRON Biotechnology) according to the manufacturer's instructions. Amplification of cDNA was performed through PCR in a CFX Connect Real-Time system (Bio-Rad Laboratories, Inc.) using the QuantiNova SYBR Green PCR Kit (Qiagen), with GAPDH as normalization control. 2^−ΔΔCt method was introduced to reflect alternations in mRNA levels.^[26]

With the employment of a mitochondrial DNA (mtDNA) isolation kit (Abnova), mtDNA copy number DNA was extracted from ATDC5 cells. PCR amplification was executed utilizing (QuantiNova SYBR Green PCR Kit; Qiagen), and analysis was performed using CFX Connect Real-Time system (Bio-Rad Laboratories, Inc.).

Immunofluorescence staining

Treated ATDC5 cells received immobilization and permeabilization, respectively, adopting 4% paraformaldehyde and 0.2% Triton X-100. One percent bovine serum albumin was used for blocking after cells were rinsed in phosphate buffer solution. Then, cells were labeled with primary antibody against Collagen II (Cat. No. ab34712; 1/100; Abcam) overnight at 4°C and goat anti-rabbit immunoglobulin G/Alexa Fluor 555 (Cat. No. bs-0295G-A555; 1/100; Beijing Biosynthesis Biotechnology Co., Ltd.) was used. Nuclear staining with DAPI was then conducted. The images were captured applying a fluorescence microscope (Nikon Eclipse 80i).

JC-1 staining

Mitochondrial membrane potential was analyzed using 5,5′, 6, 6′-tetrachloro-1,1′, 3, 3′-tetraethylbenzimidazolocarbocyanine iodide (JC-1) detection kit (Abcam). Following indicated treatment and transfection, collected ATDC5 cells from DMEM-H medium were submerged in 5 μmol/l JC-1 staining buffer at 37°C for half an hour protected from light and images of red (JC-1 aggregates)/green (JC-1 monomers) fluorescence intensity were captured under a fluorescence microscope (Nikon Eclipse 80i) and analyzed by Quantity One (version 4.0; Bio-Rad Laboratories).

Detection of adenosine triphosphate production

The luciferase-based luminescence adenosine triphosphate (ATP) detection kit (Beyotime Institute of Biotechnology) was applied as per the protocol of the manufacturer. Generally, before the supplementation of 100 μl ATP detection working solution, treated and transfected cells were subjected to centrifugation at 12,000 × g for 5 min at 4°C. Cell lysate (40 μl) was then added to the wells of the culture plate, and luminescence was measured immediately using a luminometer (Turner BioSystems).

Statistical analysis

All statistical analyses were executed using GraphPad Prism 8 software (GraphPad Software, Inc.), and continuous variables were given as mean ± standard deviation from three independent experiments. One-way analysis of variance (ANOVA) followed by Turkey's test was applied for comparisons among multiple groups. Statistical significance was identified when P < 0.05.

Results

Genkwanin treatment exacerbates the viability and reduces the apoptosis of IL-1β-challenged ATDC5 cells

To elaborate the impacts of genkwanin on OA, ATDC5 cells were treated by elevated concentrations (12.5, 25, and 50 μM) of genkwanin and then cell viability was judged. Through MTT assay, no apparent alternations were noticed in ATDC5 cell viability under the condition of genkwanin treatment [Figure 1]b. Intriguingly, following IL-1β exposure, ATDC5 cell viability was noticeably declined and genkwanin administration markedly improved the viability of IL-1β-challenged ATDC5 cells in a dose-dependent manner [Figure 1]c. Conversely, the experimental data from TUNEL assay illuminated that IL-1β exposure-stimulated apoptosis of ATDC5 cells was remarkably abrogated by increasing concentrations of genkwanin [Figure 1]d. All above data presented that genkwanin might protect against IL-1β-induced viability injury and apoptosis of ATDC5 cells.

Genkwanin administration mitigates IL-1β-elicited inflammatory response of ATDC5 cells

In addition, levels and expression of inflammatory factors were examined. As expected, the fortified TNF-α and IL-6 levels in IL-1β-treated ATDC5 cells were both decreased by genkwanin in a dose-dependent manner [Figure 2]a and [Figure 2]b. Furthermore, genkwanin treatment cut down IL-1β-enhanced Cox-2, iNOS, and p-NFκB p65 expression in ATDC5 cells [Figure 2]c and [Figure 2]d. To sum up, genkwanin relieved IL-1β-evoked inflammatory response in ATDC5 cells.

Figure 2: Genkwanin administration mitigates IL-1β elicited inflammatory response of ATDC5 cells. (a and b) ELISA analysis of TNF-α and IL-6 levels. (c and d) Western blot analysis of Cox-2, iNOS, p-NFκB p65, and NFκB p65 expression. Data from three independent replicates were presented as mean ± SD. *P < 0.05, **P < 0.01, and ***P < 0.001. IL-1β: Interleukin-1 beta, ELISA: Enzyme-linked immunosorbent assay, TNF-α: Tumor necrosis factor-alpha, SD: Standard deviation.

Click here to view

Genkwanin administration eases IL-1β-evoked extracellular matrix degradation in ATDC5 cells

Meanwhile, as presented in [Figure 3]a, IL-1β lessened Collagen II expression in ATDC5 cells and genkwanin dose-dependently augmented Collagen II expression in IL-1β-exposed ATDC5 cells. Western blot analysis also manifested that the augmented MMP13, ADAMTS-5 expression, and the declined aggrecan expression in IL-1β-challenged ATDC5 cells were reversed by genkwanin in a concentration-dependent manner [Figure 3]b. On the whole, genkwanin played a suppressive role in IL-1β-stimulated extracellular matrix (ECM) degradation in ATDC5 cells.

Figure 3: Genkwanin administration eases IL-1β-evoked ECM degradation in ATDC5 cells. (a) IF staining tested Collagen II expression (×200). (b) Western blot analysis of MMP13, ADAMTS-5, and aggrecan expression. Data from three independent replicates were presented as mean ± SD. **P < 0.01 and ***P < 0.001. IL-1β: Interleukin-1 beta, ECM: Extracellular matrix, SD: Standard deviation, MMP: Matrix metallopeptidase.

Click here to view

Genkwanin treatment elevates DUSP1 expression in IL-1β-exposed ATDC5 cells

According to STITCH database, DUSP1 was discovered to serve as a potential target of genkwanin [Figure 4]a. Moreover, GEO database (GSE169077) demonstrated that DUSP1 expression was observably decreased in cartilage tissues of OA patients. Based on reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and Western blot, DUSP1 expression was found to be cut down in IL-1β-treated ATDC5 cells while was raised following treatment with increasing concentrations of genkwanin. The effect was the most prominent upon exposure to genkwanin at 50 μM [Figure 4]b and [Figure 4]c. Thence, 50 μM of genkwanin was selected for the ensuing experiments. Anyway, genkwanin was capable of enhancing DUSP1 expression in ATDC5 cells challenged with IL-1β.

Figure 4: Genkwanin treatment elevates DUSP1 expression in IL-1β-exposed ATDC5 cells. (a) STITCH database predicated that DUSP1 was a potential target of genkwanin. (b) RT-qPCR and (c) Western blot analysis of DUSP1 expression. Data from three independent replicates were presented as mean ± SD. **P < 0.01 and ***P < 0.001. IL-1β: Interleukin-1 beta, DUSP1: Dual-specificity protein phosphatase-1, RT-qPCR: Reverse transcription-quantitative polymerase chain reaction, SD: Standard deviation.

Click here to view

Genkwanin upregulates DUSP1 expression to alleviate IL-1β-triggered inflammatory response and ECM degradation in ATDC5 cells

To identify whether genkwanin participated in the biological events during the process of OA through regulating DUSP1 expression, DUSP1 was knocked down by the transduction of si-DUSP1 and the transduction efficacy was verified by RT-qPCR and Western blot [Figure 5]a and [Figure 5]b. ELISA and Western blot analysis elucidated that genkwanin lessened IL-1β-stimulated levels and expression of inflammatory factors, whereas this impact was abrogated after DUSP1 was depleted [Figure 5]c, [Figure 5]d, [Figure 5]e. Similarly, the augmented Collagen II, aggrecan expression, and the diminished MMP13, ADAMTS-5 expression imposed by genkwanin treatment in ATDC5 cells exposed to IL-1β were all reversed by declined DUSP1 expression [Figure 5]f and [Figure 5]g. Collectively, genkwanin alleviated IL-1β-triggered inflammatory response and ECM degradation in ATDC5 cells through elevating DUSP1 expression.

Figure 5: Genkwanin upregulates DUSP1 expression to alleviate IL-1β-triggered inflammatory response and ECM degradation in ATDC5 cells. (a and b) Interference efficiency of DUSP1. (c and d) ELISA analysis of TNF-α and IL-6 levels. (e) Western blot analysis of Cox-2, iNOS, p-NFκB p65, and NFκB p65 expression. (f) IF staining tested Collagen II expression (×200). (g) Western blot analysis of MMP13, ADAMTS-5, and aggrecan expression. Data from three independent replicates were presented as mean ± SD. *P < 0.05, **P < 0.01, and ***P < 0.001. IL-1β: Interleukin-1 beta. ELISA: Enzyme-linked immunosorbent assay, ECM: Extracellular matrix, DUSP1: Dual-specificity protein phosphatase-1, TNF-α: Tumor necrosis factor-alpha, SD: Standard deviation, MMP: Matrix metallopeptidase, Cox-2: Cyclooxygenase-2, iNOS: Inducible nitric oxide synthase, NFκB: nuclear factor kappa B.

Click here to view

Genkwanin elevates DUSP1 expression to ameliorate IL-1β-triggered mitochondrial dysfunction in ATDC5 cells

Subsequently, JC-1 staining demonstrated that genkwanin prominently protected against IL-1β-impaired mitochondrial membrane potential in ATDC5 cells, and this impact was countervailed by DUSP1 deficiency [Figure 6]a. Similarly, IL-1β-suppressed ATP level in ATDC5 cells was remarkably ascending due to genkwanin administration. Under this condition, ATP level was declined again following downregulation of DUSP1 [Figure 6]b. Besides, genkwanin greatly increased IL-1β-reduced mtDNA copy number, whereas this impact was offset after DUSP1 was silenced [Figure 6]c. Taken together, genkwanin opposed IL-1β-evoked mitochondrial dysfunction and mtDNA injury in ATDC5 cells through upregulating DUSP1.

Figure 6: Genkwanin elevates DUSP1 expression to ameliorate IL-1β-triggered mitochondrial dysfunction in ATDC5 cells. (a) JC-1 staining appraised mitochondrial membrane potential (×200). (b) ATP production was tested through corresponding assay kit. (c) mtDNA copy number was examined with RT-qPCR. Data from three independent replicates were presented as mean ± SD. **P < 0.01 and ***P < 0.001. IL-1β: Interleukin-1 beta, DUSP1: Dual-specificity protein phosphatase-1, RT-qPCR: Reverse transcription-quantitative polymerase chain reaction, JC-1: 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolocarbocyanine iodide, ATP: Adenosine triphosphate, mtDNA: Mitochondrial DNA, SD: Standard deviation.

Click here to view

Discussion

OA is viewed as the most frequently occurring degenerative disease of cartilage which is primarily attributed to inflammation-mediated cartilage degeneration.^[27] Inflammatory response is a major pathological reaction in the pathogenetic process of OA which may result in chondrocyte apoptosis as well as ECM degradation, further provoking the reduction in chondrocytes and homeostasis imbalance.^[28] Multiple clinical studies have corroborated that IL-1β possessed relative higher expression in OA patients than in healthy controls.^[29],[30] Meanwhile, as a proinflammatory factor, IL-1β is regarded as an essential inducer of OA.^[21] Thereafter, this work applied IL-1β to treat murine chondrogenic ATDC5 cells to construct in vitro OA model and then chondrocyte apoptosis, inflammatory response, together with ECM degradation were investigated to measure the development of OA.

Belonging to non-glycosylated flavonoids, genkwanin has been pointed out to protect against inflammatory response, plasmodium, free radicals as well as tumors.^{[13],[14],[31],[32]} As Xu et al. proposed, IL-1β has been corroborated to reduce the viability of chondrocytes during the process of OA.^[33] In conformity with this finding, this study also exposed that IL-1β exposure prominently declined ATDC5 cell viability, while genkwanin administration dose dependently improved the viability of IL-1β-treated ATDC5 cells. Conversely, the apoptosis of ATDC5 cells was accelerated upon exposure to IL-1β and genkwanin treatment remarkably suppressed the apoptosis of IL-1β-insulted ATDC5 cells in a dose-dependent manner. TNF-α is considered a major pro-inflammatory cytokine contributing to chondrocyte apoptosis and damaging cartilage matrix to aggravate OA.^[34] Furthermore, IL-6 is a kind of pro-inflammatory factor, the expression of which may reflect the course of OA.^[35] Cox-2 can be widely expressed under the circumstance of stimulation of inflammatory factors.^[36] Furthermore, under stimulation, excessive iNOS expressed by chondrocytes may induce apoptosis and inflammatory response.^[37] NFκB is central transcription factor which may be activated by TNF-α and IL-1β to participate in inflammatory and immune response.^[38] More importantly, NFκB signaling is activated in OA.^[39] Our experimental data delineated that IL-1β treatment prominently augmented TNF-α, IL-6 levels and Cox-2, iNOS, and p-NFκB p65 expression, which was consistent with the perspective held by Guo and Wu that IL-1β enhanced the expression of inflammatory markers to drive the progression of OA.^[40] Notably, the inhibitory role of genkwanin in inflammatory response has also been underlined in adjuvant-induced rat arthritis model and LPS-activated macrophages.^[14],[15] In the same way, the present study elaborated that IL-1β-elevated TNF-α, IL-6 levels and Cox-2, iNOS, and p-NFκB p65 expression were all diminished by genkwanin in a dose-dependent manner. ECM degradation is tightly associated with joint tissue injuries in OA.^[41] As components of ECM, Collagen II and aggrecan are critical to maintain the balance between the anabolism and catabolism of ECM.^[42] During the activated metabolism process of chondrocytes in OA progression, MMP13 is produced which is deemed as a driving factor of ECM degradation.^[43] ADAMTS-5 is a cartilage-degrading enzyme playing a significant role in ECM degradation.^[44] As expected, IL-1β has been supported to function as a contributor of ECM degradation in OA.^[45] The present study also clarified that the declined Collagen II and aggrecan expression and the augmented MMP13 and ADAMTS-5 expression in IL-1β-challenged ATDC5 cells were all reversed upon genkwanin administration.

According to STITCH and GEO database, DUSP1 is a potential target of genkwanin and is lowly expressed in cartilage tissues of OA patients. In addition, it is worth mentioning that DUSP1 blocks MAPK signaling to cut down the expression of OA-associated mediators.^[20] In the same way, DUSP1 expression was noted to be decreased in IL-1β-exposed ATDC5 cells, whereas genkwanin treatment dose dependently raised DUSP1 expression. More intriguingly, the descending TNF-α, IL-6 levels and Cox-2, iNOS, and p-NFκB p65 expression in genkwanin-administrated ATDC5 cells exposed to IL-1β were aggrandized again after DUSP1 was silenced. Similarly, DUSP1 interference also restored the ascending Collagen II, aggrecan expression, and the declined MMP13 and ADAMTS-5 expression imposed by genkwanin in IL-1β-challenged ATDC5 cells. It is acknowledged that mitochondria are implicated in the plenty of biochemical processes, and mitochondrial dysfunction is closely related to the pathogenesis of OA.^[46] At the same time, DUSP1 has been disclosed to relieve mitochondrial dysfunction.^[47] ATP which is frequently supplied by mitochondria is thought to be a source of chemical energy in organisms.^[48] Through investigation, it was observed that genkwanin prominently protected against IL-1β-impaired mitochondrial membrane potential in ATDC5 cells, and this impact was countervailed by DUSP1 knockdown. Besides, the reduced ATP production and mtDNA copy number in IL-1β-induced ATDC5 cells were increased by genkwanin treatment, and these impacts were offset by DUSP1 depletion. These results corresponded to the opinion put forward by Chen et al. that genkwanin might ameliorate mitochondrial dysfunction in a murine model of colitis.^[49]

Nevertheless, future studies are required to expound the role of genkwanin in OA in vivo using an OA animal model, and this is a limitation of the present study. It has been reported that IL-1β treatment downregulated the LC3II/LC3I ratio and the expression of beclin-1 and upregulated the expression of p62 in chondrocytes compared with the control group, suggesting that IL-1β could inhibit autophagy of chondrocytes.^[50],[51] Besides, IL-1β was demonstrated to increase the levels of reactive oxygen species (ROS), lipid ROS, lipid peroxidation end product malondialdehyde (MDA), and altered ferroptosis-related protein expression in chondrocytes, indicating the induction of ferroptosis by IL-1β in chondrocytes.^[52],[53] Therefore, our next experiments will also focus on whether genkwanin can regulate the autophagy and ferroptosis in IL-1β-induced chondrocytes.

Conclusion

To sum up, genkwanin mitigated IL-1β-mediated viability injury, apoptosis, inflammation, ECM degradation, and mitochondrial dysfunction in chondrocytes in OA through elevating DUSP1 expression. To our knowledge, this study is the first to definite the suppressive role of genkwanin in OA and presents a potential regulatory mechanism involving DUSP1 in OA. Overall, our research may provide efficient therapeutic modalities for OA.

Data availability

No data were used for the research described in the article. Data will be made available on request. All data are within the manuscript and figure.

Author contributions

Conception and design: Zhongqing Wu and Kanna Xu. Administrative support: Zhongqing Wu. Provision of study materials or patients: Kanna Xu. Collection and assembly of data: Kanna Xu and Haoran Wang. Data analysis and interpretation: Kanna Xu. Manuscript writing: All authors. Final approval of manuscript: All authors.

Financial support and sponsorship

This study was supported by the Project of Zhejiang Administration of Traditional Chinese Medicine (2020ZA112).

Conflicts of interest

There are no conflicts of interest.

References

1.	Ebell MH. Osteoarthritis: Rapid evidence review. Am Fam Physician 2018;97:523-6.
2.	Hawker GA, King LK. The burden of osteoarthritis in older adults. Clin Geriatr Med 2022;38:181-92.
3.	Nedunchezhiyan U, Varughese I, Sun AR, Wu X, Crawford R, Prasadam I. Obesity, inflammation, and immune system in osteoarthritis. Front Immunol 2022;13:907750.
4.	Vina ER, Kwoh CK. Epidemiology of osteoarthritis: Literature update. Curr Opin Rheumatol 2018;30:160-7.
5.	Sharma L. Osteoarthritis of the knee. N Engl J Med 2021;384:51-9.
6.	D'Arcy Y, Mantyh P, Yaksh T, Donevan S, Hall J, Sadrarhami M, et al. Treating osteoarthritis pain: Mechanisms of action of acetaminophen, nonsteroidal anti-inflammatory drugs, opioids, and nerve growth factor antibodies. Postgrad Med 2021;133:879-94.
7.	Saltzman BM, Leroux TS, Verma NN, Romeo AA. Glenohumeral osteoarthritis in the young patient. J Am Acad Orthop Surg 2018;26:e361-70.
8.	Nian L, Zhang P, Liu X, Zhang H, Zhang Y, Li Q, et al. Application of the concept of accelerated rehabilitation surgery in rehabilitation nursing of patients with knee osteoarthritis after total knee arthroplasty. Chin J Med 2022;57:575-7.
9.	Williams A, Dunning T, Manias E. Continuity of care and general wellbeing of patients with comorbidities requiring joint replacement. J Adv Nurs 2007;57:244-56.
10.	Li XZ, Zhang SN. Recent advance in treatment of osteoarthritis by bioactive components from herbal medicine. Chin Med 2020;15:80.
11.	Yang L, Wu BY, Ma L, Li ZD, Xiong H. Comparative efficacy and safety of Chinese herbal medicine for knee osteoarthritis: A protocol for systematic review and network meta-analysis. Medicine (Baltimore) 2021;100:e26671.
12.	Kai H, Koine T, Baba M, Okuyama T. Pharmacological effects of Daphne genkwa and Chinese medical prescription, “Jyu-So-To”. Yakugaku Zasshi 2004;124:349-54.
13.	Wang X, Song ZJ, He X, Zhang RQ, Zhang CF, Li F, et al. Antitumor and immunomodulatory activity of genkwanin on colorectal cancer in the APC(Min/+) mice. Int Immunopharmacol 2015;29:701-7.
14.	Gao Y, Liu F, Fang L, Cai R, Zong C, Qi Y. Genkwanin inhibits proinflammatory mediators mainly through the regulation of miR-101/MKP-1/MAPK pathway in LPS-activated macrophages. PLoS One 2014;9:e96741.
15.	Bao Y, Sun YW, Ji J, Gan L, Zhang CF, Wang CZ, et al. Genkwanin ameliorates adjuvant-induced arthritis in rats through inhibiting JAK/STAT and NF-κB signaling pathways. Phytomedicine 2019;63:153036.
16.	Lawan A, Shi H, Gatzke F, Bennett AM. Diversity and specificity of the mitogen-activated protein kinase phosphatase-1 functions. Cell Mol Life Sci 2013;70:223-37.
17.	Lawan A, Zhang L, Gatzke F, Min K, Jurczak MJ, Al-Mutairi M, et al. Hepatic mitogen-activated protein kinase phosphatase 1 selectively regulates glucose metabolism and energy homeostasis. Mol Cell Biol 2015;35:26-40.
18.	Moosavi SM, Prabhala P, Ammit AJ. Role and regulation of MKP-1 in airway inflammation. Respir Res 2017;18:154.
19.	Low HB, Zhang Y. Regulatory roles of MAPK phosphatases in cancer. Immune Netw 2016;16:85-98.
20.	Peng HZ, Yun Z, Wang W, Ma BA. Dual specificity phosphatase 1 has a protective role in osteoarthritis fibroblast-like synoviocytes via inhibition of the MAPK signaling pathway. Mol Med Rep 2017;16:8441-7.
21.	Jenei-Lanzl Z, Meurer A, Zaucke F. Interleukin-1β signaling in osteoarthritis – Chondrocytes in focus. Cell Signal 2019;53:212-23.
22.	Zhou J, Wang Q. Daphnoretin relieves IL-1β-mediated chondrocytes apoptosis via repressing endoplasmic reticulum stress and NLRP3 inflammasome. J Orthop Surg Res 2022;17:487.
23.	Wang BW, Jiang Y, Yao ZL, Chen PS, Yu B, Wang SN. Aucubin protects chondrocytes against IL-1β-induced apoptosis in vitro and inhibits osteoarthritis in mice model. Drug Des Devel Ther 2019;13:3529-38.
24.	Zhao Z, Liu W, Cheng G, Dong S, Zhao Y, Wu H, et al. Knockdown of DAPK1 inhibits IL-1β-induced inflammation and cartilage degradation in human chondrocytes by modulating the PEDF-mediated NF-kB and NLRP3 inflammasome pathway. Innate Immun 2022; 17534259221086837.
25.	Yang G, Wang Y, Chen Y, Huang R. UFL1 attenuates IL-1β-induced inflammatory response in human osteoarthritis chondrocytes. Int Immunopharmacol 2020;81:106278.
26.	Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) method. Methods 2001;25:402-8.
27.	Greene MA, Loeser RF. Aging-related inflammation in osteoarthritis. Osteoarthritis Cartilage 2015;23:1966-71.
28.	Ryu JH, Shin Y, Huh YH, Yang S, Chun CH, Chun JS. Hypoxia-inducible factor-2α regulates Fas-mediated chondrocyte apoptosis during osteoarthritic cartilage destruction. Cell Death Differ 2012;19:440-50.
29.	Wang F, Liu J, Chen X, Zheng X, Qu N, Zhang B, et al. IL-1β receptor antagonist (IL-1Ra) combined with autophagy inducer (TAT-Beclin1) is an effective alternative for attenuating extracellular matrix degradation in rat and human osteoarthritis chondrocytes. Arthritis Res Ther 2019;21:171.
30.	Xie P, Dan F, Yu G, Ruan W, Yu H. Laquinimod mitigated IL-1β-induced impairment of the cartilage extracellular matrix in human ATDC5 chondrocytes. Chem Res Toxicol 2020;33:933-9.
31.	Zhang CF, Zhang SL, He X, Yang XL, Wu HT, Lin BQ, et al. Antioxidant effects of Genkwa flos flavonoids on Freund's adjuvant-induced rheumatoid arthritis in rats. J Ethnopharmacol 2014;153:793-800.
32.	Köhler I, Jenett-Siems K, Siems K, Hernández MA, Ibarra RA, Berendsohn WG, et al. In vitro antiplasmodial investigation of medicinal plants from El Salvador. Z Naturforsch C J Biosci 2002;57:277-81.
33.	Xu J, Pei Y, Lu J, Liang X, Li Y, Wang J, et al. LncRNA SNHG7 alleviates IL-1β-induced osteoarthritis by inhibiting miR-214-5p-mediated PPARGC1B signaling pathways. Int Immunopharmacol 2021;90:107150.
34.	Khotib J, Utami NW, Gani MA, Ardianto C. The change of proinflammatory cytokine tumor necrosis factor α level in the use of meloxicam in rat model of osteoarthritis. J Basic Clin Physiol Pharmacol 2019;30:20190331.
35.	Hou SM, Chen PC, Lin CM, Fang ML, Chi MC, Liu JF. CXCL1 contributes to IL-6 expression in osteoarthritis and rheumatoid arthritis synovial fibroblasts by CXCR2, c-Raf, MAPK, and AP-1 pathway. Arthritis Res Ther 2020;22:251.
36.	Cui J, Jia J. Natural COX-2 inhibitors as promising anti-inflammatory agents: An update. Curr Med Chem 2021;28:3622-46.
37.	Leonidou A, Lepetsos P, Mintzas M, Kenanidis E, Macheras G, Tzetis M, et al. Inducible nitric oxide synthase as a target for osteoarthritis treatment. Expert Opin Ther Targets 2018;22:299-318.
38.	Liu T, Zhang L, Joo D, Sun SC. NF-κB signaling in inflammation. Signal Transduct Target Ther 2017;2:17023.
39.	Cao Y, Tang S, Nie X, Zhou Z, Ruan G, Han W, et al. Decreased miR-214-3p activates NF-κB pathway and aggravates osteoarthritis progression. EBioMedicine 2021;65:103283.
40.	Guo X, Wu Z. GABARAP ameliorates IL-1β-induced inflammatory responses and osteogenic differentiation in bone marrow-derived stromal cells by activating autophagy. Sci Rep 2021;11:11561.
41.	Ratneswaran A, Rockel JS, Kapoor M. Understanding osteoarthritis pathogenesis: A multiomics system-based approach. Curr Opin Rheumatol 2020;32:80-91.
42.	Nielsen FK, Egund N, Jørgensen A, Jurik AG. Risk factors for joint replacement in knee osteoarthritis; a 15-year follow-up study. BMC Musculoskelet Disord 2017;18:510.
43.	Chow YY, Chin KY. The role of inflammation in the pathogenesis of osteoarthritis. Mediators Inflamm 2020;2020:8293921.
44.	Yang CY, Chanalaris A, Troeberg L. ADAMTS and ADAM metalloproteinases in osteoarthritis – Looking beyond the 'usual suspects'. Osteoarthritis Cartilage 2017;25:1000-9.
45.	Lü G, Li L, Wang B, Kuang L. LINC00623/miR-101/HRAS axis modulates IL-1β-mediated ECM degradation, apoptosis and senescence of osteoarthritis chondrocytes. Aging (Albany NY) 2020;12:3218-37.
46.	Blanco FJ, Fernández-Moreno M. Mitochondrial biogenesis: A potential therapeutic target for osteoarthritis. Osteoarthritis Cartilage 2020;28:1003-6.
47.	Lu C, Wu B, Liao Z, Xue M, Zou Z, Feng J, et al. DUSP1 overexpression attenuates renal tubular mitochondrial dysfunction by restoring Parkin-mediated mitophagy in diabetic nephropathy. Biochem Biophys Res Commun 2021;559:141-7.
48.	Annesley SJ, Fisher PR. Mitochondria in health and disease. Cells 2019;8:680.
49.	Chen Z, He Y, Hu F, Li M, Yao Y. Genkwanin alleviates mitochondrial dysfunction and oxidative stress in a murine model of experimental colitis: The participation of Sirt1. Ann Clin Lab Sci 2022;52:301-13.
50.	Li Z, Cheng J, Liu J. Baicalin protects human OA chondrocytes against IL-1β-induced apoptosis and ECM degradation by activating autophagy via MiR-766-3p/AIFM1 axis. Drug Des Devel Ther 2020;14:2645-55.
51.	Li J, Jiang M, Yu Z, Xiong C, Pan J, Cai Z, et al. Artemisinin relieves osteoarthritis by activating mitochondrial autophagy through reducing TNFSF11 expression and inhibiting PI3K/AKT/mTOR signaling in cartilage. Cell Mol Biol Lett 2022;27:62.
52.	Guo Z, Lin J, Sun K, Guo J, Yao X, Wang G, et al. Deferoxamine alleviates osteoarthritis by inhibiting chondrocyte ferroptosis and activating the Nrf2 pathway. Front Pharmacol 2022;13:791376.
53.	Xu W, Zhang B, Xi C, Qin Y, Lin X, Wang B, et al. Ferroptosis plays a role in human chondrocyte of osteoarthritis induced by IL-1β in vitro. Cartilage 2023. Doi: 10.1177/19476035221142011.