Part 3:Acteoside Counteracts Interleukin-1β-Induced Catabolic Processes Through The Modulation Of Mitogen-Activated Protein Kinases And The NFκB Cellular Signaling Pathway

Acteoside Counteracts Interleukin-1β-Induced Catabolic Processes through the Modulation of Mitogen-Activated Protein Kinases and the NFκB Cellular Signaling Pathway

HyangI Lim ,1 Do Kyung Kim ,1 Tae-Hyeon Kim ,1 Kyeong-Rok Kang ,1 Jeong-Yeon Seo ,1,2 Seung Sik Cho ,3 Younghee Yun ,4,5 Ye-yong Choi ,4,5 Jungtae Leem ,4,5 Hyoun-Woo Kim ,6 Geon-Ung Jo ,6

Chan-Jin Oh ,6 Deuk-Sil Oh ,6 Hong-Sung Chun ,2 and Jae-Sung Kim 1

Contact: joanna.jia@wecistanche.com / WhatsApp: 008618081934791

1Institute of Dental Science, Chosun University, Gwangju 61452, Republic of Korea

2Departments of Biomedical Science, Chosun University, Gwangju 61452, Republic of Korea

3Department of Biomedicine, Health & Life Convergence Sciences, BK21 Four, College of Pharmacy, Mokpo National University,

Jeonnam 58554, Republic of Korea

4Chung-Yeon Medical Institute, Gwangju 61949, Republic of Korea

5Research and Development Institute, CY Pharma Co., Seoul 06224, Republic of Korea

6Jeollanamdo Forest Resources Institute, Naju, Jeollanamdo, 58213, Republic of Korea

Correspondence should be addressed to Jae-Sung Kim; js_kim@chosun.ac.kr

Received 2 July 2020; Revised 15 February 2021; Accepted 6 March 2021; Published 25 March 2021

Academic Editor: Joël R. Drevet

Copyright © 2021 HyangI Lim et al. This is an open-access article distributed under the CreativeCommonsAttributionLicense,

which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Osteoarthritis (OA) is the most common degenerative joint disease with chronic joint pain caused by progressive degeneration of articular cartilage at synovial joints. Acteoside, a caffeoylphenylethanoid glycoside, has various biological activities such as antimicrobial, anti-inflammatory, anticancer, antioxidative, cytoprotective, and neuroprotective effect. Further, oral administration of acteoside at a high dosage does not cause genotoxicity. Therefore, the aim of the present study is to verify the anticatabolic effects of acteoside against osteoarthritis and its anticatabolic signaling pathway. Acteoside did not decrease the viabilities of mouse fibroblast L929 cells used as normal cells and primary rat chondrocytes. Acteoside counteracted the IL-1β-induced proteoglycan loss in the chondrocytes and articular cartilage by suppressing the expression and activation of cartilage-degrading enzymes such as matrix metalloproteinase- (MMP-) 13, MMP-1, and MMP-3. Furthermore, acteoside suppressed the expression of inflammatory mediators such as inducible nitric oxide synthase, cyclooxygenase-2, nitric oxide, and prostaglandin E2 in the primary rat chondrocytes treated with IL-1β. Subsequently, the expression of proinflammatory cytokines was decreased by acteoside in the primary rat chondrocytes treated with IL-1β. Moreover, acteoside suppressed not only the phosphorylation of mitogen-activated protein kinases in primary rat chondrocytes treated with IL-1β but also the translocation of NFκB from the cytosol to the nucleus through suppression of its phosphorylation. Oral administration of 5 and 10mg/kg acteoside attenuated the progressive degeneration of articular cartilage in the osteoarthritic mouse model generated by destabilization of the medial meniscus. Our findings indicate that acteoside is a promising potential anticatabolic agent or supplement to attenuate or prevent progressive degeneration of articular cartilage.

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Elevated cartilage-degrading enzymes including MMP-1, MMP-3, MMP-13, ADMITS-4, and ADAMTS-5 in the synovial fluid of patients with OA are the key enzymes responsible for the progressive degeneration of articular cartilage through degradation of collagen and ECM component [26, 27]. Hence, the inhibition of MMP expression and activation seems to be an attractive therapeutic strategy to prevent and attenuate the progressive degeneration of articular cartilage for maintaining the mechanical function of synovial joints [26]. In the present study, acteoside effectively suppressed the expression and activation of the cartilage-degrading enzymes in the primary rat chondrocytes treated with proinflammatory cytokine IL-1β as shown in Figure 4. These data indicate that acteoside may attenuate the progressive degeneration of articular cartilage by suppressing the expression and activation of articular cartilage in the synovial joint with catabolic conditions.

The inflammatory mediators such as iNOS, NO, COX-2, and PGE2 are integral to OA pathogenesis [28]. Especially, proinflammatory cytokines such as IL-1β and TNFα upregulate the production of NO and PGE2 through the increase of iNOS and COX2, respectively, in the synovial joint with OA [29, 30]. Upregulated NO inhibits the synthesis of ECM components such as type II collagen and proteoglycan. Besides, increased PGE2 inhibits the proliferation of chondrocytes and reduces the synthesis of ECM [28]. Hence, suppression of inflammatory mediators may attenuate the progressive degeneration of articular cartilage through the inhibition of ECM reduction in the synovial joint with OA. In the present study, acteoside effectively suppressed the upregulation of inflammatory mediators as shown in Figure 5. These data indicate consistently that acteoside may attenuate the progressive degeneration of articular cartilage through the suppression of inflammatory mediators in the synovial joint with OA.

acteoside in cistanche can anti-inflammatory

Moreover, the overexpression of proinflammatory cytokines by the inflamed synovium and chondrocytes is a major risk pathogenic factor in OA pathogenesis. Especially, the expression of proinflammatory cytokine is thought to be generated by the synovial membrane at the stage of OA initiation. Sequentially, upregulated proinflammatory cytokines activate chondrocytes to express their own expression and to synthesize the cartilage-degrading enzymes, chemokines, and inflammatory mediators [31]. Therefore, the suppression of proinflammatory cytokines can prevent OA and may attenuate the progressive degeneration of articular cartilage through the inhibition of other proinflammatory cytokines, inflammatory mediators, and cartilage-degrading enzymes. In the present study, acteoside suppressed the production of proinflammatory cytokines such as CINC-2, CINC-3, CNTF, fractalkine, IL-1α, IL-1β, leptin, MCP-1, MIP-3α, and β-NGF in primary rat chondrocytes treated with IL-1β compared with IL-1β alone, as shown in Figure 6.

Gouze et al. reported that CINC-2 was significantly increased in chondrocytes treated with IL-1β similar to our study [32]. However, a recent study showed that spinal processing of painful inputs is closely altered during OA pathogenesis [33]. With regard to joint pain, CINC-2 and CINC-3 were significantly upregulated in the spinal dorsal horn of OA animals generated by the intra-articular injection of monosodium iodoacetate into the knee joint [34, 35]. Although the pathophysiological role of CINC-2 and CINC-3 in OA pathogenesis is still largely unknown, these studies indicate that the expression of CINC-2 and CINC-3 in the spinal dorsal horn under OA conditions may be closely associated with the development of joint pain during OA pathogenesis.

CNTF, which is a pluripotent neurotropic factor and is related with the cytokine family that includes IL-6, IL-11, leukemia inhibitory family, and oncostatin, binds and signals to maintain the bone homeostasis through the gp130 coreceptor subunit [36]. Although the biological function of CNTF is still largely unknown in OA, recent studies have shown that CNTF-gp130 signaling may be associated with the pathologic bone remodeling evident in rheumatoid arthritis (RA), periodontal disease, spondyloarthropathies, and OA through regulating the differentiation and activity of osteoblast, osteoclast, and chondrocytes [36]. In addition, a recent study showed that β-NGF, a neurotrophic factor involved with

the physiological regulation of neuronal cells was upregulated in blood and synovial fluid in patients with OA [37]. However, several studies have reported that the blockade of NGF reduces OA pain [38–40]. Therefore, neurotropic factors including CNTF and NGF not only are considered pathogenic risk factors of OA progression but also provide the neurological linkage between the progressive degeneration of articular cartilage and the development of chronic OA pain. Furthermore, it has been considered a therapeutic targeting molecule to reduce chronic OA pain.

Fractalkine also known as chemokine CX3CL1 is exuberantly expressed in both adult human and rat articular chondrocytes treated with IL-1β [41, 42]. Recent studies have reported that fractalkine promotes the expression of MMP- 3 through the CX3CR1, c-Raf, MEK, ERK, and NFκB cellular signaling pathways in the synovial tissue obtained from the patients with OA [43]. Furthermore, the genomic-wide DNA methylation analysis in OA chondrocytes revealed that the fractalkine gene was not only hypomethylated but also constantly correlated with its mRNA expression [44]. MCP-1, a member of the chemokine family to induce the inflammation, triggers the chemotaxis, and transendothelial migration of monocyte to inflammatory lesion. Recently, Xu et al., have reported that MCP-1 and chemokine (C-C motif) receptor 2 axis are involved with the degradation of articular cartilage through the expression of MMP-13 and the increase of OA chondrocyte apoptosis [45]. Furthermore, MIP-3α also called a chemokine CCL20 is abundantly expressed in the articular cartilage of patients with OA and increases the progressive degeneration of articular cartilage through the expression of cartilage-degrading enzymes such as MMP-1 and MMP-3, inflammatory mediator such as PGE2, and proinflammatory cytokine IL-6 [46]. Hence, chemokines such as fractalkine, MCP-1, and MIP-3α have been also considered a pathophysiological risk factor to initiate the progression of OA.

Leptin is a peptide hormone belonging to adipokines, which are cytokines secreted by adipose tissue [47]. Recent studies have reported that the level of leptin is not only elevated significantly in the human body with obesity but also increased in the serum and synovial fluid collected from the patients with OA that is correlated with the severity of OA [48]. Hence, recent studies have suggested that the expressions of leptin and its receptor have been considered positively as a risk factor associated with the development of OA [49–51]. [52] IL-1 family, including IL-1α and IL-1β, is considered the most key cytokine associated with the pathogenesis of OA that induces the inflammatory catabolic process combined with other catabolic factors such as aging, obesity, and traumatic joint injury [53]. Generally, the level of IL-1 family in the synovial fluid, synovial membrane, articular cartilage, and subchondral bone is elevated in the synovial joint of patients with OA [54]. After the IL-1 family binds onto their receptors, it manifests the progressive degeneration of articular cartilage by the expression of other cytokines, chemokines, adhesion molecules, inflammatory mediators, and cartilage-degrading enzymes through the phosphorylation of cellular signaling transcriptional factors such as the NFκB and MAPKs [54]. As shown in Figure 7, acteoside not only reduced the phosphorylation of ERK1/2, p38, and JNK but also inhibited the phosphorylation of NFκB in the primary rat chondrocytes treated with IL-1β. Moreover, Figure 8 shows that acteoside inhibited the translocation of NFκB from the cytosol to the nucleus in the primary rat chondrocytes treated with IL-1β. Therefore, our results consistently indicate that acteoside counteracts the IL-1β-induced catabolic effects such as the expression of cartilage-degrading enzymes and the production of proinflammatory cytokines and inflammatory mediators through the inactivation of cellular signaling pathways such as MAPK and NFκB in the primary rat chondrocytes. Recently, similar to our study, Qiao et al. have reported that acteoside inhibits inflammatory response in OA-induced animals [55]. They showed the suppression of inflammatory cytokines through the inactivation of the JAK/STAT signaling pathway in the synovial tissue of DMM-induced OA animals that were administered intraperitoneal injection of acteoside [55]. However, to estimate the effectiveness of acteoside as an OA preventive supplement, acteoside was orally administrated to DMM-induced OA animals in the present study. Thereafter, the alteration of articular cartilage was histologically assessed as shown in Figure 9. Our histological assessment showed that the oral administration of acteoside consistently prevented the progressive degeneration of articular cartilage through the inhibition of proteoglycan loss in DMM-induced OA animals.

5. Conclusions

Our findings suggest that acteoside is capable of oral administration and may be used as an effective supplement to prevent or attenuate OA based on the biological safety and anticatabolic effects against proinflammatory cytokines.

Data

Data Availability

The data used to support the findings of this study are available from the corresponding author upon request.

Ethical Approval

Primary rat chondrocytes were isolated from the articular cartilage of rat (5-day-old; Sprague–Dawley) knee joints, in accordance with the protocol (CIACUC2019-A0027) approved by the Institutional Animal Care and Use Committee of Chosun University, Gwangju, Republic of Korea. To generate osteoarthritic animals, the medial meniscus (DMM) was surgically destabilized in the knee joints of BALB/c mice (average body weight 19:3±0:5g) in accordance with IACUC guidelines (CIACUC2019-A0029).

Conflicts of Interest

The authors declare that there is no conflict of interest regarding the publication of this article.

Authors’ Contributions

HL, THK, KRK, JYS, HWK, and GUJ carried out the cell assay, ex vivo assay, in vivo assay using animal model, data preparation, and manuscript preparation. DKK, SSC, YY, YYC, JTL, CJO, DSO, and HSC carried out data interpretation, writing, review, and editing. JSK designed and carried out supervision, investigation, formal analysis, original draft, and writing, review, and editing. Hyang Lim and Do Kyung Kim contributed equally to this study.

Acknowledgments

This study was supported by the Korean Forestry Promotion Institute (2019141A00-1921-AB02), Republic of Korea.

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