Icariin Regulates Cellular Functions and Gene Expression of Osteoarthritis Patient-Derived Human Fibroblast-Like Synoviocytes

Lianhong Pan, Yonghui Zhang, Na Chen, Li Yang, Lianhong Pan, Yonghui Zhang, Na Chen, Li Yang

Abstract

Synovial inflammation plays an important role in the pathogenesis and progress of osteoarthritis (OA). There is an urgent need to find safe and effective drugs that can reduce the inflammation and regulate the pathogenesis of cytokines of the OA disease. Here, we investigated the effect of icariin, the major pharmacological active component of herb Epimedium on human osteoarthritis fibroblast-like synoviocytes (OA-FLSs). The OA-FLSs were isolated from patients with osteoarthritis and cultured in vitro with different concentrations of icariin. Then, cell viability, proliferation, and migration were investigated; MMP14, GRP78, and IL-1β gene expression levels were detected via qRT-PCR. Icariin showed low cytotoxicity to OA-FLSs at a concentration of under 10 μM and decreased the proliferation of the cells at concentrations of 1 and 10 μM. Icariin inhibited cell migration with concentrations ranging from 0.1 to 1 μM. Also, the expression of three cytokines for the pathogenesis of OA which include IL-1β, MMP14 and GRP78 was decreased by the various concentrations of icariin. These preliminary results imply that icariin might be an effective compound for the treatment of OA disease.

Keywords: GRP78; MMP14; OA–FLSs; anti-inflammation; icariin; osteoarthritis.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Effects of icariin on cell viability of osteoarthritis fibroblast-like synoviocytes (OA–FLSs). Cells were incubated with different concentrations of icariin for 12 h; cell metabolic activity and proliferation were measured by MTS assay. * p < 0.05 were accepted as statistically significant (mean ± SD, n = 4).
Figure 2
Figure 2
The effect of different concentrations of icariin on cell proliferation of OA–FLSs for 12 h. (a) The OA–FLSs were treated with 0.1, 0.5, 1 μM and 10 μM of Icariin. The proliferation of cells was measured by ethynyl deoxyuridine (EdU) assay, EdU staining (red) and Hoechst 33342 staining (blue), scar bar: 200 μm. (b) The percentage of EdU+ cells based on (a) statistical difference when compared to the control (* p < 0.05) and significant difference when compared to the control (** p < 0.01) (mean ± SD, n = 4).
Figure 2
Figure 2
The effect of different concentrations of icariin on cell proliferation of OA–FLSs for 12 h. (a) The OA–FLSs were treated with 0.1, 0.5, 1 μM and 10 μM of Icariin. The proliferation of cells was measured by ethynyl deoxyuridine (EdU) assay, EdU staining (red) and Hoechst 33342 staining (blue), scar bar: 200 μm. (b) The percentage of EdU+ cells based on (a) statistical difference when compared to the control (* p < 0.05) and significant difference when compared to the control (** p < 0.01) (mean ± SD, n = 4).
Figure 3
Figure 3
The effect of different concentrations of icariin on cell migration of OA–FLSs for 12 h. (a) Cells were treated with 0.1, 0.5 and 1 μM of Icariin.The migrated cellst were stained with crystal violet. (b) Statistical analysis of the cell count for the migrated cells. Statistical difference when compared to the control (* p < 0.05), significant difference when compared to the control (** p < 0.01) (mean ± SD, n = 5). Scale bar: 100 μm.
Figure 4
Figure 4
Effects of icariin on MMP14, GRP78, and IL-1β expression in OA–FLSs. (a,b): The mRNA expression level of MMP14 (a) and GRP78 (b) of OA–FLSs with different concentrations of icariin treatment for 12 h. (c,d): The protein synthesis of MMP14 (c) and GRP78 (d) of OA–FLSs with different concentrations of icariin treatment for 12 h. Statistical difference when compared to the control (* p < 0.05), significant difference when compared to the control (** p < 0.01) (mean ± SD, n = 3). (e,f) Immunofluorescence of the MMP14 (e) and IL-1β (f) of OA–FLSs with different concentrations of icariin treatment for 12 h. Scale bar: 100 μm.

References

    1. Guermazi A., Roemer F.W., Burstein D., Hayashi D. Why radiography should no longer be considered a surrogate outcome measure for longitudinal assessment of cartilage in knee osteoarthritis. Arthritis Res. Ther. 2011;13:247. doi: 10.1186/ar3488.
    1. Heidari B. Knee osteoarthritis prevalence, risk factors, pathogenesis and features: Part I. Casp. J. Intern. Med. 2011;2:205–212.
    1. Benito M.J., Veale D.J., FitzGerald O., van den Berg W.B., Bresnihan B. Synovial tissue inflammation in early and late osteoarthritis. Ann. Rheum. Dis. 2005;64:1263–1267. doi: 10.1136/ard.2004.025270.
    1. Dziak R. Articular Cartilage and Osteoarthritis. Instr. Course Lect. 2005;54:465–480. doi: 10.1016/0169-6009(92)90848-8.
    1. Peter B., Novikoff A.B., David H. Electron Microscopy of the Human Synovial Membrane. J. Cell Biol. 1962;14:207–220.
    1. Pelletier J.P., Martel-Pelletier J., Abramson S.B. Osteoarthritis, an inflammatory disease: Potential implication for the selection of new therapeutic targets. Arthritis Rheumatol. 2001;44:1237–1247. doi: 10.1002/1529-0131(200106)44:6<1237::AID-ART214>;2-F.
    1. Smith M.D., Triantafillou S., Parker A., Youssef P.P., Coleman M. Synovial membrane inflammation and cytokine production in patients with early osteoarthritis. J. Rheumatol. 1997;24:365–371.
    1. Rollín R., Marco F., Jover J.A., García-Asenjo J.A., Rodríguez L., López-Durán L., Fernández-Gutiérrez B. Early lymphocyte activation in the synovial microenvironment in patients with osteoarthritis: Comparison with rheumatoid arthritis patients and healthy controls. Rheumatol. Int. 2008;28:757–764. doi: 10.1007/s00296-008-0518-7.
    1. Nair A., Kanda V., Bush-Joseph C., Verma N., Chubinskaya S., Mikecz K., Glant T.T., Malfait A.M., Crow M.K., Spear G.T., et al. Synovial fluid from patients with early osteoarthritis modulates fibroblast-like synoviocyte responses to toll-like receptor 4 and toll-like receptor 2 ligands via soluble CD14. Arthritis Rheumatol. 2012;64:2268–2277. doi: 10.1002/art.34495.
    1. Kloesch B., Liszt M., Krehan D., Broell J., Kiener H., Steiner G. High concentrations of hydrogen sulphide elevate the expression of a series of pro-inflammatory genes in fibroblast-like synoviocytes derived from rheumatoid and osteoarthritis patients. Immunol. Lett. 2012;141:197–203. doi: 10.1016/j.imlet.2011.10.004.
    1. Fu Z., Liu P., Yang D., Wang F., Yuan Y., Lin Z., Jiang J. Interleukin-18-induced inflammatory responses in synoviocytes and chondrocytes, from osteoarthritic patients. Int. J. Mol. Med. 2012;30:805–810. doi: 10.3892/ijmm.2012.1073.
    1. Pelletier J.P., Dibattista J.A., Roughley P., McCollum R., Martel-Pelletier J. Cytokines and inflammation in cartilage degradation. Rheum. Dis. Clin. N. Am. 1993;19:545–568.
    1. Goldring M.B. Osteoarthritis and cartilage: The role of cytokines. Curr. Rheumatol. Rep. 2000;2:459–465. doi: 10.1007/s11926-000-0021-y.
    1. Fernandes J.C., Martel-Pelletier J., Pelletier J.P. The role of cytokines in osteoarthritis pathophysiology. Biorheology. 2002;39:237–246.
    1. Hassanali S.H., Oyoo G.O. Osteoarthritis: A look at pathophysiology and approach to new treatments: A review. East Afr. Orthop. J. 2011;5:51–57.
    1. Hasegawa A., Nakahara H., Kinoshita M., Asahara H., Koziol J., Lotz M.K. Cellular and extracellular matrix changes in anterior cruciate ligaments during human knee aging and osteoarthritis. Arthritis Res. Ther. 2013;15:R29. doi: 10.1186/ar4165.
    1. Itoh Y., Seiki M. MT1-MMP: A potent modifier of pericellular microenvironment. J. Cell. Physiol. 2006;206:1–8. doi: 10.1002/jcp.20431.
    1. Petrow P.K., Wernicke D., Westhoff C.S., Hummel K.M., Bräuer R., Kriegsmann J., Gromnica-Ihle E., Gay R.E., Gay S. Characterisation of the cell type-specificity of collagenase 3 mRNA expression in comparison with membrane type 1 matrix metalloproteinase and gelatinase A in the synovial membrane in rheumatoid arthritis. Ann. Rheum. Dis. 2002;61:391–397. doi: 10.1136/ard.61.5.391.
    1. Kiener H.P., Watts G.F., Cui Y., Wright J., Thornhill T.S., Skold M., Behar S.M., Niederreiter B., Lu J., Cernadas M., et al. Synovial fibroblasts self-direct multicellular lining architecture and synthetic function in three-dimensional organ culture. Arthritis Rheumatol. 2010;62:742–752. doi: 10.1002/art.27285.
    1. Bánhegyi G., Baumeister P., Benedetti A., Dong D., Fu Y., Lee A.S., Li J., Mao C., Margittai E., Ni M., et al. Endoplasmic Reticulum Stress. Ann. N. Y. Acad. Sci. 2007;1113:58–71. doi: 10.1196/annals.1391.007.
    1. Kim R., Emi M., Tanabe K., Murakami S. Role of the unfolded protein response in cell death. Apoptosis. 2006;11:5–13. doi: 10.1007/s10495-005-3088-0.
    1. Feng L.J., Jiang T.C., Zhou C.Y., Yu C.L., Shen Y.J., Li J., Shen Y.X. Activated macrophage-like synoviocytes are resistant to endoplasmic reticulum stress-induced apoptosis in antigen-induced arthritis. Inflamm. Res. 2014;63:335–346. doi: 10.1007/s00011-013-0705-1.
    1. Sze S.C., Tong Y., Ng T.B., Cehng C.L., Cheung H.P. Herba epimedii: Anti-oxidative properties and its medical implications. Molecules. 2010;15:7861–7870. doi: 10.3390/molecules15117861.
    1. Xu C.Q., Liu B.J., Wu J.F., Xu Y.C., Duan X.H., Cao Y.X., Dong J.C. Icariin attenuates LPS-induced acute inflammatory responses: Involvement of PI3K/Akt and NF-κB signaling pathway. Eur. J. Pharmacol. 2010;642:146–153. doi: 10.1016/j.ejphar.2010.05.012.
    1. Hsieh T.P., Sheu S.Y., Sun J.S., Chen M.H. Icariin inhibits osteoclast differentiation and bone resorption by suppression of MAPKs/NF-κB regulated HIF-1α and PGE2 synthesis. Phytomedicine. 2011;18:176–185. doi: 10.1016/j.phymed.2010.04.003.
    1. Liu M.H., Sun J.S., Tsai S.W., Sheu S.Y., Chen M.H. Icariin protects murine chondrocytes from lipopolysaccharide-induced inflammatory responses and extracellular matrix degradation. Nutr. Res. 2010;30:57–65. doi: 10.1016/j.nutres.2009.10.020.
    1. De Lange-Brokaar B.J.E., Ioanfacsinay A., van Osch G.J.V.M., Zuurmond A.-M., Schoones J., Toes R.E.M., Huizinga T.W.J., Kloppenburg M. Synovial inflammation, immune cells and their cytokines in osteoarthritis: A review. Osteoarthr. Cartil. 2012;20:1484–1499. doi: 10.1016/j.joca.2012.08.027.
    1. Goldring M.B., Goldring S.R. Osteoarthritis. J. Cell. Physiol. 2007;213:626–634. doi: 10.1002/jcp.21258.
    1. Zwerina J., Redlich K., Polzer K., Joosten L., Krönke G., Distler J., Hess A., Pundt N., Pap T., Hoffmann O., et al. TNF-induced structural joint damage is mediated by IL-1. Proc. Natl. Acad. Sci. USA. 2007;104:11742–11747. doi: 10.1073/pnas.0610812104.
    1. Richardson D.W., Dodge G.R. Effects of interleukin-1β and tumor necrosis factor-α on expression of matrix-related genes by cultured equine articular chondrocytes. Am. J. Vet. Res. 2000;61:624–630. doi: 10.2460/ajvr.2000.61.624.
    1. Goldring M.B., Birkhead J., Sandell L.J., Kimura T., Krane S.M. Interleukin 1 suppresses expression of cartilage-specific types II and IX collagens and increases types I and III collagens in human chondrocytes. J. Clin. Investig. 1988;82:2026–2037. doi: 10.1172/JCI113823.
    1. Sadouk M.B., Pelletier J.P., Tardif G., Kiansa K., Cloutier J.M., Martel-Pelletier J. Human synovial fibroblasts coexpress IL-1 receptor type I and type II mRNA. The increased level of the IL-1 receptor in osteoarthritic cells is related to an increased level of the type I receptor. Lab. Investig. 1995;73:347–355.
    1. Johnson E.O., Charchandi A., Babis G.C., Soucacos P.N. Apoptosis in osteoarthritis: Morphology, mechanisms, and potential means for therapeutic intervention. J. Surg. Orthop. Adv. 2008;17:147–152.
    1. Zhang L., Zhang X., Li K.F., Li D.X., Xiao Y.M., Fan Y.J., Zahng X.D. Icariin promotes extracellular matrix synthesis and gene expression of chondrocytes in vitro. Phytother. Res. 2012;26:1385–1392. doi: 10.1002/ptr.3733.
    1. Xu C., Baillymaitre B., Reed J.C. Endoplasmic reticulum stress: Cell life and death decisions. Int. J. Endocrinol. Metab. 2005;115:2656–2664. doi: 10.1172/JCI26373.
    1. Rao R.V., Ellerby H.M., Bredesen D.E. Coupling endoplasmic reticulum stress to the cell death program. J. Biol. Chem. 2001;276:33869–33874. doi: 10.1074/jbc.M102225200.
    1. Liu C., Cao Y., Yang X., Shan P., Liu H. Tauroursodeoxycholic acid suppresses endoplasmic reticulum stress in the chondrocytes of patients with osteoarthritis. Int. J. Mol. Med. 2015;36:1081–1087. doi: 10.3892/ijmm.2015.2295.
    1. Wang Y., Tang Z., Xue R., Singh G.K., Lv Y., Shi K., Cai K., Deng L., Yang L. TGF-β1 promoted MMP-2 mediated wound healing of anterior cruciate ligament fibroblasts through NF-κB. Connect. Tissue Res. 2011;52:218–225. doi: 10.3109/03008207.2010.516849.
    1. Yan Y., Singh G.K., Zhang F., Wang P., Liu W., Zhong L., Yang L. Comparative study of normal and rheumatoid arthritis fibroblast-like synoviocytes proliferation under cyclic mechanical stretch: Role of prostaglandin E2. Connect. Tissue Res. 2012;53:246–254. doi: 10.3109/03008207.2011.632828.

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