Korean J Physiol Pharmacol.  2020 May;24(3):249-257. 10.4196/kjpp.2020.24.3.249.

Oxysterol 25-hydroxycholesterol as a metabolic pathophysiological factors of osteoarthritis induces apoptosis in primary rat chondrocytes

• Seo YS
• Cho IA
• Kim TH
• You JS
• Oh JS
• Lee GJ
• Kim DK
• Kim JS

• KMID: 2502507
• DOI: http://doi.org/10.4196/kjpp.2020.24.3.249

Abstract

The aim of the present study was to investigate the pathophysiological etiology of osteoarthritis that is mediated by the apoptosis of chondrocytes exposed to 25-hydroxycholesterol (25-HC), an oxysterol synthesized by the expression of cholesterol-25-hydroxylase (CH25H) under inflammatory conditions. Interleukin-1β induced the apoptosis of chondrocytes in a dose- dependent manner. Furthermore, the production of 25-HC increased in the chondrocytes treated with interleukin-1β through the expression of CH25H. 25-HC decreased the viability of chondrocytes. Chondrocytes with condensed nucleus and apoptotic populations increased by 25- HC. Moreover, the activity and expression of caspase-3 were increased by the death ligand-mediated extrinsic and mitochondria-dependent intrinsic apoptotic pathways in the chondrocytes treated with 25-HC. Finally, 25-HC induced not only caspasedependent apoptosis, but also induced proteoglycan loss in articular cartilage ex vivo cultured rat knee joints. These data indicate that 25-HC may act as a metabolic pathophysiological factor in osteoarthritis that is mediated by progressive chondrocyte death in the articular cartilage with inflammatory condition.

Keyword

Apoptosis; Cholesterol; Chondrocytes; Osteoarthritis; 25-Hydroxycholesterol

Figure

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  Fig. 1 Interleukin (IL)-1β-induced apoptotic chondrocyte death is involved partially with cholesterol metabolism. (A) IL-1β decreases the number of live chondrocytes. Primary rat chondrocytes treated with IL-1β for 24 h was stained using Cell Live/Dead assay kit composed of green calcein AM for labeling live cells and ethidium homodimer-1 for labeling dead cells. Thereafter, cells were imaged using a fluorescence microscope at ×100 magnification. (B) Viability of chondrocytes decreased in a dose-dependent manner following IL-1β treatment. (C) IL-1β-induced chondrocyte death is mediated by apoptosis. (D, E) mRNA levels of cholesterol-25-hydroxylase (CH25H) and CYP7B1 were significantly induced in the chondrocytes. (F) The expression of CH25H was increased in the chondrocytes treated with IL-1β in a dose-dependent manner. (G) The production of 25-hydroxycholesterol (25-HC) increased dose-dependently following IL-1β treatment. (H) Desmosterol, a potential inhibitor of CH25H, counteracted the IL-1β-induced chondrocyte apoptosis. *p < 0.05 and **p < 0.01 compared to non-treated.

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  Fig. 2 25-Hydroxycholesterol (25-HC) induces the apoptotic chondrocytes death. (A) 25-HC decreases the viability of chondrocytes. (B) The survival of chondrocytes was reduced by 25-HC. Primary rat chondrocytes treated with 25-HC for 24 h was stained using Cell Live/Dead assay kit composed of green calcein AM for labeling live cells and ethidium homodimer-1 for labeling dead cells. Thereafter, cells were imaged using a fluorescence microscope at ×100 magnification. (C) DAPI staining was performed to stain the nucleus of primary rat chondrocytes treated with 25-HC for 24 h. Thereafter, cells were imaged using a fluorescence microscope at X100 magnification. (D) The apoptotic population of chondrocytes increased by 25-HC. *p < 0.05 and **p < 0.01 compared to non-treated.

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  Fig. 3 25-Hydroxycholesterol (25-HC)-induced chondrocyte death is mediated by death receptor-mediated extrinsic and mitochondria-dependent apoptosis.

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  Fig. 4 25-hydroxycholesterol (25-HC) not only induced caspase-dependent apoptosis, but also induced proteoglycan loss in articular cartilage. (A) 25-HC increased the expression (upper panel) and activation (lower panel) of caspase-3 in chondrocytes. Immunocytochemostry using caspase-3 antibody (upper) and caspase-3/-7 activity staining (lower) using cell-permeable fluorogenic substrate PhiPhiLux-G1D2 was performed to verify the expression of caspase-3 and the activation of caspase-3-7, respectively, in primary rat chondrocytes treated with 25-HC for 24 h. Thereafter, cells were imaged using a fluorescence microscope at ×200 magnification. Red arrow indicate a cell positive for caspase-3. (B) Z-VAD-fmk, a pan-caspase inhibitor, counteracts 25-HC-cell death in chondrocytes. (C) Z-VAD-fmk suppresses the expression of caspase-3 in chondrocytes treated with 25-HC. (D) 25-HC induced the proteoglycan loss and the expression of caspase-3 in articular cartilage. Safranin-O & fast green staining (upper) and immunohistochemistry (lower) using caspase-3 were performed to verify the loss of proteoglycan and the expression of caspase-3, respectively, in the articular cartilage of rat knee joint. Thereafter, tissues were imaged using a fluorescence microscope at ×100 magnification. *p < 0.05 and **p < 0.01 compared to non-treated.

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