Abstract
BackgroundChondrocyte apoptosis activated by the mitochondrial dependent pathway serves a crucial role in cartilage degeneration of osteoarthritis (OA). In the present study, the protective effects of CMCS against sodium nitroprusside (SNP)-induced chondrocyte apoptosis were evaluated and the underlying molecular mechanisms were elucidated.MethodsChondrocytes were isolated from articular cartilage of SD rats and identified by type II collagen immunohistochemistry. The chondrocytes stimulated with or without SNP to induce apoptosis, were treated by CMCS for various concentrations. The cell viability were determined by MTT and LDH assays. Cell apoptotic ratio was determined by Annexin V-FITC/PI staining. Mitochondrial membrane potential (ΔΨm) was detected by using Rhodamine123 (Rho123) staining. To understand the mechanism, the mRNA expression levels of Bcl-2, Bax, cytochrome c (Cyt c) and cleaved caspase-3 were detected by real-time PCR and western blot analysis, respectively.ResultsIt was shown using the MTT and LDH assays that CMCS protected the viability of chondrocyte against SNP damage. Annexin V-FITC/PI and Rho123 staining showed that CMCS not only inhibited the cell apoptosis but also restored the reduction of the ΔΨm in chondrocytes. In SNP-induced chondrocytes, CMCS down-regulated the expression of Bax, Cyt c and cleaved caspase-3 but upregulated the expression of Bcl-2, as shown by real-time PCR and western blot.ConclusionsTaken together, these results indicated that CMCS has the protective effect on chondrocytes against SNP-induced apoptosis, at least partly, via inhibiting the mitochondrial dependent apoptotic pathway. Thus, CMCS may be potentially used as a biological agent for prevention and treatment of OA.
Highlights
Chondrocyte apoptosis activated by the mitochondrial dependent pathway serves a crucial role in cartilage degeneration of osteoarthritis (OA)
Anti-B-cell lymphoma 2 (Bcl-2) (Cat# 2870), anti-Bcl-2 associated X protein (Bax) (Cat# 5023), anti-cytochrome c (Cyt c) (Cat# 11940) and anti-cleaved caspase-3 (Cat# 9579) antibodies were purchased from Cell Signaling Technology, Inc. (USA). horseradish peroxidase (HRP) conjugated mouse anti-rabbit IgG (Cat# sc2357), anti-collagen type-2 (Cat# sc-52,658) and βactin (Cat# sc-47,778) antibodies were purchased from Santa Cruz Biotechnology, Inc. (USA)
The MTT results showed that sodium nitroprusside (SNP) could inhibit cell viability in cultured chondrocytes with a concentration dependent manner, 3.0~4.0 mM SNP treated cells have the maximum inhibitory response, there was no significant difference between 3.0 and 4.0 mM SNP-treated groups (Fig. 2a)
Summary
Chondrocyte apoptosis activated by the mitochondrial dependent pathway serves a crucial role in cartilage degeneration of osteoarthritis (OA). Osteoarthritis (OA) is characterized by degeneration of the articular cartilage and is a major cause of joint dysfunction in the elderly population, is one of the most common chronic disease, affecting an estimated 10% of man and 18% of women over the age of 60 years [1]. Numerous studies have suggested that the main cause of OA is an excessive apoptosis related loss of chondrocytes and degeneration of the. He et al BMC Complementary Medicine and Therapies (2020) 20:23 elements involves in these pathways is the activation of caspase-3 by cytochrome c (Cyt c) that is induced in mitochondria signaling pathway. We have used CMCS in cultured chondrocytes and found CMCS has the inhibitory effect on NO-induced apoptosis in cultured chondrocytes [18, 19]
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