Purpose: Superoxide dismutase 2 (SOD2) is localized in mitochondrial matrix to regulate mitochondrial superoxide. Accumulating evidence demonstrated that SOD2 expression is significantly down-regulated in the articular cartilage of osteoarthritis (OA) patients. We previously concluded that genetically-manipulated SOD2 imbalance in chondrocytes results in cartilage degeneration under mechanical overloading, suggesting that mitochondrial redox balance in chondrocytes plays a pivotal role in maintaining healthy cartilage. However, therapeutic approaches for OA progression focused on mitochondrial function of chondrocytes have not been fully accomplished yet. We previously demonstrated that oral treatment of apple polyphenols (AP) significantly extended lifespan and attenuated cardiac phenotypes in heart-specific Sod2-deficient mice. In this context, we here investigated its protective effects on cartilage of chondrocyte-specific Sod2-deficient(chondrocyte-Sod2-/-) mice.
Methods: Chondrocyte-Sod2-/- mice were generated by crossbreeding with Col2a1-Cre and Sod2fl/fl mice. Destabilization of medial meniscus (DMM) was created by surgical transection of the medial meniscotibial ligament under a microscope in knee joints of chondrocyte-Sod2-/- and Sod2fl/fl mice at eight weeks of age. AP (500 mg/kg) and procyanidin B2 (100 mg/kg), a major component of AP, were daily administered to chondrocyte-Sod2-/- and Sod2fl/fl mice for 8 weeks, and then OA pathologies of knee joints were histologically evaluated using the modified OARSI histopathology grade. To clarify the biological effect of AP for Sod2-/- chondrocytes in vitro, cellular phenotypes were evaluated in primary articular chondrocytes isolated from knee joints of neonate chondrocyte-Sod2-/- and Sod2fl/fl littermates. Cell viability was measured by a MTT assay. Using a flow cytometry, mitochondrial superoxide production and membrane potential were investigated with MitoSox and JC-1 stainings, respectively. In addition, expression profiles of anabolic-, catabolic-, and mitochondrial biogenesis-related genes were analyzed by qRT-PCR. Copy number of mitochondrial DNA was also measured by genomic PCR. Furthermore, proteoglycan levels were quantified with alcian blue staining.
Results: Histological analyses revealed that oral administration of AP significantly attenuated cartilage degeneration in the medial side of femoral condyle and tibial plateau of DMM-treated mutant mice. Moreover, oral treatment of isolated procyanidin B2 from AP also protected cartilage erosion of the DMM-treated mutant mice. Interestingly, AP and procyanidin B2 treatments did not modify body weight and locomotive activity in these mice, suggesting that AP treatment directly regulated cartilage maintenance in vivo. In vitro experiments unveiled that AP significantly promoted cell viability and proteoglycan synthesis associated with up-regulation of Aggrecan gene in Sod2-/- chondrocytes. Furthermore, AP also positively regulated mitochondrial functions with improving mitochondrial depolarization and increasing copy number of mitochondrial DNA, concomitant with up-regulation of Pgc-1α gene in Sod2-/- chondrocytes.
Conclusions: AP effectively suppressed OA progression of chondrocyte-Sod2-/- mice under an experimental-overloading condition. AP also promoted cellular function, such as viability and proteoglycan synthesis, associated with enhancing mitochondrial biogenesis in Sod2-/- chondrocytes. Thus, AP might be a healthy food material against OA progression by operating the mitochondrial condition of chondrocytes.
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