Abstract
Oxidative stress-related apoptosis and autophagy play crucial roles in the development of osteoarthritis (OA), a progressive cartilage degenerative disease with multifactorial etiologies. Here, we determined autophagic flux changes and apoptosis in human OA and tert-Butyl hydroperoxide (TBHP)-treated chondrocytes. In addition, we explored the potential protective effects of trehalose, a novel Mammalian Target of Rapamycin (mTOR)-independent autophagic inducer, in TBHP-treated mouse chondrocytes and a destabilized medial meniscus (DMM) mouse OA model. We found aberrant p62 accumulation and increased apoptosis in human OA cartilage and chondrocytes. Consistently, p62 and cleaved caspase-3 levels increased in mouse chondrocytes under oxidative stress. Furthermore, trehalose restored oxidative stress-induced autophagic flux disruption and targeted autophagy selectively by activating BCL2 interacting protein 3 (BNIP3) and Phosphoglycerate mutase family member 5 (PGAM5). Trehalose could ameliorate oxidative stress-mediated mitochondrial membrane potential collapse, ATP level decrease, dynamin-related protein 1 (drp-1) translocation into the mitochondria, and the upregulation of proteins involved in mitochondria and endoplasmic reticulum (ER) stress-related apoptosis pathway. In addition, trehalose suppressed the cleavage of caspase 3 and poly(ADP-ribose) polymerase (PARP) and prevented DNA damage under oxidative stress. However, the anti-apoptotic effects of trehalose in TBHP-treated chondrocytes were partially abolished by autophagic flux inhibitor chloroquine and BNIP3- siRNA. The protective effect of trehalose was also found in mouse OA model. Taken together, these results indicate that trehalose has anti-apoptotic effects through the suppression of oxidative stress-induced mitochondrial injury and ER stress which is dependent on the promotion of autophagic flux and the induction of selective autophagy. Thus, trehalose is a promising therapeutic agent for OA.
Highlights
Osteoarthritis (OA) is the most prevalent form of degenerative joint disease characterized by articular cartilage degeneration, joint pain and functional impairment, and affects millions of people worldwide.[1,2] The etiology of OA is multifactorial, but several pathological factors such as inflammatory cytokines, mechanical stress and senescence lead to increasing levels of reactive oxygen species (ROS).[3]
By CCK8 assay and western blot analysis we found that Tert-Butyl hydroperoxide (TBHP) decreased the viability of mouse chondrocytes (Figure 1d), and induced the accumulation of p62 and the cleavage of caspase 3 in a dose- and time-dependent manner (Figures 1e–h)
Our study demonstrated the accumulation of p62 in the cartilage and chondrocytes derived from patients with severe OA compared to controls
Summary
Blocked autophagic flux and increased apoptosis in human OA and TBHP-treated mouse chondrocytes. Western blot analysis revealed that TBHP-induced increased levels of cleaved caspase 3, cytochrome C (Cyt C, marker of Mitochondria dysfunction) and C/EBP Homologous Protein (CHOP, marker of ER-stress) in chondrocytes were antagonized by trehalose pretreatment in a dose-dependent manner (Figures 1j and k). To determine whether apoptosis is attenuated by trehalose-induced autophagic flux activation, we examined the levels of cleaved caspase-3 and PARP by western blot analysis (Figures 6a and b) and DNA damage using the tunel method (Figure 6c and d). The cartilage of OA group showed elevated positive staining for 8OHdG, p62, Cyt C, CHOP and cleaved caspase 3 compared to sham group while trehalose administration markedly decreased the staining of these proteins in a dose dependent manner (Figures 8e and f). TUNEL assay showed significantly higher number of apoptotic chondrocytes in OA group compared to sham group, while trehalose administration decreased the number of apoptotic chondrocytes in a dose-dependent manner (Figures 8e and g)
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