Abstract
This study aimed to determine whether hesperetin (HPT) has chondroprotective effects against the TNF-α-induced inflammatory response of chondrocytes and related mechanisms and clarify the impact of HPT on osteoarthritis (OA) induced by anterior cruciate ligament transection (ACLT). Under tumor necrosis factor-α (TNF-α) stimulation, rat chondrocytes were treated with or without HPT. The CCK-8 assay was used to detect viability and cytotoxicity. RT-qPCR and Western blot were used to examine the expression of aggrecan, collagen type II, and inflammatory and proliferative genes/proteins in chondrocytes. Flow cytometry was used to check the cell cycle to determine whether HPT protects chondrocytes against the inhibitory effect of TNF-α on chondrocyte proliferation. In addition, RNA sequencing was used to discover possible molecular targets and pathways and then validate these pathways with specific protein phosphorylation levels. Finally, immunofluorescence staining was used to examine the phosphorylation of the AMP-activated protein kinase (AMPK) pathway. The results showed that HPT restored the upregulation of interleukin 1β (IL-1β), PTGS2, and MMP-13 induced by TNF-α. In addition, HPT reversed the degradation of the extracellular matrix of chondrocytes induced by TNF-α. HPT also reversed the inhibitory effect of TNF-α on chondrocyte proliferation. RNA sequencing revealed 549 differentially expressed genes (DEGs), of which 105 were upregulated and 444 were downregulated, suggesting the potential importance of the AMPK pathway. Progressive analysis showed that HPT mediated the repair of TNF-α-induced chondrocyte damage through the AMPK signaling pathway. Thus, local treatment of HPT can improve OA induced by ACLT. These findings indicated that HPT has significant protective and anti-inflammatory effects on chondrocytes through the AMPK signaling pathway, effectively preventing cartilage degradation. Given the various beneficial effects of HPT, it can be used as a potential natural drug to treat OA.
Highlights
Osteoarthritis (OA) is a connective tissue degenerative disease and frequently associated with joint inflammation and substantial pain, possibly debilitating the affected patient (Dieppe and Lohmander, 2005; Neogi, 2013; Glyn-Jones et al, 2015; Lin et al, 2016; Wang et al, 2020a)
The results demonstrated that compared with the tumor necrosis factor-α (TNF-α) group, there were many differentially expressed genes (DEGs) in chondrocytes treated with tumor necrosis factor- (TNF-)α and 10 μM HPT, of which 105 genes were upregulated and 444 genes were downregulated (Figure 4A)
To determine the role of the AMPactivated protein kinase (AMPK) signaling pathway in HPT-induced alleviation of inflammation, we examined the effect of HPT on TNF-α-induced inflammation with or without compound C, a selective
Summary
Osteoarthritis (OA) is a connective tissue degenerative disease and frequently associated with joint inflammation and substantial pain, possibly debilitating the affected patient (Dieppe and Lohmander, 2005; Neogi, 2013; Glyn-Jones et al, 2015; Lin et al, 2016; Wang et al, 2020a) It is characterized by the loss of the articular cartilage and subchondral bone reactive hyperplasia due to repeated inflammatory responses (Chen et al, 2017). Available therapeutic strategies for OA include injectable glucocorticoid compounds and nonsteroidal anti-inflammatory drugs (NSAIDs) (Deyle et al, 2020; Kolasinski et al, 2020). These drugs only offer symptomatic relief, with no effect on disease modification or progression. It is imperative to seek effective medicines to prevent OA
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