RESUMO
BACKGROUND:Studies have shown that hesperetin exerts protective effects on chondrocytes through a variety of mechanisms or signaling pathways,but the protective mechanisms have not been fully elucidated. OBJECTIVE:To investigate the effect of hesperetin on lipopolysaccharide-induced inflammatory degeneration of chondrocytes. METHODS:Knee joint chondrocytes from suckling Sprague-Dawley rats were isolated and cultured in vitro,and identified by Safranine O staining.The cytotoxicity of hesperetin on chondrocytes was determined by the MTT assay to ensure the optimal concentration of hesperetin.Chondrocytes were randomly divided into three groups,including the control group,model group,and experimental group.The cellular model of osteoarthritis was established in the latter two groups by simulating chondrocytes with lipopolysaccharide.Chondrocytes in the experimental group was then intervened with hesperetin for 24 hours.Calcein-AM/EthD-I staining was used to detect cell viability.Immunohistochemical staining was performed to determine the expression of type Ⅱ collagen in chondrocytes.Intracellular reactive oxygen species level was detected by a reactive oxygen species detection kit.Total glutathione level was detected by ELISA.Real-time fluorescent PCR was employed to detect the expression of interleukin 1β,interleukin 6,tumor necrosis factor α and type Ⅱ collagen. RESULTS AND CONCLUSION:Safranine O staining results showed that the cells extracted were chondrocytes.Cytotoxicity test results showed 0.5 μmol/L hesperetin had the most significant effect on chondrocyte vitality.Compared with the control group,the model group showed a decrease in chondrocyte proliferation ability,an increase in reactive oxygen species levels,a decrease in total glutathione levels,an increase in type Ⅱ collagen degradation,an increase in the levels of interleukin 1β,interleukin 6,and tumor necrosis factor α,and a decrease in the expression of type Ⅱ collagen(P＜0.05).Compared with the model group,in the experimental group,the proliferation ability of chondrocytes increased,reactive oxygen species levels decreased,total glutathione levels increased,and type Ⅱ collagen degradation decreased,levels of interleukin 1β,interleukin 6,and tumor necrosis factor α downregulated,and the expression of type Ⅱ collagen upregulated(P＜0.05).To conclude,hesperetin has a protective effect on lipopolysaccharide-induced inflammatory degeneration of osteoarthritic chondrocytes,and the mechanism may be associated with inhibition of reactive oxygen species-mediated oxidative stress.