Mechanism underlying the effect of formononetin on interleukin-1beta-induced chondrocyte injury

Shan Jixin; Ye Ruibin; Ju Shaohua; Wang Qiang

doi:10.12307/2025.388

Abstract：BACKGROUND:Formononetin is an isoflavonoid compound widely found in red clover,astragalus,and chickweed,which has the ability to inhibit oxidative stress,inflammatory factor release,and apoptosis. OBJECTIVE:To investigate the effect of formononetin on interleukin-1β-induced chondrocyte injury and its mechanism. METHODS:(1) Cartilage tissues from patients with osteoarthritis and patients with simple meniscus injury were collected,and real-time quantitative PCR was used to detect miR-135b-5p expression. (2) Human chondrocytes were cultured in vitro,and then divided them into nine groups:cells in normal control group were cultured for 48 hours with no treatment;cells in interleukin-1β group were treated with interleukin-1β for 48 hours;cells in interleukin-1β+low-dose formononetin group were treated with 25 μmol/L formononetin for 24 hours followed by treatment with interleukin-1β for 48 hours;cells in interleukin-1β+middle-dose formononetin group were treated with 50 μmol/L formononetin for 24 hours followed by treatment with interleukin-1β for 48 hours;cells in interleukin-1β+high-dose formononetin group were treated with 100 μmol/L formononetin for 24 hours followed by treatment with interleukin-1β for 48 hours;cells in interleukin-1β+miR NC group were treated with miR NC for 6 hours followed by treatment with interleukin-1β for 48 hours;cells in interleukin-1β+miR-135b-5p group were treated with miR-135b-5p mimics for 6 hours followed by treatment with interleukin-1β for 48 hours;cells in interleukin-1β+high dose formononetin+anti-miR-NC group were treated with anti-miR-NC for 6 hours,then treated with 100 μmol/L formononetin for 24 hours,and finally treate with interleukin-1β for 48 hours;cells in interleukin-1β+high-dose formononetin+anti-miR-135b-5p group were treated with anti-miR-135b-5p for 6 hours,the treated with 100 μmol/L formononetin for 24 hours,and finally treated with interleukin-1β for 48 hours. Relevant tests are performed after treatment. RESULTS AND CONCLUSION:The expression level of miR-135b-5p in cartilage tissue of patients with osteoarthritis was significantly lower than that of patients with simple meniscus injury (P<0.05). Compared with the normal control group,the expression level of miR-135b-5p,proliferative ability,activity of superoxid dismutase,and expression levels of collagenase Ⅱ protein and Bcl-2 protein in chondrocytes were lower in the interleukin-1β group (P<0.05),while apoptotic rate,lactate dehydrogenase activity,malondialdehyde level,levels of proinflammatory factors,and expression levels of matrix metalloproteinase-13 protein and Bax protein were higher in the interleukin-1β group (P<0.05). Formononetin inhibited chondrocyte damage caused by interleukin-1β in a concentration-dependent manner. Transfection of miR-135b-5p mimics elevated miR-135b-5p expression in the interleukin-1β group and inhibited chondrocyte damage induced by interleukin-1β;transfection of anti-miR-135b-5p decreased miR-135b-5p expression in the interleukin-1β+high-dose formononetin group and inhibited the effect of formononetin on chondrocytes. To conclude,the protective effect of formononetin on chondrocyte injury induced by interleukin-1β may be related to the regulation of miR-135b-5p expression.