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 treated 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, then 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 superoxide 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.

Key words: formononetin, miR-135b-5p, chondrocyte, oxidative stress, inflammation, proliferation, apoptosis