Puerarin attenuates Thiram-induced cytotoxicity in cultured chicken growth plate chondrocytes: insights into the in vitro therapeutic efficacy by modulating HIF-1α, TIMP-3, and BCL-2 expressions

Endochondral ossification is a crucial process in longitudinal bone growth, relying on chondrocytes proliferation, hypertrophy and cartilage matrix secretion. Disruption in this process, particularly due to cytotoxic agents like tetramethyl thiuram disulfide (Thiram), can impair skeletal development and induce apoptosis. The present research aimed to explore the in vitro protective role of Puerarin, a reputable bioactive isoflavone from Traditional Chinese Medicine on the growth plate (GP) chondrocyte’s morphology and survival, as well as mRNA and protein expressions of hypoxia-inducible factor-1α (HIF-1α), tissue inhibitor of metalloproteinase-3 (TIMP-3), and B-cell lymphoma-2 (BCL-2) against Thiram-induced cytotoxicity in chicken growth plate chondrocytes. The chondrocytes from chicken tibial growth plates were obtained, cultured, refined, and divided into Control, Thiram and Puerarin groups. The chondrocytes in Thiram and Puerarin groups were subsequently treated with a sub-lethal dose of Thiram at 2.5 µg/mL to cause cytotoxicity, followed by an optimal dose of puerarin at 2.5 µg/mL to Puerarin group. Microscopy, RT-qPCR and Western blotting were used to investigate chondrocyte morphology and viability, and molecular expressions of key regulators i.e., HIF-1α, TIMP-3 and BCL-2. Thiram exposure resulted in diminished survival and drastic structural anomalies of chondrocytes, upregulated HIF-1α, and downregulated TIMP-3 and BCL-2. Nonetheless, puerarin treatment efficiently counteracted these Thiram-induced structural and molecular alterations (P<0.05). These outcomes support puerarin being a protective agent and presents innovative therapeutic strategies against Thiram-induced cytotoxicity in chickens GP chondrocytes.