Shikonin targets to m6A-modified oxidative damage pathway to alleviate benzene-induced testicular injury

Benzene exposure causes reproductive toxicity through oxidative damage. However, the specific mechanisms of benzene-induced testicular damage and the potential therapeutic drugs remain poorly understood. In the present study, C57BL/6J mice have been exposed to 0 and 150 mg/kg benzene for four weeks. Then, we found that benzene exposure induced testicular damage in mice, mainly manifested by decreased testicular coefficients, abnormal semen parameters and HE-stained intraepithelial vacuolation. On the mechanism, benzene exposure activated Kelch Like ECH Associated Protein 1/Nuclear factor-erythroid 2 related factor 2 (Keap1/Nrf2) and NF-κB signaling pathway, then promoted apoptosis and inflammatory responses in testes. In vitro, massive reactive oxygen species (ROS) production and a large number of apoptotic cells were observed after 1,4-BQ treatment of GC-2 cells. Furthermore, benzene altered the expression of three important RNA methylation modulator genes, methyltransferase-like 3 (Mettl3), AlkB homolog 5 (Alkbh5) and YTH domain containing 2 (Ythdc2). Moreover, both m6A modification and mRNA levels of NF-κB increased with benzene exposure. Inspiringly, shikonin alleviated benzene-induced male reproductive damage by targeting m6A-modified NF-κB in mice testes. Our study provides new insights into molecular mechanisms of RNA m6A modification in benzene-induced reproductive injury and directions to find potential drugs for the treatment of male infertility.