High-fat diet led to testicular inflammation and ferroptosis via dysbiosis of gut microbes

•HFD-feeding induced the damage of testis by disrupting gut microbes;•HFD-induced obesity could disorder gut microbiota homeostasis, leading to the overproduction of lipopolysaccharides (LPS) in the bloodstream;•The over-accumulation of LPS in the bloodstream triggered an inflammatory response and ferroptosis by activating TLR4 in testis;•Inhibition of TLR4 with Resatorvid (TAK-242) alleviated obesity-induced testis injury through suppression of inflammation and ferroptosis. The disorder of gut microbiota has negative impact on male reproductive, and testicular damage is associated with obesity. However, the detailed mechanism of gut microbiota on the obesity-induced testis injury are still unknown. Therefore, we constructed a mouse model to investigate the effects of obesity on testis injury. In this study, we found that HFD-induced obesity could disorder gut microbiota homeostasis, which increased the abundance of Brevundimonas, Desulfovibrionaceae_unclassified and Ralstonia, ultimately leading to the overproduction of lipopolysaccharides (LPS). Meanwhile, HFD-feeding promoted intestinal permeability via inhibiting expression of tight junction proteins (ZO-1, Occludin and Claudin) and reducing excretion of mucus, leading to translocation of LPS. The over-accumulation of LPS in the bloodstream triggered an inflammatory response by activating TLR4/NF-κB pathway in testis. On the other hand, the gut microbiota produced-LPS also could induce ferroptosis in testis, as reflected by enhancing iron content and lipid peroxidation (MDA), as well as decreasing ferroptosis-related proteins, including GPX4, FTH1 and SLC1A11. Moreover, inhibition of LPS ligand (TLR4) with Resatorvid (TAK-242) alleviated obesity-induced testis injury through suppression of inflammation and ferroptosis. In conclusion, this study provides novel insights into the underlying mechanisms of obesity–related testis injury induced by gut microbiota disorder via the gut–testis axis, thus offering potential targets to counteract obesity-induced male reproductive disorder.