Effects of Wuzi Yanzong Pill on Mitochondrial Homeostasis and the cGAS‐STING Signaling Pathway in Rats Under Testicular Heat Stress

The incidence of male infertility has remained notably high in recent years, with testicular inflammation being a major contributing factor. Testicular heat stress (THS) is a common inducer of testicular inflammation. Testicular tissue is rich in mitochondria, and disruptions in mitochondrial homeostasis may play a key role in driving inflammation. Wuzi Yanzong Pill (WZYZ Pill), a traditional Chinese medicine (TCM) formula, has long been used in treating male infertility and may inhibit testicular inflammation caused by heat stress. In this study, a rat model of THS was established, and WZYZ Pill was administered at different doses. The effects of WZYZ Pill on sperm parameters, testicular histology, and the secretion of inflammatory factors were assessed. The study also examined the link between mitochondrial imbalance and inflammation, aiming to clarify whether the WZYZ Pill interferes with this pathway. Western blotting, immunohistochemistry, and real‐time PCR (RT‐PCR) were used to assess mitochondrial DNA (mtDNA) leakage and the expression of key regulators of mitochondrial homeostasis and the cyclic GMP–AMP synthase (cGAS)‐stimulator of interferon gene (STING) signaling pathway. The results showed that heat stress disrupted mitochondrial homeostasis, leading to mtDNA leakage and activation of the cGAS‐STING pathway, which contributed to testicular inflammation. WZYZ Pill significantly improved mitochondrial homeostasis, reduced mtDNA leakage, suppressed the expression of cGAS‐STING‐related molecules, and decreased the secretion of inflammatory factors in testicular tissue, ultimately alleviating the adverse effects of heat stress on both testicular structure and sperm production.