Indigo, An Extract from Indigo Naturalis, Improves Busulfan-Induced Oligoasthenospermia in Mice by Regulating the Testicular Immune Environment

introduction: Indigo Naturalis, a traditional Chinese medicine primarily composed of indigo, has documented anti-inflammatory properties. This study investigates the therapeutic potential and regulatory mechanisms of indigo in busulfan-induced oligoasthenospermia in mice. materials and methods: Male mice were divided into three groups: normal control, busulfan-induced model, and indigo-treated (100 mg/kg). After establishing oligoasthenospermia via busulfan injection, treatments were administered for 28 days. Assessments included sperm motility analysis, histopathological evaluation (HE staining) of testis/epididymis, immunofluorescence staining, serum ELISA for sex hormones, and testicular RNA sequencing. results: 1.Phenotypic Restoration: Indigo significantly improved sperm count and motility while increasing spermatogenic cells. 2.Cellular Protection: Restored cytoskeletal integrity, reduced testicular malondialdehyde (MDA) and reactive oxygen species (ROS). 3.Hormonal Regulation: Elevated serum testosterone and other sex hormones. 4.Transcriptomic Analysis: RNA-seq revealed enrichment in collagen catabolism and immune-related pathways. Indigo upregulated anti-inflammatory cytokines while downregulating pro-inflammatory factors . discussion: The regulated genes encode proteins essential for cytoskeletal organization and testicular immune microenvironment homeostasis. Indigo's efficacy correlates with its dual modulation of oxidative stress and inflammatory cascades, particularly through suppression of pro-inflammatory mediators and ECM remodeling. conclusion: Indigo alleviates busulfan-induced oligoasthenospermia primarily by rebalancing the testicular immune microenvironment, demonstrating therapeutic potential for male infertility.