Modified Liuwei Dihuang Decoction Ameliorates Oligoasthenozoospermia in Mice via Modulation of the PI3K/AKT/Nrf2 Signaling Pathway

<b>Background</b>: Oligoasthenozoospermia (OA) is a common cause of male infertility. Modified Liuwei Dihuang Decoction (MLWDH) is an improved version of Liuwei Dihuang Decoction (LWDH), a traditional Chinese medicine prescription, which has demonstrated significant therapeutic effects against OA. This study aims to evaluate the protective effects of MLWDH against OA and elucidate its underlying molecular mechanisms. <b>Methods</b>: The constituents of MLWDH were identified via UPLC-HRMS and compound databases (TCMSP, HERB). Network pharmacology analysis was conducted to predict potential therapeutic targets and associated signaling pathways. In vivo, a CP-induced mouse model of OA was established to evaluate the therapeutic efficacy of MWDH by assessing testicular and epididymal indices, sperm quality, histopathological changes and serum hormone levels. Oxidative stress markers, including MDA, SOD, GSH and NO, were measured using commercial assay kits. The underlying molecular mechanisms, particularly those related to oxidative stress and inflammation (PI3K, Akt, Nrf2, Keap1, HO-1, NQO1, NF-κB, TNF-α, IL-6), were further elucidated by RT-qPCR, Western blot, and immunofluorescence. <b>Results</b>: A total of 345 major bioactive compounds were identified in MLWDH. Network pharmacology and molecular docking analyses indicated that MLWDH exerts its effects primarily through the PI3K/AKT signaling pathway. MLWDH administration in vivo significantly improved sperm count, motility, and morphology, while also increasing serum levels of testosterone, FSH, and LH. Moreover, MLWDH significantly mitigated oxidative damage, as evidenced by decreased MDA concentrations and elevated levels of GSH, NO and SOD. Mechanistic investigations further substantiated that MLWDH enhanced PI3K/AKT/Nrf2 signaling while inhibiting NF-κB signaling in OA mice. <b>Conclusions</b>: Our findings suggest that MLWDH ameliorates OA in a preclinical mouse model by improving sperm quality and testicular function, potentially via activation of the PI3K/AKT/Nrf2 signaling pathway and the inhibition of NF-κB signaling, thereby alleviating oxidative stress and inflammatory responses.