Guilu Erxian oral liquid alleviates oligoasthenospermia via the mitochondria-complement-NETs network

Guilu Erxian Oral Liquid (GLEX) is a traditional Chinese medicinal formula first recorded in Yi Bian, a medical classic written during the Ming Dynasty, and has been conventionally used to treat male infertility based on its kidney tonification and is now widely recognized for its therapeutic effects against oligoasthenospermia (OAS).

This study aims to investigate the impact of GLEX on cyclophosphamide (CTX)-induced OAS-like mice and to explore its underlying mechanisms, utilizing pharmacodynamics, multi-omics, and molecular biological techniques.

The chemical constituents of GLEX were identified using ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). The binding affinity between GLEX constituents and DJ-1 was determined by molecular docking and molecular dynamics simulations. A mouse model of OAS was induced using 60 mg/kg CTX for 5 days. ICR mice were randomly divided into seven groups: Control group, Model group, low (1.93 mL/kg)-, medium (3.86 mL/kg)-, and high (7.72 mL/kg)-dose groups of GLEX, Wu Zi Yan Zong pill group, and L-Carnitine group. Each group received consecutive gavage for 28 days. Semen quality parameters were assessed using a sperm quality analyzer, along with morphological assessment by hematoxylin and eosin (HE) staining. Serum sex hormone levels were measured using an enzyme-linked immunosorbent assay (ELISA). Gut microbiota was profiled via 16S rRNA gene sequencing. RNA-Seq and DIA proteomics were used to screen the potential pathways and targets associated with OAS pathogenesis and GLEX treatment. Subsequently, the ultrastructure of testis was examined under a transmission electron microscope. Testicular oxidative stress markers and Na⁺-K⁺-ATPase activity were measured by biochemical analysis and fluorescence microscopy. The Mitochondria-Complement-NETs network targets (DJ-1, UQCRQ, C3, FGG, FGA, NE-DNA, MPO-DNA, citH3, and PAD4) were assessed by immunohistochemistry detection, immunofluorescence staining, ELISA, and western blotting.

A total of 84 components were detected by UPLC-MS/MS analysis. After merging with the 15 components predicted from the BATMAN-TCM database and screening against the TCMSP database, 13 were structurally identified. Molecular docking and molecular dynamics simulations revealed a stable binding between GLEX constituents and DJ-1. CTX-induced OAS-like mice showed markedly reduced sperm density and motility and imbalanced serum sex hormone levels, along with structural damage in testicular and epididymal tissues. GLEX effectively restored these alterations. Besides, GLEX remodeled the gut microbiota composition and function in OAS mice. Multi-omics analyses further revealed that the therapeutic mechanisms of GLEX were associated with mitochondria, energy metabolism, oxidative phosphorylation, complement and coagulation cascades, and neutrophil extracellular trap (NET) formation pathways. OAS mice performed disrupted testicular mitochondrial ultrastructure, decreased activities of Na⁺-K⁺-ATPase, T-SOD, GSH-Px, CAT, DJ-1, and UQCRQ, and elevated levels of ROS, MDA, C3, FGA, FGG, MPO-DNA, NE-DNA, citH3, and PAD4. GLEX significantly reversed them via the Mitochondria-Complement-NETs network, exhibiting mitochondrial protection, complement inhibition, and NETs blockade.

GLEX exerts protective effects on semen quality, endocrine function, and histopathological injury of CTX-induced OAS-like mouse model, accompanied by the coordinated modulation of mitochondrial function, complement overactivation, and NETs release. These findings delineate a proposed Mitochondria-Complement-NETs network, which warrants further investigation and may inform future research on interventions for chemotherapy-related reproductive impairment.