ShenRongGuBenHuanShao Pill Ameliorates Hydrocortisone-Induced Erectile Dysfunction: Insights from Integrated Multi-Omics Analysis

Background/Objectives: ShenRongGuBenHuanShao Pill (SRGBHSP) is a traditional Chinese medicine (TCM) used for the treatment of erectile dysfunction (ED). However, its active constituents and pharmacological mechanisms remain unclear. This study characterized the flavonoid constituents of SRGBHSP and investigated its effects on HC-associated ED-related alterations and potential molecular mechanisms using an integrated multi-omics strategy. Methods: SRGBHSP constituents were characterized by UPLC-Q-TOF-MS/MS, and representative flavonoids were quantitatively analyzed by UPLC-MS/MS. Rats exposed to HC received SRGBHSP (1.08, 2.16, or 4.32 g/kg), Yougui Pill, or sildenafil concurrently with HC administration. Pharmacological effects were evaluated using APO-induced erectile responses, histopathology, sperm quality, reproductive hormones, oxidative stress indices, and serum NO/GC/cGMP-related parameters. Integrated 16S rRNA sequencing, untargeted metabolomics, transcriptomics, and DIA-based proteomics were combined with RT-qPCR and Western blotting to investigate potential regulatory mechanisms. Results: A total of 131 flavonoids were characterized in SRGBHSP, including flavones, isoflavones, flavonols, flavanones, chalcones, flavans, anthocyanidins, and aurones. Quantitative analysis identified baicalin as the predominant flavonoid constituent. APO-induced erection frequency decreased from 3.50 ± 1.29 in the control group to 0.50 ± 0.58 in the model group and increased to 4.50 ± 1.29 following high-dose SRGBHSP intervention. SRGBHSP also improved reproductive hormone homeostasis, sperm quality, and oxidative stress status in HC-treated rats. SRGBHSP increased serum NOS, NO, GC, and cGMP levels and increased the mRNA and total protein expression of PI3K, Akt, and mTOR in penile tissue. Integrated multi-omics analyses revealed coordinated alterations in gut microbiota, glycerophospholipid metabolism, and penile transcriptomic and proteomic profiles. Conclusions: SRGBHSP attenuated multiple ED-related alterations associated with HC exposure, accompanied by improvements in reproductive function, oxidative stress status, and serum NO/GC/cGMP-related parameters. Integrated multi-omics analyses together with expression-level validation revealed coordinated alterations in gut microbiota, glycerophospholipid metabolism, and PI3K/Akt/mTOR-related molecules following SRGBHSP intervention. These findings suggest the potential involvement of PI3K/Akt/mTOR-related signaling in the response to SRGBHSP, although its activation status and causal contribution remain to be established. Flavonoids may represent important bioactive constituents associated with the observed effects of SRGBHSP. Overall, this study provides an integrated pharmacological and multi-omics framework for understanding the effects of SRGBHSP on HC-associated ED-related alterations.