Shiwei Hezi Pill alleviates cisplatin-induced acute kidney injury through integrative network pharmacology, transcriptomics and experimental validation.

ETHNOPHARMACOLOGICAL RELEVANCE: Shiwei Hezi Pill (SHP) is a classical Tibetan polyherbal remedy traditionally used to treat nephritis and urinary tract disorders. It is known in Tibetan medicine for its effects in clearing "renal heat" and promoting diuresis. Despite its long-standing clinical application, the pharmacological mechanisms underlying SHP's potential protective effects against cisplatin-induced acute kidney injury (cis-AKI) remain poorly understood.

AIM OF THE STUDY: This study aimed to elucidate the protective mechanisms of SHP in cisplatin-induced AKI, focusing on inflammation and apoptosis, and to integrate systems-level prediction with molecular and histological validation.

METHODS

Experimental validation was performed to identify the active compounds in SHP and their potential targets, utilizing the TCMSP, HERB, and SwissTargetPrediction databases. AKI-related genes were sourced from GeneCards and OMIM, and the overlapping targets were analyzed through protein-protein interaction (PPI) networks, Gene Ontology (GO), and KEGG pathway enrichment analysis. Key compound-target interactions were further validated using molecular docking techniques. Constituents were characterized by UPLC-QTOF-MS. An Cis-induced AKI mouse model was validated by histopathology (H&E), immunohistochemistry (IHC), TUNEL, ELISA, and Western blot (WB). In vitro, Cis-injured HK-2 cells were evaluated using CCK-8, scratch wound-healing, and Annexin V-FITC/PI flow cytometry. Mechanistically, RT-qPCR, WB, and reference-based transcriptome sequencing of kidney tissue were employed.

RESULTS

Network pharmacology identified 166 overlapping targets between SHP and AKI, enriched in inflammation- and apoptosis-related pathways, particularly JAK-STAT signaling. Molecular docking showed strong binding of β-sitosterol with JAK2, STAT3, EGFR, and BCL-2. In vivo, SHP improved renal function, reduced tubular injury, and lowered inflammatory cytokines. TUNEL staining confirmed a significant reduction in tubular apoptosis. Transcriptomic analysis revealed that SHP reversed cisplatin-induced gene expression changes, consistent with network pharmacology findings. In cell experiments, SHP protected HK-2 cells from cisplatin-induced apoptosis, as confirmed by flow cytometry. Western blotting and qPCR showed downregulation of BAX and upregulation of BCL-2, supporting SHP's anti-apoptotic effects. Both in vitro and in vivo, SHP inhibited JAK2 and STAT3 phosphorylation, validating the involvement of the JAK2/STAT3 pathway in its protective effects.

CONCLUSION

SHP protects against cisplatin-induced acute kidney injury (AKI) by inhibiting the activation of the JAK2-STAT3 signaling pathway and preventing apoptosis. These findings highlight SHP's potential for preventing nephrotoxicity.