Hai Zao Xiao Liu Jiao Nang ameliorated hypothyroidism in rat models by targeting ferroptosis pathway: Integrated network study of multi-omics.

ETHNOPHARMACOLOGICAL RELEVANCE: Hai Zao Xiao Liu Jiao Nang (HZXLJN) is a brown granular capsule formulation derived from the classical TCM prescription "Hai Zao Yu Hu Tang" for thyroid disorders. This unique herbal compound has been clinically applied for years in treating thyroid diseases, demonstrating remarkable therapeutic efficacy with low adverse effects and recurrence rates. However, its underlying mechanisms remain unclear.

AIMS: This study elucidates the therapeutic effects of HZXLJN on hypothyroidism (HT) via the ferroptosis pathway using an integrated strategy of network pharmacology, multi-omics analysis, and in vitro experiments.

METHODS: The chemical composition of HZXLJN was analyzed using mass spectrometry. Network pharmacology, proteomics, and metabolomics were integrated in a multi-omics approach to investigate the molecular mechanisms underlying HZXLJN's therapeutic effects on HT. Animal experiments were conducted to screen potential therapeutic pathways. A hypothyroidism rat model was established in SD rats using propylthiouracil (PTU), followed by intervention with either HZXLJN, levothyroxine sodium (LS), or Ferrostatin-1 (Fer-1), with subsequent observation of rat conditions; Histopathological evaluation of thyroid tissue was performed using hematoxylin-eosin (HE) staining. Serum hormone levels and biochemical parameters were assessed by enzyme-linked immunosorbent assay (ELISA) and biochemical assays. Immunohistochemistry (IHC) was employed to examine GPX4 protein expression in thyroid tissue. The mitochondrial status of thyroid follicular cells was observed via transmission electron microscopy (TEM). Western blot (WB) analysis was conducted to detect the expression of pathway-related proteins in thyroid tissue. In vitro, a ferroptosis cell model was established in Nthy-ori 3-1 cells by Erastin induction to screen for the specific active components of HZXLJN, followed by investigation of the underlying mechanisms through immunofluorescence (IF) and Western blot (WB) analyses.

RESULTS: HZXLJN contains nine active components. Network pharmacology predicted AKT and p53 as its primary therapeutic targets. Proteomic and metabolomic analyses suggested its therapeutic effects may be associated with ferroptosis, preliminarily identifying five ferroptosis-related pathways: glutathione metabolism, lipid metabolism, mitochondrial function, heme metabolism, and autophagolysosome activity. Animal experiments demonstrated that HZXLJN ameliorated PTU-induced hypothyroidism in SD rats. Through GPX4 level assessment and transmission electron microscopy, we confirmed that HZXLJN improved hypothyroidism by inhibiting ferroptosis. Further validation via Western blot and biochemical assays of the multi-omics-identified ferroptosis pathways determined that HZXLJN primarily exerts its effects by modulating the glutathione metabolism pathway. In vitro experiments demonstrated that the active components of HZXLJN are Nobiletin, Hesperetin, and Berberine, which inhibit ferroptosis primarily by suppressing the p53 pathway.

CONCLUSION: Hai Zao Xiao Liu Jiao Nang (HZXLJN) may alleviate hypothyroidism (HT) by inhibiting ferroptosis, primarily through three active components - Nobiletin, Hesperetin, and Berberine. These bioactive compounds exert their therapeutic effects mainly by suppressing p53 signaling, partially activating AKT, and enhancing glutathione metabolic pathways to promote cell survival and prevent ferroptosis.