A new approach for treating AD: Guifu Dihuang Pills improves brain insulin resistance by promoting NrCAM to activate the EGFR/PI3K/Akt signaling pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: Guifu Dihuang Pills (GFDHP), a classical Chinese herbal formula originally recorded in the Synopsis of the Golden Chamber (Jingui Yaolüe), significantly ameliorate cognitive dysfunction in patients with Alzheimer's disease (AD). However, the precise molecular mechanisms underlying its therapeutic effects, particularly those involving the regulation of cerebral insulin resistance (IR), are not yet fully elucidated.

AIM OF THE STUDY: This study systematically investigated the neuroprotective mechanism of the traditional Chinese medicine (TCM) compound GFDHP in alleviating AD by integrating RNA-seq transcriptomics, surface plasmon resonance (SPR) analysis, and bioinformatic analysis. The aim was to clarify its key targets and signaling pathways involved in the modulation of cerebral IR by in vitro and in vivo experiments.

METHODS

The chemical components of GFDHP were characterized using ultra-performance liquid chromatography quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF/MS). An AD model with cerebral IR was established by an intracerebroventricular streptozotocin (ICV-STZ) injection. Cognitive function was assessed using the Morris water maze (MWM) test, while hippocampal damage was evaluated by histological staining. The insulin level in hippocampal tissues was quantified using enzyme-linked immunosorbent assay (ELISA). AD-related proteins were analyzed using immunohistochemistry and Western blot. Integrated transcriptome sequencing, AlphaFold3 prediction, and SPR analysis confirmed the interaction between neuronal cell adhesion molecule (NrCAM) and epidermal growth factor receptor (EGFR). In vitro experiments were performed using HT22 cells treated with dexamethasone (DXM) to induce an IR model, followed by evaluation of IR and AD-related markers. Lentivirus-mediated shRNA knockdown of NrCAM was performed to systematically examine its regulatory effect on the EGFR/PI3K/Akt signaling pathway and GFDHP's therapeutic intervention.

RESULTS

GFDHP significantly attenuated ICV-STZ-induced IR and AD-related neuropathological alterations. SPR analysis further revealed that NrCAM mediated the neuroprotective effect through the specific binding to EGFR, thereby activating the downstream PI3K/Akt signaling cascade. NrCAM silencing not only exacerbated both IR and AD pathology but also suppressed the EGFR/PI3K/Akt pathway and reduced the therapeutic effect of GFDHP.

CONCLUSIONS

This study demonstrated that GFDHP alleviated IR by upregulating NrCAM expression and subsequently activating the EGFR/PI3K/Akt signaling pathway, thereby ameliorating AD. This is the first report identifying NrCAM as the key molecular target mediating the neuroprotective effect of this herbal formulation, providing a novel therapeutic strategy for AD treatment.