Zexieyin formula ameliorates high-fat diet-induced cognitive impairment via pericyte-associated LRP1 modulating...

The global rise in obesity and metabolic syndrome is increasingly recognized as a major risk factor for cognitive decline and neurodegenerative diseases. Chronic high-fat diet (HFD) consumption disrupts blood-brain barrier (BBB) integrity, a pivotal pathological event that facilitates neurotoxic infiltration, neuroinflammation, and neuronal injury. Brain pericytes play a central role in maintaining BBB function and are particularly vulnerable to HFD-induced metabolic stress. Loss or dysfunction of pericytes contributes to BBB breakdown. Low-density lipoprotein receptor-related protein 1 (LRP1), which is highly expressed in pericytes, is a critical regulator of neurovascular integrity; its downregulation activates the CypA/NF-κB/MMP-9 signaling cascade, thereby exacerbating BBB permeability.

This study aimed to investigate whether the traditional Chinese medicine formula Zexieyin Formula (ZXYF) ameliorates HFD-induced cognitive impairment by targeting pericyte dysfunction. We hypothesized that ZXYF exerts neuroprotective effects by restoring pericyte-associated LRP1 expression, suppressing activation of the CypA/NF-κB/MMP-9 pathway, preserving BBB integrity, and consequently attenuating hippocampal neuronal damage. Both in vivo and in vitro models were employed to elucidate the underlying neurovascular mechanisms.

In vivo, C57BL/6 mice were fed an HFD for 16 weeks to induce cognitive impairment, followed by a 4-week intervention with ZXYF; atorvastatin (ATO) served as a positive control. Cognitive function was evaluated using a battery of behavioral tests. BBB integrity was assessed by transmission electron microscopy (TEM) and Western blot analysis of the tight junction proteins ZO-1 and Claudin-5. Cerebrovascular permeability was further evaluated using sodium fluorescein extravasation assays combined with co-localization analysis with the endothelial marker CD31. Pericyte-associated LRP1 expression in the hippocampus was examined by immunofluorescent co-localization of LRP1 with the pericyte marker PDGFR-β. Activation of the CypA/NF-κB/MMP-9 signaling pathway in hippocampal tissue was analyzed by Western blotting. In vitro, mouse brain microvascular pericytes (MBVPs) were exposed to free fatty acids (FFA) to model metabolic stress. LRP1 expression and localization were assessed by confocal microscopy, and protein levels of the CypA/NF-κB/MMP-9 pathway were determined by Western blotting. An LRP1-knockdown MBVPs cell line was generated via lentiviral transduction to evaluate LRP1 dependence.

HFD-fed mice exhibited pronounced cognitive deficits, which were significantly ameliorated by ZXYF treatment. TEM and Western blot analyses revealed marked BBB disruption in HFD-fed mice, characterized by ultrastructural abnormalities and reduced expression of ZO-1 and Claudin-5. Immunofluorescence demonstrated increased sodium fluorescein leakage in the hippocampus, indicating cerebrovascular damage, which was partially reversed by ZXYF. Mechanistically, confocal imaging showed a significant reduction in LRP1 expression in hippocampal pericytes of HFD-fed mice, accompanied by activation of the CypA/NF-κB/MMP-9 pathway. ZXYF intervention restored pericyte-associated LRP1 expression and suppressed pathway activation. Consistently, lentivirus-mediated LRP1 knockdown in MBVPs abolished the inhibitory effects of ZXYF on CypA/NF-κB/MMP-9 signaling, demonstrating that ZXYF-mediated pathway regulation is LRP1-dependent.

This study demonstrates that ZXYF alleviates HFD-induced cognitive impairment by targeting BBB function. ZXYF restores pericyte-associated LRP1 expression, thereby inhibiting the CypA/NF-κB/MMP-9 signaling pathway, preserving BBB integrity, and protecting hippocampal neurons. These findings identify pericytes and the LRP1/CypA/NF-κB/MMP-9 axis as critical therapeutic targets in metabolic cognitive disorders and provide a novel mechanistic basis for the neuroprotective effects of traditional Chinese medicine.