Zhimu-Huangbo ameliorates AQP4 depolarization in diabetic cognitive impairment rats by targeting the serum-derived exosomal TIMP3-mediated MMP9/β-DG pathway

Aquaporin-4 (AQP4) depolarization-mediated glymphatic system (GS) dysfunction is a critical pathological mechanism in diabetic cognitive impairment (DCI). However, its underlying mechanism has not been clarified. The Zhimu-Huangbo (ZMHB) herb pair, a classic formulation historically employed in traditional Chinese medicine, demonstrates neuroprotective effects, including the amelioration of cognitive dysfunction. Emerging evidence suggests that the exosomal pathway may regulate abnormalities in diabetes-induced AQP4 polarization. Nevertheless, whether the neuroprotective effects of ZMHB mediate the restoration of AQP4 polarity through the exosomal pathway remains to be elucidated. This study takes the exosomal pathway as the entry point to elucidate the mechanism of AQP4 depolarization in DCI and determine how ZMHB restores AQP4 polarization and improves cognitive function. ZMHB significantly improved cognitive function, effectively restored GS function and maintained AQP4 polarization. Pharmacological blockade of exosomal pathways partially reduced the therapeutic effects of ZMHB, demonstrating that its efficacy depends on exosome-mediated signaling; proteomics combined with western blotting validation revealed tissue inhibitor of metalloproteinase 3 (TIMP3) as a pivotal protein modulated by ZMHB, which restored AQP4 polarization by inhibiting matrix metalloproteinase-9 (MMP9) activity and blocking β-dystroglycan (β-DG) cleavage in astrocytes. In vitro studies further demonstrated that both ZMHB-treated serum exosomes (ZMHB-sExos) and the TIMP3 agonist MPT0B390 restored astrocytic AQP4 polarization by modulating the MMP9/β-DG pathway. We demonstrated that ZMHB restored AQP4 depolarization in DCI rats by modulating the exosomal TIMP3-MMP9/β-DG pathway.