Internalization and transport mechanisms of the walnut-derived peptide in bEnd.3 cells

<p class="0000" style="margin-top: 6.0pt;"><span lang="EN-US">The blood–brain barrier (BBB) is a major challenge in drug delivery for the treatment of central nervous system diseases. <a name="_Hlk163636866"></a>Walnut derived peptide TWLPLPR (TW-7) has been proved to promote neuronal mitochondrial autophagy and enhance hippocampal neuronal synaptic plasticity, thereby improving learning and memory abilities in mice. We investigated the <a name="_Hlk157457195"></a>internalization mechanism and intracellular transport pathway for the walnut-derived peptide, TWLPLPR (TW-7), using bEnd.3 cells in an <em>in vitro</em> BBB model system. <a name="_Hlk157457892"></a>TW-7 was taken up by the bEnd.3 cells in a concentration-, temperature-, and energy-dependent manner; <a name="_Hlk157457969"></a>this involved increases in caveolin-1 and caveolin-2 protein expression and phosphorylation and inhibition of P-glycoprotein-mediated efflux. <a name="_Hlk157457990"></a>Subcellular localization of TW-7 in bEnd.3 cells was observed, indicating that the plasma membrane, endoplasmic reticulum, Golgi, lysosomes, and mitochondria participated in intracellular trafficking and that the peptide escaped from lysosomes over time. <a name="_Hlk153566620"></a>Caveolae may be critical for TW-7 uptake by brain microvascular endothelial cells, assisting TW-7 to cross the BBB. The results of this study provide a theoretical basis for the mechanism of active peptide penetrating the BBB, and provide a reference for developing neuroprotective active peptide products.</span>