Role of PCBP2 in regulating nanovesicles loaded with curcumin to mitigate neuroferroptosis in neural damage caused by heat stroke.

Abstract

Objective: This study aims to elucidate the mechanisms by which nanovesicles (NVs) transport curcumin(CUR) across the blood-brain barrier to treat hypothalamic neural damage induced by heat stroke by regulating the expression of poly(c)-binding protein 2 (PCBP2).

Methods: Initially, NVs were prepared from macrophages using a continuous extrusion method. Subsequently, CUR was loaded into NVs using sonication, yielding engineered cell membrane Nanovesicles loaded with curcumin (NVs-CUR), which were characterized and subjected to in vitro and in vivo tracking analysis. Evaluations included assessing the toxicity of NVs-CUR using the MTT assay, evaluating neuroprotection of NVs-CUR against H₂O₂-induced oxidative stress damage in PC12 cells, examining effects on cell morphology and quantity, and detecting ferroptosis-related markers through Western blot and transmission electron microscopy (TEM). Proteomic analysis was conducted on PC12 cells treated with NVs (n = 3) and NVs-CUR (n = 3) to identify downstream key factors. Subsequently, the expression of key factors was modulated, and rescue experiments were performed to validate the impact of NVs-CUR through the regulation of key factor expression. Furthermore, a mouse model of hypothalamic neural damage induced by heat stroke was established, where CUR, NVs-CUR, and ferroptosis inducer Erastin were administered to observe mouse survival rates, conduct nerve function deficit scoring, perform histological staining, and measure levels of inflammation and oxidative stress factors in hypothalamic tissue.

Results: NVs-CUR was successfully prepared with excellent stability, serving as an advantageous drug delivery system that effectively targets brain injury sites or neurons both in vitro and in vivo. Subsequent in vitro cell experiments demonstrated the biocompatibility of NVs-CUR, showing superior protective effects against H₂O₂-induced PC12 cell damage and reduced ferroptosis compared to CUR. Moreover, in the mouse model of hypothalamic neural damage induced by heat stroke, NVs-CUR exhibited enhanced therapeutic effects. Proteomic analysis revealed that NVs-CUR exerted its effects through the regulation of key protein PCBP2; silencing PCBP2 reversed the protective effect of NVs-CUR on neural damage and its inhibition of ferroptosis. Additionally, NVs-CUR regulated solute carrier family 7 member 11 (SLC7A11) expression by PCBP2; overexpression of SLC7A11 reversed the promotion of neural damage and ferroptosis by silencing PCBP2. Animal experiments indicated that ferroptosis inducers reversed the improved survival and nerve function observed with NVs-CUR, silencing PCBP2 reversed the ameliorative effects of NVs-CUR on hypothalamic neural injury induced by heat stroke, and overexpression of SLC7A11 further reversed the adverse effects of silencing PCBP2 on hypothalamic neural injury induced by heat stroke. This suggests that NVs-CUR alleviates hypothalamic neural damage induced by heat stroke by targeting the PCBP2/SLC7A11 axis to reduce neuronal ferroptosis.

Conclusion: This study successfully developed engineered cell membrane NVs-CUR with neuron-targeting properties. NVs-CUR increased the expression of PCBP2, maintained the stability of SLC7A11 mRNA, reduced ferroptosis, and ultimately alleviated hypothalamic neuroinflammation induced by heatstroke.