Targeted delivery of USP10 via neutrophil membrane-coated biomimetic nanoparticle facilitates cardiac repair through AMPK-dependent anti-apoptotic and pro-angiogenic pathways

Fig. 1. USP10 is involved in cardiomyocyte apoptosis during MI. (A–D) IL-1β, IL-6, TNF-α, and ROS concentrations in the infarcted myocardial tissue were quantified by ELISA. (E) Serum LDH activity was measured on day 3 following MI induction. (F, G) TUNEL-positive cardiomyocytes in the infarct region were detected by immunofluorescence staining. Scale bar = 100 μm. (H, I) Immunofluorescence staining was also performed for determining USP10 expression in the infarcted area. Scale bar = 100 μm. (J–M) Relationship between USP10 expression and apoptosis-related genes, including Casp3, Casp11, Bax, and Bcl2, was analyzed. N = 4–6 mice per group. Quantitative data are presented as mean ± SD. Statistical significance was evaluated by using Student’s t-test in (A-E, G and I), Pearson’s correlation analysis in (J-M)

Fig. 2. Wu-5 inhibits the activity of cardiomyocytes and induces their apoptosis. (A) Timeline of the in vivo experimental procedure. (B) ROS levels in the infarcted myocardial tissue were quantified by ELISA. (C) Serum LDH activity was measured on day 3 after MI induction. (D, E) Immunofluorescence staining was conducted for detecting TUNEL-positive cardiomyocytes in the infarct zone. Scale bar = 100 μm. (F–H) Casp3, Bax, and Bcl2 expression was assessed by RT-qPCR. N = 4–6 mice per group. Quantitative data are presented as mean ± SD. Statistical significance was evaluated by using one-way ANOVA followed by Tukey’s post-hoc test in (B, C and E-H)

Fig. 3. Synthesis and characterization of NP NM-NP-USP10. (A) Schematic representation of the structure of NM-NP-USP10. (B) TEM was used to examine NP morphology. Scale bar = 100 nm. (C, D) DLS was performed for determining particle size distribution and zeta potential. (E) NP stability was evaluated in serum. (F, G) Immunofluorescence staining was conducted to confirm cellular internalization of NPs. Scale bar = 100 μm. N = 4 independent experiments per group. Quantitative data are presented as mean ± SD. Statistical significance was evaluated by using using Student’s t-test in (C, D and G)

Fig. 4. NM-NP-USP10 preferentially accumulates in the ischemic myocardium with favorable biosafety. (A) The in vivo experimental protocol timeline. (B) RT-qPCR was conducted for estimating Usp10 gene expression in the myocardial tissue. (C, D) Immunofluorescence analysis of NM-NP-USP10 distribution in the infarcted myocardium. Scale bar = 100 μm. (E) The biodistribution of USP10 plasmids in the heart, liver, spleen, lung, and kidney was quantified using RT-qPCR. (F) Serum biochemical analyses, including measurements of alanine aminotransferase (ALT), aspartate aminotransferase (AST), urea, and creatinine levels, were conducted to assess the systemic biosafety of NM-NP-USP10. N = 4 mice per group. Quantitative data are presented as mean ± SD. Statistical significance was determined using Student’s t-test for panels (B, D, E, and G)

Fig. 5. NM-NP-USP10 facilitates cardiac repair after myocardial injury. (A) Kaplan–Meier survival curve. (B) HW/BW ratio. (C–G) Cardiac function was evaluated by echocardiography, including determination of EF (%), LVIDs (mm), LVIDd (mm), and FS (%). (H–J) Ventricular wall thickness and fibrotic area were quantified by Masson’s trichrome staining. (K, L) Col1a1 and Col3a1 gene expression levels were quantified by RT-qPCR. Statistical comparisons were achieved with one-way ANOVA followed by Bonferroni’s post hoc test. N = 4–10 mice per group. Quantitative data are presented as mean ± SD. Statistical significance was evaluated by using one-way ANOVA followed by Tukey’s post-hoc test in (B, D-G, I, J, K, and L)

Fig. 6. NM-NP-USP10 activates the AMPK/eNOS signaling pathway. (A–F) USP10, AMPK, p-AMPK, p-Akt, and eNOS protein levels were determined by western blot analysis across the specified treatment groups, including NM-NP-USP10 and NM-NP-USP10 combined with the AMPK inhibitor Compound C. N = 4 mice per group. Quantitative data are presented as mean ± SD. Statistical significance was evaluated by using one-way ANOVA followed by Tukey’s post-hoc test in (B-F)

Fig. 7. NM-NP-USP10 inhibits cardiomyocyte apoptosis through AMPK signaling. (A, B) Apoptotic cardiomyocytes within the infarct region were identified using TUNEL staining. Scale bar = 100 μm. (C–E) The expression levels of Casp3, Bax, and Bcl2 were assessed via RT-qPCR. (F) Representative Western blot images of pro-caspase-3 and cleaved caspase-3. (G, H) Quantitative analysis of pro-caspase-3 and cleaved caspase-3 protein expression. N = 4–6 independent experiments per group. Quantitative data are presented as mean ± SD. Statistical significance was evaluated using one-way ANOVA followed by Tukey’s post-hoc test in (B-E, G and H)

Fig. 8. NM-NP-USP10 promotes angiogenesis through AMPK signaling. (A) Representative immunofluorescence images depicting α-SMA-positive vessels within the infarct region are presented alongside a quantitative analysis of arteriole density. (B) Representative immunofluorescence images of CD31-positive capillaries in the infarct region are shown with corresponding quantitative analysis of capillary density. Quantification of CD31-positive and α-SMA-positive vessel densities was conducted by counting positive vessels in three randomly selected fields per section, with the average value calculated for each mouse. Scale bar = 100 μm. (C, D) Representative images and quantitative analyses of endothelial cell migration, assessed via wound-healing assay, and endothelial tube formation are provided. Scale bar = 100 μm. (E–H) Expression levels of Vegf, Ang1, Del-1, and Fgf were measured using RT-qPCR. N = 4 independent experiments per group. Quantitative data are presented as mean ± SD. Statistical significance was evaluated using one-way ANOVA followed by Tukey’s post-hoc test in (A-H)