NIPAAm-MMA nanoparticle-encapsulated visnagin ameliorates myocardial ischemia/reperfusion injury through the promotion of autophagy and the inhibition of apoptosis

Figures

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  Figure 1.. Visnagin-loaded NP synthesis. (A) Synthesis of NP. NIPAAm and MMA were polymerized with DMAEMA and BPO in an N2 atmosphere at 70°C. (B) Chemical structure of visnagin. (C) Scanning electron microscopy image of the NP. NP were microspherical in shape and their size was approaching 100 nm. (D) NP-visnagin release curve. NP-visnagin was dissolved in NaH2PO4-Na2HPO4 solution at 37°C and at each time point, the sample was analyzed with an ultraviolet-visible spectrometer. NP, nanoparticles; NIPA Am, N-isopropylacrylamide; MAA, methacrylic acid; DMAEMA, 2-(dimethylamino) ethyl methacrylate; BPO, Benzoyl peroxide.

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  Figure 2.. NP-visnagin targeted to the reperfused myocardium and exerted cardioprotective effects. (A) Following the intravenous injection of PBS (baseline), FITC or NP-FITC, in vivo BLI was performed at each time point. (B) Quantitative analysis of BLI data for NP-FITC compared with FITC alone. (C) Fluorescence images of heart cross-sections at 12 h after the intravenous injection of PBS, FITC or NP-FITC. (D) Quantification of the fluorescence intensity of the non-ischemic myocardium and AAR. (E) Representative stereomicrographs of heart sections double stained with TTC and Evans blue at 72 h after intravenous injection. (F) Quantitative analysis of IF/AAR. (G) Quantitative analysis of AAR/LV. *P<0.05; ***P<0.005. NP, nanoparticles; FITC, fluorescein isothiocyanate; BLI, bioluminescence imaging; TTC, 2,3,5-triphenyltetrazolium chloride; IF, infarct area; AAR, area at risk; LV, left ventricle; NS, not significant.

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  Figure 3.. NP-visnagin ameliorated cardiac dysfunction 4 weeks after ischemia/reperfusion. (A) M-mode echocardiography was performed for rats at 4 weeks after intravenous injection with NP-visnagin and other treatments. (B) Effect of NP-visnagin on LVEDD, LVESD, LVEF and LVFS. (C) Systolic blood pressure was detected through the micromanometer catheter method, and the LVSP, the rate of rise of left ventricular pressure (dP/dt) and LVEDP were calculated. (D) Based on Masson staining, the effects of NP-visnagin and the other treatments on fibrosis at 4 weeks after intravenous injection were detected; representative images are shown. (E) Quantification of fibrosis. *P<0.05; **P<0.01; ***P<0.005. NP, nanoparticles; LVEDD, left ventricular end-diastolic dimension; LVESD, left ventricular end-systolic dimension; LVEF, left ventricular ejection fraction; LVFS, left ventricular fractional shortening; LVSP, left ventricular systolic pressure; LVEDP, left ventricular end-diastolic pressure; MI, myocardial infarction.

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  Figure 4.. NP-visnagin enhanced the rate of autophagy in the ischemic region after ischemia/reperfusion. (A) Western blotting was performed for the detection of autophagic flux; LC3II was detected in protein harvested from the treated hearts at 24 h after intravenous injection. GAPDH was detected as an internal standard. (B) Quantification of the expression of LC3II relative to GAPDH. (C) Representative images of the immunofluorescence detection of Troponin-I and LC3. Green dyeing represents Troponin-I, indicative of cardiomyocytes, whereas red dots represent autophagosomes. (D) Quantification of LC3 immunofluorescence. (E) Detection of double membrane autophagosomes with transmission electron microscopy. *P<0.05; **P<0.01; ***P<0.005. NP, nanoparticles; LC, light chain.

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  Figure 5.. NP-visnagin inhibited apoptosis in the ischemic region subsequent to IR. (A) In order to examine the effect of NP-visnagin on the rate of apoptosis in IR injury, a TUNEL assay was performed; green fluorescence indicates TUNEL-positive nuclei, indicating apoptotic cells, whereas blue fluorescence (DAPI) indicates all cells. (B) Quantification of apoptotic cells. (C) Caspase-3 and. (D) caspase-9 activity of the myocardium subsequent to IR. **P<0.01; ***P<0.005. NP, nanoparticles; IR, ischemia/reperfusion; TUNEL, terminal deoxynucleotidyl transferase dUTP nick end labeling.