Effects of quercetin-conjugated with superparamagnetic iron oxide nanoparticles on learning and memory improvement through targeting microRNAs/NF-κB pathway

Abstract

Quercetin-conjugated superparamagnetic iron oxide nanoparticles (QCSPIONs) have an ameliorative effect on diabetes-induced memory impairment. The current study aimed to compare the effect of quercetin (QC) and QCSPIONs on inflammation-related microRNAs and NF-κB signaling pathways in the hippocampus of diabetic rats. The expression levels of miR-146a, miR-9, NF-κB, and NF-κB-related downstream genes, including TNF-α, BACE1, AβPP, Bax, and Bcl-2 were measured using quantitative real-time PCR. To determine the NF-κB activity, immunohistochemical expression of NF-κB/p65 phosphorylation was employed. Computer simulated docking analysis also performed to find the QC target proteins involved in the NF-κB pathway. Results indicate that diabetes significantly upregulated the expression levels of miR-146a, miR-9, TNF-α, NF-κB, and subsequently AβPP, BACE1, and Bax. Expression analysis shows that QCSPIONs are more effective than pure QC in reducing the expression of miR-9. Interestingly, QCSPIONs reduce the pathological activity of NF-κB and subsequently normalize BACE1, AβPP, and the ratio of Bax/Bcl-2 expression better than pure QC. Comparative docking analyses also show the stronger binding affinity of QC to IKK and BACE1 proteins compared to specific inhibitors of each protein. In conclusion, our study suggests the potent efficacy of QCSPIONs as a promising drug delivery system in memory improvement through targeting the NF-κB pathway.

Figure 1

Results of Prussian blue staining and ICP-AES. (A) Prussian blue staining of pancreas, liver, kidney, and hippocampus tissues of NDC and DC treated with SPION. (B) ICP results obtained of hippocampus tissue in NDC and DC treated with SPION and QCSPION. (C) Schematic picture of Prussian blue staining and ICP assay. NDC: non-diabetic control, DC, diabetic control, DC + SPION: diabetic treated with superparamagnetic iron oxide nanoparticle, DC + QCSPION: diabetic treated with quercetin-conjugated superparamagnetic iron oxide nanoparticle. *** P < 0.001 versus the diabetic control group (one-way ANOVA, Tukey’s multiple comparison tests). Arrow represents the position of the iron oxide nanoparticles. (scale bar: 30 μm, magnification 40X).

Figure 2

The graph of relative expression of (A) miR-146a, (B) miR-9 (C) NF-κB, (D) TNF-α, (E) BACE1 (F) AβPP (G) Bax, and (H) Bcl-2 in the hippocampus of experimental groups. NDC: non-diabetic control, DC: diabetic control, DC + SPION: diabetic treated with superparamagnetic iron oxide nanoparticle, DC + QC: diabetic treated with quercetin, DC + QCSPION: diabetic treated with quercetin-conjugated superparamagnetic iron oxide nanoparticle. * P < 0.05, ** P < 0.01 and *** P < 0.001 and P < 0.0001 versus diabetic control group (one-way ANOVA, Tukey’s multiple comparison tests). The expression levels are investigated by quantitative real-time PCR and ΔΔCt method. Data expressed as mean ± S.E.M.

Figure 3

Representative photomicrographs of immunohistochemistry staining with antiphospho-NF-κB p65 antibody in the hippocampus of different groups. (A) NDC rats showing no phospho-p65 positive cells, (B) DC rats showing a significant increase in a number of phospho-p65 positive cells, (C) DC + QC demonstrating a reduction in NF-κB immunoreactivity, (D) DC + QCSPION showing a significant reduction in activated NF-κB signal. (E) Schematic picture of IHC. NDC: non-diabetic control, DC: diabetic control, DC + QC: diabetic treated with quercetin, DC + QCSPION: diabetic treated with quercetin-conjugated superparamagnetic iron oxide nanoparticle. (scale bar: 20 μm, magnification 40X). Brown color indicates NF-κB positivity.

Figure 4

General view of QC-IKK and Inhibitor VII-IKK complexes. (A) The structure of all specific inhibitors of IKK are represented. (B) Interactions of Inhibitor VII-IKK complex include one hydrogen bond. (C) Interactions of the QC-IKK complex contain seven hydrogen bonds.

Figure 5

Schematic representation of QC-BACE1 and Lanabecestat-BACE1 complexes. (A) The structure of all specific inhibitors of BACE1 are represented. (B) Interactions of the Lanabecestat-IKK complex include three hydrogen bonds. (C) Interactions of the QC-BACE1 complex contain six hydrogen bonds.

Figure 6

Schematic picture of the beneficial effect of QC after released from SPIONs in neural cells on the miRNAs/NF-κB-dependent inflammatory pathway in the hippocampus of diabetic rats. STZ, streptozotocin; TLR, Toll-like receptors; MYD88, Myeloid differentiation primary response gene 88; IRAK, Interleukin 1 Receptor Associated Kinase 1; TRAF6, TNF receptor-associated factor 6; IKK, Inhibitor of nuclear factor Kappa-B kinase; NF-κB, Nuclear factor-kappa BAPP, Amyloid precursor protein; BACE1, β-site amyloid precursor protein cleaving enzyme 1; BCL-2, B-cell lymphoma 2; BAX, BCL2-associated X protein.