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. 2024 Jul 1;22(1):382.
doi: 10.1186/s12951-024-02631-0.

Targeting delivery of miR-146a via IMTP modified milk exosomes exerted cardioprotective effects by inhibiting NF-κB signaling pathway after myocardial ischemia-reperfusion injury

Wan-Ting Meng  1 , Jing Zhu  1 , Ya-Chao Wang  1 , Chang-le Shao  1 , Xiu-Ya Li  1 , Ping-Ping Lu  1 , Meng-Ying Huang  1 , Fang-Fang Mou  2 , Hai-Dong Guo  3 , Guang Ji  4
Affiliations

Targeting delivery of miR-146a via IMTP modified milk exosomes exerted cardioprotective effects by inhibiting NF-κB signaling pathway after myocardial ischemia-reperfusion injury

Wan-Ting Meng et al. J Nanobiotechnology. .

Abstract

Reperfusion therapy is critical for saving heart muscle after myocardial infarction, but the process of restoring blood flow can itself exacerbate injury to the myocardium. This phenomenon is known as myocardial ischemia-reperfusion injury (MIRI), which includes oxidative stress, inflammation, and further cell death. microRNA-146a (miR-146a) is known to play a significant role in regulating the immune response and inflammation, and has been studied for its potential impact on the improvement of heart function after myocardial injury. However, the delivery of miR-146a to the heart in a specific and efficient manner remains a challenge as extracellular RNAs are unstable and rapidly degraded. Milk exosomes (MEs) have been proposed as ideal delivery platform for miRNA-based therapy as they can protect miRNAs from RNase degradation. In this study, the effects of miR-146a containing MEs (MEs-miR-146a) on improvement of cardiac function were examined in a rat model of MIRI. To enhance the targeting delivery of MEs-miR-146a to the site of myocardial injury, the ischemic myocardium-targeted peptide IMTP was modified onto the surfaces, and whether the modified MEs-miR-146a could exert a better therapeutic role was examined by echocardiography, myocardial injury indicators and the levels of inflammatory factors. Furthermore, the expressions of miR-146a mediated NF-κB signaling pathway-related proteins were detected by western blotting and qRT-PCR to further elucidate its mechanisms. MiR-146 mimics were successfully loaded into the MEs by electroporation at a square wave 1000 V voltage and 0.1 ms pulse duration. MEs-miR-146a can be up-taken by cardiomyocytes and protected the cells from oxygen glucose deprivation/reperfusion induced damage in vitro. Oral administration of MEs-miR-146a decreased myocardial tissue apoptosis and the expression of inflammatory factors and improved cardiac function after MIRI. The miR-146a level in myocardium tissues was significantly increased after the administration IMTP modified MEs-miR-146a, which was higher than that of the MEs-miR-146a group. In addition, intravenous injection of IMTP modified MEs-miR-146a enhanced the targeting to heart, improved cardiac function, reduced myocardial tissue apoptosis and suppressed inflammation after MIRI, which was more effective than the MEs-miR-146a treatment. Moreover, IMTP modified MEs-miR-146a reduced the protein levels of IRAK1, TRAF6 and p-p65. Therefore, IMTP modified MEs-miR-146a exerted their anti-inflammatory effect by inhibiting the IRAK1/TRAF6/NF-κB signaling pathway. Taken together, our findings suggested miR-146a containing MEs may be a promising strategy for the treatment of MIRI with better outcome after modification with ischemic myocardium-targeted peptide, which was expected to be applied in clinical practice in future.

Keywords: Inflammatory factors; Milk exosome; Myocardial ischemia-reperfusion injury; NF-κB signaling pathway; Targeting delivery; microRNA-146a.

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Conflict of interest statement

TThe authors declare that they have no conflicts of interest.

Figures

Fig. 1
Fig. 1
Preparation and characterization of MEs and MEs-miR-146a. (A) Representative TEM image of MEs. Scale bar = 100 nm. (B) Size diameters distribution of MEs as detected by NTA. (C) The typical exosome biomarkers of MEs and milk were measured by Western blotting. (D) The efficiency and the best parameters of MEs-miR-146a for electroporation were measured by qRT–PCR. (E) Representative TEM image of MEs-miR-146a. Scale bar = 100 nm. (F) Size of MEs-miR-146a detected by NTA
Fig. 2
Fig. 2
MEs-miR-146a protects H9c2 cells from OGD/R induced damage. (A) After 24 h of incubation, PKH26 labeled exosomes could be uptaken up by H9c2. Scale bar = 20 μm. (B) MEs-miR-146a decreased OGD/R induced cell apoptosis of H9c2 according to TUNEL assay. Scale bar = 50 μm. *P < 0.05, **P < 0.01 versus the control group; &P < 0.05, &&P < 0.01 versus the OGD/R group; ▲P < 0.05 versus the OGD/R-MEs group. n = 6
Fig. 3
Fig. 3
MEs-miR-146a protects NRCM cells from OGD/R induced damage. (A) Representative fluorescence images indicated that PKH26 labeled exosomes were taken up by NRCM cells after 24 h of incubation. Scale bar = 20 μm. (B) MEs-miR-146a decreased OGD/R induced cell apoptosis of NRCM according to TUNEL assay. Scale bar = 50 μm. **P < 0.01 versus the control group; &P < 0.05, &&P < 0.01 versus the OGD/R group; ▲P < 0.05 versus the OGD/R-MEs group. n = 6
Fig. 4
Fig. 4
Oral administration of MEs-miR-146a improved cardiac function after MIRI. (A) The preparation flow of MEs-miR-146a and the schematic illustration of the treatment plan. (B) Representative echocardiography images. (C) The values of EF and FS. *P < 0.05, **P < 0.01 versus the Sham group; &&P < 0.01 versus the MIRI group; ▲▲P < 0.01 versus the MIRI-MEs-miR-146a group. (D) Representative H&E images of heart tissue from each group. Scale bar = 50 μm. (E) Detection of miR-146a expression in rat myocardium by qRT-PCR. (F-H) Quantification of CK-MB, LDH and SOD levels in the rat serum after MIRI. *P < 0.05, **P < 0.01 versus the Sham group; &&P < 0.01 versus the MIRI group; ▲▲P < 0.01 versus the MIRI-MEs-miR-146a group. n = 5
Fig. 5
Fig. 5
MEs-miR-146a exhibited better therapeutic efficacy in decreasing apoptotic cells and limiting inflammation than MEs. (A-B) Representative TUNEL staining images and quantification of apoptotic radio. **P < 0.01 versus the Sham group; &P < 0.05 and &&P < 0.01 versus the MIRI group. (C-F) The expression of inflammatory factors of rat serum were detected by ELISA. *P < 0.05, **P < 0.01 versus the Sham group; &P < 0.05, &&P < 0.01 versus the MIRI group; ▲P < 0.05, ▲▲P < 0.01 versus the MIRI-MEs-miR-146a group. n = 5
Fig. 6
Fig. 6
MEs-miR-146a protected myocardium from MIRI through suppressing NF-κB activation and IRAK1 / TRAF6 expression. (A-C) Changes in intracellular IRAK1, TRAF6, p65, p-p65, ikBα and p-ikBα protein levels after treatment of MEs-miR-146a. (D) The expression of IRAK1, TRAF6 and p65 mRNA level were examined by qRT-PCR. *P < 0.05, **P < 0.01 versus the Sham group; &P < 0.05, &&P < 0.01 versus the MIRI group; ▲P < 0.05, ▲▲P < 0.01 versus the MIRI-MEs-miR-146a group. n = 5
Fig. 7
Fig. 7
Preparation and characterization of IMTP-MEs-miR-146a. (A) Schematic of the IMTP-MEs-miR-146a used for MIRI therapy. (B) A standard curve was generated based on different IMTP-MEs-miR-146a concentrations and their corresponding fluorescence intensities. (C) The morphology of IMTP-MEs-miR-146a was observed by TEM. Scale bar = 100 nm. (D) Characterization of IMTP-MEs-miR-146a by NTA
Fig. 8
Fig. 8
The biodistribution of MEs in MIRI rat. In vitro imaging analyses of organs 24 h after the intravenous injection of MEs. *P < 0.05, **P < 0.01 versus the MIRI group; &&P < 0.01 versus the MIRI-MEs group; ▲▲P < 0.01 versus the MIRI-MEs-miR-146a group
Fig. 9
Fig. 9
Tail vein injection of IMTP-MEs-miR-146a exhibited superior therapeutic efficacy in improving cardiac function. (A) Quantification of miR-146a mRNA level in myocardial tissue. *P < 0.05, **P < 0.01 versus the MIRI group; &&P < 0.01 versus the MIRI-MEs-miR-146a group. n = 8. (B-C) The expression of SDF-1 and CK-MB in the rat serum were detected by ELISA. *P < 0.05, **P < 0.01 versus the MIRI group; &&P < 0.01 versus the MIRI-MEs-miR-146a group. n = 8. (D-E) Representative echocardiography images from each group and quantitative analysis of EF and FS. **P < 0.01 versus the MIRI group; ▲▲P < 0.01 versus the MIRI-MEs-miR-146a group. n = 8
Fig. 10
Fig. 10
Tail vein injection of IMTP-MEs-miR-146a significantly ameliorated myocardium apoptosis and attenuated inflammation at the early stage of MIRI. (A) Representative H&E images of heart tissue from each group. Scale bar = 50 μm. (B-C) Representative images of TUNEL staining 24 h after MIRI and quantitative analysis of apoptotic radio. *P < 0.05 and **P < 0.01 versus the MIRI group; &P < 0.05 versus the MIRI-MEs-miR-146a group. n = 8. (D-G) The expression of inflammatory factors of rat serum were detected by ELISA. ∆∆P < 0.01 versus the MIRI group; ▲▲P < 0.01 versus the MIRI-MEs-miR-146a group. n = 8
Fig. 11
Fig. 11
Intravenous injection of IMTP-MEs-miR-146a protected myocardium from MIRI through targeting IRAK1/TRAF6 and suppressing NF-κB activation. (A-B) Changes in intracellular IRAK1, TRAF6, p65, p-p65, ikBα and p-ikBα protein levels after treatment of MEs-miR-146a and IMTP-MEs-miR-146a. (C) The expression of IRAK1, TRAF6 and p65 mRNA level were detected by qRT-PCR. *P < 0.05, **P < 0.01 versus the MIRI group; &P < 0.05, &&P < 0.01 versus the MIRI-MEs-miR-146a group. n = 8
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