RP105 plays a cardioprotective role in myocardial ischemia reperfusion injury by regulating the Toll‑like receptor 2/4 signaling pathways

Abstract

The revascularization of blood vessels after myocardial infarction can lead to serious myocardial damage. Previous studies showed that radioprotective 105 kDa protein (RP105) is a specific negative regulator of myocardial ischemia reperfusion injury (MIRI). RP105 can modulate the Toll‑like receptor (TLR)2/TLR4 signaling pathways. However, the synergistic effect of TLR2/4 regulated by RP105 during MIRI requires further investigation. To determine this effect, a MIRI model was established in rats in the present study. The expression of RP105 was depleted by transfecting RP105‑siRNA and then detected using western blotting. Furthermore, the myocardium tissue was stained with the hematoxylin and eosin staining. Knockdown of RP105 promoted the activity of serum myocardial enzymes during MIRI and increased myocardial infarction. The present results indicated that knockdown of RP105 activated the TLR2/4 signaling pathway by modulating the myeloid differentiation primary response 88 and NF‑κB signaling pathways. Furthermore, decreased expression of RP105 promoted myocardial cell apoptosis, which induced the damage of myocardial ischemic reperfusion. The present results suggested both TLR2 and TLR4 as key targets of RP105, thus RP105 may be a promising candidate to facilitate the development of novel therapeutic strategies for MIRI.

Keywords: radioprotective 105 kda protein; anti-inflammation; Toll-like receptor 4; Toll-like receptor 2; myocardial ischemia reperfusion injury.

Figure 1.

Overexpression or knockdown of RP105 in rat cardiac muscle tissue. (A) Expression levels of EGFP, with the indicated adenovirus gene injection for RP105 overexpression and RP105-siRNA, and DAPI-labeled nuclei density of cardiomyocytes. Scale bar, 50 µm. (B) mRNA expression of RP105 in rat cardiac muscle tissues was detected by reverse transcription-quantitative PCR after transfection with the indicated vector. (C) Protein expression levels of RP105 in rat cardiac muscle tissue. *P<0.05, **P<0.01, ***P<0.001. Ad, adenovirus; RP105, radioprotective 105 kDa protein; siRNA, small interfering RNA; EGFP, enhanced green fluorescent protein; sham, control sham group; I/R, ischemic and reperfusion; Ad-R, Ad-RP105 group; Ad-R-siRNA, Ad-RP105-siRNA group; Ad-G-siRNA, Ad-EGFP-siRNA group.

Figure 2.

Knockdown of RP105 promotes the activity of serum myocardial enzymes during MIRI. Levels of serum (A) CK-MB, (B) LDH and (C) CK in sham, I/R, Ad-R, Ad-R-siRNA and Ad-G-siRNA groups. *P<0.05, **P<0.01, ***P<0.001. Ad, adenovirus; RP105, radioprotective 105 kDa protein; siRNA, small interfering RNA; EGFP, enhanced green fluorescent protein; CK, creatine kinase; CK-MB, MB isoenzyme of CK; LDH, lactate dehydrogenase; sham, control sham group; I/R, ischemic and reperfusion; Ad-R, Ad-RP105 group; Ad-R-siRNA, Ad-RP105-siRNA group; Ad-G-siRNA, Ad-EGFP-siRNA group.

Figure 3.

Silencing of RP105 increases myocardial infarction during MIRI. (A) Area of myocardial infarction in each group was imaged and analyzed using Image-Pro Plus 5. The injury areas were highlighted with white arrows. (B) Statistical analysis of the myocardial infarction of each group. (C) Hematoxylin and eosin staining of the myocardium tissue slices in each group. Scale bar, 25 µm. ***P<0.001. Ad, adenovirus; RP105, radioprotective 105 kDa protein; siRNA, small interfering RNA; EGFP, enhanced green fluorescent protein; sham, control sham group; I/R, ischemic and reperfusion; Ad-R, Ad-RP105 group; Ad-R-siRNA, Ad-RP105-siRNA group; Ad-G-siRNA, Ad-EGFP-siRNA group; IA, infarct area; AAR, area at risk.

Figure 4.

Knockdown of RP105 increases the expression levels of TLR2 and TLR4 in MIRI. Protein expression levels of serum (A) TLR2 and (B) TLR4 in each group. (C) Western blotting for the expression levels of TLR2 and TLR4 in each group. The interactions of MD2 with TLR2 and TLR4 following overexpression or knockdown of RP105 were investigated. Expression level of (D) TNF-α and (E) IL-6 in each group. (F) Western blotting bands of TNF-α and IL-6. *P<0.05, **P<0.01. Ad, adenovirus; RP105, radioprotective 105 kDa protein; siRNA, small interfering RNA; EGFP, enhanced green fluorescent protein; sham, control sham group; I/R, ischemic and reperfusion; Ad-R, Ad-RP105 group; Ad-R-siRNA, Ad-RP105-siRNA group; Ad-G-siRNA, Ad-EGFP-siRNA group; TLR2/4, Toll-like receptor 2/4; TNF-α, tumor necrosis factor; IL-6, interleukin 6; MD2, myeloid differentiation protein 2; IP, immunoprecipitation.

Figure 5.

Silencing of RP105 increases myocardial apoptosis. Protein expression levels of serum (A) Bcl-2 and (B) cleaved caspase-3 in each group. (C) Western blotting bands of Bcl-2 and cleaved caspase-3. *P<0.05, **P<0.01. Ad, adenovirus; RP105, radioprotective 105 kDa protein; siRNA, small interfering RNA; EGFP, enhanced green fluorescent protein; sham, control sham group; I/R, ischemic and reperfusion; Ad-R, Ad-RP105 group; Ad-R-siRNA, Ad-RP105-siRNA group; Ad-G-siRNA, Ad-EGFP-siRNA group.

Figure 6.

Knockdown of RP105 regulates the MyD88 signaling pathway. Protein expression levels of serum (A) MyD88 and (B) NF-κB in each group. (C) Western blotting bands of MyD88 and NF-κB in each group. *P<0.05. Ad, adenovirus; RP105, radioprotective 105 kDa protein; siRNA, small interfering RNA; EGFP, enhanced green fluorescent protein; sham, control sham group; I/R, ischemic and reperfusion; Ad-R, Ad-RP105 group; Ad-R-siRNA, Ad-RP105-siRNA group; Ad-G-siRNA, Ad-EGFP-siRNA group; MyD88, myeloid differentiation factor 88.