Crocetin alleviates myocardial ischemia/reperfusion injury by regulating inflammation and the unfolded protein response

Abstract

Crocetin, a natural compound, has been demonstrated to exhibit beneficial effects in cardiovascular diseases. Previous studies demonstrated that crocetin reduced ischemia/reperfusion (I/R) injury by attenuating cytotoxicity and cellular apoptosis. However, the previous mechanistic studies did not fully elucidate its pharmacological effects on cardiac damage, especially I/R injury. The present study verified its cardioprotective effects in a Langendorff perfusion system, an ex vivo model of I/R. It was demonstrated that crocetin significantly attenuated the activities of pro‑inflammatory cytokines and nuclear factor erythroid‑2 related factor 2 (Nrf2)/heme oxygenase‑1 signaling. The present study provided novel insight that crocetin regulated the unfolded protein response (UPR) and decreased associated protein levels to protect the heart. Furthermore, it was identified that Nrf2 played a key role in the cardioprotective effect of crocetin by attenuating inflammation and the UPR.

Keywords: crocetin; ischemia/reperfusion injury; inflammation; unfolded protein response; nuclear factor erythroid‑2 related factor 2.

Figure 1.

Cardiac protective effect of CRO in the Langendorff perfusion system. (A) Schematic representation of the experimental protocol. Sham: No occlusion; I/R: Surgery with 30 min occlusion, followed by 120 min reperfusion; CRO: 10 min pretreatment of CRO followed by I/R. Effect of CRO on cardiac functional parameters, including (B) heart rate, (C) dp/dt_max and (D) LVDP. Data (n=8) are presented as the mean ± standard error of the mean. CRO, crocetin; I/R, ischemia/reperfusion; dp/dt_max, maximal rate of the increase of left ventricular pressure; LVDP, left ventricular developed pressure; Ctrl, control.

Figure 2.

CRO alleviates myocardial I/R injury via regulation of cardiac enzyme activities. (A) Captured images and (B) analysis of the effect of CRO on the infarct size of the heart induced by I/R injury. Effect of CRO on (C) CK and (D) LDH of the heart induced by I/R injury. Effect of CRO on (E) SOD, (F) MDA and (G) GSH-PX of heart induced by I/R injury. Data (n=8) are presented as the mean ± standard error of the mean. *P<0.05, **P<0.01 and ***P<0.001. CRO, crocetin; I/R, ischemia/reperfusion; CK, creatine kinase; LDH, lactate dehydrogenase; SOD, superoxide dismutase; MDA, malondialdehyde; GSH-PX, glutathione peroxidase.

Figure 3.

CRO alleviates myocardial I/R injury via regulation of the inflammation response. (A) Effect of CRO on pro-inflammatory cytokines of the heart induced by I/R injury. (B) Effect of CRO on Nrf2, HO-1 and NQO1 of the heart induced by I/R injury. Data (n=8) are presented as the mean ± standard error of the mean. *P<0.05, **P<0.01 and ***P<0.001. CRO, crocetin; I/R, ischemia/reperfusion; UPR, unfolded protein response; Nrf2, nuclear factor erythroid-2 related factor 2; HO-1, heme oxygenase-1; NQO1, NAD(P)H: quinine oxidoreductase 1.

Figure 4.

CRO alleviates myocardial I/R injury via regulation of UPR. (A) Western blotting and (B) analysis of the effect of CRO on UPR signaling proteins of the heart induced by I/R injury. Data (n=8) are presented as the mean ± standard error of the mean. *P<0.05, **P<0.01 and ***P<0.001. CRO, crocetin; I/R, ischemia/reperfusion; UPR, unfolded protein response; p-PERK, phosphorylated-protein kinase-like ER kinase; IRE1, inositol requiring enzyme 1; ATF6, Activating transcription factor 6; XBP1, X-box binding protein 1.

Figure 5.

CRO alleviates myocardial I/R injury possibly in a Nrf2-dependent manner. (A and B) Effect of Nrf2 siRNA on protein and mRNA expression of Nrf2 in H9c2 cells. (C) Nrf2 siRNA treatment reduced the effect of CRO on UPR protein expressions. (D) Nrf2 siRNA treatment reduced the effect of CRO on pro-inflammatory cytokinases. (E) Nrf2 siRNA treatment reduced the effect of CRO on cell survival. Data (n=6) are presented as the mean ± standard error of the mean. *P<0.05, **P<0.01 and ***P<0.001. CRO, crocetin; I/R, ischemia/reperfusion; Nrf2, nuclear factor erythroid-2 related factor 2; siRNA, small interfering RNA; UPR, unfolded protein response; NS, no significance.