Roflumilast reduces myocardial ischemia reperfusion injury in vivo and in vitro by activating the AMPK signaling pathway

Figure 1. Roflumilast decreases MI/R-induced myocardial infarction. (A) Chemical structure of roflumilast was obtained from PubChem. (B) Representative images of myocardial infarct size demonstrated by TTC staining. The non-stained areas (white or pale) indicate infarct areas; TTC-stained areas (red) indicate non-infarct areas. The activities of (C) CK-MB, (D) LDH and (E) AST in rat serum were detected by corresponding assay kits. ***P<0.001 vs. Control. ##P<0.01 and ###P<0.001 vs. MI/R. ++P<0.01 and +++P<0.001 vs. MI/R + roflumilast (1 mg/kg). MI/R, myocardial ischemia/reperfusion; TTC, 2,3,5-triphenyltetrazolium chloride; CK-MB, creatine kinase-myocardial band; LDH, lactate dehydrogenase; AST, aspartate transaminase; MI, myocardial infarction.

Figure 2. Roflumilast attenuates MI/R-induced myocardial injury. (A) Changes of pathological injury of myocardial tissue were detected by H&E staining. The arrows indicated hemocyte infiltration. (B) Inflammatory factors (IL-1β and IFN-γ) in myocardial tissue were detected by corresponding assay kits. ***P<0.001 vs. Control. #P<0.05 and ###P<0.001 vs. MI/R. ++P<0.01 vs. MI/R + roflumilast (1 mg/kg). MI/R, myocardial ischemia/reperfusion.

Figure 3. Roflumilast attenuates myocardial mitochondrial damage induced by MI/R. (A) Changes in mitochondrial membrane potential were detected by JC-1 staining. (B) Levels of oxidative stress markers in myocardial tissue were detected by assay kits. ***P<0.001 vs. Control. ##P<0.01 and ###P<0.001 vs. MI/R. +++P<0.001 vs. MI/R + roflumilast (1 mg/kg). MI/R, myocardial ischemia/reperfusion; MDA, malondialdehyde; SOD, superoxide dismutase.

Figure 4. Roflumilast activates the AMPK signaling pathway in MI/R injury. The expression of AMPK signaling pathway-associated proteins in myocardial tissue was detected by western blotting. ***P<0.001 vs. Control. #P<0.05 and ###P<0.001 vs. MI/R. ++P<0.01 vs. MI/R + roflumilast (1 mg/kg). AMPK, AMP-activated protein kinase; MI/R, myocardial ischemia/reperfusion; p, phosphorylated; SIRT1, sirtuin 1.

Figure 5. Roflumilast mitigates damage of H/R to H9C2 cell viability by activating the AMPK signaling pathway. (A) Effect roflumilast on H9C2 cell viability was analyzed using CCK-8 assay. (B) Viability of H/R-induced H9C2 cells treated with roflumilast was analyzed by CCK-8 assay. (C) Expression AMPK signaling pathway-related proteins in H/R-induced H9C2 cells with roflumilast treatment was detected by western blotting. (D) Apoptosis of H/R-induced H9C2 cells with roflumilast and compound C treatment was analyzed by TUNEL assay. (E) Expression of apoptosis-related proteins in H/R-induced H9C2 cells with roflumilast and compound C treatment was detected by western blotting. ***P<0.001 vs. Control. #P<0.05, ##P<0.01 and ###P<0.001 vs. H/R. ++P<0.01 and +++P<0.001 vs. H/R + roflumilast (5 µM). H/R, hypoxia/reoxygenation; AMPK, AMP-activated protein kinase; CCK-8, Cell Counting Kit-8; p, phosphorylated; SIRT1, sirtuin 1.

Figure 6. Roflumilast activates the AMP-activated protein kinase signaling pathway to alleviate oxidative stress and the inflammatory response of H9C2 cells induced by H/R. (A) Oxidative stress in H/R-induced H9C2 cells with roflumilast and compound C treatment were detected by assay kits. (B) Inflammatory factors in H/R-induced H9C2 cells with roflumilast and compound C treatment were detected by assay kits. ***P<0.001 vs. Control. ###P<0.001 vs. H/R. ++P<0.01 and +++P<0.001 vs. H/R + roflumilast (5 µM). H/R, hypoxia/reoxygenation; MDA, malondialdehyde; SOD, superoxide dismutase.

Figure 7. Roflumilast activates the AMP-activated protein kinase signaling pathway to decrease mitochondrial damage of H9C2 induced by H/R. (A) Changes of mitochondrial membrane potential in H/R-induced H9C2 cells with roflumilast and compound C treatment were detected by JC-1 staining. (B) ATP levels in H/R-induced H9C2 cells with roflumilast and compound C treatment were detected by ATP assay kit. (C) mPTP in H/R-induced H9C2 cells with roflumilast and compound C treatment was examined via a calcein-loading/CoCl2-quenching system (magnification, x100). (D) Quantification of mPTP opening level. (E) Expression of mitochondrial regulatory proteins in H/R-induced H9C2 cells with roflumilast and compound C treatment was determined by western blotting. ***P<0.001 vs. Control. ###P<0.001 vs. H/R. +P<0.05 and +++P<0.001 vs. H/R + roflumilast (5 µM) group. H/R, hypoxia/reoxygenation; mPTP, mitochondrial permeability transition pore; PINK1, PTEN-induced kinase 1; p, phosphorylated; DRP1, dynamin-related protein 1.