Tilianin Protects against Ischemia/Reperfusion-Induced Myocardial Injury through the Inhibition of the Ca2+/Calmodulin-Dependent Protein Kinase II-Dependent Apoptotic and Inflammatory Signaling Pathways

Abstract

Tilianin is a naturally occurring phenolic compound with a cardioprotective effect against myocardial ischemia/reperfusion injury (MIRI). The aim of our study was to determine the potential targets and mechanism of action of tilianin against cardiac injury induced by MIRI. An in silico docking model was used in this study for binding mode analysis between tilianin and Ca²⁺/calmodulin-dependent protein kinase II (CaMKII). Oxygen-glucose deprivation/reperfusion- (OGD/R-) injured H9c2 cardiomyocytes and ischemia/reperfusion- (I/R-) injured isolated rat hearts were developed as in vitro and ex vivo models, respectively, which were both treated with tilianin in the absence or presence of a specific CaMKII inhibitor KN93 for target verification and mechanistic exploration. Results demonstrated the ability of tilianin to facilitater the recovery of OGD/R-induced cardiomyocyte injury and the maintenance of cardiac function in I/R-injured hearts. Tilianin interacted with CaMKIIδ with an efficient binding performance, a favorable binding score, and restraining p-CaMKII and ox-CaMKII expression in cardiomyocytes injured by MIRI. Importantly, inhibition of CaMKII abolished tilianin-mediated recovery of OGD/R-induced cardiomyocyte injury and maintenance of cardiac function in I/R-injured hearts, accompanied by the disability to protect mitochondrial function. Furthermore, the protective effects of tilianin towards mitochondrion-associated proapoptotic and antiapoptotic protein counterbalance and c-Jun N-terminal kinase (JNK)/nuclear factor- (NF-) κB-related inflammation suppression were both abolished after pharmacological inhibition of CaMKII. Our investigation indicated that the inhibition of CaMKII-mediated mitochondrial apoptosis and JNK/NF-κB inflammation might be considered as a pivotal mechanism used by tilianin to exert its protective effects on MIRI cardiac damage.

Figure 1

Chemical structure of tilianin. The molecular formula of tilianin is C₂₂H₂₂O₁₀.

Figure 2

Ex vivo experimental procedure of the isolated rat hearts subjected to I/R and treated with tilianin and/or KN93. Isolated rat hearts were randomly divided into control, control+tilianin, I/R, I/R+tilianin, I/R+tilianin/KN93, and I/R+KN93 groups. The hearts of the control group were perfused with KHB for 30 min and then for the same period of time as the global I/R. The hearts of the control+tilianin group received 10 min perfusion with KHB and then 20 min KHB containing 4 μM tilianin and continuous perfusion with oxygenated KHB for the same period of time as the control group. The hearts of the I/R group were subjected to global ischemia by interrupting KHB perfusion for 45 min, followed by reperfusion for 60 min. The hearts of the I/R+tilianin group were perfused with KHB containing 4 μM tilianin for 20 min before I/R. The hearts of the I/R+KN93/tilianin group were perfused with 2.5 μM KN93 for 10 min before tilianin perfusion. The hearts of the I/R+KN93 group were perfused with 2.5 μM KN93 for 10 min and KHB for 20 min before I/R. After monitoring of the heart cardiodynamic parameters during I/R procedure, the isolated perfused rat heart was placed in ice-cold KHB for the subsequent measurement of western blot assay and other biochemical indicators.

Figure 3

Tilianin treatment protects H9c2 cardiomyocytes against OGD/R-induced cytotoxicity. (a) Tilianin increased cell viability as evaluated by MTS assay. (b) Tilianin decreased the release of lactate dehydrogenase (LDH) after OGD/R injury. (c) Representative images of nuclei stained with Hoechst 33342 (20×). (d) Tilianin decreased the mean fluorescence intensity of nuclei stained with Hoechst in H9c2 cells after OGD/R injury. (e) Representative images of the microtubule morphology stained with β-tubulin (20×). (f) Tilianin increased mean fluorescence intensity of β-tubulin in H9c2 cells after OGD/R injury. Results are expressed as the mean ± S.E.M.n = 6. ^###P < 0.001vs. control, ^∗P < 0.05, ^∗∗P < 0.01, and ^∗∗∗P < 0.001vs. OGD/R.

Figure 4

Prediction of CaMKIIδ/tilianin binding ability and inhibitory effect of tilianin on CaMKIIδ kinase activity. (a) Three-dimensional model of tilianin binding with the binding domain of CaMKIIδ. (b) Two-dimensional ligand interaction diagram of tilianin and CaMKIIδ. (c) Inhibitory effect of tilianin on CaMKIIδ kinase activity in vitro (n = 5). (d) Double-reciprocal analysis of the inhibitory effect. (e) Representative images of p-CaMKIIδ and ox-CaMKIIδ staining (20×). (f, g) Mean fluorescence intensity of p-CaMKIIδ and ox-CaMKIIδ (n = 6). (h) Representative western blot bands of the expression of p-CaMKIIδ and ox-CaMKIIδ in H9c2 cells. (i) Quantitative analysis indicated that tilianin inhibited the p-CaMKIIδ/CaMKIIδ and ox-CaMKIIδ/CaMKIIδ ratios (n = 4). Results are expressed as the mean ± S.E.M.^###P < 0.001vs. control, ^∗P < 0.05, ^∗∗P < 0.01, and ^∗∗∗P < 0.001vs. OGD/R.

Figure 5

Tilianin exerts a protective effect against OGD/R injury through mitochondrion preservation via CaMKII inhibition. (a) The effect of tilianin treatment on increasing cell viability against OGD/R-induced toxicity was blocked by CaMKII pharmacological inhibition using KN93. (b) The effect of tilianin treatment on decreasing LDH release on OGD/R-injured H9c2 cells was reduced due to KN93 treatment. (c) Representative images of MMP staining by Rh123 (20×) and superoxide by MitoSOX in mitochondria (20x). (d, e) The protective effect of tilianin on mitochondrial function evaluated by Rh123 staining (d) and MitoSOX staining (e) was abolished when OGD/R-injured H9c2 cells were pretreated with KN93. Results are expressed as the mean ± S.E.M.n = 6. ^###P < 0.001vs. control, ^∗P < 0.05, ^∗∗∗P < 0.001vs. OGD/R; ^$P < 0.05, ^$$P < 0.01, and ^$$$P < 0.001vs. OGD/R+tilianin.

Figure 6

Tilianin protects H9c2 cardiomyocytes against OGD/R injury through mitochondrion-associated apoptotic signaling via CaMKII inhibition. (a) Representative images of cytochrome c, Bax, and Bcl-2 expression by immunohistochemistry and nuclear changes stained by Hoechst 33342 (20×). (b–e) Restorative effects of tilianin treatment on the expression of cytochrome c, Bax, and Bcl-2 and nucleus damage caused by OGD/R-induced toxicity were abolished by KN93. (f, g) The inhibitory effect on caspase-3/9 activity by tilianin was reduced due to KN93 treatment. Results are expressed as the mean ± S.E.M.n = 6. ^###P < 0.001vs. control, ^∗∗P < 0.01, ^∗∗∗P < 0.001vs. OGD/R; ^$P < 0.05, ^$$P < 0.01, and ^$$$P < 0.001vs. OGD/R+tilianin.

Figure 7

Tilianin protects H9c2 cardiomyocytes against OGD/R injury through JNK/NF-κB-related inflammatory suppression via CaMKII inhibition. (a) Representative immunohistochemical images of p-JNK, p-c-Jun, and p-p65 expression (20×). (b–d) Ameliorative effects of tilianin on JNK/c-Jun and p-p65 activation were reduced by KN93. (e, f) Inhibitory effects on TNF-α and IL-6 release by tilianin were prevented by KN93 treatment. Results are expressed as the mean ± S.E.M.n = 6. ^###P < 0.001vs. control, ^∗P < 0.05, ^∗∗P < 0.01, and ^∗∗∗P < 0.001vs. OGD/R; ^$P < 0.05, ^$$P < 0.01, ^$$$P < 0.001vs. OGD/R+tilianin.

Figure 8

CaMKII contributes to tilianin protection against ischemia/reperfusion (I/R) injury in heart tissue. (a–e) LVSP (a), LVDP (b), +dp/dt_max (c), −dp/dt_min (d), and heart rate (e) values recovered after tilianin treatment, effects that were prevented when the tissue was pretreated with KN93 (n = 12). (f, g) Na⁺-K⁺-ATPase activity and ATP concentration increased following tilianin treatment but weakened after pretreatment with KN93 (n = 5). (h) Representative immunoblots illustrating the expression of cytochrome c, Bax, Bcl-2, p-JNK, JNK, p-p65, and p65 (n = 5). (i, j) Quantitative analysis indicating that restoration of the cytochrome c expression and Bcl-2/Bax, p-JNK/JNK, and p-p65/p65 ratios by tilianin was reversed by pretreatment with KN93. (k, l) Inhibitory effect on caspase-9 (k) and caspase-3 (l) activity by tilianin blocked by KN93 pretreatment (n = 5). Results are expressed as the mean ± S.D.^#P < 0.05, ^##P < 0.01, and ^###P < 0.001vs. control; ^∗P < 0.05, ^∗∗P < 0.01, ^∗∗∗P < 0.001vs. I/R; ^$P < 0.05, ^$$P < 0.01, ^$$$P < 0.001vs. I/R+tilianin.

Figure 9

Tilianin exerts myocardial protection against I/R injury through its action on mitochondrion-related apoptosis and JNK/NF-κB-activated inflammation via CaMKII. Bax: Bcl-2-associated X protein; Bcl-2: B cell lymphoma-2; CaMKII: Ca²⁺/calmodulin-dependent protein kinase II; IL-6: interleukin-6; JNK: c-Jun N-terminal protein kinase; NF-κB: nuclear factor kappa-B; TNF-α: tumor necrosis factor alpha.