Cardioprotective Effect of Paeonol on Chronic Heart Failure Induced by Doxorubicin via Regulating the miR-21-5p/S-Phase Kinase-Associated Protein 2 Axis

FIGURE 1. Paeonol improved the cardiac function and ameliorated the cardiac damage of CHF rats. (A–E) The parameters underlying the cardiac function of the rats, including LVSP (A), LVEDP (B), +dp/dtmax (C), –dp/dtmax (D), and HR (E), were detected using RM6240 series multichannel physiological signal acquisition apparatus. (F–K) The concentration of several specific markers related to the myocardial damage in the rats, including BNP (F), LDH (G), RE (H), Ang II (I), ALD (J), and ET-1 (K), were evaluated using a Synergy H1 Hybrid Multi-Mode Reader. ∧∧P < 0.01, ∧∧∧P < 0.001, vs. Sham; *P < 0.05, **P < 0.01, ***P < 0.001, vs. DOX. DOX, doxorubicin; LVSP, left ventricular systolic pressure; LVEDP, left ventricle end diastolic pressure; +dp/dtmax, +left ventricle dp/dtmax; –dp/dtmax, –left ventricle dp/dtmax; HR, heart rate; BNP, brain natriuretic peptide; LDH, lactate dehydrogenase; RE, renin; Ang II, angiotensin II; ALD, aldosterone; ET-1, endothelin 1.

FIGURE 2. Paeonol ameliorated histopathological injury, suppressed apoptosis, and downregulated the miR-21-5p expression in the myocardium of CHF rats. (A) The myocardial histopathology of the rats was detected by hematoxylin–eosin staining (magnification of ×200). (B) The myocardial apoptosis of the rats was detected by TUNEL staining (Magnification: ×200). (C,D) The levels of Bcl-2, Bax, caspase-3, C-caspase-3, and Cyt C in the myocardium of rats were detected by Western blot. GAPDH was used as an internal control. (E) The expression of miR-21-5p in the myocardium of rats was detected by RT-qPCR, with U6 serving as an internal control. ∧P < 0.05, ∧∧P < 0.01, ∧∧∧P < 0.001, vs. Sham; *P < 0.05, **P < 0.01, ***P < 0.001 vs. DOX. DOX, doxorubicin.

FIGURE 3. Paeonol increased the MMP and viability, while decreasing the apoptosis and ROS, and suppressing miR-21-5p expression in the DOX-induced cardiomyocytes. (A) The viability of H9c2 cells treated with DOX or paeonol was detected by CCK-8 assay. (B,C) The apoptosis of H9c2 cells treated with DOX or paeonol was determined via flow cytometry. (D,F) The level of ROS in the H9c2 cells treated with DOX or paeonol was visualized by H2DCFDA staining. (E,G) The MMP in the H9c2 cells received the treatment with DOX or paeonol and was detected using TMRM staining. (H) The expression of miR-21-5p in H9c2 cells treated with DOX or paeonol was quantified with the help of RT-qPCR. U6 served as an internal control. ∧P < 0.05, ∧∧∧P < 0.001 vs. Control; **P < 0.01, ***P < 0.001 vs. DOX. DOX, doxorubicin; MMP, mitochondrial membrane potential; ROS, reactive oxygen species.

FIGURE 4. miR-21-5p mimic counteracted the effects of paeonol on the viability, apoptosis, and miR-21-5p expression of the DOX-induced cardiomyocytes. (A) The expression of miR-21-5p in DOX-induced H9c2 cells treated with miR-21-5p mimic or paeonol-M was quantified by RT-qPCR. U6 served as an internal control. (B) The viability of DOX-induced H9c2 cells treated with miR-21-5p mimic or paeonol-M was detected by CCK-8 assay. (C,D) The apoptosis of DOX-induced H9c2 cells treated with miR-21-5p mimic or paeonol-M was unveiled using flow cytometry. *P < 0.05, **P < 0.01, ***P < 0.001 vs. DOX + MC; ##P < 0.01, ###P < 0.001, vs. DOX + M;∧P < 0.05, ∧∧P < 0.01, ∧∧∧P < 0.001 vs. DOX + Paeonol-M + MC). DOX, doxorubicin; M, miR-21-5p mimic; MC, mimic control.

FIGURE 5. miR-21-5p mimic counteracted the effects of paeonol on the MMP and ROS of the DOX-induced cardiomyocytes. (A,B) The ROS level in the DOX-induced H9c2 cells treated with miR-21-5p mimic or Paeonol-M was observed by H2DCFDA staining. (C,D) The MMP in the DOX-induced H9c2 cells treated with miR-21-5p mimic or Paeonol-M was determined by TMRM staining. **P < 0.01, ***P < 0.001 vs. DOX + MC;###P < 0.001, vs. DOX + M;∧∧∧P < 0.001 vs. DOX + Paeonol-M + MC. DOX, doxorubicin; MMP, mitochondrial membrane potential; ROS, reactive oxygen species; M, miR-21-5p mimic; MC, mimic control.

FIGURE 6. SKP2 was targeted by miR-21-5p and was downregulated in CHF rats and DOX-induced cardiomyocytes. (A) The binding sites between miR-21-5p and SKP2 were predicted by the TargetScan database. (B) Dual-luciferase reporter assay was employed to verify the targeting relationship between miR-21-5p and SKP2. (C,D) The expression of SKP2 in the myocardium of CHF rats was detected by Western blot, with GAPDH serving as an internal control. (E) The expression of SKP2 in the myocardium of CHF rats was detected by RT-qPCR. GAPDH was used as an internal control. (F,G) The expression of SKP2 in the DOX-induced H9c2 cells treated with PAE was detected by immunocytochemistry. (H) The expression of SKP2 in the DOX-induced H9c2 cells treated with miR-21-5p mimic or Paeonol-M was quantified by RT-qPCR. GAPDH served as an internal control. ††P < 0.01 vs. MC; Δ Δ P < 0.01, Δ Δ Δ P < 0.001 vs. Sham; ξ P < 0.05, ξ ξ P < 0.01, ξ ξ ξ P < 0.001 vs. DOX; *P < 0.05, ***P < 0.001 vs. DOX + MC;###P < 0.001 vs. DOX + M;∧∧∧P < 0.001 vs. DOX + PAE-M + MC. DOX, doxorubicin; M, miR-21-5p mimic; MC, mimic control.

FIGURE 7. (A) The expression of SKP2 in the miR-21-5p-treated DOX-induced H9c2 cells was calculated using RT-qPCR. GAPDH was used as an internal control. (B,C) The expression of SKP2 in the miR-21-5p-treated DOX-induced H9c2 cells was detected by western blot, where GAPDH was used as an internal control. (D) The levle of SKP2 in the miR-21-5p-treated DOX-induced H9c2 cells was calculated using RT-qPCR. GAPDH was used as an internal control. (E) The viability of the miR-21-5p-treated DOX-induced H9c2 cells was evaluated based on CCK-8 assays. (F,G) The apoptosis of the miR-21-5p-treated DOX-induced H9c2 cells was determined by flow cytometry. (H,I) The ROS of the miR-21-5p-treated DOX-induced H9c2 cells was observable following the H2DCFDA staining (Magnification: ’200). (ΔP < 0.05, ΔΔΔP < 0.001 vs. Control; *P < 0.05, ***P < 0.001 vs. DOX+shNC; ###P < 0.001, vs. DOX+Paeonol+shNC; +P < 0.05, ++P < 0.01 vs. DOX; ∧∧∧P < 0.001, vs. DOX+M+NC). (DOX, doxorubicin; ROS, reactive oxygen species; M, miR-21-5p mimic; NC, negative control).

FIGURE 8. SKP2 overexpression increased the MMP and regulated the apoptosis-related factors in miR-21-5p-treated DOX-induced cardiomyocytes. (A,B) The MMP in the miR-21-5p-treated DOX-induced H9c2 cells was determined based on the assay of TMRM staining (Magnification: ×200). (C,D) The expression of Bcl-2, Bax, and C-caspase-3 in the miR-21-5p-treated DOX-induced H9c2 cells was calculated by Western blot. GAPDH served as an internal control. (E,F) The expression of cytoplasm or mitochondrion Cyt c in the miR-21-5p-treated DOX-induced H9c2 cells was detected via Western blot. GAPDH served as an internal control for cytoplasm Cyt c, and COXIV was used as an internal control for mitochondrion Cyt c. Δ P < 0.05, Δ Δ Δ P < 0.001 vs. Control;+P < 0.05, ++P < 0.01, +++P < 0.001 vs. DOX;∧P < 0.05, ∧∧P < 0.01, ∧∧∧P < 0.001 vs. DOX + M + NC. DOX, doxorubicin; MMP, mitochondrial membrane potential; M, miR-21-5p mimic; NC, negative control.