BYD Ameliorates Oxidative Stress-Induced Myocardial Apoptosis in Heart Failure Post-Acute Myocardial Infarction via the P38 MAPK-CRYAB Signaling Pathway

Figures

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  FIGURE 1. BYD improves cardiac function. (A) Representative images of echocardiography exhibiting the changes in cardiac function in each group. (B) Echocardiography results showed that BYD improved EF and FS and decreased LVIDs. (C) Haemodynamic results showed that BYD up-regulated the levels of MBP and +dp/dt max and down-regulated the level of –dp/dt max, suggesting that BYD could improve cardiac contraction function. All the data were presented as the means ± SEM from independent experiments performed in triplicate. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001 vs. model group. N = 10 per group.

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  FIGURE 2. BYD inhibits inflammatory cell infiltration and protects against cardiac injury. (A) HE showed that BYD preserved cardiomyocyte architecture and inhibited inflammatory cell infiltration. (B) The concentrations of LDH and CK-MB in serum in the model group were increased compared with those in the sham group and could be up-regulated by BYD. (C) BYD treatment up-regulated the level of T-SOD and down-regulated the level of MDA in serum compared with model group. All the data were presented as the means ± SEM from independent experiments performed in triplicate. ∗∗P < 0.01, ∗∗∗P < 0.001 vs. the model group. N = 6 per group.

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  FIGURE 3. BYD regulates the levels of OS mediators in vitro. (A) The doses involved in this study showed no cytotoxicity at 24 h. (B) BYD showed a protective effect on LPS-induced RAW 264.7 cells. (C) BYD significantly decreased the release of NO induced by LPS. (D) The results of ROS determination showed that BYD treatment decreased the green fluorescent intensity. All the data were presented as the means ± SEM from independent experiments performed in triplicate. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001 vs. the model group. N = 6 per group.

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  FIGURE 4. BYD inhibited apoptosis and increased CRYAB in HF post-AMI rats. (A) TUNEL results showed that BYD attenuated the apoptosis of cardiomyocytes. (B) Western blot analysis showed that BYD decreased the expression of Bax, cleaved-caspase 9 and cleaved-caspase 3 and increased the expressions of Bcl-2 in cardiac tissue compared with that in the model group. (C) Immunohistochemistry results showed that BYD up-regulated the expression of p-CRYAB in cardiac tissue. (D) Western blot analysis results showed that BYD up-regulated the expression of p-CRYAB in cardiac tissue compared with that in the model group. The same GAPDH band was selected due to reusing the membrane by stripping solution. All the data were presented as the means ± SEM from independent experiments performed in triplicate. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001 vs. the model group. N = 3 per group. The original bands of Western blot were showed in Supplementary Figure 1.

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  FIGURE 5. BYD inhibits apoptosis in CM-induced H9C2 cells. (A) Flow diagram of CM-induced H9C2 cell injury. (B) BYD showed a significant protective effect on CM-induced H9C2 cell injury. (C) BYD could increase the release of T-SOD from H9C2 injuried by CM. (D) Hoechst 33258 staining results showed that BYD attenuated apoptosis in CM-induced H9C2 cells. (E) BYD could up-regulate the expression of p-CRYAB in vitro. (F) Different doses of BYD treatments down-regulated the increased expression of Bax, cleaved-caspase 9 and cleaved-caspase 3 compared with that in the model group. The same GAPDH band was selected due to reusing the membrane by stripping solution. All the data were presented as the means ± SEM from independent experiments performed in triplicate. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001 vs. the model group. N = 3 per group for western blotting and Hoechst 33258 staining. N = 6 per group in the CCK-8 and T-SOD test. The original bands of Western blot were showed in Supplementary Figure 2.

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  FIGURE 6. BYD exerts its anti-apoptosis effect through the P38 MAPK-CRYAB pathway. (A) BYD activated the P38 MAPK cascade pathway to activate CRYAB in vivo. (B) The Hoechst 33258 staining assay showed that, after SB203580 treatment, the effect of BYD on anti-apoptosis was inhibited. (C) Western blot results showed that, compared with the model group, the expression of p-CRYAB was down regulated in both the SB203580 + model group and BYD + SB203580 group. All the data were presented as the means ± SEM from independent experiments performed in triplicate. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001 vs. model group #P < 0.05, ###P < 0.001 vs. CM+BYD group. N = 3 per group. The original bands of Western blot were showed in Supplementary Figure 3.

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  FIGURE 7. Potential mechanism underlying how BYD exerts a cardioprotective effect.