Mechanisms of Chinese Medicine Xinmailong’s protection against heart failure in pressure-overloaded mice and cultured cardiomyocytes

Figure 1. XML is extracted from Periplaneta Americana (a). The main active components of XML include adenosine (b), inosine (c), protocatechuic acid (d), and pyroglutamate dipeptides (e).

Figure 2. Dynamic changes in cardiac structure and function were detected by echocardiography in the SHAM, TAC, XML, and MET groups. (a) LVPWd (b) EF (c) FS and (d) LVIDd were observed dynamically after 0, 1, and 2 weeks. **P < 0.01 and ***P < 0.001 indicate significant differences from SHAM. #P < 0.05 indicates significant difference from TAC group.

Figure 3. Anatomical data in SHAM, TAC, MET, and XML mice. (a) HW, (b) the ratio of LV weight (LW) to tibial length (TL), (c) the ratio of lung weight to TL, and (d) the ratio of liver weight to TL were compared. *P < 0.05, **P < 0.01, ***P < 0.001 indicate significant difference. n.s.: no significant difference.

Figure 4. Dye-stained HF heart sections. (a) H&E-stained (upper) and (b) Sirius red-stained (lower) sections of representative hearts from SHAM, TAC, MET and XML mice. Scale at bottom is in cm.

Figure 5. Positive linear correlation between the weights of echocardiographic left ventricular myocardium (Echo LVM) and anatomical LVM in four models (SHAM, TAC, MET, and XML mice).

Figure 6. XML inhibited the phosphorylation of ERK1/2 and AKT after pressure overload in vivo. (a and b) Phosphorylated (p)-Thr202/204 extracellular signal regulated kinase (ERK) 1/2. (c and d) p-Ser473 protein kinase (b) (AKT). Data (mean ± SEM, n = 3) were expressed as fold changes from phosphorylated and total protein (ERK1/2, AKT). *P < 0.05, **P < 0.01.

Figure 7. XML inhibited the proliferation of H9C2 cell line in vitro. (a) microscopic photography, (b) BrdU and (c,d) MTT assays were detected in different XML concentrations from 0.1 to 5 g/ml. (e) Dose-dependent curve was obtained to normalize the inhibition effect of XML on H9C2, IC50 = 2.92 mg/ml.

Figure 8. XML inhibited the phosphorylation of ERK1/2, and AKT after ISO stimulation. (a) Western blotting was performed to detect the phosphor-ERK1/2, JNK, p38, and AKT. Total proteins and GAPDH were used as loading control. (b) p-ERK1/2, (c) p-P38, (d) p-JNK, and (e) p-AKT (mean ± SEM, n = 3) were expressed as fold changes from phosphorylated and total proteins. *P < 0.05, **P < 0.01, n.s.: no significant difference.

Figure 9. XML inhibited the phosphorylation of GSK3β and nucleus protein expression of GATA4. (a and c) Phosphorylated (p)- GSK3β and (b and d) nucleus GATA4. Data (mean ± SEM, n = 3) were expressed as fold changes from GSK3β phosphorylated/total protein and GATA3/H3. **P < 0.01, ***P < 0.001.

Figure 10. A model of pathways in the cardio-protection of XML in response to pressure stress overload. TAC (stress overload) could activate phosphorylation of the protein kinases of ERK1/2, AKT and GSK3β, enhance the expression of GATA4, promote the transcription of HF gene, and result in heart failure. XML could inhibit the phosphorylation of ERK1/2 and AKT, decreasing the expression of GSK3β and GATA4, leading to inhibit heart failure. ⊗Denotes inhibition of protein kinase by XML treatment.