DanHong injection targets endothelin receptor type B and angiotensin II receptor type 1 in protection against cardiac hypertrophy

Figures

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  Figure 1. DH modulate beating patterns of hiPS-CMs in CH (A) Representative beating traces captured at selected time points of hiPS-CMs treated by DH. (B) Representative contraction profiles treated by various dose (0.04, 0.08, 0.16, 0.32, 0.63, 1.25, 2.5 and 5.0 μL/mL) of DH. Four CM beating parameters, including beating rate, amplitude, rising slope, and falling slope were used for evaluation 3.0 hours after DH treatment. (C) DH reduced the quantity of total protein on hypertrophic hiPS-CMs. **P < 0.05: versus ET-1 group; ##P < 0.05: versus vehicle group.

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  Figure 2. DH attenuates ET-1 induced cell area enlargement and elevation of ACTN2 and TNNT2 contents on hypertrophic hiPS-CMs (A) Representative images of the HCA analysis of hypertrophic hiPS-CMs treated by a various dose of DH for the evaluation of cell area, ACTN2 and TNNT2 contents. (B) Cell area of hypertrophic hiPS-CMs were alleviated by DH treatment at 1.25, 2.5 and 5.0 μL/mL. (C) ACTN2 content of hypertrophic hiPS-CMs were suppressed by DH treatment at 1.25, 2.5 and 5.0 μL/mL. (D) TNNT2 content of hypertrophic hiPS-CMs were suppressed by DH treatment at 0.63, 1.25, 2.5 and 5.0 μL/mL. **P < 0.05: versus ET-1 group; ##P < 0.05: versus vehicle group.

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  Figure 3. DH attenuated elevation of NPPB gene expression and BNP protein levels in CH (A) NPPB gene expression of hypertrophic hiPS-CMs were suppressed by DH treatment at 2.5 and 5.0 μL/mL. (B) Representative images of the HCA analysis of hiPS-CMs treated by DH for the evaluation of BNP content. (C) BNP content of hypertrophic hiPS-CMs were suppressed by DH treatment at 1.25, 2.5 and 5.0 μL/mL. **P < 0.05: versus ET-1 group; ##P < 0.05: versus vehicle group.

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  Figure 4. ET-1 related receptors and their interacting proteins Yellow nodes represent the receptors, blue nodes represent the interacting proteins. Red nodes represent differentially expressed proteins after DH treatment.

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  Figure 5. Agonist and antagonist assays of DH on HEK293/Gα15/ETB cell line (A) Agonist assay of the HEK293/Gα15/ETB cell line. Dose-response curve for ET-1, with EC50 value of 11.6 nM. (B) Antagonist assay of the HEK293/Gα15/ETB cell line. Dose-response curve for bosentan, with IC50 value of 616.5 nM. (C) Antagonist assay of DH on HEK293/Gα15/ETB cell line. Dose-response curve for DH, with IC50 value of 25.67 μL/mL.

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  Figure 6. Agonist and antagonist assay of DH on HEK293/Gα15/AT1 cell line (A) Agonist assay of the HEK293/Gα15/AT1 cell line. Dose-response curve for angiotensin II, with EC50 value of 2.34 nM. (B) Antagonist assay of the HEK293/Gα15/AT1 cell line. Dose-response curve for telmisartan, with IC50 value of 69.82 nM. (C) Antagonist assay of DH on HEK293/Gα15/AT1 cell line. Dose-response curve for DH, with IC50 value of 1.10 μL/mL.

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  Figure 7. Protein expression of hypertrophic hiPS-CMs with different treatments Red dots represent differentially expressed proteins (fold change > 2 and p-value < 0.05), while green dots represent proteins without significant difference. CH related proteins and their interacting proteins are marked as blue. (A) Differential protein expression in ET1-induced CH model against control. (B) Differential protein expression after Bosentan treatment against ET1-induced CH model. (C) Differential protein expression after DH treatment against ET1-induced CH model.

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  Figure 8. Biological process for differentially expressed proteins of hypertrophic hiPS-CMs Red nodes indicate that over 70% of the differentially expressed proteins associated with the term were up-regulated, and green nodes mean 70% of the differentially expressed proteins in this term were down-regulated. The color gradient of node represents the significance of over-representation, and size of node represents the number of protein in this term. (A) Over-represented GO terms (Biological Process at 4th level) for differentially expressed proteins identified from vehicle control and ET1-induced CH model. (B) Over-represented GO terms (Biological Process at 4th level) for differentially expressed proteins identified from model group and bosentan treatment by ClueGO. (C) Over-represented GO terms (Biological Process at 4th level) for differentially expressed proteins identified from model group and DH treatment by ClueGO. (D) Hierarchical clustering of the quantitative information from significantly differentially expressed proteins, expression level of proteins were normalized using z-score.

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  Figure 9. Schematic representation of DH-mediated protection from ET-1-induced CH on hiPS-CMs Signaling network after DH treatment. DH attenuated CH by blocking ETBR and AT1R, and then suppressing the downstream signaling pathways related to cell cycle, RNA processing, cytoskeleton and mitochondria. Blue nodes present the key proteins related to CH, and purple nodes presents proteins related to CH. Green node presents the biological processes.

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  Figure 10. Possible mechanism of DH in treating cardiovascular and cerebrovascular diseases DTs: direct targets.