An Integrative Pharmacology-Based Strategy to Uncover the Mechanism of Zuogui Jiangtang Shuxin Formula in Diabetic Cardiomyopathy

Figure 1. A schematic representation shows the outlines on studying the potential molecular mechanisms of action of Zuogui Jiangtang Shuxin formula (ZGJTSXF). The main active compounds of ZGJTSXF were identified by UPLC-Q-Exactive-Orbitrap-MS analysis of medicated rat serum. The potential targets for ZGJTSXF in treating DCM were also identified through analysis of multiple databases. The common targets were subjected to in silicon network pharmacology (C-T network and PPI network) analysis and pathway enrichment analysis. The mouse DCM model was established to experimentally validate the predicted targets and pathways through multiple cellular and molecular approaches.

Figure 2. Total ion chromatography of the blood-entry compounds of ZGJTSXF in the positive and negative ion mode by UHPLC-Q-Orbitrap-MS.

Figure 3. The component-target network of ZGJTSXF in the treatment of DCM.

Figure 4. The PPI network was constructed using the common targets to predict core target proteins of ZGJTSXF.The color and thickness of the configuration edge follow the combine_score to continuously change, and finally the target genes were laid out into six concentric circles according to the node degree. The degree of the target gene node is between 21–32; the degree of the node in the fourth layer is between 33 and 40; the degree of the node in the third layer is between 41 and 60; the degree of the node in the second layer is between 60 and 100; and the degree of the node in the innermost layer is between 33 and 40. The larger the value of the parameter Degree in the figure, the larger the shape of the corresponding node and the darker the color.

Figure 5. GO enrichment and KEGG pathway analysis identified critical pathways associated with ZGJTSXF. (A) The top 10 enrichment items of Biological Process, Molecular Function and Cellular Components from GO enrichment analysis. (B) The top 35 pathways of KEGG pathway analysis.

Figure 6. ZGJTSXF administration changed myocardial function and myocardial histology in DCM mice. (A–C) Echocardiographic observation of changes in cardiac function in various groups of mice. (A) Representative M-mode echocardiograms in mice of the indicated groups. (B) Summary on the left ventricular fractional shortening (FS). (C) Summary on the left ventricular ejection fraction (EF). ##P<0.01, compared with the CON group; ▲▲P<0.01, compared with the DCM group; **P<0.01, compared with the ZGJTSXFL group; ΔΔP<0.01, compared with the ZGJTSXFH group. (D) Histopathological changes in myocardial tissues of mice in the indicated groups were observed by HE staining (Scale bar, 20 μm). (a) CON group; (b) DCM group; (c) Met group, (d) ZGJTSXFL group; (e) ZGJTSXFM group; (f) ZGJTSXFH group. Green arrow, cardiomyocyte hypertrophy; red arrow, small focal inflammatory cell infiltrates in the interstitial myocardium; blue arrow, vacuolar degeneration of cardiomyocytes; yellow arrow, thickening of the capillary basement membrane.

Figure 7. Apoptosis of mouse myocardial tissues was evaluated by TUNEL staining. (A) Representative images of apoptosis of mice myocardial tissues after TUNEL staining (Scale bar, 20 μm). (B) Apoptosis rate of cardiomyocytes in mice of the indicated group was summarized. n=6 for each group; ##P<0.01, compared with the CON group; ▲▲P<0.01, compared with the DCM group; **P<0.01, *P<0.05, compared with the ZGJTSXFM group.

Figure 8. The expression levels of PI3K-AKT pathway molecules and apoptosis-related molecules in mouse myocardial tissues were quantitated. (A–D) The mRNA levels of PI3K (A), AKT (B), Bcl-2 (C), and Bcl-xL (D) in mouse myocardial tissues of the indicated groups were quantitated by qPCR. n=6 for each group; ##P<0.01, Compared with the CON group; ▲▲P<0.01, compared with the DCM group; **P<0.01, compared with the ZGJTSXFL group; ΔΔP<0.01, compared with the ZGJTSXFH group. (E–I) The protein levels of phosphorylated PI3K (p-PI3K; (F), phosphorylated AKT (p-AKT; (G), Bcl-2 (H), and Bcl-xL (I) in mouse myocardial tissues of the indicated groups were quantitated by Western blot assay. Representative images of Western blot bands are shown (E), and the relative expressions were summarized. n=6 for each group; ##P<0.01, compared with the CON group; n=6 for each group; ▲▲P<0.01, ▲P<0.05, compared with the DCM group.

Figure 9. ZGJTSXF inhibits cardiomyocyte apoptosis by regulating PI3K/Akt signaling pathway.