Guanxinjing ameliorates coronary microvascular dysfunction in myocardial ischemia-reperfusion injury by alleviating inflammation and restoring endothelial function

Figures

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  Figure 1. Network pharmacology diagram of GXJC for the treatment of MIRI. (A) The total Ion Chromatogram of GXJC at positive mode by UHPLC-Q-Exactive HRMS. (B) The total Ion Chromatogram of GXJC at negative mode by UHPLC-Q-Exactive HRMS. (C) Venn diagram of the drug targets of GXJC and the disease targets of MIRI. (D) The PPI networks after three rounds of screening based on topological algorithms (DC, CC, BC, NC, EC, LAC). (E) The herb-compound-Targets gene network constructed using core genes and corresponding compounds. Detailed compound information is available in Supplementary Table 3. (F) KEGG Pathways. (G) GO terms, including biological process (BP), cellular component (CC), and molecular function (MF).

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  Figure 2. GXJC attenuates MIRI-induced myocardial injury in mice. (A) Flowchart of Animal Experiments and Establishment of the MIRI Model. (B) The area of myocardial infarction was detected by Evans blue-TTC Double Staining. The nonischemic section shown in blue area, red represent risk area, and the infarct region is stained white. (C, D) The INF/AAR and AAR/LV ratios were quantified (n ≥ 3). (E) H&E staining of myocardial tissue sections, scale bar = 100 μm. (F, G) Apoptosis was evaluated via TUNEL staining and quantitative analysis of cardiomyocyte apoptosis by measuring the TUNEL fluorescence intensity within cTnI+ areas (green: cTnI, blue: DAPI-stained nuclei, red: TUNEL, scale bar = 50 μm) (n = 3). (H) NO content in the serum, measured by the Griess assay (n = 4). #P < 0.05, ##P < 0.01 vs. Sham group. *P < 0.05, **P < 0.01 vs. Model group.

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  Figure 3. GXJC alleviates MIRI-induced inflammation. A-B The expression of IL-1β (A), IL-6 (B) were detected by WB, (n ≥ 4). C-F Protein levels of IL-6 (C), IL-1β (D), IL-10 (E), and CCL2 (F) in serum, measured by ELISA (n≥4). #P < 0.05, ##P < 0.01 vs. Sham group. *P < 0.05, **P < 0.01 vs. Model group.

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  Figure 4. GXJC ameliorated vascular endothelial function following MIRI injury. (A, B) Representative immunofluorescence images of α-SMA in myocardial tissues and quantitative analysis, scale bar = 50 μm (n = 3). (C, D) Representative immunofluorescence images of CD31 in myocardial tissues and quantitative analysis, scale bar = 50 μm (n = 3). (E, F) Representative immunofluorescence images of ET-1 in myocardial tissues and quantitative analysis, scale bar = 50 μm (n = 3). (G, H) Representative IF images of p-selectin in myocardial tissues and quantitative analysis, scale bar = 50 μm (n = 3). (I) The expression of FGL2 and eNOS were detected by WB. (n = 3). #P < 0.05, ##P < 0.01 vs. Sham group. *P < 0.05, **P < 0.01 vs. Model group.

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  Figure 5. GXJC inhibited TNF-α-induced inflammation in HUVECs. (A) Cell viability of HUVECs treated with various concentrations of GXJC, assessed by CCK-8 assay (n=5). (B) Representative fluorescence images of the F-actin cytoskeleton in HUVECs stained with phalloidin. Images are representative of three independent experiments with similar results. Scale bar = 20 μm. (C-H) Relative mRNA expression levels of IL-6, IL-10, IL-1β, ICAM-1, VCAM-1, and CCL2 in HUVECs, measured by RT-qPCR (n=3). I-K Protein levels of IL-6, CCL2, and ICAM-1 in HUVECs, measured by ELISA (n=3). L-M The expression of IL-1β were detected by WB in HUVECs (n = 3). #P < 0.05, ##P < 0.01 vs. Control group. *P < 0.05, **P < 0.01 vs. Model group.

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  Figure 6. GXJC alleviated TNF-α-induced endothelial cell dysfunction in HUVECs. (A, B) The expression of FGL2, ET-1, VEGF were detected by WB after TNF-α stimulation, (n = 3). (C, D) Representative immunofluorescence images of p-selectin in HUVECs and quantitative analysis. Scale bar =200 μm (n = 3). (E, F) Representative immunofluorescence images of eNOS in HUVECs and quantitative analysis. Scale bar = 200 μm (n = 3). (G) Determination of NO Content in Culture Medium Supernatant by Griess method (n = 3). (H–L) Relative mRNA expression levels of FGL2, ET-1, VEGF, p-selectin and eNOS were measured by RT-qPCR in HUVECs (n = 3). #P < 0.05, ##P < 0.01 vs. Control group. *P < 0.05, **P < 0.01 vs. Model group.

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  Figure 7. The potential mechanism of GXJC in treating MIRI-induced CMD.