Shexiang Tongxin Dropping Pills Promote Macrophage Polarization-Induced Angiogenesis Against Coronary Microvascular Dysfunction via PI3K/Akt/mTORC1 Pathway

Figures

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  FIGURE 1. Effects of Shexiang Tongxin dropping pills (STDPs) on cardiac function. (A) The experimental protocol for STDP study in left anterior descending (LAD) ligation induced the coronary microvascular dysfunction (CMD) model rats. (B) Representative images of echocardiography in the sham, model, STDP, and TLM groups. (C) Echocardiographic parameter analysis in different groups. N = 6 per group. (D) Representative H and E staining of paraffin sections. Scale bar = 20 μm. (E) The serum troponin (cTnI) in different groups. N = 6 per group. *p < 0.05, **p < 0.01, ***p < 0.001 vs. the model group; # p < 0.05, # # p < 0.01, # # # p < 0.001 vs. the sham group.

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  FIGURE 2. STDPs promoted angiogenesis by inducing polarization of M2 macrophages in ischemic myocardial tissues. (A) Representative real-time perfusion images of cardiac vessels by optical microangiography (OMAG). (B) Vessel diameter index (VDI), vessel area density (VAD), vessel skeleton density (VSD), vessel perimeter index, and vessel complexity index analysis in different groups. N = 3 per group. (C) IF staining of CD206 and CD31 in different groups. N = 3 per group. (D) Immunoblots and the quantification of Arg-1, CD206, and VEGF-A, N = 6 per group. (E) The level of IL-10 in serum. N = 6 per group. *p < 0.05, **p < 0.01 vs. the model group; # p < 0.05, # # p < 0.01 vs. the sham group.

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  FIGURE 3. STDPs upregulated the PI3K/Akt/mTORC1 pathway in ischemic heart of rats. (A–C) Representative immunoblots and analysis for p-PI3K, p-Akt, and mTOCR1 in the sham, model, STDP, and TLM groups. N = 3 per group. *p < 0.05, **p < 0.01, ***p < 0.001 vs. the model group; # p < 0.05, # # p < 0.01 vs. the sham group.

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  FIGURE 4. STDPs enhanced the proliferation, migration, and tube-forming capacity of HUVECs. (A) STDPs (800 ng/ml) have no cytotoxicity on HUVECs. (B) Bone marrow-derived macrophages (BMDMs) were treated with various concentrations of STDPs for 24 h (C) STDPs (100 ng/ml) promoted the cell viability in OGD/R-induced HUVECs. VEGF-A (20 ng/ml) promoted the proliferation capacity of HUVECs. N = 6 per group. (D) STDPs promoted cell migration in OGD/R-induced HUVECs. N = 3 per group. (E) STDPs promoted tube formation in OGD/R-induced HUVECs. N = 3 per group. VEGF-A was used as the positive control. **p < 0.01, ***p < 0.001 vs. the model group; # # # p < 0.001 vs. the sham group.

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  FIGURE 5. STDPs regulated the PI3K/AKT/mTORC1 pathway in BMDMs. (A) STDPs (10, 100, and 1,000 μg/ml) had no cytotoxicity on BMDMs. N = 6 per group. (B, C) NO level in the cell supernatant in the different groups. N = 6 per group. (D–F) Representative immunoblots and analysis for p-PI3K, p-Akt, and mTOCR1 in the control, model, STDP, LY29, and LY29+STDP groups. N = 3 per group. *p < 0.05, **p < 0.01, ***p < 0.001.

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  FIGURE 6. STDPs modulated M2 macrophage polarization and increased VEGF-A release. (A) IF staining of CD206 in the different groups. Scale bar = 20 μm. (B) Flow cytometry results showed that CD206-positive cells were upregulated after STDP treatment. N = 3 per group. (C, D) Representative immunoblots and analysis for Arg-1 and VEGF-A in different groups. N = 3 per group. (E) The release of VEGF-A in BMDM supernatant detected by ELISA. N = 3 per group. *p < 0.05, **p < 0.01, ***p < 0.001.

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  FIGURE 7. Potential mechanism of STDPs on CMD. The red arrows represent the change trend of key factors in the pathological environment, and the blue arrows represent the change trend of key factors after the STDPs administration.