Baicalin Depresses the Sympathoexcitatory Reflex Induced by Myocardial Ischemia via the Dorsal Root Ganglia

Figures

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  FIGURE 1. Baicalin prevented the upregulation of P2Y12 receptor in MI rats. (A) Real-time PCR assay showed that the expression levels of P2Y12 receptor mRNA in the DRG were higher in the MI group than in the sham group (p < 0.01). No statistically significant difference was found between MI and MI + NS rats. In MI rats treated with baicalin, the expression level of P2Y12 mRNA was lower than that in untreated MI rats (p < 0.01). No statistically significant difference was found between control + baicalin and sham rats. ± s, n = 8. ∗∗p < 0.01 compared with the sham group. ##p < 0.01 compared with the MI group. (B). Western blotting assay indicated that expression levels of P2Y12 protein in the DRG were higher in the MI group than in the sham group (p < 0.001). No statistically significant difference was found between control + baicalin and sham rats. In MI rats treated with baicalin, the expression of P2Y12 receptor protein was lower than that in the untreated MI rats (p < 0.01). No statistically significant difference was found between MI and MI + NS rats. ± s, n = 8. ∗∗∗p < 0.01 compared with the sham group. ##p < 0.01 compared with the MI group. (C) Western blotting assay indicated that…

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  FIGURE 2. Baicalin relieved pathological changes in sympathetic nerve activity, and ECGs in MI rats. (A) The sympathetic nerve activity was significant enhanced after MI injury, and was reduced with baicalin treatment. (B) On day 14 after MI injury, there were obvious abnormal Q-waves in the MI and MI + NS groups. In MI rats treated with baicalin, Q-wave abnormalities were improved.

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  FIGURE 3. Baicalin decreased the coexpression values for P2Y12 receptor and GFAP in the MI rats. (A) Coexpression values for P2Y12 receptor and GFAP in the DRG SCGs were assessed by double-labeled immunofluorescence. Coexpression values for the P2Y12 receptor with GFAP in the MI group were higher than those in the sham group. No statistical difference was found between control + baicalin and sham rats. Coexpression values in the MI + baicalin group were lower than those in the untreated MI group. No statistical difference was found between MI and MI + NS rats. The green signal represents GFAP staining with FITC, and the red signal indicates P2Y12 receptor staining with TRITC. Merge represents the P2Y12 receptor and GFAP double-staining image. Histogram showed that the number of neurons surrounded with GFAP and P2Y12 positive SGCs in the SCGs. Scale bar: 20 μm. (B) Western blotting assay to show that the expression levels of the GFAP protein in the DRG were higher in the MI group than in the sham group (p < 0.01). No statistical difference was found between control + baicalin and sham rats. Baicalin treatment in MI rats appeared to decrease expression levels of GFAP in the DRG compared with…

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  FIGURE 4. Baicalin decreased the expression levels of IL-1β in the DRG of MI rats. Western blotting assay showed that expression of IL-1β in the MI group was significantly elevated compared with the sham group (p < 0.01). No statistical difference was found between control + baicalin and sham rats. Relative expression of IL-1β was lower in the MI rats treated with baicalin compared with untreated MI rats (p < 0.05). There was no statistical difference between the MI group and the MI + NS group. ±s, n = 8. ∗∗p < 0.01 compared with the control group, #p < 0.05 compared with the MI group.

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  FIGURE 5. Baicalin decreased the activation of Akt in the DRG of MI rats. Western blotting assay showed that there is no statistical difference for the IOD ratio of AKT/β-actin between the MI rats and the sham rats (A,B). The expression levels of p-AKT to AKT were significantly increased in the MI rats in comparison with that in the sham rats (p < 0.01) (A,C). No statistical difference was found between control + baicalin and sham rats. In the MI rats treated with baicalin, the expression levels of p-AKT to AKT were significantly reduced compared to untreated MI rats (p < 0.01) (A,C). No statistical difference was found between MI and MI + NS groups. ± s, n = 8. ∗∗p < 0.01 compared with the control group, ##p < 0.01 compared with the MI group.

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  FIGURE 6. Baicalin inhibited the P2Y12-agonist-activated [Ca2+]i in HEK239 cells transfected with the P2Y12 receptor and DRG SGCs. (A) Baicalin inhibited the P2Y12-agonist-activated [Ca2+]i in HEK239 cells transfected with the P2Y12 receptor. The ratio of peak/baseline fluorescence of [Ca2+]i activated by the P2Y12 agonist, 2Me-SADP, in HEK293 cells transfected with a P2Y12 receptor plasmid was recorded by Fluo 3-AM calcium fluorescence imaging. 24 h treatment with baicalin inhibited 2Me-SADP-mediated increases of [Ca2+]i in HEK293 cells transfected with P2Y12 receptor plasmid. (B) Baicalin reduced the P2Y12-agonist-activated [Ca2+]i in DRG SGCs. P2Y12-agonist-activated [Ca2+]i in the DRG SGCs was recorded by calcium fluorescence imaging. The ratio of peak/baseline fluorescence of [Ca2+]i activated by the P2Y12 agonist 2Me-SADP (100 μM) in DRG SGCs without treated with baicalin was larger than that in cells treated with baicalin group. Baicalin treatment inhibited 2Me-SADP-induced [Ca2+]i in DRG SGCs. (C) The full concentration-response curve of baicalin reducing the P2Y12-agonist-activated [Ca2+]i in DRG SGCs. EC50 and Emax of control + baicalin are 4.2498 ± 0.2902 and 1.5098 ± 0.0156,…

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  FIGURE 7. Molecular docking of baicalin on the human P2Y12 protein. Simulated model of baicalin docked with the human P2Y12 (hP2Y12) protein generated using a computer. Structures in (A) (forward map) and (B) (top view) show that the best docking position between baicalin and hP2Y12. The docking position was in the outside of the cell membrane and the entrance of the channel. (C) Was the docking pocket. (C) Showed that the yellow dotted line was a hydrogen bond between the residues on the chains of P2Y12 protein and baicalin. The photo indicated that there was strong binding energy between baicalin and GLN101, ARG 171, and HIS 193 in left chain, LYS 286 in right chain. The results revealed that baicalin could interact with the hP2Y12 protein.

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  FIGURE 8. Comparison of the P2Y12 receptor expression in C1-T2 with lumbar DRG1-4. (A) Real-time PCR assay showed that the expression levels of P2Y12 receptor mRNA increased only in C1-T2 DRGs, with no changes in lumbar DRGs following MI. ± s, n = 5. ∗∗∗p < 0.001 compared with the sham group. (B) Western blotting assay showed that the expression levels of the P2Y12 protein were significantly increased in C1-T2 DRGs, with no change in the lumbar DRGs post-MI. ± s, n = 5. ∗∗p < 0.01 compared with the sham group.