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Matrine-type alkaloids inhibit advanced glycation end products induced reactive oxygen species-mediated apoptosis of aortic endothelial cells in vivo and in vitro by targeting MKK3 and p38MAPK signaling

Journal of the American Heart Association (2017) 6(12)
DOI: 10.1161/JAHA.117.007441
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Abstract

Background-The matrine-type alkaloids are bioactive components extracted from Sophora flavescens, which is used in treatment of diabetes mellitus in traditional Chinese medicine. Advanced glycation end products mediate diabetic vascular complications. This study was aimed to investigate the protective effects and molecular mechanisms of matrine-type alkaloids on advanced glycation end products-induced reactive oxygen species-mediated endothelial apoptosis. Methods and Results-Rats aorta and cultured rat aortic endothelial cells were exposed to advanced glycation end products. Matrine-type alkaloids, p38 mitogen-activated protein kinase (MAPK) inhibitor, and small interference RNAs against p38 MAPK kinases MAPK kinase kinase (MKK)3 and MKK6 were administrated. Intracellular reactive oxygen species production, cell apoptosis, phosphorylation of MKKs/p38 MAPK, and expression levels of heme oxygenase/NADPH quinone oxidoreductase were assessed. The nuclear factor erythroid 2-related factor 2 nuclear translocation and the binding activity of nuclear factor erythroid 2-related factor 2 with antioxidant response element were also evaluated. Matrine-type alkaloids suppressed intracellular reactive oxygen species production and inhibited endothelial cell apoptosis in vivo and in vitro by recovering phosphorylation of MKK3/6 and p38 MAPK, nuclear factor erythroid 2-related factor 2 nuclear translocation, and antioxidant response element binding activity, as well as the expression levels of heme oxygenase/NADPH quinone oxidoreductase. p38 MAPK inhibitor treatment impaired the effects of matrine-type alkaloids in vivo and in vitro. MKK3/6 silencing impaired the effects of matrine-type alkaloids in vitro. Conclusions-Matrine-type alkaloids exert endothelial protective effects against advanced glycation end products induced reactive oxygen species-mediated apoptosis by targeting MKK3/6 and enhancing their phosphorylation.

Figures

  • Figure 1. A, The left panel demonstrates the captured fluorescent images of aorta stained with DHE. ROS (reactive oxygen species) was positively stained. Columns on the right panel indicated the mean fluorescence intensity (MFI) of DHE stain in Control, AGEs (advanced glycation end products), AGEs+Mat, AGEs+Oxy, AGEs+Soh, AGEs+Mat+SB, AGEs+Oxy+SB, and AGEs+Soh+SB, respectively. B, Columns indicate the detected total antioxidant capacity (TAC) of aorta harvested from experimental animals. C, The left panel demonstrates the captured fluorescent images of TUNEL (terminal transferase UTP nick end labeling) assay of aorta. Apoptotic cells were positively stained. Columns on the right panel indicate the MFI of TUNEL staining in Control, AGEs, AGEs+Mat, AGEs+Oxy, AGEs+Soh, AGEs+Mat+SB, AGEs+Oxy+SB, and AGEs+Soh+SB, respectively (3 independent experiments were performed; *P<0.05; **P<0.01; ***P<0.001).
  • Figure 2. A, The immunoblots of Nrf2 in nuclear protein when histone H3 was used as the internal reference. B, The immunoblots of phosphorylated MKK3 (p-MKK3), MKK3, p-MKK6, MKK6, p-p38, p38, HO1 (heme oxygenase 1), and NQO1 (NADPH quinone oxidoreductase 1) in total protein when GAPDH was introduced as the internal reference. C through H, Columns in these panels indicate the relative expression level of Nrf2 (Nrf2/histon H3), phosphorylation level of MKK3 (p-MKK3/MKK3), phosporylation level of MKK6 (p-MKK6/MKK6), phosphorylation level of p38 MAPK, the relative expression levels of HO1 (HO1/GAPDH), and the relative expression level of NQO1 (NQO1/GAPDH), respectively (3 independent experiments were performed; *P<0.05; **P<0.01; ***P<0.001; #P>0.05).
  • Figure 3. A, Results of MTT assay are indicated by the line chart in this figure. Cultured RAECs (rat aortic endothelial cells) were incubated with matrine, oxymatrine, and sophocarpine at serial diluted concentrations. B, Columns indicate the TAC (total antioxidant capacity) levels in cultured RAECs in Control, AGEs, AGEs+Mat, AGEs+Oxy, AGEs+Soh, AGEs+Mat+SB, AGEs+Oxy+SB, AGEs+Soh+SB, AGEs+Mat+mkk3 , AGEs+Mat+mkk6 , AGEs+Oxy+mkk3 , AGEs+Oxy+mkk6 , AGEs+Soh+mkk3 , and AGEs+Soh+mkk6 , respectively. C, Left panel demonstrates the captured fluorescent images of cultured RAECs stained with DCFH-DA. ROS (reactive oxygen species) was positively stained. Columns on the right panel indicate the MFI of DCFH-DA stain in Control, AGEs, AGEs+Mat, AGEs+Oxy, AGEs+Soh, AGEs+Mat+SB, AGEs+Oxy+SB, AGEs+Soh+SB, AGEs+Mat+mkk3 , AGEs+Mat+mkk6 , AGEs+Oxy+mkk3 , AGEs+Oxy+mkk6 , AGEs+Soh+mkk3 , and AGEs+Soh+mkk6 , respectively (3 independent experiments were performed; *P<0.05; **P<0.01; ***P<0.001).
  • Figure 4. Charts on the upper panel of this figure demonstrate PI/Annexin V double staining detected by a flow cytometer. Columns on the lower panel indicate the apoptotic percentage in Control, AGEs, AGEs+Mat, AGEs+Oxy, AGEs+Soh, AGEs+Mat+SB, AGEs+Oxy+SB, AGEs+Soh+SB, AGEs+Mat+mkk3 , AGEs+Mat+mkk6 , AGEs+Oxy+mkk3 , AGEs+Oxy+mkk6 , AGEs+Soh+mkk3 , and AGEs+Soh+mkk6 , respectively (3 independent experiments were performed; **P<0.01; ***P<0.001).
  • Figure 5. A, This panel demonstrates the captured images of Nrf2 and DAPI fluorescent immunochemistry staining and their merged images in cultured RAECs (rat aortic endothelial cells). B, Columns indicate the ratio of Nrf2 nuclear translocated cell count against total cell count in RAECs in Control, AGEs, AGEs+Mat, AGEs+Oxy, AGEs+Soh, AGEs+Mat+SB, AGEs+Oxy+SB, AGEs+Soh+SB, AGEs+Mat+mkk3 , AGEs+Mat+mkk6 , AGEs+Oxy+mkk3 , AGEs+Oxy+mkk6 , AGEs+Soh+mkk3 , and AGEs+Soh+mkk6 , respectively. C, Columns indicate the ARE-luciferase activities in RAECs in Control, AGEs, AGEs+Mat, AGEs+Oxy, AGEs+Soh, AGEs+Mat+SB, AGEs+Oxy+SB, AGEs+Soh+SB, AGEs+Mat+mkk3 , AGEs+Mat+mkk6 , AGEs+Oxy+mkk3 , AGEs+Oxy+mkk6 , AGEs+Soh+mkk3 , and AGEs+Soh+mkk6 , respectively (3 independent experiments were performed; *P<0.05; **P<0.01; ***P<0.001).
  • Figure 6. A, The immunoblots of Nrf2 in nuclear protein when histoneH3was used as the internal reference. B, The immunoblots of phosphorylated MKK3 (p-MKK3), MKK3, p-MKK6, MKK6, p-p38, p38, HO1 (heme oxygenase 1), and NQO1 (NADPH quinone oxidoreductase 1) in total protein when GAPDH was introduced as the internal reference. C through H, Columns in these panels indicate the relative expression level of Nrf2 (Nrf2/histone H3), phosphorylation level of MKK3 (p-MKK3/MKK3), phosporylation level of MKK6 (p-MKK6/ MKK6), phosphorylation level of p38 MAPK, the relative expression levels of HO1 (HO1/GAPDH), and the relative expression level of NQO1 (NQO1/GAPDH), respectively (3 independent experiments were performed; *P<0.05; **P<0.01; ***P<0.001; #P>0.05).
  • Figure 7. The schematic diagram of the antioxidant molecular mechanisms of matrine-type alkaloids. AGEs (advanced glycation end products) trigger intracellular ROS (reactive oxygen species) production, which induces apoptosis of aortic endothelial cells. The matrine-type alkaloids administration promotes the phosphorylation of MKK3 and MKK6. As the kinases of p38 MAPK, phosphorylated MKK3 and MKK6 further phosphorylate p38 MAPK, which facilitate the nuclear translocation of Nrf2. Binding with ARE (antioxidant response element), the nuclear transcription factor Nrf2 initiates the transcriptions of antioxidant proteins such as NQO1 (NADPH quinone oxidoreductase 1) and HO1 (heme oxygenase 1). Thus, the intracellular ROS accumulation is alleviated and apoptosis is suppressed.

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Liu, Z., Lv, Y., Zhang, Y., Liu, F., Zhu, L., Pan, S., … Wang, J. (2017). Matrine-type alkaloids inhibit advanced glycation end products induced reactive oxygen species-mediated apoptosis of aortic endothelial cells in vivo and in vitro by targeting MKK3 and p38MAPK signaling. Journal of the American Heart Association, 6(12). https://doi.org/10.1161/JAHA.117.007441

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