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. 2019 Oct 7:2019:6168340.
doi: 10.1155/2019/6168340. eCollection 2019.

Andrographolide Protects against HG-Induced Inflammation, Apoptosis, Migration, and Impairment of Angiogenesis via PI3K/AKT-eNOS Signalling in HUVECs

Ming-Xia Duan  1   2   3 , Heng Zhou  1   2   3 , Qing-Qing Wu  1   2   3 , Chen Liu  1   2   3 , Yang Xiao  1   2   3 , Wei Deng  1   2   3 , Qi-Zhu Tang  1   2   3
Affiliations

Andrographolide Protects against HG-Induced Inflammation, Apoptosis, Migration, and Impairment of Angiogenesis via PI3K/AKT-eNOS Signalling in HUVECs

Ming-Xia Duan et al. Mediators Inflamm. .

Abstract

Andrographolide (Andr) is a major component isolated from the plant Andrographis paniculata. Inflammation, apoptosis, and impaired angiogenesis are implicated in the pathogenesis of high glucose (HG)-induced injury of vascular endotheliocytes. Our study is aimed at evaluating the effect of Andr on HG-induced HUVEC injury and the underlying mechanism. HUVECs were exposed to HG levels (33 mM) and treated with Andr (0, 12.5, 25, and 50 μM). Western blot analysis, real-time PCR, immunofluorescence staining, the scratch test, and the tube formation assay were performed to assess the effects of Andr. We discovered that Andr inhibited the inflammatory response (IL-1β, IL-6, and TNFα), decreased the apoptosis ratio and cell migration, and promoted tube formation in response to HG stimulation. Andr ameliorated the levels of phosphorylated PI3K (p-PI3K), phosphorylated AKT (p-AKT), and phosphorylated eNOS (p-eNOS). The expression of vascular endothelial growth factor (VEGF) protein, a vital factor in angiogenesis, was improved by Andr treatment under HG stimulation. LY294002 is a blocker of PI3K, MK-2206 2HCI (MK-2206) is a highly selective AKT inhibitor, and L-NAME is a suppressor of eNOS, all of which significantly reduce Andr-mediated protective effects in vitro. Hence, Andr may be involved in regulating HG-induced injury by activating PI3K/AKT-eNOS signalling in HUVECs.

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Conflict of interest statement

The authors declare that they have no competing interests.

Figures

Figure 1
Figure 1
Andr attenuates HG-induced inflammation and apoptosis in HUVECs. HUVECs were exposed to HG (33 mM) and treated with different concentrations of Andr (0, 12.5, 25, and 50 μM) for 24/36 hours. (a, b) The cell counting kit-8 assay was used to assess cell viability in each group (n = 6). (c) The effect of Andr was assessed at different time points in each group (n = 6). (d, e) LDH leakage (d) and MDA concentration (e) in each group (n = 6). (f–h) The mRNA levels of IL-1β, IL-6, and TNFα in HUVECs in each group (n = 6). (i, j) TUNEL staining and quantitative analysis of apoptotic cells in each group (n = 6). (k, l) The expression of Bcl-2, Bax, and C-caspase3 protein and quantitative analysis in each group (n = 6). ∗P < 0.05 vs. the control group. #P < 0.05 vs. the HG group.
Figure 2
Figure 2
Andr attenuates HG-induced cell migration and impairment of angiogenesis in HUVECs. (a, b) In the scratch assay, HUVECs were treated with Andr (50 μM) and/or HG for 24 hours and photographed at 0, 12, and 24 hours (n = 6). (c, d) In the tube formation assay, HUVECs were treated with Andr (50 μM) and/or HG. After 24 hours, images were acquired by inverted microscopy, and the results were analysed in each group (n = 6). (e, f) The expression of VEGF protein and the quantitative analysis in each group (n = 6). ∗P < 0.05 vs. the control group. #P < 0.05 vs. the HG group.
Figure 3
Figure 3
Andr regulates PI3K/AKT-eNOS signalling in vitro. (a) The expression of P-PI3K, T-PI3K, P-AKT, T-AKT, P-eNOS, and T-eNOS protein in each group (n = 6). (b–d) Quantification analysis of p-PI3K/PI3K protein, p-AKT/AKT protein, and p-eNOS/eNOS protein. (e, f) Effects of LY294002 at different concentrations on P-P13K and quantitative analysis of p-PI3K/PI3K protein (n = 6). (g, h) Effects of MK-2206 at different concentrations on P-AKT and quantitative analysis of p-AKT/AKT protein (n = 6). (i, j) Effects of L-NAME at different concentrations on P-eNOS and quantitative analysis of p-eNOS/eNOS protein (n = 6). ∗P < 0.05 vs. the control group. #P < 0.05 vs. the HG group.
Figure 4
Figure 4
Andr attenuates HG-induced inflammation, apoptosis, cell migration, and impairment of angiogenesis by PI3K/AKT-eNOS signalling in vitro. HUVECs were stimulated with HG and treated with Andr (50 μM), LY294002 (10 μM), MK-2206 (100 nM), and L-NAME (100 μM) for 24 hours. (a–c) Cell viability, MDA concentration, and LDH leakage in each group (n = 6). (d–f) The mRNA levels of IL-1β, IL-6, and TNFα in HUVECs in each group (n = 6). (g, h) TUNEL staining and quantitative analysis of apoptotic cells in each group (n = 6). (i, j) Scratch assay and the number of migrated cells in each group (n = 6). (k, l) Tube formation and quantitative analysis in each group (n = 6). (m, n) The expression of VEGF protein and quantitative analysis in each group (n = 6). ∗P < 0.05 vs. the control group. #P < 0.05 vs. the HG group.
Figure 5
Figure 5
Graphical abstract. Andrographolide (Andr) attenuates HG-induced vascular endothelial dysfunction. Andr increases the expression of P-PI3K, P-AKT, and P-eNOS that was inhibited by HG, thus suppressing the gene expression of IL-1β, IL-6, and TNFα and the expression of VEGF. Furthermore, this effect inhibits HUVECs and promotes tubule formation, consequently decreasing the levels of LDH and MDA induced by HG.
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References

    1. Bugger H., Abel E. D. Molecular mechanisms of diabetic cardiomyopathy. Diabetologia. 2014;57(4):660–671. doi: 10.1007/s00125-014-3171-6. - DOI - PMC - PubMed
    1. Varga Z. V., Giricz Z., Liaudet L., Haskó G., Ferdinandy P., Pacher P. Interplay of oxidative, nitrosative/nitrative stress, inflammation, cell death and autophagy in diabetic cardiomyopathy. Biochimica et Biophysica Acta-Molecular Basis of Disease. 2015;1852(2):232–242. doi: 10.1016/j.bbadis.2014.06.030. - DOI - PMC - PubMed
    1. Lin Y., Berg A. H., Iyengar P., et al. The hyperglycemia-induced inflammatory response in adipocytes: the role of reactive oxygen species. Journal of Biological Chemistry. 2005;280(6):4617–4626. doi: 10.1074/jbc.M411863200. - DOI - PubMed
    1. Giacco F., Brownlee M. Oxidative stress and diabetic complications. Circulation Research. 2010;107(9):1058–1070. doi: 10.1161/CIRCRESAHA.110.223545. - DOI - PMC - PubMed
    1. Costa P. Z., Soares R. Neovascularization in diabetes and its complications. Unraveling the angiogenic paradox. Life Sciences. 2013;92(22):1037–1045. doi: 10.1016/j.lfs.2013.04.001. - DOI - PubMed

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