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

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.

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

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

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

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

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.