Improvement of vascular dysfunction by argirein through inhibiting endothelial cell apoptosis associated with ET-1/Nox4 signal pathway in diabetic rats - PubMed (original) (raw)

Improvement of vascular dysfunction by argirein through inhibiting endothelial cell apoptosis associated with ET-1/Nox4 signal pathway in diabetic rats

Jie Su et al. Sci Rep. 2018.

Abstract

Endothelial cell apoptosis plays an important role in the pathophysiological mechanism of vascular complications in type 2 diabetes mellitus (T2DM). Argirein, a new synthetic compound was demonstrated to inactivate NADPH oxidase to alleviate cardiac dysfunction in T2DM. Here, we investigated whether argirein medication attenuated the vascular dysfunction in T2DM by inhibiting endothelial cell apoptosis which was associated with NADPH oxidase. The rat aortic endothelial cells (RAECs) were incubated with glucose (30 mM) for 48 hour in vitro. It was shown that high glucose significantly increased the protein expression of BAX (Bcl-2 Associated X protein) and Caspase-3 and decreased Bcl2 (B-Cell Leukemia/Lymphoma 2) protein level in RAECs, which was normalized by argirein medication. The annexin V-FITC bound cell percentage and DNA fragments in agarose electrophoresis were markedly suppressed by argirein to confirm the anti-apoptotic property of argirein in RAECs. Furthermore, we found that argirein blocked the endothelin (ET)-1/Nox4 signal-dependent superoxide (O2-.) generation, which regulated endothelial cell apoptosis in RAECs. In vivo, argirein intervention relieved the vasodilatory response to acetylcholine and restored the expressions of Nox4 and BAX in the aorta endothelium of high-fat diet (HFD)-fed rats following streptozocin (STZ) injection. For the first time, we demonstrated that argirein could inhibit vascular endothelial cell apoptosis, which was attributed to blocking ET-1/Nox4 signal-dependent O2- generation in RAECs. This current study revealed the therapeutic effects of argirein to prevent the vascular complication in T2DM through inhibiting endothelial cell apoptosis which was associated with the anti-oxidative property of argirein.

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Conflict of interest statement

The authors declare no competing interests.

Figures

Figure 1

Figure 1

The chemical structure of argirein compound shows a hydrogen bond connecting the two moieties.

Figure 2

Figure 2

Primary endothelial cell characterization. (a) Cell morphology of RAECs under light microscopy showed the typical two-demensional cobblestone. (b) Immunocytochemistry of RAECs that was stained with anti-vWF antibody followed by a dylight 594-conjugated second antibody, and photographs were at 400x magnification (n = 4).

Figure 3

Figure 3

High glucose induced apoptosis-related protein expression in different time. RAECs were incubated with glucose at the dose of 30 mM for 24, 48, 72 hours. Representative western blot gel documents and summarized data showing the protein expression of BAX, Bcl-2 and caspase-3 (a–c). The original gel documents of western blot were shown in Fig. 3 in Supplementary Information. *P < 0.05 vs. Control (Ctrl) (n = 4).

Figure 4

Figure 4

Argirein inhibited the apoptosis of RAECs under high glucose. RAECs were incubated with high glucose (HG, 30 mM) for 48 h in the presence or absence of argirein (AR, 1 μM) treatment for the last 12 h. Representative western blot gel documents (a and Fig. 4a in Supplementary Information.) showing the protein expression of BAX, Bcl-2 and Caspase-3. Representative plots (b) and summarized data (c) showing the apoptotic populations of RAECs (the bottom right of the plots) sorted by flow cytometry after they were double-stained with FITC-annexin V and propidium iodide (PI). (d) DNA ladders showing DNA fragments in 1% agarose gel electrophoresis. Representative fluorescent images of TUNEL assay (e) and summarized data (f) showing the TUNEL positive cells. Photographs were at 400x magnification. *P < 0.05 vs. Vehicle (Vehl) Ctrl; #P < 0.05 vs. HG alone treated group (n = 4).

Figure 5

Figure 5

Argirein decreased O2−. production under high glucose. Typical representative fluorescent images for DHE staining (a) and summarized data (b) showing the roles of argirein (AR, 1 μM) and on O2−. production in RAECs incubated with high glucose (HG, 30 mM), and photographs were at 100 × magnification. Representative ESR spectrographs of O2−. trapped by CMH (c) and summarized data (d) showing O2−. production dependent on NADPH oxidase in the RAECs. *P < 0.05 vs. Vehl Ctrl; #P < 0.05 vs. HG alone treated group (n = 4).

Figure 6

Figure 6

Nox4 mediated anti-apoptotic role of argirein. (a) Representative western blot gel documents and summarized data showing the roles of argirein (AR, 1 μM) on Nox4 protein expression. After Nox4 siRNA transfection, Nox4 protein expression (b) and O2−. production with DHE staining (c) and the apoptotic-related protein expression including BAX, Bcl-2, and Caspase-3 (d–g) in RAECs incubated with high glucose (HG, 30 mM). Representative western blot gel documents and summarized data showing the roles of AR on ETAR protein expression (h,i) and the roles of specific ETAR inhibitor BQ123 on Nox4 protein expression (h,j) in RAECs. The original gel documents of western blot were shown in Fig. 6a,b,d,h in Supplementary Information. *P < 0.05 vs. Vehl or Scramble (Scr) Ctrl; #P < 0.05 vs. HG alone treated group (n = 4).

Figure 7

Figure 7

Argirein attenuated vascular dysfunction in T2DM rats. Male Sprague-Dawley rats were fed HFD and a low dose of STZ (40 mg/kg, i.p.) to develop a rat model of type2 diabetes. Diabetic rats were intervened with argirein (AR, 200 mg/kg, ip) for 3 weeks. Summarized data showing the fasting blood glucose (FBG) (a) and homeostasis model assessment of insulin resistance (HOMA-IR) (b). Representative fluorescent images of TUNEL assay (c) on the aorta showing the TUNEL positive cells on endothelium, and photographs were at 400x magnification (n = 4). Representative immunohistochemistry images showing Nox4 and BAX in the aorta, and photographs were at 100x magnification (n = 4) (d). Vascular relaxant response to acetylcholine (Ach, 10−10–10−4M) and sodium nitroprusside (SNP,10−10–10−4M) showing the role of AR on thoracic aorta from diabetic rats (e,f) and the role of Nox4 inhibitor GKT137831(10−5 M, 0.5 h) on aorta rings incubated with HG(50 mM, 3.5 h) (g,h). *P < 0.05 vs. Ctrl; #P < 0.05 vs. T2DM or HG group (n = 6).

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