SKF38393 prevents high glucose (HG)-induced endothelial dysfunction by inhibiting the effects of HG on cystathionine γ-lyase/hydrogen sulfide activity and via a RhoA/ROCK1 pathway

Gui-Quan Chang^1,†, Shu-Zhi Bai^1,†, Feng-Qi Sun¹, Ren Wu¹, Can Wei¹, Xin Wen¹, Yu-Xin Xi¹, Jing-Hui Hao¹, Altaany Zaid², Hong-Zhu Li^1,3,*

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¹ Department of Pathophysiology, Harbin Medical University, 150081 Harbin, Heilongjiang, China

² Faculty of Medicine, Yarmouk University, 21163 Irbid, Jordan

³ School of Medicine, Xiamen University, 361100 Xiamen, Fujian, China

^*Correspondence: lhz2020@xmu.edu.cn (Hong-Zhu Li)
^†These authors contributed equally.
Academic Editor: Graham Pawelec

Front. Biosci. (Landmark Ed) 2022, 27(2), 49; https://doi.org/10.31083/j.fbl2702049

Submitted: 2 September 2021 | Revised: 22 November 2021 | Accepted: 23 November 2021 | Published: 11 February 2022

This is an open access article under the CC BY 4.0 license.

Abstract

Background: Endothelial dysfunction plays a crucial role in diabetic vascular complications. A decrease in hydrogen sulfide (H ${}_{2}$ S) levels is increasingly becoming a vital factor contributing to high glucose (HG)-induced endothelial dysfunction. Dopamine D1-like receptors (DR1) activation has important physiological functions in the cardiovascular system. H ${}_{2}$ S decreases the dysfunction of vascular endothelial cells. However, no studies have reported whether DR1 protects the function of vascular endothelial cells by regulating H ${}_{2}$ S levels. Aim: The present study aimed to determine whether DR1 regulates the levels of endogenous H ${}_{2}$ S, which exerts protective effects against HG-induced injury of human umbilical vein endothelial cells (HUVECs) via Ras homolog gene family member A (RhoA)/Rho-associated coiled-coil containing kinase 1 (ROCK1) signalling. Methods: HUVECs were exposed to HG (30 mM) or normal glucose (5.5 mM) after different treatments. Cell viability, proliferation and migration were measured by Cell Counting Kit-8, EdU cell proliferation assay, transwell assay and wound healing assay, respectively. H ${}_{2}$ S probe (7-Azido-4-Methylcoumarin) was used to detect levels of H ${}_{2}$ S. The intracellular calcium concentration ([Ca ${}^{2+}$ ] ${}_{i}$ ) were measured using Fluo-4 AM. The protein expressions were quantified by Western blot. Results: We found that HG decreased the expression of DR1 and cystathionine $\gamma$ -lyase (CSE) and H ${}_{2}$ S production. The DR1 agonist SKF38393 significantly increased DR1 and CSE expression and H ${}_{2}$ S production, whereas NaHS (a H ${}_{2}$ S donor) only increased CSE expression and H ${}_{2}$ S production but had no effect on DR1 expression. Meanwhile, SKF38393 further increased the [Ca ${}^{2+}$ ] ${}_{i}$ induced by HG. In addition, HG reduced cell viability and the expression of Cyclin D1 and proliferating cell nuclear antigen and increased the expression of p21 ${}^{Cip/WAF-1}$ , collagen I, collagen III, matrix metalloproteinase 9, osteopontin and α-smooth muscle actin and the activity of phosphorylated RhoA and ROCK1. SKF38393 and NaHS reversed these effects of HG. PPG (a CSE inhibitor) abolished the beneficial effect of SKF38393. These effects of SKF38393 were similar to those of Y-27632 (a ROCK inhibitor). Conclusion: Taken together, our results suggest that DR1 activation upregulates the CSE/H ${}_{2}$ S pathway by increasing the [Ca ${}^{2+}$ ] ${}_{i}$ , which protects endothelial cells from HG-induced injury by inhibiting the RhoA/ROCK1 pathway.

Keywords

Dopamine D1-like receptors

Endothelial cells

Hydrogen sulfide

High glucose

RhoA/ROCK1 pathway

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Fig. 1.

DR1 activation upregulates the CSE/H ${}_{2}$ S pathway in HG-induced HUVECs. (A) The expression DR1 and CSE was determined using western blot (n = 5). The intensity of each band was quantified by densitometry, and data was normalized to the $\beta$ -actin signal. (B) Fluorescence microscopy was used to detect H ${}_{2}$ S levels in the HUVECs (n = 4, magnification $\times$ 200; scale bar, 200 $\mu$ m). The results were expressed as the mean $\pm$ SEM. * p $<$ 0.05 vs. control group; ** p $<$ 0.01 vs. control group; # p $<$ 0.05 vs. HG group; ## p $<$ 0.01 vs. HG group.

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