Korean J Physiol Pharmacol.  2023 Nov;27(6):533-540. 10.4196/kjpp.2023.27.6.533.

Sweroside plays a role in mitigating high glucose-induced damage in human renal tubular epithelial HK-2 cells by regulating the SIRT1/NF-κB signaling pathway

• Ma X
• Guo Z
• Zhao W
• Chen L

• KMID: 2547319
• DOI: http://doi.org/10.4196/kjpp.2023.27.6.533

Abstract

Sweroside is a natural monoterpene derived from Swertia pseudochinensis Hara. Recently, studies have shown that sweroside exhibits a variety of biological activities, such as anti-inflammatory, antioxidant, and hypoglycemic effects. However, its role and mechanisms in high glucose (HG)-induced renal injury remain unclear. Herein, we established a renal injury model in vitro by inducing human renal tubular epithelial cell (HK-2 cells) injury by HG. Then, the effects of sweroside on HK-2 cell activity, inflammation, reactive oxygen species (ROS) production, and epithelial mesenchymal transition (EMT) were observed. As a result, sweroside treatment ameliorated the viability, inhibited the secretion of inflammatory cytokines (TNF-α, IL-1β, and VCAM-1), reduced the generation of ROS, and inhibited EMT in HK-2 cells. Moreover, the protein expression of SIRT1 was increased and the acetylation of p65 NFkB was decreased in HK-2 cells with sweroside treatment. More importantly, EX527, an inhibitor of SIRT1, that inactivated SIRT1, abolished the improvement effects of sweroside on HK-2 cells. Our findings suggested that sweroside may mitigate HGcaused injury in HK-2 cells by promoting SIRT1-mediated deacetylation of p65 NF-kB.

Keyword

Epithelial mesenchymal transition; High glucose; HK-2 cells; Inflammation; Oxidative stress; Sweroside

Figure

• 

  Fig. 1 Sweroside promotes the cell viability of HK-2 cells exposed to HG. (A) The chemical structure of sweroside. (B) The activity of cells treated with sweroside (0, 25, 50, 100 µM) under non-HG conditions were determined by CCK8 assay. (C) CCK8 assay was performed under HG exposure. (D, E) Flow cytometry analysis of cell apoptosis under HG exposure. Values are presented as mean ± SD. SW, sweroside; HK-2 cells, human renal tubular epithelial cell; HG, high glucose; Ctrl, control. NSp > 0.05, compared with non-SW group or HG group; ***p < 0.001, compared with the Ctrl group; ###p < 0.001, compared with the HG group.

• 

  Fig. 2 Sweroside attenuates HG-induced inflammation and oxidative stress in HK-2 cells. (A) The mRNA levels of TNF-α, IL-1β, and VCAM-1 were detected by qRT-PCR. (B) The concentrations of TNF-α, IL-1β, and VCAM-1 in the culture medium were examined by ELISA. (C) Representative images of DHE fluorescent staining showing ROS production. Scale bar = 50 µM. Values are presented as mean ± SD. SW, sweroside; HK-2 cells, human renal tubular epithelial cell; HG, high glucose; Ctrl, control; TNF-α, tumor necrosis factor-α; IL, interleukin; qRT-PCR, quantitative real-time polymerase chain reaction; ROS, reactive oxygen species. ***p < 0.001, compared with the Ctrl group; NSp > 0.05, #p < 0.05, ##p < 0.01, ###p < 0.001, compared with the HG group.

• 

  Fig. 3 Sweroside reverses EMT-related proteins expression in HK-2 cells under HG condition. (A) Blot images were gained from the Western blot membrane. (B) Quantification of the Western blot assay. Values are presented as mean ± SD. SW, sweroside; HK-2 cells, human renal tubular epithelial cell; HG, high glucose; Ctrl, control; EMT, epithelial mesenchymal transition. ***p < 0.001, compared with the Ctrl group; ##p < 0.01, ###p < 0.001, compared with the HG group.

• 

  Fig. 4 Sweroside regulates SIRT1/NF-kB signaling pathway in HG-induced HK-2 cells. (A) SIRT1 protein level in HK-2 cells under HG condition was examined by Western blot. (B) Ac-p65, p65, and β-actin protein expression were detected by Western blot. Values are presented as mean ± SD. SW, sweroside; HK-2 cells, human renal tubular epithelial cell; HG, high glucose; Ctrl, control; SIRT1, silent information regulator 2 homolog 1. ***p < 0.001, compared with the Ctrl group; NSp > 0.05, ###p < 0.001, compared with the HG group.

• 

  Fig. 5 EX527 reversed the alleviating effects of sweroside on HG-treated HK-2 cells. (A) Western blot assay for SIRT1 protein expression in HK-2 cells of Ctrl group, HG group, HG + SW (100 µM) group and HG + SW (100 µM) + EX527 group. (B) CCK8 assay for cell viability. (C) ELISA was performed to analyze TNF-α, IL-1β, and VCAM-1 concentration. (D) Detection of ROS in different groups of HK-2 cells by DHE fluorescent staining. Scale bar = 50 µM. (E) Western blot assays for ZO-1, Vimentin, α-SMA, and Snail protein expression. Values are presented as mean ± SD. SW, sweroside; HK-2 cells, human renal tubular epithelial cell; HG, high glucose; Ctrl, control; SIRT1, silent information regulator 2 homolog 1; TNF-α, tumor necrosis factor-α; IL, interleukin; ROS, reactive oxygen species. ***p < 0.001, compared with the control group; ##p < 0.01, ###p < 0.001, compared with the HG group; ^^p < 0.01, ^^^p < 0.001, compared with the HG + SW (100 µM) group.

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