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. 2022 Feb;13(2):3486-3502.
doi: 10.1080/21655979.2022.2026552.

Salvianolic acid B alleviates diabetic endothelial and mitochondrial dysfunction by down-regulating apoptosis and mitophagy of endothelial cells

Jie Xiang  1 , Chunling Zhang  2 , Tietao Di  3 , Lu Chen  4 , Wei Zhao  4 , Lianggang Wei  5 , Shiyong Zhou  6 , Xueli Wu  7 , Gengxin Wang  8 , Yun Zhang  8
Affiliations

Salvianolic acid B alleviates diabetic endothelial and mitochondrial dysfunction by down-regulating apoptosis and mitophagy of endothelial cells

Jie Xiang et al. Bioengineered. 2022 Feb.

Abstract

Endothelial dysfunction is a critical mediator in the pathogenesis of vascular complications of diabetes. Herein, this study was conducted to investigate the therapeutic effects of Salvianolic acid B (Sal B) on diabetes-induced endothelial dysfunction and the underlying mechanisms. Diabetic models were established both in db/db mice and high glucose (HG)-induced human umbilical vein endothelial cells (HUVECs). Moreover, HUVECs were exposed to carbonyl cyanide m-chlorophenyl hydrazone (CCCP) to induce endothelial cell damage. Following Sal B treatment, pathological changes of thoracic aorta were investigated by hematoxylin and eosin, alcian blue (AB), elastic fiber, Masson, and reticular fiber staining. BCL2-associated X (BAX), B-cell lymphoma-2 (Bcl-2), Beclin1, Parkin and PTEN Induced Kinase 1 (Pink1) expression was detected by Western blot, immunohistochemistry, and immunofluorescence in thoracic aorta, HG- and CCCP-induced HUVECs. Cell scratch test, MitoTracker Red CMXRos staining and Flou-4 AM staining were separately presented to detect migration, mitochondrial activity and intracellular Ca2+ in HUVECs. Our results showed that Sal B significantly ameliorated hyperlipidemia, hyperglycemia, hyperinsulinemia, and insulin resistance in db/db mice. Furthermore, it significantly alleviated diabetes-induced vascular endothelial dysfunction according to histopathology analysis. In diabetic thoracic aorta, HG- and CCCP-induced HUVECs, Sal B distinctly increased Bcl-2 expression and reduced BAX, Beclin1, Parkin and Pink1 expression, thereby protecting endothelial cells from apoptosis and mitophagy. Moreover, Sal B markedly enhanced migration, mitochondrial activity and intracellular Ca2+ levels both in HG- and CCCP-induced HUVECs. Collectively, Sal B exhibited a potential to improve diabetes-induced endothelial and mitochondrial dysfunction through down-regulating apoptosis and mitophagy of endothelial cells.Abbreviations: DM: diabetes mellitus; T2DM: type 2 diabetes mellitus; Sal B: Salvianolic acid B; HG: high glucose; FBG: fasting blood glucose; TC: total cholesterol; TG: triglycerides; LDL-C: low-density lipoprotein cholesterol; HDL-C: high-density lipoprotein cholesterol; FINS: fasting insulin; HOMA-IR: homeostasis model assessment insulin resistance; QUICKI: quantitative insulin-sensitivity check index; H&E: hematoxylin and eosin; HUVECs: human umbilical vein endothelial cells; IHC: immunohistochemistry; CCCP: carbonyl cyanide m-chlorophenyl hydrazone; FCM: flow cytometry; CCK-8: cell counting kit-8.

Keywords: Salvianolic acid B; apoptosis; diabetes; endothelial dysfunction; mitophagy.

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Conflict of interest statement

No potential conflict of interest was reported by the authors.

Figures

Figure 1.
Figure 1.
Sal B ameliorates diabetes-induced vascular pathology in db/db mice. (a–e) Representative images of (a) H&E staining, (b) AB staining, (c) elastic fiber staining (d) Masson staining, and (e) reticular fiber staining of thoracic aortas of db/m mice, db/db mice as well as db/db mice that were orally administrated 50 mg/kg Sal B for six weeks. Scale bar, 20 μm. Arrows indicate the abnormalities as claimed.
Figure 2.
Figure 2.
Sal B protects against apoptosis and mitophagy in thoracic aortas of db/db mice. (a–f) Western blot detecting BAX, Bcl-2, Beclin1, Parkin, and Pink1 expression in thoracic aortas of db/m mice, db/db mice as well as db/db mice that were orally administrated 50 mg/kg Sal B for six weeks. (g–l) IHC staining of BAX, Bcl-2, Beclin1, Parkin, and Pink1 expression in thoracic aortas of above mice. Scale bar, 20 μm. *P < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001. Arrows indicate the abnormalities as claimed.
Figure 3.
Figure 3.
Effects of Sal B on apoptosis and mitophagy in thoracic aortas of db/db mice. (a) Representative images of immunofluorescence of BAX, Bcl-2, Beclin1, Parkin, and Pink1 in thoracic aortas from db/m mice, db/db mice as well as db/db mice that were orally administrated 50 mg/kg Sal B for 6 weeks. Scale bar, 20 μm. Arrows indicate the abnormalities as claimed. (b–f) Quantification of the expression of BAX, Bcl-2, Beclin1, Parkin, and Pink1 in thoracic aorta of above mice. *P < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.
Figure 4.
Figure 4.
Sal B enhances migration and mitochondrial activity and alleviates apoptosis and mitophagy in HG-induced HUVECs. (a) FCM detecting the apoptosis of HUVECs treated by 0, 10, 20, 30, 50 and 100 μM Sal B. (b) CCK-8 examining the cell viability of HUVECs treated by 0, 5, 10, 20, 30, 50, 80 and 100 μM Sal B. (c, d) Cell scratch test detecting the wound distance of HUVECs in four groups: control, DMSO, HG and HG + Sal B. Scale bar, 200 μm. (e, f) Detection of mitochondrial activity of HUVECs in above groups through MitoTracker Red CMXRos staining. Scale bar, 20 μm. (g–l) Western blot detecting the expression of BAX, Bcl-2, Beclin1, Parkin and Pink1 in HUVECs of above groups. *P < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.
Figure 5.
Figure 5.
Sal B alleviates apoptosis and mitophagy in HG-induced HUVECs. (a–f) IHC detecting the expression of BAX, Bcl-2, Beclin1, Parkin and Pink1 in HUVECs of four groups: control, DMSO, HG and HG + Sal B. Scale bar, 20 μm. (g–l) Immunofluorescence examining the expression of BAX, Bcl-2, Beclin1, Parkin and Pink1 in HUVECs of above groups. Scale bar, 20 μm. *P < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001. Arrows indicate the abnormalities as claimed.
Figure 6.
Figure 6.
Sal B enhances migration and mitochondrial activity of CCCP-induced HUVECs and increases intracellular Ca2+ levels both in HG- and CCCP-induced HUVECs. (a, b) Cell scratch test detecting the wound distance of HUVECs in four groups: control, DMSO, CCCP and CCCP + Sal B. Scale bar, 200 μm. (c, d) Measurement of mitochondrial activity of HUVECs in above groups through MitoTracker Red CMXRos staining. Scale bar, 20 μm. (e, f) Detection of intracellular Ca2+ levels of HUVECs in control, DMSO, HG and HG + Sal B groups. Scale bar, 20 μm. (g, h) Detection of intracellular Ca2+ levels of HUVECs in control, DMSO, CCCP and CCCP + Sal B groups. Scale bar, 20 μm. **P < 0.01; ***p < 0.001; ****p < 0.0001.
Figure 7.
Figure 7.
Sal B treatment alleviates apoptosis and mitophagy in CCCP-induced HUVECs. (a–f) Western blot measuring BAX, Bcl-2, Beclin1, Parkin and Pink1 expression in HUVECs of control, DMSO, CCCP and CCCP + Sal B groups. (g–l) IHC staining assessing the expression of BAX, Bcl-2, Beclin1, Parkin and Pink1 in HUVECs of above groups. Scale bar, 20 μm. *P < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001. Arrows indicate the abnormalities as claimed.
Figure 8.
Figure 8.
Effects of Sal B treatment on apoptosis and mitophagy in CCCP-induced HUVECs. (a) Representative images of immunofluorescence staining for BAX, Bcl-2, Beclin1, Parkin and Pink1 expression in HUVECs of control, DMSO, CCCP and CCCP + Sal B groups. (b–f) Quantification of BAX, Bcl-2, Beclin1, Parkin and Pink1 expression in HUVECs of above groups. Scale bar, 20 μm. *P < 0.05; **p < 0.01; ****p < 0.0001. Arrows indicate the abnormalities as claimed.
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This work was funded by Innovation Group Major Research Project of Education Department of Guizhou Province in 2017 (Qianjiaohe KY [2017] No.042); National Natural Science Foundation of China [81960805].