Notoginsenoside Fc Accelerates Reendothelialization following Vascular Injury in Diabetic Rats by Promoting Endothelial Cell Autophagy

Abstract

Interventional therapies, such as percutaneous transluminal angioplasty and endovascular stent implantation, are used widely for the treatment of diabetic peripheral vascular complications. Reendothelialization is an essential process in vascular injury following interventional therapy, and hyperglycemia in diabetes mellitus (DM) plays an important role in damaging endothelial layer integrity, leading to the retardance of reendothelialization and excessive neointimal formation. Notoginsenoside Fc (Fc), a novel saponin isolated from Panax notoginseng, effectively counteracts platelet aggregation. Nevertheless, the potential effects and molecular mechanisms of Fc on reendothelialization have yet to be explored. In this study, we present novel findings that show the benefit of Fc in accelerating reendothelialization and alleviating excessive neointimal formation following carotid artery injury in diabetic Sprague-Dawley rats in vivo. Simultaneously, the decreased autophagy of the injured carotid artery in diabetic rats was restored by Fc treatment. Our in vitro results also demonstrated that Fc promoted endothelial cell proliferation and migration under high-glucose treatment by increasing autophagy. In summary, this study supported the notion that Fc could accelerate reendothelialization following vascular injury in diabetic rats by promoting autophagy, suggesting that Fc may exert therapeutic benefits for early endothelial injury and restenosis following intervention in diabetes-associated vascular diseases.

Figure 1

(a) Molecular structure of notoginsenoside Fc (Fc). (b) Blood glucose levels in the different rat groups at days 14 and 28. DM represents diabetes mellitus. (c) A schematic diagram illustrating the experimental animal groups and different treatments. STZ represents streptozotocin.

Figure 2

Effects of notoginsenoside Fc (Fc) on reendothelialization following carotid artery injury. Reendothelialization was quantified in Evans blue-stained carotid arteries at 14 and 28 days after vascular wire injury. (a) Blue staining indicates endothelial denudation. (b) The reendothelialization rates in the rats across treatment groups were analyzed statistically. The images are at 10x magnification. Scale bar = 1 mm. Data are shown as the mean ± standard deviation. ^#P < 0.05 and ^##P < 0.01 vs. the control group; ^∗P < 0.05 vs. the diabetes mellitus (DM) group. n = 6.

Figure 3

Effects of notoginsenoside Fc (Fc) on pathomorphology and intimal hyperplasia following carotid artery injury. (a) Representative photographs of carotid arteries by hematoxylin-eosin staining at 14 days are shown. Images are at 50x magnification and 400x magnification. Scale bars = 200 μm and 25 μm. Black arrows indicate elastic lamellae. I refers to intima and M refers to media. (b) Neointimal area of carotid arteries within each treatment group. (c) Media area of carotid arteries within each treatment group. (d) Neointima-to-media area ratios of carotid arteries within each treatment group. Data are shown as the mean ± standard deviation. ^##P < 0.01 vs. the control group; ^∗∗P < 0.01 vs. the diabetes mellitus (DM) group. n = 6.

Figure 4

Effects of notoginsenoside Fc (Fc) on Beclin 1 protein of the intima following carotid artery injury. (a) Representative images by immunohistochemistry showing Beclin 1 protein in brown. Beclin 1 protein was found predominantly in the intima. L represents lumen. Images are at 200x and 400x magnification. Scale bars = 50 μm and 25 μm. (b) The number of Beclin 1-positive cells was counted and analyzed statistically in five random high-power fields. Data are shown as the mean ± standard deviation. ^##P < 0.01 vs. the control group; ^∗P < 0.05 vs. the diabetes mellitus (DM) group. n = 6.

Figure 5

Notoginsenoside Fc (Fc) prevents HG-induced autophagy reduction in rat aortic endothelial cells. (a, b) Expression of LC3B, Beclin 1, and p62 proteins was detected by Western blot analysis. (c) Expression of LC3B, Beclin 1, and p62 mRNA was detected by RT-qPCR. ^#P < 0.05 and ^##P < 0.01 vs. the normal-glucose (NG) group; ^∗P < 0.05 vs. the high-glucose (HG) group. n = 3.

Figure 6

Notoginsenoside Fc (Fc) prevents HG-induced autophagy reduction in rat aortic endothelial cells. (a, b) The mRFP-eGFP-LC3B plasmid was transfected into cells and visualized by confocal microscopy. Representative images show puncta formation in different groups and the quantitative analysis of three types of puncta (8 cells per group). Scale bar = 10 μm. (c, d) Representative images and quantitative analysis of autophagosomes in different groups assessed by electron microscopy (6 cells per group). Black arrows indicate autolysosomes/amphisomes and red arrows indicate autophagosomes. Scale bars = 2 μm and 1 μm. ^#P < 0.05 and ^##P < 0.01 vs. the normal-glucose (NG) group; ^∗P < 0.05 and ^∗∗P < 0.01 vs. the high-glucose (HG) group. n = 3.

Figure 7

Notoginsenoside Fc (Fc) promotes proliferation and migration via autophagy. (a, b) Cell cycle progression analysis of rat aortic endothelial cells (RAOECs) and G1 arrest rate across treatment groups, as quantified by flow cytometry. (c, d) Representative images of the wound healing assay and wound closure rate in RAOECs across treatment groups. Scale bar = 200 μm. (e) Expression of proliferating cell nuclear antigen (PCNA) protein was detected by Western blot analysis. ^#P < 0.05 vs. the normal-glucose (NG) group; ^##P < 0.01 vs. the NG group; ^∗P < 0.05 vs. the high glucose (HG) group; ⁺P < 0.05 vs. the HG+Fc group. n = 3.