Ginsenoside Rg1 ameliorates chronic intermittent hypoxia-induced vascular endothelial dysfunction by suppressing the formation of mitochondrial reactive oxygen species through the calpain-1 pathway

Figures

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  Fig. 1. Ginsenoside Rg1 and Calpain-1 Knockout Protected Against CIH-induced Vascular Endothelial Dysfunction. (A) Representative images of the impaired endothelial-dependent relaxation and vascular contraction response. (B) The effect of ginsenoside Rg1 treatment on Ach-induced vasodilation in aortas of mice exposed to CIH (n = 5). (C) The effect of ginsenoside Rg1 treatment on the vascular contraction response to PE in aortas of mice exposed to CIH (n = 5). (D) The effect of L-NAME treatment on the vasodilation responses of all groups (n = 5). (E, G) Serum NO levels detected by the nitrate reductase method (n = 8). (F) Fluorescence intensity of p-eNOS in aortic sections (n = 3). (H) The percentage of p-eNOS-positive cells was determined by immunohistochemistry staining (n = 3). (I) The effects of calpain-1 knockout on Ach-induced vasodilation, the PE-induced vascular contraction response and the vasodilation responses after incubation with L-NAME in the aortas of mice exposed to CIH (n = 5). Data are presented as the mean ± SD (∗∗P < 0.01 compared with the CIH group).

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  Fig. 2. Ginsenoside Rg1 and Calpain-1 Knockout improved CIH-induced oxidative stress and mitochondrial dysfunction. (A) Representative fluorescence images intercellular ROS and mitoROS in aortas (n = 3). (B) MDA levels in the serum of mice subjected to CIH were determined by an MDA assay kit (n = 8). (C) Drp1 and Mfn2 protein expression in the aortas of mice exposed to CIH was examined by western blotting (n = 3). Data are presented as the mean ± SD (∗∗P < 0.01, ∗P < 0.05 compared with the CIH group).

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  Fig. 3. The activation of the calpain-1/PP2A/eNOS signaling pathway and vascular reactivity studies in aortas of mice exposed to CIH. (A-D) The levels of calpain-1, PP2A and p-eNOS were examined by western blot analysis (n = 3). (E) The percentage of cells positive for calpain-1 and PP2A was determined by immunohistochemistry staining (n = 3). (F) The effects of PP2A treatment on Ach-induced vasodilation, the PE-induced vascular contraction response and the vasodilation responses after incubation with L-NAME in the aortas of mice exposed to CIH (n = 5). (G-L) The effects of tiron, PEG-Cat, NAC, TEMPOL, rotenone and 5-HD treatment on the impaired endothelium-dependent relaxation in aortas of mice with CIH exposure (n = 5). Data are presented as the mean ± SD (∗∗P < 0.01, ∗P < 0.05 compared with the CIH group; ##P < 0.01 compared with the control group).

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  Fig. 4. Ginsenoside Rg1 improved CIH-induced vascular endothelial dysfunction and oxidative stress through the calpain-1/PP2A/eNOS signaling pathway in HACECs. (A) Cell viability of HCAECs in different groups (n = 8). (B) Level of NO in HCAEC supernatant in different groups (n = 8). (C) Level of MDA in HCAEC supernatant in different groups (n = 8). (D-G) Representative fluorescence images and fluorescence intensity of intercellular ROS and p-eNOS in HCAECs exposed to CIH (n = 3). (H–K) Western blotting was conducted to examine the levels of calpain-1, PP2A and p-eNOS in HCAECs exposed to CIH (n = 3). Data are presented as the mean ± SD (∗∗P < 0.01 compared with the CIH group).

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  Fig. 5. Ginsenoside Rg1 alleviated CIH-induced mitochondrial dysfunction via the calpain-1/PP2A/eNOS signaling pathway in HACECs exposed to CIH. (A-D) Representative fluorescence images and fluorescence intensity of mitoROS and ΔΨm in HCAECs subjected to CIH (n = 3). (E-G) Western blotting was conducted to examine the levels of Drp1 and Mfn2 in HCAECs exposed to CIH (n = 3). Data are presented as the mean ± SD (∗∗P < 0.01 compared with the CIH group).

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  Fig. 6. Effect of calpain-1 overexpression on CIH-induced vascular endothelial dysfunction, oxidative stress and mitochondrial dysfunction in HACECs. (A-B) Western blotting was conducted to examine the levels of calpain-1, PP2A and p-eNOS in HCAECs exposed to CIH (n = 3). (C) Cell viability of HCAECs in different groups (n = 8). (D) Level of NO in HCAEC supernatant in different groups (n = 8). (E) Level of MDA in HCAEC supernatant in different groups (n = 8). (F) Level of intracellular ROS in HCAECs exposed to CIH (n = 3). (G-H) Representative fluorescence images and fluorescence intensity of mitoROS in HCAECs subjected to CIH (n = 3). (I–K) Western blotting was conducted to examine the levels of Drp1 and Mfn2 in HCAECs exposed to CIH (n = 3). Data are presented as the mean ± SD (∗∗P < 0.01, ∗P < 0.05 compared with the CIH group; ##P < 0.01 compared with the control group).