Ginkgolide B Mediated Alleviation of Inflammatory Cascades and Altered Lipid Metabolism in HUVECs via Targeting PCSK-9 Expression and Functionality

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  Figure 1. (a) Ox-LDL alters the expression of SREBP-2, PCSK-9, and LDL-R in HUVECs. The HUVECs were cultured in Dulbecco's Modified Eagle's Medium (DMEM) containing 10% heat-inactivated FBS, 2 mM glutamine, and antibiotics at 37°C in a humidified 5% CO2 atmosphere. Thus, cultured cells were either treated with native LDL or Ox-LDL at different concentrations 25, 50, 75, and 100μg/ml designated as Ox-LDL 25, Ox-LDL 50, Ox-LDL 75, and Ox-LDL 100, respectively, for gene expression analysis. Significantly different from native LDL at Ap<0.001. Nonsignificantly different from native LDL at Bp>0.05. (b) Ginkgolide B amends the Ox-LDL-induced altered expression of SREBP-2, PCSK-9, and LDL-R in HUVECs. The HUVECs were pretreated with 20-100 μg/ml of Ginkgolide B (designated as GB 20, GB 40, GB 80, and GB 100) for 24 h and then all the groups except control were exposed to Ox-LDL (75μg/ml) for another 24 h followed by m-RNA extraction for qRT-PCR analysis. Significantly different from control HUVECs at Ap<0.001. Significantly different from control HUVECs at Bp<0.01. Significantly different from control HUVECs at Cp<0.05. Nonsignificantly different from control HUVECs at Dp>0.05. Significantly…

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  Figure 2. (a) Ginkgolide B decreases Ox-LDL-induced total cholesterol and free cholesterol deposition in HUVECs. The HUVECs were cultured in DMEM containing 10% heat-inactivated FBS, 2 mM glutamine, and antibiotics at 37°C in a humidified 5% CO2 atmosphere. Thus, cultured cells were pretreated with 20-100 μg/ml of Ginkgolide B for 24 h and then exposed to Ox-LDL (75μg/ml) for another 24 h for quantitation of TC and FC. Significantly different from control HUVECs at Ap<0.001. Significantly different from control HUVECs at Bp<0.01. Nonsignificantly different from control HUVECs at Cp>0.05. Significantly different from Ox-LDL at Dp<0.001. (b) Ox-LDL raises the level of PCSK-9 in HUVECs. The cultured HUVECs cells were either treated with native LDL or Ox-LDL (25, 50, 75, and 100 μg/ml) for determination of PCSK-9 level by ELISA. Significantly different from native LDL at Ap<0.001. (c) Ginkgolide B lowers the Ox-LDL-induced PCSK-9 level in HUVECs. The cultured HUVECs cells were pretreated with 20-100 μg/ml of Ginkgolide B for 24 h and then exposed to Ox-LDL (75μg/ml) for another 24 h for quantitation of PCSK-9 via ELISA. Significantly different from control HUVECs at Ap<0.001. Significantly…

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  Figure 3. (a) Ox-LDL treatment induces the ROS generation in HUVECs. The cultured HUVECs cells were either treated with native LDL or Ox-LDL (25, 50, 75, and 100 μg/ml) for determination of ROS generation in terms of % DCFDA fluorescence. Significantly different from native LDL at Ap<0.001. (b) Ginkgolide B suppresses ROS generation in Ox-LDL-treated HUVECs. ROS level in terms of %DCFDA fluorescence was quantified by the pretreatment of HUVECs with different concentrations of Ginkgolide B (20-100μg/ml) for 24 h followed by the treatment of Ox-LDL (75μg/ml) for another 6 h. Significantly different from control HUVECs at Ap<0.001. Significantly different from control HUVECs at Bp<0.01. Nonsignificantly different from control HUVECs at Cp>0.05. Significantly different from Ox-LDL at Dp<0.001. Significantly different from Ox-LDL at Ep<0.01. (c) Ox-LDL treatment upregulates the expression of ICAM-1 and VCAM-1 in HUVECs. The cultured HUVECs cells were either treated with native LDL or Ox-LDL (25, 50, 75, and 100 μg/ml) for the ICAM-1 and VCAM-1 expression analysis. Significantly different from native LDL at Ap<0.001. Significantly different from native LDL at Bp<0.01. (d) Ginkgolide B…

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  Figure 4. (a) Ginkgolide B downregulates the expression of inflammatory cytokines and chemokines in Ox-LDL-treated HUVECs. HUVECs were grown in six-well plates for 24 h and treated with different concentrations of Ginkgolide B (20-100 μg/ml) for 24 h followed by the treatment of Ox-LDL (75μg/ml) for another 24 h. The cells were harvested and the expression of cytokines (IL-1α, IL-1β, & IL-6) and chemokines (MCP-1, CXCL-1, & CXCL-2) was examined by qRT-PCR analysis. (b) Ginkgolide B inhibited inflammatory protein expression in Ox-LDL-treated HUVECs. Cells were grown in six-well plates for 24 h and treated with different concentrations of Ginkgolide B (20-100 μg/ml) for 24 h followed by the treatment of Ox-LDL (75μg/ml) for another 24 h. The cells were harvested and cell lysates were prepared. Protein levels of different cytokines (IL-1α, IL-1β, & IL-6) and chemokines (MCP-1, CXCL-1, & CXCL-2) were examined by ELISA kits. The levels of statistical significance for both (a) and (b) are significantly different from control HUVECs at Ap<0.001. Significantly different from control HUVECs at Bp<0.01. Significantly different from control HUVECs at Cp<0.05. Nonsignificantly different from control…

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  Figure 5. Molecular informatics studies of Ginkgolide B and Atorvastatin with PCSK-9. (a) Interaction of Ginkgolide B (represented as red ball-stick model) with PCSK-9 (PDB I.D.: 2p4e) residues showed the binding energy (ΔG) of –7.13 Kcal/mol. (b) Interaction of Atorvastatin (represented as red ball-stick model) with the residues of PCSK-9 showed the binding energy (ΔG) of –7.02 Kcal/mol.

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  Figure 6. Ginkgolide B reduces the affinity of PCSK-9 to complex with EGF-A-like repeats of LDL-R. (a) PCSK-9-EGF-A-complex (PCSK-9 chains have been represented as ribbon structures in various colors, while EGF-A of LDL-R has been represented as purple ribbon-like structure). (b) Ginkgolide B-PCSK-9-EGF-A-complex (Ginkgolide B has been represented in yellow as ball-stick molecule). (c) Atorvastatin-PCSK-9-EGF-A-complex (Atorvastatin has been represented in yellow as ball-stick molecule).