Biological evaluation of linalool on the function of blood vessels

Figures

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  Figure 1.. Linalool inhibits the proliferation of Ang II-induced vascular smooth muscle cells. (A) The viability of cells treated with different concentrations of Ang II was detected using a CCK-8 assay. **P<0.01, ***P<0.001 vs. control (n ≥3). (B) The viability of cells treated with different concentrations (50, 100, 150 µmol/l) of linalool was detected using a CCK-8 assay. (C) An MTT assay was used to detect proliferation of A7r5 cells treated by Ang II and linalool (n ≥3). *P<0.05 vs. control; #P<0.05, ##P<0.01 vs. Ang II (n ≥3). (D) The expression of PCNA, which is relevant to cell proliferation, was detected via western blotting. ***P<0.001 vs. control; ###P<0.001 vs. Ang II (n ≥3). Ang II, angiotensin II; CCK-8; Cell Counting Kit-8; PCNA, proliferating cell nuclear antigen; Lina, linalool.

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  Figure 2.. Linalool inhibits the migration of Ang II-induced vascular smooth muscle cells. (A) Wound-healing and (B) Transwell assays were applied to evaluate cell migration. Scale bar, 100 µm. (C) Quantitative results of wound-healing and (D) Transwell assay. (E and F) The expression levels of MMP2 and MMP9, which are involved in cell migration were detected by western blotting. ***P<0.001 vs. control; #P<0.05, ##P<0.01, ###P<0.001 vs. Ang II (n ≥3). Ang II, angiotensin II.

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  Figure 3.. Linalool downregulates CHRM3 expression in Ang II-induced vascular smooth muscle cells. (A) Reverse transcription-quantitative PCR and (B) western blotting were used to detect CHRM3 expression in cells treated with Ang II and different concentrations of linalool. ***P<0.001 vs. control; ##P<0.01, ###P<0.001 vs. Ang II (n ≥3). Ang II, angiotensin II; CHRM3, cholinergic receptor muscarinic 3.

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  Figure 4.. Linalool inhibits the proliferation of Ang II-induced vascular smooth muscle cells via downregulating CHRM3 expression. (A) Reverse transcription-quantitative PCR and (B)western blotting were employed to detect the expression level of CHRM3 in transfected cells. ***P<0.001 vs. pcDNA3.1-NC (n≥3). (C) MTT assay was used to detect cell proliferation. *P<0.05, **P<0.01 vs. Ang II; #P<0.05, ##P<0.01 vs. Ang II+linalool+pcDNA3.1-NC (n ≥3). (D) The expression of PCNA, which is relevant to cell proliferation, was detected via western blotting. ***P<0.001 vs. control; ###P<0.001 vs. Ang II; &&&P<0.001 vs. Ang II+linalool+pcDNA3.1-NC (n ≥3). Ang II, angiotensin II; CHRM3, cholinergic receptor muscarinic 3; NC, negative control; PCNA, proliferating cell nuclear antigen; NC, negative control.

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  Figure 5.. Linalool inhibits the migration of Ang II-induced vascular smooth muscle cells via downregulating the expression of CHRM3. (A) Wound-healing and (B) Transwell assays were applied to determinate cell migration. Scale bar, 100 µm. (C) Quantitative results of wound-healing and (D) Transwell assay. (E and F) The expression levels of MMP2 and MMP9, which are relevant to cell migration, were detected by western blotting. ***P<0.001 vs. control; ###P<0.001 vs. Ang II; &P<0.05, &&P<0.01, &&&P<0.001 vs. Ang II+linalool+pcDNA3.1-NC (n ≥3). Ang II, angiotensin II; CHRM3, cholinergic receptor muscarinic 3; NC, negative control.

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  Figure 6.. Linalool blocks the MAPK signaling pathway via downregulating the expression of CHRM3. (A) Western blotting was applied to detect the expression of proteins related to the MAPK signaling pathway in A7r5 and transfected cells. (B) The semi-quantitative graphs of western blotting. ***P<0.001 vs. control; ###P<0.001 vs. Ang II; &&&P<0.01 vs. Ang II+linalool+pcDNA3.1-NC (n ≥3). Ang II, angiotensin II; CHRM3, cholinergic receptor muscarinic 3; NC, negative control; p-, phosphorylated.