Korean J Physiol Pharmacol.  2024 Jan;28(1):39-48. 10.4196/kjpp.2024.28.1.39.

Wogonin attenuates vascular remodeling by inhibiting smooth muscle cell proliferation and migration in hypertensive rat

• Yang Y
• Huang S
• Wang J
• Nie X
• Huang L
• Li T

• KMID: 2550287
• DOI: http://doi.org/10.4196/kjpp.2024.28.1.39

Abstract

Wogonin, extracted from the roots of Scutellaria baicalensis Georgi, has been shown to suppress collagen deposition in spontaneously hypertensive rats (SHRs). This study was performed to investigate the role and mechanism of wogonin underlying vascular remodeling in SHRs. After injection of SHRs with 40 mg/kg of wogonin, blood pressure in rats was measured once a week. Masson's trichrome staining was conducted to observe the changes in aortas and mesenteric arteries. Vascular smooth muscle cells (VSMCs) isolated from rat thoracic aortas were treated with Angiotensin II (Ang II; 100 nM) in the presence or absence of varying concentrations of wogonin. The viability and proliferation of VSMCs were examined using Cell Counting Kit-8 assay and 5-ethynyl-2’-deoxyuridine assay, respectively. The migration of VSMCs was examined using wound healing assay and transwell assay. We found that wogonin administration alleviated hypertension, increased lumen diameter, and reduced the thickness of the arterial media in SHRs. Ang II treatment enhanced the viability of VSMCs, which was inhibited by wogonin in a concentration-dependent manner. Wogonin reversed Ang II-induced increases in the viability, proliferation, and migration of VSMCs. Moreover, wogonin inhibited Ang II-induced activation of mitogen-activated protein kinase (MAPK) signaling in VSMCs. Overall, wogonin repressed the proliferative and migratory capacity of VSMCs by regulating the MAPK signaling pathway, thereby attenuating vascular remodeling in hypertensive rats, indicating that wogonin might be a therapeutic agent for the treatment of vascular diseases.

Keyword

Angiotensins; Flavonoids; Hypertension; Vascular remodeling

Figure

• 

  Fig. 1 Wogonin alleviates vascular remodeling in SHRs. The rats were intravenously injected with wogonin (40 mg/kg) or NS every other day for 3 weeks. (A–C) The SBP, DBP, and MAP were measured in each conscious rat once a week within 3 weeks. (D) Representative images of aortas and MAs in WKYs and SHRs by Masson’s trichrome staining. Scale bar = 100 μm (aorta), Scale bar = 200 μm (MA). (E–G) Quantification of media thickness, lumen diameter, and the ratio of media thickness/lumen diameter in aortas and MAs of SHRs and WKYs based on Masson’s staining results. N = 8 in each group. Measurement data are expressed as the mean ± SD. SHRs, spontaneously hypertensive rats; WKYs, Wistar-Kyoto rats; NS, normal saline; MAs, mesenteric arteries; SBP, systolic blood pressure; DBP, diastolic blood pressure; MAP, mean arterial pressure. *p < 0.05, **p < 0.01, ***p < 0.001.

• 

  Fig. 2 Effects of wogonin on the viability of VSMCs treated with Ang II. (A) CCK-8 kit was used to measure the viability of VSMCs that were treated with Ang II (100 nM) for 6, 12, and 24 h. (B) Chemical structure of wogonin. (C) VSMCs were treated with Ang II (100 nM) alone or in combination with 0, 0.1, 1, 3, 10, or 20 μM of wogonin for 24 h. Cell viability was examined using CCK-8 assay. N = 3. Measurement data are expressed as the mean ± SD. VSMCs, vascular smooth muscle cells; Ang II, angiotensin II; CCK-8, Cell Counting Kit-8; OD, optical density. **p < 0.01.

• 

  Fig. 3 Wogonin administration suppresses the proliferation and migration of Ang II-treated VSMCs. VSMCs were treated with Ang II (100 nM) in the presence or absence of wogonin (10 μM) or NS for 24 h. (A) The viability of VSMCs in the groups of Control, Ang II, Ang II + NS, and Ang II + Wogonin was assessed using CCK8 assay. (B) EdU assay was performed for cell proliferation assessment. Scale bar = 100 μM. (C) Bar graph showing the percentage of EdU-positive cells. (D) Representative images of migrated cells from wound healing assay. (E) Representative images of migrated cells from transwell assay. Scale bar = 200 μM. (F) Bar graph showing the wound closure rate. (G) Quantification of migrated cells from transwell assay. Measurement data are expressed as the mean ± SD. VSMCs, vascular smooth muscle cells; Ang II, angiotensin II; CCK-8, Cell Counting Kit-8; EdU, 5-ethynyl-2’-deoxyuridine; NS, normal saline. **p < 0.01, ***p < 0.001.

• 

  Fig. 4 Effects of wogonin on the phosphorylation of MAPKs. VSMCs were treated with Ang II (100 nM) in the presence or absence of wogonin (10 μM) or NS for 24 h. (A) The levels of the key proteins in the MAPK signaling pathway in Control, Ang II, Ang II + NS, and Ang II + Wogonin groups were examined using Western blot analysis. (B–D) The ratio of p-ERK/ERK, p-JNK/JNK, and p-p38/p38 was calculated. N = 3. (E) The protein levels of CDK4, Cyclin D1, MMP2, and MMP9 in the Control, Ang II, Ang II + NS, and Ang II + Wogonin groups were examined using Western blot analysis. (F–I) The protein levels of CDK4, Cyclin D1, MMP2, and MMP9 were quantified. N = 3. Measurement data are expressed as the mean ± SD. MAPK, mitogen-activated protein kinase; VSMCs, vascular smooth muscle cells; Ang II, angiotensin II; NS, normal saline; ERK, extracellular signal-regulated kinase; JNK, c-Jun N-terminal kinase; CDK4, cyclin-dependent kinase4; MMP2, matrix metalloproteinase2; MMP9, matrix metalloproteinase9. **p < 0.01.

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