Hypertension is the main cause of cardiovascular diseases and affects more than 25% of the global population [1]. During vascular injury, vascular smooth muscle cells (VSMCs) switch from a quiescent "contractile" phenotype to a highly migratory and proliferative "synthetic" phenotype [2]. Increasing studies have demonstrated that abnormal proliferation and migration of VSMCs play key roles in the pathogenesis and development of cardiovascular disorders, such as diabetic vascular complications, transplantation arteriopathy, atherosclerosis, and hypertension [3,4]. Vascular remodeling is considered to be protective in the short term but harmful in the long term, and these pathological effects of vascular remodeling are related to increased risks of cardiovascular diseases [5]. Therefore, reversing hypertension-related vascular remodeling is of great importance for the prevention of hypertension progression.

Hypertension-induced vascular remodeling can be mediated by activation of the renin-angiotensin system [6]. It has been demonstrated that the renin-angiotensin system plays an important role during vascular remodeling. As a multi-functional hormone, angiotensin II (Ang II) is crucial for vascular homoeostasis via angiotensin II receptor type 1 (AT1R) or AT2R [7]. Studies have shown that Ang II has a high level in the serum of spontaneously hypertensive rats (SHRs) [8,9]. Ang II regulates vascular function, including contraction, growth, and fibrosis, and contributes to increased vascular permeability by initiating inflammatory responses [10]. It is also a powerful vasoconstrictive factor that induces the phenotypic conversion of SMCs and participates in vascular remodeling [10,11]. Therefore, Ang II has been commonly used to establish the in vitro models of SAHs [12,13].

Scutellaria baicalensis Georgi has a long history in the treatment of inflammation and hypertension in Eastern Asian countries [14]. Wogonin is one of the flavonoids extracted from the dried root of Scutellaria baicalensis Georgi and possesses multiple pharmacological and biological effects including neuroprotective, anti-inflammation, antioxidation, anti-angiogenesis, and anticancer activities [14-17]. Previous reports demonstrated that wogonin treatment inhibits serum-induced rabbit VSMC proliferation [18], TNF-alpha-induced human aortic SMC (HASMC) migration [19], and lipotoxicity-induced apoptosis of VSMCs [20]. In addition, the anti-angiogenesis role of wogonin has also been found in tumors and interleukin-6-induced human umbilical vein endothelial cells [21,22]. Moreover, wogonin represses collagen deposition in cardiac fibroblasts of Wistar-Kyoto rats (WKYs) and SHRs [23], and also exerts vasodilatory effect in male Wistar rats [24]. A recent study shows that wogonin attenuates cisplatin-induced cardiotoxicity in animal models [25]. However, the role of wogonin in vascular remodeling remains unclear.

The mitogen-activated protein kinase (MAPK) signaling is an important pathway consisting of three family members, p38 MAPK, c-Jun N-terminal kinase (JNK), and extracellular signal-regulated kinase (ERK) [26]. It has been reported that activation of MAPK signaling promotes hypoxic pulmonary vascular remodeling via stimulation of the migration and proliferation as well as reduction of the apoptosis of pulmonary artery SMCs [27]. Wogonin has been shown to suppress TNF-alpha-induced migration of HASMCs in vitro by suppressing MAPK signaling [19]. Therefore, we hypothesized that wogonin also regulates MAPK pathway in Ang II-induced VSMCs.

The purpose of the present study was to investigate the role and mechanism of wogonin in SHRs. We hypothesized that wogonin may alleviate vascular remodeling by suppressing VSMC proliferation and migration by regulating MAPK pathway, which may provide a potential therapeutic agent to ameliorate vascular remodeling.

Animal procedures

Nine-week-old male SHRs and WKYs were purchased from BioLasco Co., Ltd. All rats had free access to drinking water and normal rat chow in a temperature-controlled (24°C ± 2°C) room with a 12-h dark/light cycle. SHRs with systolic blood pressure (SBP) higher than 150 mmHg were selected for this study. Animal experiment was performed in accordance with the principles of the Guide for the Care and Use of Laboratory Animals (National Institutes of Health [NIH], 8th edition, 2011). In addition, we ensure that the manuscript report conforms to the ARRIVE guidelines for animal experiment reports. All animal experiments were approved by the Ethical Review System for Laboratory Animal Welfare of the Wuhan Myhalic Biotechnology Co., Ltd (approval number: 202205041). After acclimation for seven days, WKYs or SHRs were intravenously injected with wogonin (purity ≥ 99.0%; Yunnan Yunyao Laboratory Company Limited) at a dose of 40 mg/kg or equal amount of normal saline every other day for three weeks. The dose of wogonin was selected according to a previous study [28]. Three weeks later, all rats were euthanized with 200 mg/kg pentobarbital sodium (Scrbio) and the aortas and mesenteric arteries (MAs) were collected for further use.

Measurement of blood pressure

After treatment, animal models were placed in a warm room, and the SBP, diastolic blood pressure (DBP), and mean arterial pressure (MAP) of the tail artery were measured once a week by the tail-cuff method using a noninvasive computerized tail-cuff system (NIBP, ADInstruments).

Masson's trichrome staining

The aortas and MAs isolated from WKYs and SHRs were fixed with formalin, embedded in paraffin and sliced into 2-mm sections. The sections were deparaffinized in xylene and then rehydrated in alcohol gradient. Masson's trichrome dye was used to stain the sections and the images were captured under a microscope. Evaluation of vascular remodeling was performed according to the media cross-sectional area, lumen diameter, media thickness, media thicknesses/lumen diameter ratio, and outer diameter.

Cell culture and treatment

Primary VSMCs were isolated from rat thoracic aortas as previously performed [29]. The obtained VSMCs were maintained in Dulbecco's modified Eagle's medium (DMEM, YT8231; YITA Bio) containing 10% fetal bovine serum (FBS, 76294-180; AVANTOR), 1% penicillin-streptomycin (15140-122; Gibco) at 37°C in the presence of 5% CO₂ and 95% O₂. VSMCs were treated with Ang II (100 nM; Sigma) alone for 6, 12, and 24 h, coincubated with Ang II (100 nM) and wogonin (0.1, 1, 3, 10, and 20 μM) for 24 h, or co-incubated with Ang II (100 nM) and wogonin (10 μM) for 24 h.

Cell Counting Kit-8 (CCK-8) assay

VSMCs (7 × 10³ cells/well) were plated in 96well plates and were treated with Ang II or/and wogonin for 24 h. Next, 10 μl of CCK-8 solution (PUFFE Biotechnology Co., Ltd.) was added to each well of the plates to incubate the cells for 4 h. A microplate reader was used to detect the absorbance of each well at 450 nm.

5-ethynyl-2’-deoxyuridine (EdU) assay

EdU assay was performed to detect the proliferation of VSMCs following the product manuals. Cells were fixed with 4% paraformaldehyde and then incubated with EdU working solution for 2 h. 4’,6-diamidino2-phenylindole (D8200-10; Solarbio) was used to stain the cells for 15 min in the dark. Cell proliferation was evaluated by calculating the percentage of EdU-positive cells using a fluorescence microscope (Nikon).

Transwell assay

Cells cultured serumfree DMEM medium were placed in the upper chamber and DMEM supplemented with 10% FBS was placed in its lower chamber. After incubation for 24 h at 37°C, a cotton swab was used to remove the cells on the upper surface of the membrane, and the cells that had migrated to the lower surface were fixed and stained with 0.1% crystal violet. The number of migrated cells was measured by counting under a light microscope.

Wound healing assay

Cells in 6well plates were cultured at 37°C for 24 h and allowed to reach 80% confluence. The cell monolayer was wounded by scratching using a sterile micropipette tip. After incubation in serum-free DMEM for 24 h, an inverted microscope was used, and wound healing percentages were analyzed with ImageJ software (NIH).

Western blot analysis

Total proteins were extracted by lysing VSMCs in RIPA lysis buffer (R0020-100; Solarbio), followed by quantification of the protein content using a BCA protein quantification kit (E112-01; Vazyme Biotechnology Co., Ltd.). Afterwards, equal amounts of protein samples (50 μg) were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred on a PVDF membrane (Millipore). After blocking with 5% skimmed milk, all membranes were probed with primary antibodies, including anti-phospho (p)-p38 (ab4822, 1:1,000), anti-p38 (ab170099, 1:5,000), anti-p-JNK (ab76572, 1:5,000), anti-JNK (ab208035, 1:2,000), anti-p-ERK (ab201015, 1:1,000), anti-ERK (ab184699, 1:10,000), anti-cyclin-dependent kinase4 (CDK4, ab199728, 1:1,000), anti-Cyclin D1 (ab16663, 1:200), anti-matrix metalloproteinase2 (MMP2, ab92536, 1:1,000), anti-matrix metalloproteinase9 (MMP9, ab76003, 1:1,000), and anti-GAPDH (ab181603, 1:1,000) at 4°C overnight. The membranes were rinsed with Tris Buffered Saline with Tween three times and incubated with anti-rabbit secondary antibody (ab96899, 1:10,000) at room temperature for 1 h. Protein bands were visualized with an ECL kit and quantified with ImageJ software (NIH). GAPDH acted as loading control.

Statistical analysis

All data were analyzed using SPSS 20.0 software (IBM Corp.) and are displayed as the mean ± SD. For multiple group comparisons, one-way analysis of variance together with Tukey’s post-hoc test was used. Each experiment was performed at least in triplicate. p < 0.05 was considered statistically significant.

Wogonin attenuates vascular remodeling in hypertensive rats

High blood pressure indices, including SBP, DBP, and MAP were measured in WKYs and SHRs. We observed that SBP, DBP, and MAP were increased in SHRs compared to WKYs. Wogonin administration reduced the SBP, DBP, and MAP of SHRs two weeks after injection, and exerted no significant effect on blood pressure during the first week (Fig. 1A–C). Masson’s staining was performed to evaluate vascular remodeling in the aortas and MAs of WKYs and SHRs (Fig. 1D–G). Compared to WKYs, significant increases in the thickness of media and the media thickness/lumen diameter ratio were found in the aortas and MAs of SHRs. However, wogonin administration significantly reversed increased thickness of media and media thickness/lumen diameter ratio in SHRs. A notable reduction in lumen diameter was found in the MAs of SHRs, which was restored after the treatment of wogonin. Additionally, the images of the aorta showed less blue color levels in the wogonin treatment group compared to the control group, both in WKYs and SHRs, demonstrating the inhibitory effect of wogonin on collagen deposition in the aortas and MAs of hypertensive rats.

Figure 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.

Wogonin suppresses the viability of Ang II-treated VSMCs

Ang II-stimulated VSMCs have been widely used to establish the in vitro models of SAHs [12,13,30]. To investigate the effect of wogonin on cell viability, VSMCs were treated with 100 nM Ang II for 6, 12, and 24 h, respectively. The concentrations of Ang II were used according to previous studies [31,32]. The CCK-8 assay results showed that the viability of VSMCs was enhanced by Ang II treatment in a time-dependent manner, compared to the control group (Fig. 2A). As previously studies reported [19], wogonin (Fig. 2B) under 20 μM of concentration had no toxicity on VSMCs. We found that wogonin inhibited the viability of Ang II-treated VSMCs in a concentration-dependent manner, while the viability was slightly increased when the concentration of wogonin was 20 μM (Fig. 2C). Therefore, we chose 10 μM of wogonin for the subsequent investigations.

Figure 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.

Administration of wogonin inhibits the proliferation and migration of VSMCs

CCK-8 assay was performed to detect the effect of wogonin on VSMC viability, and the results revealed that wogonin treatment reversed Ang II-mediated increase in the viability of VSMCs (Fig. 3A). Consistent with the CCK-8 results, wogonin treatment notably reduced the percentage of EdU positive cells in Ang II-treated VSMCs, demonstrating that wogonin inhibited Ang II-induced cell proliferation (Fig. 3B, C). Wound healing assay and transwell assay were conducted to assess the impact of wogonin on VSMC migration. In wound healing assay, Ang II stimulation notably enhanced wound closure rate in VSMCs, and this effect was reversed by wogonin (Fig. 3D, F). In transwell assay, wogonin treatment significantly reduced the number of migrated cells in Ang II-treated VSMCs (Fig. 3E, G), indicating that wogonin inhibited the migration and invasion of VSMCs.

Figure 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.

Wogonin inhibits Ang II-induced activation of MAPK signaling in VSMCs

A previous study has shown that wogonin protects against endotoxin-induced acute lung injury by reducing the phosphorylation of MAPKs [14]. Therefore, we investigated whether wogonin also regulates the MAPK signaling in Ang II-induced VSMCs. The results of Western blot showed that Ang II significantly enhanced the phosphorylation levels of ERK, JNK, and p38 in VSMCs, which were reduced by treatment with wogonin (Fig. 4A–D). Therefore, wogonin administration leads to suppression of MAPK signaling pathway in Ang II-induced VSMCs. To further verify the anti-proliferation and anti-migration effects of wogonin, we examined the expression of proliferation- and migration-related markers. The results of western blot revealed that the protein levels of CDK4, Cyclin D1, matrix metalloproteinases MMP2, and MMP9 were upregulated by Ang II in VSMCs, and wogonin treatment reversed the effect of Ang II on these markers (Fig. 4E–I).

Figure 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.

Vascular remodeling is related to hypertension that is an important predisposing factor for cardiovascular diseases [29]. Wogonin is a flavonoid extracted from the root of Scutellaria baicalensis Georgi and has high potential to treat cardiovascular diseases. Our study revealed the role of wogonin in vascular remodeling. We found that wogonin ameliorated vascular remodeling in SHRs by inhibiting VSMC proliferation and migration via suppression of the MAPK signaling pathway.

Almost all organs and tissues can be affected by vascular remodeling in patients with hypertension. Vascular remodeling is characterized by reduced vascular compliance, enhanced media thickness, and intimal injury [33]. Due to the extensive use of SHRs in the evaluation of the effect of drugs on hypertensive vascular remodeling [34], this animal model was used to investigate the role of wogonin in our study. We found that wogonin (40 mg/kg) decreased SBP, DBP, and MAP in SHRs. In addition, significant improvements were observed in the vascular wall of aortas and MAs, as manifested by reduced media thickness and increased lumen diameter, suggesting the beneficial benefits of wogonin in improving vascular remodeling in SHRs.

Phenotypic transition of VSMCs is a key determinant of vascular homeostasis. Unlike skeletal or cardiac muscle cells, VSMCs switches from contractile phenotype to a highly migratory and proliferative synthetic phenotype in response to injured stimuli [35]. Increasing evidence has shown that hyperproliferation and abnormal migration of VSMCs promote vascular remodeling and facilitate the process of various vascular diseases [36-38]. Previous studies have confirmed that Ang II plays a key role in the proliferation and migration of VSMCs and is widely used for the exploration of the mechanisms of cardiovascular diseases [39]. In our current study, Ang II at a dose of 100 nM was used to treat VSMCs for 6, 12, and 24 h. We found that Ang II significantly enhanced the viability of VSMCs in a time-dependent manner. Notably, wogonin treatment inhibited Ang II-promoted viability of VSMCs. Moreover, the effects of Ang II on the proliferation and migration of VSMCs were significantly reversed by wogonin administration. These findings are consistent with the wogonin’s effects on serum-induced rabbit VSMC and TNF-alpha-induced HASMC [18,19] as well as in a serious of human cancers (e.g., hepatocellular carcinoma, gastric cancer, melanoma, and renal cell carcinoma) [40-43].

The MAPK signaling pathway has been known to modulate cell proliferation, apoptosis, transformation, and differentiation [44]. Previous reports have revealed that the MAPK signaling stimulates the proliferation and migration whereas decreases the apoptosis of SMCs to contribute to vascular remodeling. For example, TCONS_00034812 depletion promotes the proliferation but suppresses the apoptosis of SMCs by activating the MAPK signaling [45]. Srolo Bzhtang suppresses vascular remodeling and pulmonary arterial pressure by inhibiting MAPK signaling in rat models [46]. Additionally, the suppressive effect of wogonin on MAPK signaling has been verified in acute lung injury [14], TNF-alpha-induced HASMCs [19], intervertebral disc degeneration [47], and rheumatoid arthritis [48]. Consistent with previous studies, our study showed that the Ang II-induced increased phosphorylation levels of ERK, JNK, and p38 MAPK were inhibited by treatment with wogonin, suggesting that wogonin blocked the MAPK signaling to suppress the proliferation and migration of VSMCs.

In conclusion, our current investigation demonstrated that wogonin attenuated vascular remodeling in SHRs by restraining Ang II-induced proliferation and migration of VSMCs via inhibition of the MAPK signaling pathway, suggesting that wogonin might be a novel therapeutic agent for the treatment of vascular diseases. It has been demonstrated that after intragastric administration of 5 mg/kg wogonin, little wogonin is detected in the plasma of rat models. It suggests that intravenous administration might be the clinical administration method of wogonin [49]. Although wogonin offers a wide margin of safety [50], the extract dosage of wogonin applied in clinical patients deserves careful confirmation in future studies.

The authors appreciate all the participants providing supports for this study.

This work is supported by National Natural Science Foundation of China (Approval number: 81860075).

The authors declare no conflicts of interest.