Rutaecarpine prevents hypertensive cardiac hypertrophy involving the inhibition of Nox4‐ROS‐ADAM17 pathway

Abstract Rutaecarpine attenuates hypertensive cardiac hypertrophy in the rats with abdominal artery constriction (AAC); however, its mechanism of action remains largely unknown. Our previous study indicated that NADPH oxidase 4 (Nox4) promotes angiotensin II (Ang II)‐induced cardiac hypertrophy through the pathway between reactive oxygen species (ROS) and a disintegrin and metalloproteinase‐17 (ADAM17) in primary cardiomyocytes. This research aimed to determine whether the Nox4‐ROS‐ADAM17 pathway is involved in the protective action of rutaecarpine against hypertensive cardiac hypertrophy. AAC‐induced hypertensive rats were adopted to evaluate the role of rutaecarpine in hypertensive cardiac hypertrophy. Western blotting and real‐time PCR were used to detect gene expression. Rutaecarpine inhibited hypertensive cardiac hypertrophy in AAC‐induced hypertensive rats. These findings were confirmed by the results of in vitro experiments that rutaecarpine significantly inhibited Ang II‐induced cardiac hypertrophy in primary cardiomyocytes. Likewise, rutaecarpine significantly suppressed the Nox4‐ROS‐ADAM17 pathway and over‐activation of extracellular signal‐regulated kinase (ERK) 1/2 pathway in the left ventricle of AAC‐induced hypertensive rats and primary cardiomyocytes stimulated with Ang II. The inhibition of Nox4‐ROS‐ADAM17 pathway and over‐activation of ERK1/2 might be associated with the beneficial role of rutaecarpine in hypertensive cardiac hypertrophy, thus providing additional evidence for preventing hypertensive cardiac hypertrophy with rutaecarpine.


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pathological cardiac hypertrophy may slow or prevent the progress of hypertension to heart failure.
Recently, rutaecarpine was shown to alleviate hypertensive cardiac hypertrophy in the rats subjected to abdominal artery constriction (AAC). 9 High blood pressure contributes to the develop- Therefore, this study aimed to determine whether the Nox4-ROS-ADAM17 pathway is involved in the protective role of rutaecarpine in hypertensive cardiac hypertrophy in the rats subjected to AAC.

| Animal model of AAC-induced cardiac hypertrophy
Abdominal artery constriction was carried out to induce pathological cardiac hypertrophy through pressure overload, as previously described. 12,13 In brief, the following procedures were described: rats were anaesthetized with sodium pentobarbital through intraperitoneal injection before the surgery until toe pinch reflex dis-

| Measurement of blood pressure
Systolic blood pressure (SBP) was measured in conscious state before the treatment with rutaecarpine, at the 2nd week and the 4th week after the treatment using tail-cuff method. 14

| Haemodynamic measurement
Haemodynamic measurement was implemented in the rats as previously described. 12 After the rats were anaesthetized with sodium pentobarbital (ip, 45 mg/kg), a 24-gauge polyethylene catheter filled with heparin was introduced into the right carotid artery of rats, and systolic arterial blood pressure (SABP), diastolic arterial blood pressure (DABP) were measured using a BL-420S system (Chengdu Tai-meng Technology Co., Ltd, Sichuan, China). Next, the 24-gauge polyethylene catheter was further introduced into the left ventricle of rats. Finally, the BL-420S system was used to measure the maximal rate of left ventricular pressure increase (dp/dtmax) and decrease (dp/dtmin), and heart rate.

| Histological analysis
The hearts were arrested in diastole using potassium chloride cross-sectional area was measured in 10 randomly chosen nonrepeating fields in cross-sections stained with H&E using Image-pro plus 6.0 according to the method described previously. 16 Finally, interstitial fibrosis was quantified as the percentage of fibrotic area over the total myocardial area in five randomly chosen nonrepeating visual fields (excluding the fields that contained a coronary artery) of sections stained with Masson's trichrome reagent using Image-pro plus 6.0.

| Immunohistochemistry
Immunohistochemistry was used to detect the protein levels of col I, col III and TNF-α in the left ventricle as described previously. 17

| RNA isolation and quantitative real-time quantitative PCR
RNA extraction and quantitative real-time PCR were performed as described previously. 18 Real-time PCR used primers for atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP), Col I, Col III, Nox4, ADAM17, TNF-α and the gene-specific for GADPH

| Western blotting
Western blotting was executed according to the standard procedures previously described. 11

| Cell surface area
After fixed with 4% paraform, primary cardiomyocytes were permeabilized using 0.5% Triton X-100 and then incubated with TRITC-labeled phalloidin for 30 minutes at room temperature. Next, the cell surface area was automatically analysed in 40 nonrepeated fields of the cardiomyocytes using a Cellomics/High Content Screening (Thermo Scientific, Shanghai, China) as previously described. 11,13

| Enzyme-linked immunosorbent assay
After cardiomyocytes were stimulated with Ang II for 24 hours, the conditioned DMEM was collected to detect the mature TNF-α released from myocardial cells. These samples were used to detect the TNF-α level by a TNF-α enzyme-linked immunosorbent assay kit (CSB-E11987r; Cusabio Biotech., Co. Ltd., Wuhan, China) according to the manufacturer's instructions.

| Statistical analysis
Data are expressed as mean ± SD. Statistical analysis was performed using one-way or two-way ANOVA followed by Bonferroni's post hoc test among at least three groups, and P < 0.05 was considered to have statistical significance.

| Effect of rutaecarpine on blood pressure in the rats subjected to AAC
For evaluating the effect of rutaecarpine on blood pressure, we compared SBP, SABP, and DABP obtained from the four groups.
As shown in Table 1 (Tables 1 and 2). Likewise, the AAC-induced hypertensive rats treated with low or high dosage of rutaecarpine exhibited increases in dp/dtmax and dp/dtmin, but no significant change in heart rate compared with the AAC group. Accordingly, rutaecarpine decreased blood pressure in AAC-induced hypertensive rats, consistently with its depressor effect reported by previous studies. 5-9

| Rutaecarpine suppressed hypertensive cardiac hypertrophy
We next investigated the role of rutaecarpine in hypertensive cardiac hypertrophy in AAC-induced hypertensive rats. After 4 weeks of AAC induction, the ratio between heart weight and body weight (HW/BW), and the ratio between left ventricular weight and body weight (LVW/BW) were markedly elevated in the AAC rats compared with those of the Sham group ( Figure 1A  and systole (LVPWs) compared with the Sham group (Table 2).
Concurrently, the mRNA levels of hypertrophic genes, such as ANP and BNP, were significantly higher in the AAC group than those of the Sham group ( Figure 1D). These evidences suggest that hypertensive cardiac hypertrophy is induced through AAC induction for 4 weeks. In contrast to the results observed in the AAC rats, treatment with low or high dosages of rutaecarpine remarkably attenuated hypertensive cardiac hypertrophy, as indicated by a decrease in the HW/BW, the LVW/BW, the myocyte cross-sectional area, the LVAWd, the LVAWs, the LVPWd, the LVPWs and the mRNA levels of hypertrophic genes, compared with those of the AAC group ( Figure 1 and Table 2). Moreover, low or high dosage of rutaecarpine significantly decreased the fibrotic area and the protein and mRNA levels of col I and col III in the left ventricle of AAC-induced hypertensive rats ( Figure 2).

F I G U R E 1 Rutaecarpine (Rut) inhibited hypertensive cardiac hypertrophy in the rats subjected to abdominal artery constriction (AAC).
A, HW/BW (n = 9-10 per group). B, LVW/BW (n = 9-10 per group). C, Cross-sections of the hearts from the rats subjected to AAC or sham operation were stained with haematoxylin & eosin to analyse myocyte cross-sectional area (n = 5 per group). D, Real-time PCR analyses of hypertrophic markers (ANP and BNP, n = 5 in each group). Rut (L) represents low dosage of rutaecarpine; Rut (H) represents high dosage of rutaecarpine; HW/BW represents the ratio between heart weight and body weight; LVW/BW represents the ratio between left ventricular weight and body weight. # P < 0.05 vs Sham group, *P < 0.05 vs SHR group. One-way ANOVA followed by Bonferroni's post hoc test was carried out for the statistical tests

| Rutaecarpine prevented Ang II-induced cardiac hypertrophy in primary cardiomyocytes
We subsequently observed the effect of rutaecarpine on pathological cardiac hypertrophy in vitro. In cardiomyocytes, pathological cardiac hypertrophy could be modeled through Ang II stimulation. 11,16,23 Exposure to Ang II (100 nmol/L) increased cell surface area and the ex-

| Rutaecarpine inhibited the Nox4-ROS-ADAM17 pathway in hypertrophic cardiomyocytes
To further delineate mechanisms underlying the inhibitory effect of rutaecarpine against hypertensive cardiac hypertrophy, we determined whether rutaecarpine suppressed the Nox4-ROS-ADAM17 pathway in hypertrophic cardiomyocytes. Firstly, we examined the hypothesis in primary cardiomyocytes. As can be seen from F I G U R E 2 Rutaecarpine inhibited cardiac fibrosis in the rats subjected to abdominal artery constriction (AAC). A, Representative microphotographs of Masson staining of heart. B, Fibrosis area (n = 5 per group). C, Col I positive area (n = 5 per group). D, Representative microphotographs of immunochemistry for col I and col III. E, Col III positive area (n = 5 per group). F, mRNA levels of col I and col III in the left ventricle (n = 4 per group). One-way ANOVA followed by Bonferroni's post hoc test was used for the statistical tests. Rut (L) represents low dosage of rutaecarpine; Rut (H) represents high dosage of rutaecarpine; Col I represents collagen I; col III represents collagen III. # P < 0.05 vs Control group; *P < 0.05 vs AAC group Figure 4A and B, rutaecarpine down-regulated the ROS produc-

| Rutaecarpine blocked over-activation of the ERK1/2 pathway
We next determined whether rutaecarpine influenced the ERK1/2 pathway in hypertrophic cardiomyocytes because the ERK1/2 pathway has been proved to play a critical role in pathological cardiac hypertrophy. [26][27][28] The left ventricle of the AAC rats displayed increased activity of ERK1/2 compared with the Sham group, whereas low or F I G U R E 3 Rutaecarpine (Rut) alleviated angiotensin II (Ang II)induced cardiac hypertrophy in primary cardiomyocytes. Primary cardiomyocytes were treated with 100 nmol/L angiotensin II for 24 h after pretreating with rutaecarpine (10 µmol/L) for 60 min. A, Cell surface area. B and C, The mRNA levels of hypertrophic genes ANP (B) and BNP (C). # P < 0.05 vs control group; *P < 0.05 vs Ang II group. The statistical tests were undertaken by one-way ANOVA followed by Bonferroni's post hoc test. n = 3 independent experiments high dosage of rutaecarpine remarkably decreased the phosphorylation level of ERK1/2 in the left ventricle of AAC-induced hypertensive rats ( Figure 6A). Similarly, rutaecarpine down-regulated the ERK1/2 activity in cardiomyocytes stimulated with 100 nmol/L Ang II for 24 hours ( Figure 6B). Therefore, rutaecarpine suppresses over-active ERK1/2 pathway in hypertrophic cardiomyocytes. . After being pretreated with Rut (10 µmol/L) for 60 min, cardiomyocytes were then stimulated with 100 nmol/L Ang II for 24 h. # P < 0.05 vs Control group; *P < 0.05 vs Ang II group. The statistical analyses were executed using one-way ANOVA followed by Bonferroni's post hoc test cardiac hypertrophy after 14 days of Ang II or phenylephrine infusion in mice. 47 The possible explanation for this inconsistency might be that Ang II or phenylephrine infusion induces hypertensive cardiac hypertrophy through other signaling pathways such as p38 MAPK and JNK1/2 in mice with ERK1/2 deletion in the heart. However, ERK1/2 activation, induced by overexpression of MAPK1 within the heart, advances concentric cardiac hypertrophy. 26,47 Hence, ERK1/2 plays an important role in hypertensive cardiac hypertrophy. In the current study, we discovered that rutaecarpine markedly reduced ERK1/2 activation in the left ventricle of AAC rats and primary cardiomyocytes stimulated with Ang II. Overall, the inhibition of ERK1/2 pathway is associated the protective action of rutaecarpine against hypertensive cardiac hypertrophy.
In conclusion, we revealed that rutaecarpine attenuates hypertensive cardiac hypertrophy in AAC-induced hypertensive rats; and inhibiting the Nox4-ROS-ADAM17 pathway and over-activa-

| DATA AVAIL AB ILIT Y
All data used to support the findings of this study are available from the corresponding authors upon request.

ACK N OWLED G EM ENTS
The authors thank Ms. Menzhen Zhang for her technical assistance General Hospital (grant number YQ2015-008).

CO N FLI C T S O F I NTE R E S T
The authors have no conflict of interest to disclose.