Research ArticleZi Shen Huo Luo Formula Prevents Aldosterone-InducedCardiomyocyte Hypertrophy and Cardiac FibroblastProliferation by Regulating the Striatin-MediatedMR/EGFR/ERKSignaling Pathway

Weike Feng,1 Yue Zhao,1 Xiaotong Song,1 Yuan Wang,1,2 Qian Chen,1,2 Haijun Zhao,1,2

Guangying Lu,1 Hongyan Yuan,3 Zhichun Wu ,1,2 and Huayun Yu 1,2

1Shandong University of Traditional Chinese Medicine, Jinan 250355, China2Shandong Co-Innovation Center of Classic TCM Formula, Jinan 250355, China3Shandong Institute of Commerce & Technology, Jinan 250103, China

Correspondence should be addressed to Zhichun Wu; wuzhichun@sdutcm.edu.cn and Huayun Yu; 01000029@sdutcm.edu.cn

Weike Feng and Yue Zhao contributed equally to this work.

Received 21 February 2020; Revised 1 August 2020; Accepted 1 September 2020; Published 16 September 2020

Academic Editor: Francesca Mancianti

Copyright © 2020 Weike Feng et al. ,is is an open access article distributed under the Creative Commons Attribution License,which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Inappropriate activation of the renin-angiotensin-aldosterone system (RAAS) is an important factor in the development of hy-pertension. Excessive aldosterone can lead to myocardial extracellular matrix collagen proliferation, fibrosis, and cardiomyocytehypertrophy and aggravate maladaptive remodeling. ,e results of our previous clinical and animal experiments suggested that ZiShen Huo Luo Formula (ZSHLF) combined with perindopril can effectively control the process of left ventricular hypertrophy(LVH).,e purpose of this study was to investigate whether ZSHLF-treated serum inhibits themembrane localization of the striatin-mediated mineralocorticoid receptor (MR) and affects MR-mediated nongenomic effects and the downstream epidermal growthfactor receptor (EGFR)/extracellular regulated kinase (ERK) signaling pathways, thereby improving aldosterone-inducedmyocardialremodeling. Serum containing ZSHLF was prepared and used to treat rat cardiomyocytes and cardiac fibroblasts in vitro afteraldosterone induction and striatin knockdown by small interfering RNA (siRNA). Cell-based assays were carried out to determinethe cardiomyocyte surface area and assess the proliferation rate and hydroxyproline secretion of cardiac fibroblasts. Quantitativereal-time PCR (qRT-PCR), immunoprecipitation (IP), and Western blotting were performed to evaluate the striatin-mediated MR/EGFR/ERK signaling pathway. In the present study, ZSHLF attenuated the aldosterone-induced hypertrophy of cardiomyocytes andinhibited the proliferation and collagen synthesis of cardiac fibroblasts. ZSHLF also reduced striatin mRNA expression and inhibitedstriatin and MR binding, membrane MR protein expression, and EGFR and ERK1/2 phosphorylation. Furthermore, after striatinsilencing with siRNA, some of the effects of ZSHLF were not changed significantly. In conclusion, ZSHLF inhibits the downstreamEGFR/ERK signaling pathway by blocking the striatin-mediated membrane localization of MR, which may be an importantmolecular mechanism by which ZSHLF improves aldosterone-induced myocardial remodeling.

1. Introduction

Hypertension is one of the most common non-communicable diseases in the world, and approximately 1billion patients have currently been diagnosed with hy-pertension worldwide. Left ventricular hypertrophy (LVH),the most common complication of hypertension, is mainly

characterized by cardiomyocyte hypertrophy and the dis-proportionate proliferation of collagen fibers, and it evolvesinto progressive left ventricular dysfunction and heart failure[1, 2]. ,e renin-angiotensin-aldosterone system (RAAS), ahormonal signaling cascade with broad activity in the heart,blood vessels, kidney, and adrenal gland, plays a critical rolein maintaining normal blood pressure and electrolyte

HindawiEvidence-Based Complementary and Alternative MedicineVolume 2020, Article ID 9028047, 14 pageshttps://doi.org/10.1155/2020/9028047

balance [3, 4]. Angiotensin converting enzyme inhibitors(ACEIs) are some of the most commonly used drugs in thetreatment of hypertensive LVH. However, excessive aldo-sterone levels caused by long-term treatment with ACEIscan cause myocardial extracellular matrix deposition, fi-brosis, and cardiomyocyte hypertrophy and affect myo-cardial remodeling [5, 6].

Recent studies have shown that the rapid nongenomiceffects of aldosterone are closely related to aldosterone-in-duced myocardial remodeling [7, 8]. Aldosterone binds andactivates the cytoplasmic mineralocorticoid receptor (MR),which acts as a nuclear transcription factor to induce theexpression of specific mRNAs [9, 10]. However, the non-genomic effects of MR have been confirmed to originatefromMR near the plasma membrane, although MRmay notbe directly inserted into the plasma membrane because MRlacks palmitoylation sites similar to those identified astransmembrane domains in other steroid receptors [11, 12].Striatin is an important mediator of the nongenomic effectsof aldosterone. Aldosterone can bind MR attached to theplasma membrane through scaffold proteins, such as striatinand Cav1, and interacts with receptor tyrosine kinases, suchas the epidermal growth factor receptor (EGFR), platelet-derived growth factor receptor, insulin-like growth factor 1receptor, angiotensin II receptor 1, and G1 protein-coupledreceptors, to mediate nongenomic effects through MR[13, 14].

Zi Shen Huo Luo Formula (ZSHLF) is an empiricalprescription for the treatment of hypertensive LVH thatcontains 6 traditional Chinese medicine compounds.Previous studies suggested that combined treatment withZSHLF and the ACEI perindopril reduced the left ven-tricular mass index, reversed left ventricular remodeling[15], corrected the imbalance between type I collagensynthesis and degradation, inhibited cardiac hypertrophyand cardiac fibrosis in spontaneously hypertensive rats(SHRs), and effectively regulated the process of LVH.However, the molecular mechanism of ZSHLF in thetreatment of hypertensive LVH is still unclear. In thisstudy, we observed the nongenomic effects of aldosteroneon cardiomyocyte hypertrophy, cardiac fibroblast pro-liferation, and collagen synthesis. We furthermore ob-served the effects of ZSHLF on aldosterone-inducedmyocardial remodeling, the interaction between striatinand MR, and the expression of downstream EGFR/ex-tracellular regulated kinase (ERK) signaling pathwaymolecules. ,e purpose of this study was to investigatewhether ZSHLF affects the nongenomic effects of aldo-sterone and improves myocardial remodeling by regu-lating the striatin-mediated MR/EGFR/ERK signalingpathway.

2. Materials and Methods

2.1. Preparation of ZSHLF-Containing Serum. All studieswere carried out following the guidelines of the NationalResearch Council of China and approved by the AnimalEthics Committee of Shandong University of TraditionalChinese Medicine. ,e herbal components of ZSHLF were

purchased from the Shandong Hospital of TraditionalChinese Medicine (Jinan, China). ,e following 6 crudedrug materials fixed at a ratio of 20 :15 :12 :12 : 20 : 3 wereboiled twice in an 8-fold volume of ddH2O for 1 h per wash:Scrophularia ningpoensis Hemsl., Achyranthes bidentata Bl.,the dried rhizome of Coptis chinensis Franch., the dried rootcortex of Paeonia suffruticosa Andr., the dried aerial part ofLeonurus japonicus Houtt., and the dried bark of Cinna-momum cassia Presl., respectively. Twenty Sprague-Dawley(SD) rats (half male and half female, weighing 200± 20 g)were purchased from the Beijing Weitong Lihua Experi-mental Animal Center (Beijing, China; license No. SCXK(Jing) 2012-0001). ,e rats were randomly divided into twogroups of 10 rats each, with one group receiving ZSHLF at adose of 8.2 g/kg by intragastric administration twice a day for5 days and the other group receiving the same amount ofnormal saline. ,e rats were starved for 12 h before the lastadministration of ZSHLF or saline and then given the dosefor one day. One hour after the last drug administration,blood was collected, and then, the serum was separated,inactivated at 56°C, and filtered with a 0.22 μm membrane.,e serum was kept frozen at −80°C until use.

2.2. Identification of the Main Chemical Constituents ofZSHLF-Containing Serum. ,e main chemical componentswere identified using an UltiMate 3000 RS ultraperformanceliquid chromatography (UPLC) system coupled to aQ-Exactive mass spectrometer (,ermo Fisher Scientific,MA, USA). Each serum sample (200 μl) was mixed well with1ml of methanol: water (8 : 2, v : v), vortexed, and centri-fuged for 10min at 20000×g and 4°C. ,e supernatant wasfiltered through a 0.22 μm membrane, and the filtrate wasanalyzed with a computer. ,e analytes were separated on aWelch XB-C18 UPLC column (250mm× 4.6mm, 5 um) at35°C under the following conditions: aqueous phase, 0.1%formic acid aqueous solution; organic phase, 0.1% formicacid in acetonitrile; and flow rate, 1.2ml/min. ,e massspectrometer parameters were set as follows: ion source,electrospray ionization (ESI); scanning mode, positive andnegative ion switching; resolution, 70000 (full mass) and17500 (dd-MS2); scanning range, 100.0–1500.0m/z; sprayvoltage, 3.8 kV (positive); capillary temperature, 300°C;collision gas, high purity argon; sheath gas, nitrogen, 40Arb;auxiliary gas (auxiliary gas heater temperature), nitrogen,350°C; and data acquisition time, 30.0min.

2.3. Cell Culture and Treatment. H9c2 (2-1) rat car-diomyocytes were purchased from the Shanghai Cell Bank ofthe Chinese Academy of Sciences (Shanghai, China); ratcardiac fibroblasts were purchased from Wuhan PunuosaiLife Technology (Wuhan, China). Cardiomyocytes andcardiac fibroblasts were cultured in Dulbecco’s modifiedEagle’s medium (DMEM) (Invitrogen, CA, USA) containing10% fetal bovine serum (GIBCO, MD, USA), 1% penicillin,and streptomycin mixture in a 5% CO2 incubator at 37°C. Toobserve the effect of aldosterone, we incubated car-diomyocytes with 10−9mol/l aldosterone and cardiac fi-broblasts with 10−7mol/l aldosterone. To observe the effect

2 Evidence-Based Complementary and Alternative Medicine

of ZSHLF, we pretreated the two cell types with ZSHLF-containing serum for 2 h and then incubated the cells withaldosterone at the above concentrations.

2.4. Observation and Measurement of the CardiomyocyteSurface Area. Cardiomyocytes were collected and treatedwith 4% paraformaldehyde, 0.5% Triton X-100, and 3%methanol hydrogen peroxide at room temperature for20min, and then, they were treated with a sealing solutioncontaining goat serum at room temperature for 20min. ,ecells were incubated with anti-α-actin primary antibody (1 :60, Abcam, UK) overnight at 4°C and then incubated withfluorescein isothiocyanate- (FITC-) labeled secondary an-tibody (Beijing Solaibao Technology, China) for 20min at37°C. Finally, the cells were stained with 4′,6-diamidino-2-phenylindole (DAPI) (1 :1000, Beijing Solaibao Technology,China) for 10min and observed and photographed with anIX71 fluorescence microscope (Olympus Corporation, Ja-pan). ,e surface area of all cells in the visual field wasmeasured by ImageJ software, and the average values weretaken as the results.

2.5. Detection of the Proliferation of Cardiac Fibroblasts.Cell Counting Kit-8 (CCK-8) reagent (SAB, MD, USA) andserum-free basic medium were mixed at a 1 :10 volume ratioand added to the cardiac fibroblasts, which were incubatedin a 5% CO2 incubator at 37°C for 1 h. ,e absorbance at awavelength of 450 nm was determined with an MK3microplate reader (,ermo Fisher Scientific).

2.6. Detection of Collagen Synthesis in Cardiac Fibroblasts.,e hydroxyproline content in the cell culture medium wasused to evaluate collagen production in the cardiac fibro-blasts [16], and it was measured according to the instructionsof a hydroxyproline kit (Nanjing Construction Bioengi-neering Research Institute, China). A UV-5200 ultravioletspectrophotometer (Shanghai Yuanxi Instrument, China)was used to determine the absorbance at a wavelength of550 nm. ,e hydroxyproline content was calculated andused to detect collagen synthesis.

2.7. Immunoprecipitation (IP). Cardiomyocytes and cardiacfibroblasts were lysed with NETcell buffer (containing 1mMPMSF and 1mM protease inhibitor) by ultrasound on ice for5min. ,e lysate was centrifuged at 12000×g for 20min at4°C. ,e supernatant combined with lysis buffer (20mMTris-HCl, pH 7.5, 150mM NaCl, 1% Triton X-100, 1mMEDTA, and protease inhibitor) was centrifuged at 13000 rpmfor 10min at 4°C.,e protein content of the supernatant wasquantified by the bicinchoninic acid (BCA) method (4–8 μg/μl). Proteins (500 μg) were incubated with primary antibody(antistriatin, anti-MR, 1 : 60, Abcam, UK) for precipitationovernight at 4°C and then incubated with protein G-agarosebeads overnight at 4°C. ,e mixture was centrifuged at2500 rpm for 1min at 4°C, and the sediment was supple-mented with protein sample buffer, boiled for 5min, and

centrifuged at 2500 rpm for 1min. Proteins in the super-natant were detected by Western blotting.

2.8. Small Interfering RNA (siRNA) Transfection. ,reesiRNAs against striatin (siRNA STRs) were synthesized byShanghai Jima Pharmaceutical Technology (Shanghai,China). ,e si-striatin-1 target sequence was GCA-GACTCATTAGCTTATG, the si-striatin-2 target sequencewas GCGCTGACTGTATTTAATG, and the si-striatin-3target sequence was GCAGCGAATTCTCACGTTA. Non-target siRNA was used as a negative control (NC) sequence.Cardiomyocytes and cardiac fibroblasts were transfectedwith the three siRNAs against striatin according to a pre-viously described method [17], and cells were collected forPCR to identify the interference efficiency (SupplementaryFigure 1).

2.9. Quantitative Real-Time PCR (qRT-PCR). Primer se-quences were designed based on GenBank sequences withPrimer Premier 5.0 software (Table 1). ,e PCR primerswere synthesized by Shanghai Jima Pharmaceutical Tech-nology (Shanghai, China). mRNA was extracted usingTRIzol (,ermo Scientific), and RNA was reverse tran-scribed with a TIANScript RT Kit (Tiangen Biotechnology,Beijing, China) according to the manufacturer’s instruc-tions. ,e reaction system was prepared with a SYBR GreenPCR kit (Tiangen Biotechnology), and mRNA amplificationand quantification were performed using an ABI 7500fluorescence quantitative PCR instrument (Applied Bio-systems, Foster, USA) according to the instructions. ,ereaction procedure was as follows: preincubation at 95°C for5min, amplification at 95°C for 10 s and 60°C for 60 s for 40cycles, and separation at 95°C for 10 s and 60°C for 60 s.Target gene expression was normalized to the expression ofglyceraldehyde-3-phosphate dehydrogenase (GAPDH), andrelative expression was determined using the 2−ΔΔct method.

2.10. Western Blotting. Cardiomyocytes and cardiac fibro-blasts were collected and lysed to extract the proteins. ,emembrane proteins were extracted with a cell membraneprotein extraction kit (Biyuntian Institute of Biotechnology,Shanghai, China) following the manufacturer’s instructions.,e protein concentration was detected using a BCA assay.,e proteins were resolved by 10% sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) andtransferred to polyvinylidene fluoride (PVDF) membranes.,en, the PVDF membranes were blocked with 5% skimmilk for 1 h and incubated with antibodies against MR (1 :60, Abcam), EGFR (1 :1200, Abcam), pEGFR (1 : 800,Abcam), ERK1/2 (1 :1000, Abcam), pERK1/2 (1 : 600, CellSignaling Technology, Inc., USA), Na-K ATPase (1 :1000,Abcam), and β-actin (1 :1000, Abcam) overnight at 4°C.After the membranes were washed with TBST, they wereincubated with secondary antibodies for 4 h at room tem-perature. Finally, immunoreactivity was visualized with anECL kit (Millipore, MA, USA). ,e relative expression levels

Evidence-Based Complementary and Alternative Medicine 3

(ratio compared to Na-K ATPase or β-actin expression) ofthe target proteins were quantified by ImageJ software.

2.11. Statistical Analysis. ,e results are presented as themean± standard deviation of at least three independentexperiments. Data were analyzed by one-way ANOVA formultiple comparisons with SPSS 22.0 software. Differenceswere considered statistically significant at P< 0.05.

3. Results

3.1. Analysis of the Main Chemical Components of ZSHLF-Containing Serum. As shown in Figure 1, UPLC was used toanalyze the ZSHLF-containing serum, andmass spectrometrywas used to identify the five main chemical components:betaine, caffeic acid, DL-stachydrine, catechin, and coumarin.

3.2. Effects of Aldosterone Induction Time on CardiomyocyteSurface Area and the Proliferation and Hydroxyproline Se-cretion of Cardiac Fibroblasts. As shown in Figure 2(a),α-actinin immunofluorescence staining revealed adherentcardiomyocytes with a variety of shapes, including fusiformand polygonal cardiomyocytes. ,e cardiomyocyte surfacearea after induction with aldosterone for 15min, 30min, and2 h was significantly greater than that of uninduced car-diomyocytes (P< 0.05), with the most obvious increaseobserved in the group treated for 30min (Figure 2(b)).

In the CCK-8 assay, which was used to detect theproliferation of cardiac fibroblasts, the optical density (OD)values of the groups induced with aldosterone for 15min,30min, and 2 h were significantly higher than that of theuninduced group (P< 0.05). ,e OD value of the groupinduced with aldosterone for 30min was significantly higherthan that of the other groups (P< 0.05) (Figure 2(c)).

Detection of the hydroxyproline content in the cardiacfibroblast culture supernatant revealed a significant increasein hydroxyproline secretion after 5min of aldosterone in-duction (P< 0.05), and the hydroxyproline concentration ofeach group induced with aldosterone was significantlyhigher than that of the uninduced group (P< 0.05). ,ehydroxyproline content was significantly higher after 30minof induction than after 15min and 1 h of induction and after2 h of induction than after 5min, 15min, and 1 h of in-duction (P< 0.05) (Figure 2(d)).

,ese results suggested that 30min of aldosteronetreatment induced substantial cardiomyocyte hypertrophyand increased the proliferation and hydroxyproline secre-tion of cardiac fibroblasts. ,ese short-term effects areconsistent with nongenomic effects. Because this studyaimed to explore the nongenomic effects of aldosterone, an

aldosterone induction time of 30min was used in the fol-lowing experiments.

3.3. Effects of ZSHLF on the Cardiomyocyte Surface Area andthe Proliferation and Hydroxyproline Secretion of CardiacFibroblasts. As shown in Figure 3, the cardiomyocyte sur-face area, OD value, and hydroxyproline content of thesupernatant of cardiac fibroblasts in the ZSHLF group weresignificantly lower than those in the aldosterone group(P< 0.05), suggesting that ZSHLF effectively attenuated thealdosterone-induced hypertrophy of rat cardiomyocytes andinhibited the proliferation and collagen synthesis of cardiacfibroblasts.

3.4. Effect of ZSHLF on Striatin mRNA Expression and In-teraction between Striatin and the MR Protein in Car-diomyocytes and Cardiac Fibroblasts. As shown inFigure 4(a), qRT-PCR was used to determine the mRNAexpression level of striatin in cardiomyocytes and cardiacfibroblasts. In both cell types, the striatin mRNA expressionlevel in the aldosterone group was significantly higher thanthat in the blank group (P< 0.05), while that in the ZSHLFgroupwas lower than that in the aldosterone group (P< 0.05).

To detect the interaction between striatin and MR, IPexperiments were carried out with antibodies against striatinand MR. As shown in Figures 4(b)–4(e), the MR protein wascoprecipitated with antistriatin antibody in the car-diomyocytes and cardiac fibroblasts, and the expression ofcoprecipitated MR in both cell types pretreated with ZSHLFwas significantly lower than that in cells treated with al-dosterone alone (P< 0.05). In addition, anti-MR antibodycoprecipitated with striatin and the expression level ofstriatin in both cell types pretreated with ZSHLF was lowerthan that in cells treated with aldosterone alone (P< 0.05).

3.5. Functional identification of the Role of Striatin in theNongenomic Effects of Aldosterone on Cardiomyocytes andCardiac Fibroblasts by siRNA-Mediated Knockdown. Tofurther verify the role of striatin in the nongenomic effects ofaldosterone, we designed and screened three siRNAs againststriatin (siRNA STRs), as described in Supplementary Fig-ure 1 and transfected cardiomyocytes and cardiac fibroblastswith the most effective siRNA. ,e cardiomyocyte surfacearea in the siRNA STR group was significantly lower thanthat in the control group (P< 0.05), although the car-diomyocyte surface area did not significantly differ betweenthe siRNA STR group and the siRNA+ZSHLF group(P> 0.05) (Figures 5(a) and 5(b)). Among cardiac fibro-blasts, the OD value in the siRNA STR group was lower thanthat in the control group, while the OD value in the siR-NA+ZSHLF group was lower than that in the siRNA STRgroup (P< 0.05) (Figure 5(c)). ,e hydroxyproline contentin the siRNA+ZSHLF group was not significantly differentfrom that in the control group (P> 0.05), while thehydroxyproline content in the siRNA+ZSHLF group waslower than that in the siRNA STR group (P< 0.05)(Figure 5(d)).

Table 1: Primer sequences for qRT-PCR assay.

Primer name Primer sequence (5′–3′) Product lengthStriatin-F GAGACAACGAGTCCAGAAG 222Striatin-R GCAAGGAGGGAAGTTCATCGADPH-F GTCGGTGTGAACGGATTTG 181GADPH-R TCCCATTCTCAGCCTTGAC

4 Evidence-Based Complementary and Alternative Medicine

3.6. Effect of ZSHLF on Membrane-Localized MR ProteinExpression Levels in Cardiomyocytes and Cardiac Fibroblasts.After the extraction of membrane proteins from car-diomyocytes and cardiac fibroblasts, Western blotting wasused to study the role of striatin in regulating theMR proteinexpression level and the regulatory mechanism of ZSHLF(Figure 6). In both cell types, the MR expression level waslower in the ZSHLF group and the siRNA STR group than inthe control group (P< 0.05), but the MR expression was notsignificantly different between the siRNA STR group and thesiRNA+ZSHLF group (P> 0.05).

3.7. Effect of ZSHLFonEGFR,ERK, pEGFR, and pERKProteinExpression in Cardiomyocytes and Cardiac Fibroblasts.,eWestern blot analysis of cardiomyocytes did not identifysignificant differences in EGFR protein expression among thegroups (P> 0.05). ,e pEGFR and pERK expression levelswere lower in the ZSHLF group and siRNA STR group thanthe control group, the ERK expression levels were signifi-cantly lower in the ZSHLF group than the control group(P< 0.05), and the pEGFR, ERK, or pERK expression levelsdid not significantly differ between the siRNA STR group andsiRNA+ZSHLF group (P> 0.05) (Figures 7(a) and 7(c)).

N+OH

O

70605040302010

0Inte

nsity

(cou

nts)

(10^

6) 605040302010

0Inte

nsity

(cou

nts)

(10^

6)

0 5 10 15 20 25 30RT (min)

118.08632(M + H) + 1

140.06801(M + Na) + 1

156.04189(M + K) + 1

120.06558119.08967

129.06580

132.06544130.04982

147.07623

143.10654

148.06018

154.98997

120 130 140 150 160m (z)

2.291

Betaine

(a)

5040302010

0Inte

nsity

(cou

nts)

(10^

6)

Inte

nsity

(cou

nts)

(10^

6)

0 5 10 15 20 25RT (min) m (z)

180 181 182 183 184 185

4

3

2

1

0

181.04918(M + H) + 1

181.03818180.02797

182.02324181.15849182.05263

182.11700 183.10132181.98686

184.99504 184.88861184.98524184.10466

HO

HOOH

O18.422

15.432

Caffeic acid

(b)

N+

HO

O

Inte

nsity

(cou

nts)

(10^

6)

Inte

nsity

(cou

nts)

(10^

6)

0 5 10 15 20 25

RT (min) m (z)

25

20

15

10

5

0

3025201510

50

144.10173(M + H) + 1

144.06566143.10661

145.04941 146.10593145.98969

147.11259147.07620

148.09654148.06024145.10507

143 144 145 146 147 148

1.533

DL-stachydrine

(c)

HO O

OHOH

OH

Inte

nsity

(cou

nts)

(10^

6)

Inte

nsity

(cou

nts)

(10^

6)

0 5 10 15 20 25 30RT (min) m (z)

289.07114(M – H) – 1

289.16486288.05884 290.07446

289.93600

291.07635 292.08188291.01392 293.10193

293.04825293.08691

288 289 290 291 292 293

4

3

2

1

0

6.761

Catechin

6543210

(d)

O O

Inte

nsity

(cou

nts)

(10^

6)

Inte

nsity

(cou

nts)

(10^

6)

0 5 10 15 20 25RT (min) m (z)

4

3

2

1

0

147.04408(M + H) + 1

147.07642146.06007

148.04736148.03935

147.06531 149.05978

149.02341

148.97711150.03987

150.02670151.07545151.11183

146 147 148 149 150 151

2.505

Coumarin

3.02.52.01.51.00.50.0

(e)

Figure 1: UPLC chromatograms and mass spectrometry results for ZSHLF-containing serum. Betaine (a), caffeic acid (b), DL-stachydrine(c), catechin (d), and coumarin (e) were identified as the main chemical components of ZSHLF.

Evidence-Based Complementary and Alternative Medicine 5

,e Western blot analysis of cardiac fibroblasts revealedthat the EGFR and ERK expression levels did not signifi-cantly differ among the groups (P> 0.05). ,e pEGFR andpERK expression levels were lower in the ZSHLF group andsiRNA STR group than in the control group (P< 0.05), thepERK expression level was lower in the siRNA+ZSHLFgroup than the siRNA STR group (P< 0.05), and the pEGFRexpression did not obviously differ between the siR-NA+ZSHLF group and the siRNA STR group (P> 0.05)(Figures 7(b) and 7(d)).

4. Discussion

Long-standing hypertension leads to LVH, a recognized riskfactor for heart failure, myocardial infarction, arrhythmia,sudden cardiac death, and stroke [18, 19]. ,e reversal of

LVH as antihypertensive therapy can improve the prognosisand reduce the morbidity and mortality of cardiovasculardisease [20]. ZSHLF is an empirical prescription for theclinical treatment of hypertension. Animal and clinicalstudies have shown that the combination of ZSHLF andperindopril can steadily reduce blood pressure and inhibitthe pathological changes in myocardial hypertrophy andmyocardial fibrosis, which effectively control the LVHprocess [15]. In this study, ZSHLF significantly inhibitedaldosterone-induced cardiomyocyte hypertrophy and pro-liferation and collagen synthesis in cardiac fibroblasts, andthe associated mechanism may be closely related to thenongenomic effect of aldosterone, which affects thedownstream EGFR/ERK signaling pathway by regulating theinteraction between striatin andMR.We identified five mainchemical components of ZSHLF-containing serum as

0min

30min

5min

60min

15min

120min

20μm 20μm 20μm

20μm 20μm 20μm

(a)

Surfa

ce ar

ea o

f car

dioy

ocyt

es(c

ontr

ol g

roup

)

0.0

0.5

1.0

1.5

2.0

2.5

5min

15m

in

30m

in

60m

in

0min

120m

in

5min15min

0min 30min60min120min

∗

∗

∗

(b)

0.000.42

0.47

0.52

OD

(CCK

-8)

5min

15m

in

30m

in

60m

in

0min

120m

in

5min15min

0min 30min60min120min

∗

∗

∗

(c)

0

1

2

3

4

Hyd

roxy

prol

ine (μg

/ml)

5min

15m

in

30m

in

60m

in

0min

120m

in

5min15min

0min 30min60min120min

∗

∗

∗

∗

∗

(d)

Figure 2: Effects of aldosterone induction time on the cardiomyocyte surface area and proliferation and hydroxyproline secretion of cardiacfibroblasts. Cardiomyocytes were treated with 10−9mol/l aldosterone, and cardiac fibroblasts were treated with 10−7mol/l aldosterone for 0,5, 15, 30, 60 or 120min. (a) Cells were immunofluorescence stained with DAPI (blue), and cardiomyocytes were detected by immu-nofluorescence staining using FITC-conjugated α-actinin (green). (b) Cardiomyocyte surface area (relative to that in the untreated group)was determined by α-actinin immunofluorescence staining. (c) ,e OD values of cardiac fibroblasts were detected by a CCK-8 assay. (d)Hydroxyproline content in the cardiac fibroblast culture supernatant. ∗P< 0.05 vs. the untreated group; ◇P< 0.05 vs. the group treated for5min; ◆P< 0.05 vs. the group treated for 15min; ☆P< 0.05 vs. the group treated for 30min; and ★P< 0.05 vs. the group treated for 1 h.

6 Evidence-Based Complementary and Alternative Medicine

betaine, caffeic acid, DL-stachydrine, catechin, and cou-marin; however, the components associated with the im-provement of aldosterone-induced myocardial remodelingneed to be further identified.

,e RAAS is an important link between the regulation ofblood pressure and electrolyte balance in the human body[21]. A sustained increase in aldosterone after chronic ac-tivation of the RAAS can lead to hypertension, thrombosis,and atherosclerosis and promote inflammation [22, 23]. Inthe classical pathway, aldosterone binds cytoplasmic MR,which is transferred to the nucleus, where it dimerizes andexerts genomic effects as a transcription factor. In recentyears, a great deal of evidence has demonstrated that al-dosterone also triggers a very rapid response in the typicalepithelial target organs of MR and nonepithelial tissues[17, 24]. As this reaction is often completed in a few minutesand does not require transcription or translation, it is calleda nongenomic effect [25]. In this study, cardiomyocytes andcardiac fibroblasts were treated with aldosterone. Aldoste-rone induced cardiomyocyte hypertrophy, fibroblast

proliferation, and hydroxyproline secretion, and these ef-fects were initially observed at 5min and peaked at 30min.,e short-term nature of these effects is consistent with thecharacteristics of a nongenomic action. However, in all threegroups, the effect of aldosterone induction at 2 h wasstronger than that at 1 h, suggesting a simultaneous genomiceffect. ,e main purpose of this study was to explore thenongenomic effect of ZSHLF; therefore, an induction time of30min was chosen to further study the regulatory effect ofZSHLF. ZSHLF had an obvious inhibitory effect on thealdosterone-induced increase in cardiomyocyte surface areaand the proliferation and hydroxyproline secretion of car-diac fibroblasts, suggesting that ZSHLF can improve myo-cardial remodeling by regulating the nongenomic effect ofaldosterone.

,e striatin protein, which is widespread in the heart,kidney, and blood vessels, is a multidomain scaffold proteinthat can interact with and activate different signal trans-duction molecules [26], participate in membrane-relatedsignal transduction, and regulate the rapid nongenomic and

Blank ALD ZSHLF20μm 20μm 20μm

(a)

Surfa

ce ar

ea o

f car

diom

yocy

tes

(bla

nk g

roup

)

0.0

1.0

2.0

ALD

gro

up

ZSH

LF g

roup

Blan

k gr

oup

ALD groupZSHLF group

Blank group

(b)

0.000.44

0.48

0.52

0.56

OD

(CCK

-8)

ALD

gro

up

ZSH

LF g

roup

Blan

k gr

oup

ALD groupZSHLF group

Blank group

(c)

0

1

2

3

4

Hyd

roxy

prol

ine (μg

/ml)

ALD

gro

up

ZSH

LF g

roup

Blan

k gr

oup

ALD groupZSHLF group

Blank group

(d)

Figure 3: Effects of ZSHLF on the cardiomyocyte surface area and the proliferation and hydroxyproline secretion of cardiac fibroblasts.Cardiomyocytes and cardiac fibroblasts in the control group were pretreated with blank serum. In the aldosterone (ALD) group, car-diomyocytes and cardiac fibroblasts were pretreated with blank serum, and the cardiomyocytes were then incubated with 10−9mol/laldosterone for 30min, and the cardiac fibroblasts were incubated with 10−7mol/l aldosterone for 30min. In the ZSHLF group, car-diomyocytes and cardiac fibroblasts were pretreated with 10% ZSHLF-containing serum for 2 h and then incubated with aldosterone, asdescribed for the ALD group. (a) Cells subjected to immunofluorescent assays using DAPI (blue) and FITC (green). (b) Cardiomyocytesurface area (relative to that in the control group) was determined by α-actinin immunofluorescence staining. (c) OD values of cardiacfibroblasts were detected by a CCK-8 assay. (d) Hydroxyproline content in the culture supernatant of cardiac fibroblasts. △P< 0.05 vs. theblank group; and ▲P< 0.05 vs. the ALD group.

Evidence-Based Complementary and Alternative Medicine 7

genomic effects of steroids. Striatin is an important inter-mediary of the nongenomic effect of aldosterone [27], andevidence suggests that the cytosolic MR protein is partiallytransported near the cell membrane after it binds striatin[28, 29]. Formation of a complex between striatin andMR inendothelial cells and mouse heart tissues was shown to berelated to the nongenomic effect of aldosterone [30, 31]. Wespeculated that ZSHLF inhibits aldosterone-induced myo-cardial remodeling by regulating striatin gene expression

and interacting with MR. ,e qRT-PCR assay showed thatin both cardiomyocytes and cardiac fibroblasts, striatinmRNA expression was increased in response to aldosteroneinduction; however, ZSHLF pretreatment restrained thischange to some extent. ,e IP assay confirmed that striatinand MR interacted in rat cardiomyocytes and cardiac fi-broblasts and MR/striatin complex formation was weak-ened by ZSHLF pretreatment. To further verify the role ofstriatin in the nongenomic effects of aldosterone on

mRN

A (2

−∆∆c

t )

Cardiomyocytes Cardiac fibroblasts0

2

4

6

8

Blank groupALD groupZSHLF group

(a)

IP: striatin

Striatin

MR

IP: MRMR

Striatin

Celllysate IgG Blank

groupALDgroup

ZSHLFgroup

(b)

IP: striatin IP: MR

Blankgroup

ALDgroup

ZSHLFgroup

Blankgroup

ALDgroup

ZSHLFgroup

0

50

100

150

MR

(% o

f bla

nk g

roup

)

0

50

100

150

Stria

tin (%

of b

lank

gro

up)

(c)

Striatin

MR

IP: striatin

IP: MRMR

Striatin

Celllysate IgG Blank

groupALDgroup

ZSHLFgroup

(d)

IP: striatin IP: MR

Blankgroup

ALDgroup

ZSHLFgroup

Blankgroup

ALDgroup

ZSHLFgroup

0

50

100

150

Stria

tin (%

of b

lank

gro

up)

0

50

100

150

MR

(% o

f bla

nk g

roup

)

(e)

Figure 4: Effect of ZSHLF on striatin mRNA expression and interaction between the striatin and MR proteins in cardiomyocytes andcardiac fibroblasts. In the blank group, the cells were incubated with blank serum-containing medium. In the ALD group, cardiomyocytesand cardiac fibroblasts were pretreated with blank serum, the cardiomyocytes were then incubated with 10−9mol/l aldosterone for 30min,and the cardiac fibroblasts were incubated with 10−7mol/l aldosterone for 30min. In the ZSHLF group, cardiomyocytes and cardiacfibroblasts were pretreated with 10% ZSHLF-containing serum for 2 h and then incubated with aldosterone as described for the ALD group.(a) mRNA expression levels of striatin in cardiomyocytes and cardiac fibroblasts were detected by qRT-PCR.,e 2−ΔΔct method was used forrelative quantitative analysis of mRNA expression levels. (b) Protein bands containing striatin and MR from cardiomyocytes were detectedby IP. (c) Protein bands containing striatin and MR from the cardiac fibroblasts were detected by IP. (d) Protein expression of MR andstriatin in the cardiomyocytes. (e) Protein expression of MR and striatin in cardiac fibroblasts. △P< 0.05 vs. the blank group. ▲P< 0.05 vs.the ALD group.

8 Evidence-Based Complementary and Alternative Medicine

cardiomyocytes and cardiac fibroblasts, we used siRNA toreduce striatin gene expression. Striatin silencing decreasedthe cardiomyocyte surface area and the proliferation rate ofcardiac fibroblasts but had no significant effect on thehydroxyproline content. ,e suppressive effect of ZSHLFtreatment combined with siRNA transfection on the car-diomyocyte surface area was not significantly differentfrom that of siRNA transfection alone, whereas the effecton the proliferation rate and hydroxyproline content ofcardiac fibroblasts was more pronounced under thecombination of ZSHLF treatment and siRNA transfectionthan with siRNA transfection alone. ,ese observationssuggest that aldosterone induces cardiomyocyte hyper-trophy and cardiac fibroblast proliferation through thestriatin/MR complex, and that ZSHLF inhibits car-diomyocyte hypertrophy and cardiac fibroblast prolifera-tion by reducing the binding of striatin and MR.Furthermore, ZSHLF may also regulate cardiac fibroblastproliferation and collagen synthesis through targets in-dependent of striatin.

EGFR is a member of the receptor tyrosine kinasefamily and plays a key role in regulating cell proliferation,differentiation, and survival. Its interaction with aldoste-rone/MR has been identified as a potential cause of itsnongenomic effect, which likely proceeds through a mo-lecular mechanism that mediates the physiological andpathological effects of aldosterone [32]. MR is transportednear the cell membrane by striatin, where it forms a sig-naling complex with caveolin, src, and EGFR [28, 29],which induces the crosslinking of ERK1/2 and the phos-phoinositide-3-kinase/mechanistic target of rapamycin byactivating EGFR signaling and induces fibroblast prolif-eration, thereby further aggravating fibrosis [13, 33]. Inaddition, in human and mouse endothelial cells, the rapideffects of MR on ERK phosphorylation require striatin, anda decrease in striatin levels leads to a reduction in aldo-sterone/MR-dependent ERK phosphorylation [17]. Basedon the above studies, we examined the role of the MR/EGFR/ERK signaling pathway in the striatin-mediatednongenomic effects of aldosterone and the regulatory effect

Control group 20μm siRNA STR 20μm siRNA + ZSHLF 20μm

(a)

siRNA +ZSHLF

siRNASTR

Controlgroup

0.0

0.5

1.0

1.5

Surfa

ce ar

ea o

f car

diom

yocy

tes

(con

trol

gro

up)

Control groupsiRNA STRsiRNA + ZSHLF

(b)

siRNA +ZSHLF

siRNASTR

Controlgroup

0.00

0.34

0.44

0.54

OD

(CCK

-8)

Control groupsiRNA STRsiRNA + ZSHLF

(c)

siRNA +ZSHLF

siRNASTR

Controlgroup

0

1

2

3

4

Hyd

roxy

prol

ine (μg

/ml)

Control groupsiRNA STRsiRNA + ZSHLF

(d)

Figure 5: Functional identification of the role of striatin in the nongenomic effects of aldosterone on cardiomyocytes and cardiac fibroblastsby siRNA. In the control group, cardiomyocytes and cardiac fibroblasts were pretreated with blank serum, the cardiomyocytes were thenincubated with 10−9mol/l aldosterone for 30min, and the cardiac fibroblasts were incubated with 10−7mol/l aldosterone for 30min. Cells inthe siRNA STR group were transfected with siRNA STR, pretreated with blank serum for 2 h, and incubated with aldosterone. Cells in thesiRNA+ZSHLF group were transfected with siRNA STR, pretreated with ZSHLF-containing serum for 2 h, and then incubated withaldosterone. (a) Cells subjected to immunofluorescent assays using DAPI (blue) and FITC (green). (b) Cardiomyocyte surface area (relativeto the control group) determined by α-actinin immunofluorescence staining. (c) OD values of cardiac fibroblasts detected by the CCK-8assay. (d) Hydroxyproline content in the cardiac fibroblast culture supernatant. ▲P< 0.05 vs. the control group; ○P< 0.05 vs. the siRNA STRgroup.

Evidence-Based Complementary and Alternative Medicine 9

EGFR

pEGFR

ERK

pERK

β-Actin

ZSHLFgroup

siRNASTR

siRNA +ZSHLF

Controlgroup

(a)

EGFR

pEGFR

ERK

pERK

β-Actin

ZSHLFgroup

siRNASTR

siRNA +ZSHLF

Controlgroup

(b)

Figure 7: Continued.

ZSHLFgroup

siRNASTR

siRNA +ZSHLF

Controlgroup

MR

Na-KATPase

(a)

ZSHLFgroup

siRNASTR

siRNA +ZSHLF

Controlgroup

MR

Na-KATPase

(b)

MR

prot

ein

expr

essio

n(N

a-K

ATP

ase)

Cardiac fibroblastsCardiomyocytes0.0

0.5

1.0

Control groupZSHLF group

siRNA STRsiRNA + ZSHLF

(c)

Figure 6: Effect of ZSHLF on membrane-localized MR protein expression levels in cardiomyocytes and cardiac fibroblasts. MR expressionlevels in cardiomyocytes (a) and cardiac fibroblasts (b) were detected byWestern blotting, and the ratio relative to Na-K ATPase expressionwas used as the relative target protein content (c). ▲P< 0.05 vs. the control group; ○P< 0.05 vs. the siRNA STR group.

10 Evidence-Based Complementary and Alternative Medicine

of ZSHLF in cardiomyocytes and cardiac fibroblasts. Afterstriatin silencing, the protein expression levels of mem-brane-localized MR and the levels of phosphorylated EGFRand ERK1/2 in cardiomyocytes and cardiac fibroblastsdecreased. ,e ZSHLF treatment of striatin-silenced cellsdid not significantly alter the protein expression level ofMR, the level of EGFR phosphorylation in cardiomyocytesor cardiac fibroblasts, or the levels of ERK1/2 phosphor-ylation in cardiomyocytes, whereas it did reduce ERK1/2phosphorylation in cardiac fibroblasts. ,erefore, we

speculate that striatin induces the phosphorylation ofEGFR and ERK by mediating the membrane localization ofMR, which results in aldosterone-induced cardiomyocytehypertrophy and cardiac fibroblast proliferation (Figure 8).ZSHLF inhibits cardiomyocyte hypertrophy and cardiacfibroblast proliferation through the MR/EGFR/ERK sig-naling pathway mediated by striatin. In addition, ZSHLFmay inhibit the proliferation and collagen synthesis ofcardiac fibroblasts by regulating the ERK signaling pathwaythrough other targets independent of striatin.

EGFR ERK

Prot

ein

expr

essio

n(β

-Act

in)

0.0

0.6

1.2

1.8

pEGFR pERK

Control groupZSHLF group

siRNA STRsiRNA + ZSHLF

(c)

Prot

ein

expr

essio

n(β

-Act

in)

EGFR ERKpEGFR pERK0.0

0.6

1.2

1.8

Control groupZSHLF group

siRNA STRsiRNA + ZSHLF

(d)

Figure 7: Effects of ZSHLF on the protein expression levels of EGFR, ERK, pEGFR, and pERK in cardiomyocytes and cardiac fibroblasts.Expression levels of EGFR, ERK, and phosphorylated proteins in cardiomyocytes (a) and cardiac fibroblasts (b) were detected by Westernblotting, and the ratio relative to β-actin expression was used as the relative target protein content (c). ▲P< 0.05 vs. the control group;○P< 0.05 vs. the siRNA STR group (d).

Evidence-Based Complementary and Alternative Medicine 11

5. Conclusion

In summary, ZSHLF-containing serum effectively preventedaldosterone-induced cardiomyocyte hypertrophy, cardiacfibroblast proliferation, and collagen synthesis; inhibited thedownstream EGFR/ERK signaling pathway by blockingstriatin-mediated localization of MR to the membrane; andaltered the nongenomic effects of aldosterone, and theseeffects may all represent important molecular mechanismsby which ZSHLF improves hypertensive myocardialremodeling. Notably, ZSHLF also inhibited the proliferationand collagen synthesis of cardiac fibroblasts by regulatingother targets independent of striatin. ,ese additional tar-gets and signaling mechanisms are worthy of further studyand discussion. ,e identification of the main componentsof ZSHLF associated with the improvement of aldosterone-induced myocardial remodeling is also one of the directionsof our next work.

Abbreviations

ZSHLF: Zi Shen Huo Luo FormulaMR: Mineralocorticoid receptorGFR: Epidermal growth factor receptorERK: Extracellular signal-regulated kinaseLVH: Left ventricular hypertrophysiRNA: Small interfering RNARAAS: Renin-angiotensin-aldosterone systemACEI: Angiotensin converting enzyme inhibitor

qRT-PCR: Quantitative real-time PCRIP: ImmunoprecipitationUPLC: Ultraperformance liquid chromatographyMS: Mass spectrometer.

Data Availability

,e data used to support the findings of this study areavailable from the corresponding author upon request.

Conflicts of Interest

,e authors declare that they have no conflicts of interest.

Authors’ Contributions

Weike Feng and Yue Zhao contributed equally to this work.All coauthors participated and agreed with the content andconclusions.

Acknowledgments

,e authors would like to thank the National Natural Sci-ence Foundation of China (Grant no. 81503539) for theirsupport in this study.

Supplementary Materials

To verify the role of striatin in the nongenomic effects ofaldosterone, we designed and screened three siRNAs against

Aldo

Striatin

MR

Cell membrane

Nucleus

ERK1/2

Cell proliferationfibrosis

Genomic effects

Nongenomic effectsP P

EGFR

AldoAldo

Aldo

MR

Aldo

MR

Cav-

1Aldo

MRStriatin

Figure 8: Hypothetical model describing the striatin-mediated signaling pathway. After binding striatin, MR is translocated to andassociated with the cell membrane to form a signaling complex. On aldosterone stimulation, the EGFR/ERK signaling pathway is activated,resulting in cell proliferation and fibrosis.

12 Evidence-Based Complementary and Alternative Medicine

striatin. Because the purpose of this experiment is to screenthe most effective siRNA, which could be used in subsequentexperiments to knockdown striatin expression. Supple-mental Figure 1: identification and screening of striatinsiRNA interference efficiency. (Supplementary Materials)

References

[1] C. Cuspidi, C. Sala, F. Negri, G. Mancia, and A. Morganti,“Prevalence of left-ventricular hypertrophy in hypertension:an updated review of echocardiographic studies,” Journal ofHuman Hypertension, vol. 26, no. 6, pp. 343–349, 2012.

[2] C. Cuspidi, M. Rescaldani, C. Sala, F. Negri, G. Grassi, andG. Mancia, “Prevalence of electrocardiographic left ventric-ular hypertrophy in human hypertension: an updated review,”Journal of Hypertension, vol. 30, no. 11, pp. 2066–2073, 2012.

[3] A. Santoro and M. Mandreoli, “Hyperkalemia as a limitingfactor in the use of drugs that block the renin angiotensinaldosterone system (RAAS),” Giornale Italiano di Nefrologia:Organo Ufficiale Della Societa Italiana di Nefrologia, vol. 35,no. 3, 2018.

[4] Y. P. Ng, G. P. Balasubramanian, Y. P. Heng et al., “Reninangiotensin-aldosterone system (RAAS) blockers usageamong type II diabetes mellitus patients-a retrospectivestudy,” Diabetes & Metabolic Syndrome: Clinical Research &Reviews, vol. 12, no. 3, pp. 305–308, 2018.

[5] F. Ivanes, S. Susen, F. Mouquet et al., “Aldosterone, mortality,and acute ischaemic events in coronary artery disease patientsoutside the setting of acute myocardial infarction or heartfailure,” European Heart Journal, vol. 33, no. 2, pp. 191–202,2012.

[6] K. Ikeda, T. Isaka, K. Fujioka, Y. Manome, and K. Tojo,“Suppression of aldosterone synthesis and secretion by Ca2+

channel antagonists,” International Journal of Endocrinology,vol. 2012, Article ID 519467, 6 pages, 2012.

[7] A. W. Norman, M. T. Mizwicki, and D. P. G. Norman,“Steroid-hormone rapid actions, membrane receptors and aconformational ensemble model,” Nature Reviews DrugDiscovery, vol. 3, no. 1, pp. 27–41, 2004.

[8] P. Kolodziejczyk, A. Gromotowicz-Poplawska, M. Aleksiejczuk,E. Chabielska, P. Tutka, andW.Miltyk, “New sides of aldosteroneaction in cardiovascular system as potential targets for thera-peutic intervention,” Current Drug Targets, vol. 19, no. 16,pp. 1968–1979, 2018.

[9] A. P. Harvey, A. C. Montezano, K. Y. Hood et al., “Vasculardysfunction and fibrosis in stroke-prone spontaneously hy-pertensive rats: the aldosterone-mineralocorticoid receptor-Nox1 axis,” Life Sciences, vol. 179, pp. 110–119, 2017.

[10] A. Nguyen Dinh Cat, G. E. Callera, M. Friederich-Perssonet al., “Vascular dysfunction in obese diabetic db/db miceinvolves the interplay between aldosterone/mineralocorticoidreceptor and rho kinase signaling,” Scientific Reports, vol. 8,no. 1, Article ID 2952, 2018.

[11] G. E. Callera, A. Yogi, A. M. Briones et al., “Vascularproinflammatory responses by aldosterone are mediated viac-Src trafficking to cholesterol-rich microdomains: role ofPDGFR,” Cardiovascular Research, vol. 91, no. 4, pp. 720–731,2011.

[12] C. Grossmann, B. Husse, S. Mildenberger, B. Schreier,K. Schuman, and M. Gekle, “Colocalization of mineralo-corticoid and EGF receptor at the plasma membrane,” Bio-chimica et Biophysica Acta-Molecular Cell Research, vol. 1803,no. 5, pp. 584–590, 2010.

[13] M. Gekle, S. Mildenberger, R. Freudinger, and C. Grossmann,“Altered collagen homeostasis in human aortic smoothmuscle cells (HAoSMCs) induced by aldosterone,” PflugersArchiv: European Journal of Physiology, vol. 454, no. 3,pp. 403–413, 2007.

[14] J. L. Morgado-Pascual, S. Rayego-Mateos, J. M. Valdivielso,A. Ortiz, J. Egido, and M. Ruiz-Ortega, “Paricalcitol inhibitsaldosterone-induced proinflammatory factors by modulatingepidermal growth factor receptor pathway in cultured tubularepithelial cells,” BioMed Research International, vol. 2015,Article ID 783538, 13 pages, 2015.

[15] H. Wang and H. Gao, “Clinical study of Zishen Huoluodecoction on left ventricular hypertrophy in hypertension,”Lishizhen Medicine and Materia Medica Research, vol. 24,no. 10, pp. 2435-2436, 2013.

[16] M. Lu, Q. Qin, J. Yao, L. Sun, and X. Qin, “Induction of LOXby TGF-β1/Smad/AP-1 signaling aggravates rat myocardialfibrosis and heart failure,” IUBMB Life, vol. 71, no. 11,pp. 1729–1739, 2019.

[17] P. Coutinho, C. Vega, L. H. Pojoga et al., “Aldosterone’s rapid,nongenomic effects are mediated by striatin: a modulator ofaldosterone’s effect on estrogen action,” Endocrinology,vol. 155, no. 6, pp. 2233–2243, 2014.

[18] M. Shenasa and H. Shenasa, “Hypertension, left ventricularhypertrophy, and sudden cardiac death,” InternationalJournal of Cardiology, vol. 237, pp. 60–63, 2017.

[19] D. Lovic, P. Narayan, A. Pittaras, C. Faselis, M. Doumas, andP. Kokkinos, “Left ventricular hypertrophy in athletes andhypertensive patients,” Journal of Clinical Hypertension(Greenwich, Conn), vol. 19, no. 4, pp. 413–417, 2017.

[20] E. Z. Soliman and R. J. Prineas, “Antihypertensive therapiesand left ventricular hypertrophy,” Current Hypertension Re-ports, vol. 19, no. 10, p. 79, 2017.

[21] C. Borghi and F. Rossi, “Role of the renin-angiotensin-aldo-sterone system and its pharmacological inhibitors in cardio-vascular diseases: complex and critical issues,” High BloodPressure & Cardiovascular Prevention:Ke Official Journal of theItalian Society of Hypertension, vol. 22, no. 4, pp. 429–444, 2015.

[22] M.M.Hermidorff,G.deOliveira Faria,G. deCassia SousaAmancio,L. V. M. de Assis, and M. C. Isoldi, “Non-genomic effects ofspironolactone and eplerenone in cardiomyocytes of neonatalWistar rats: do they evoke cardioprotective pathways?” Bio-chemistry and Cell Biology, vol. 93, no. 1, pp. 83–93, 2015.

[23] J. Vıtovec, J. Spinar, and L. Spinarova, “Inhibition of renin-angiotensin-aldosterone system in heart failure, or fromCONSENSUS to PARADIGM-HF,” Vnitrni Lekarstvi, vol. 61,no. 5, pp. 470–474, 2015.

[24] M. M. Hermidorff, L. V. M. de Assis, and M. C. Isoldi,“Genomic and rapid effects of aldosterone: what we know anddo not know thus far,” Heart Failure Reviews, vol. 22, no. 1,pp. 65–89, 2017.

[25] S. Ruhs, A. Nolze, R. Hubschmann, and C. Grossmann, “30years of the mineralocorticoid receptor: nongenomic effectsvia the mineralocorticoid receptor,” Ke Journal of Endocri-nology, vol. 234, no. 1, pp. T107–T124, 2017.

[26] Q. Lu, D. C. Pallas, H. K. Surks, W. E. Baur,M. E. Mendelsohn, and R. H. Karas, “Striatin assembles amembrane signaling complex necessary for rapid, non-genomic activation of endothelial NO synthase by estrogenreceptor α,” Proceedings of the National Academy of Sciences ofthe United States of America, vol. 101, no. 49, pp. 17126–17131,2004.

[27] R. Baudrand, L. H. Pojoga, J. R. Romero, and G. H. Williams,“Aldosterone’s mechanism of action: roles of lysine-specific

Evidence-Based Complementary and Alternative Medicine 13

demethylase 1, caveolin and striatin,” Current Opinion inNephrology and Hypertension, vol. 23, no. 1, pp. 32–37, 2014.

[28] L. Michea, A. M. Delpiano, C. Hitschfeld, L. Lobos,S. Lavandero, and E. T. Marusic, “Eplerenone blocks non-genomic effects of aldosterone on the Na+/H+ exchanger,intracellular Ca2+ levels, and vasoconstriction in mesentericresistance vessels,” Endocrinology, vol. 146, no. 3, pp. 973–980,2005.

[29] A. S. Mihailidou, “Nongenomic actions of aldosterone:physiological or pathophysiological role?” Steroids, vol. 71,no. 4, pp. 277–280, 2006.

[30] A.W. Ashton, T. Y. L. Le, C. E. Gomez-Sanchez et al., “Role ofnongenomic signaling pathways activated by aldosteroneduring cardiac reperfusion injury,” Molecular Endocrinology,vol. 29, no. 8, pp. 1144–1155, 2015.

[31] L. H. Pojoga, P. Coutinho, A. Rivera et al., “Activation of themineralocorticoid receptor increases striatin levels,” Ameri-can Journal of Hypertension, vol. 25, no. 2, pp. 243–249, 2012.

[32] C. Grossmann and M. Gekle, “Nongenotropic aldosteroneeffects and the EGFR: interaction and biological relevance,”Steroids, vol. 73, no. 9-10, pp. 973–978, 2008.

[33] D. Chen, Z. Chen, C. Park et al., “Aldosterone stimulatesfibronectin synthesis in renal fibroblasts through mineralo-corticoid receptor-dependent and independent mechanisms,”Gene, vol. 531, no. 1, pp. 23–30, 2013.

14 Evidence-Based Complementary and Alternative Medicine