Abstract

   Astragaloside IV (AS-IV) has been used to treat cardiovascular disease.
   However, whether AS-IV exerts a protective effect against hypertensive
   heart disease has not been investigated. This study aimed to
   investigate the antihypertensive and cardioprotective effects of AS-IV
   on L-NAME-induced hypertensive rats via network pharmacology and
   experimental pharmacology. The network pharmacology and bioinformatics
   analyses were performed to obtain the potential targets of AS-IV and
   hypertensive heart disease. The rat hypertension model was established
   by administrated 50 mg/kg/day of L-NAME for 5 weeks. Meanwhile,
   hypertension rats were intragastrically administrated with vehicle or
   AS-IV or fosinopril for 5 weeks. Cardiovascular parameters (systolic
   blood pressure, diastolic blood pressure, mean arterial pressure, heart
   rates, and body weight), cardiac function parameters (LVEDd, LVEDs, and
   fractional shortening), cardiac marker enzymes (creatine kinase, CK-MB,
   and lactate dehydrogenase), cardiac hypertrophy markers (atrial
   natriuretic peptide and brain natriuretic peptide), endothelial
   function biomarkers (nitric oxide and eNOS), inflammation biomarkers
   (IL-6 and TNF-α) and oxidative stress biomarkers (SOD, MDA, and GSH)
   were measured and cardiac tissue histology performed. Network
   pharmacological analysis screened the top 20 key genes in the treatment
   of hypertensive heart disease treated with AS-IV. Besides, AS-IV
   exerted a beneficial effect on cardiovascular and cardiac function
   parameters. Moreover, AS-IV alleviated cardiac hypertrophy via
   down-regulating the expression of ANP and BNP and improved
   histopathology changes of cardiac tissue. AS-IV improved endothelial
   function via the up-regulation of eNOS expression, alleviated oxidative
   stress via increasing antioxidant enzymes activities, and inhibited
   cardiac inflammation via down-regulating IL-6 and TNF-α expression. Our
   findings suggested that AS-IV is a potential therapeutic drug to
   improve L-NAME-induced hypertensive heart disease partly mediated via
   modulation of eNOS and oxidative stress.

   Keywords: cardiac damage, astragaloside IV, hypertensive heart disease,
   network pharmacology, inflammation, antioxidant

Introduction

   Hypertension is a common cardiovascular disease and a primary
   contributory factor for pathological cardiac dysfunction and
   remodeling, which seriously harms the structure and function of the
   heart ([42]Santos and Shah, 2014). Besides, persistent hypertension may
   cause fibrosis and left ventricle hypertrophy, which even resulting in
   heart failure and renal injury ([43]Gradman and Alfayoumi, 2006;
   [44]Uraizee et al., 2013). Loss of nitric oxide (NO) bioavailability
   and deficiency in endogenous NO synthesis are thought to underlie
   functional and histological cardiac injury during this process
   ([45]Yang et al., 2015). Abnormal changes in NO bioavailability or
   synthesis evokes endothelial dysfunction, which is also related to the
   progression of diabetes, heart failure, and hypertension ([46]Moncada,
   1992). It has been reported that NO exerts the cardioprotective effect
   by alleviating cardiac apoptosis and remodeling after myocardial
   infarction via inhibition of oxidative stress ([47]Smith et al., 2005).
   Moreover, Up-regulation of eNOS expression declined fructose-evoked
   insulin resistance and hypertension in rats (C. X. [48]Zhao et al.,
   2009). The pathogenesis of hypertension involves complex interplays of
   pathophysiologic, environmental, and genetic factors. Oxidative stress
   plays a vital role in the pathophysiologic process of hypertension
   ([49]Touyz et al., 2020). It contributes to renal injury and vascular
   dysfunction associated with hypertension ([50]Small et al., 2018).
   Besides, oxidative stress could decrease the bioavailability of NO,
   leading to vasoconstriction, which could even cause hypertension
   ([51]Harrison et al., 2003). The increasing evidence indicated that
   inflammatory cytokines, including IL-6, TNF-α, IFN-γ, and IL-17
   secreted from T cells, contributed to both vascular and renal injury
   and dysfunction, causing organ injury, high blood pressure, and
   oxidative stress ([52]McMaster et al., 2015; [53]Zimmer et al., 2020).
   N^ω-nitro-L-arginine methyl ester (L-NAME), a nitric oxide synthase
   inhibitor, obviously causes NO deficiency and evokes high blood
   pressure in an animal model ([54]Biwer et al., 2013). Chronic
   administration of L-NAME could cause cardiac hypertrophy via
   up-regulation of brain natriuretic peptide (BNP) and atrial natriuretic
   peptide (ANP) in vivo ([55]Suo et al., 2002). Treating rats with L-NAME
   could induce vascular endothelial injury, and this animal model is
   widely used in the study of cardiovascular and hypertension diseases
   ([56]Ribeiro et al., 1992). Additionally, declined antioxidant defense
   systems and increased production of reactive oxygen species are present
   in L-NAME-induced hypertensive rat model ([57]Rincón et al., 2015;
   [58]Zambrano et al., 2013). Therefore, developing new active
   ingredients with antioxidant effects that could improve endothelial
   function, and reduce oxidative stress and inflammation might be
   beneficial for preventing and treating hypertensive heart disease.

   Astragaloside IV (AS-IV) is a major active compound of Astragalus
   membranaceus. It has been useful in the treatment of nonalcoholic fatty
   liver disease via regulating inflammatory cytokines ([59]Liu et al.,
   2020). Additionally, it has been reported that AS-IV could decrease
   obesity-associated hypertension via improving leptin resistance and
   suppressing inflammatory reactions ([60]Jiang et al., 2018). AS-IV
   could alleviate cardiac hypertrophy and improve cardiac function via
   activating Nrf2 ([61]Nie et al., 2019). However, the effects of AS-IV
   against hypertension-associated cardiac damage are poorly investigated.
   Thus, the antihypertensive and cardioprotective effects of AS-IV were
   explored in the L-NAME-evoked hypertensive model, and the underlying
   mechanism actions of protection effects were evaluated by measuring
   oxidative stress and endothelial dysfunction-related biomarkers.

Materials and Methods

Prediction of AS-IV-Associated Targets

   The CTD database ([62]http://ctdbase.org/), PubChem database
   ([63]https://pubchem.ncbi.nlm.nih.gov), and Swiss Target Prediction
   database ([64]http://www.swisstargetprediction.ch/) were used to
   identify potential targets of AS-IV.

Prediction of Hypertensive Heart Disease-Associated Targets

   The CTD database ([65]http://ctdbase.org/) and Genecards database
   ([66]https://www.genecards.org/) were used to identify potential
   targets of hypertensive heart disease. The hypertensive heart
   disease-associated targets were obtained by searching the keyword
   “hypertensive heart disease” in these databases.

   Construction of protein-protein interaction (PPI) network and core
   genes identification.

   A Venny2.1.0 tool was used to collect the common targets of the AS-IV
   and hypertensive heart disease. Then, the PPI network of these common
   targets was constructed using the STRING database
   ([67]https://stringdb.org/). Then, Cytoscape software
   ([68]www.cytoscape.org/) was used to visualize and integrate the
   topological parameters of common targets in the PPI network. The degree
   of each protein node was calculated using the CytoHubba plugin. Then,
   the top 20 genes were identified as core genes.

Enrichment Analysis and Construction of the Compound-Targets-Pathways-Disease
Network

   KEGG pathway analysis was performed using the Metascape platform
   ([69]http://metascape.org/gp/#/main/step1) to obtain the AS-IV-mediated
   pathways against hypertensive heart disease. Cytoscape software
   ([70]www.cytoscape.org/) was used to construct a
   compound-targets-pathways-disease network based on the results of PPI
   and KEGG analysis.

Experimental Verification

Animal Experimental Protocol

   Male Sprague-Dawley rats (6–8 weeks old and 180–220 g) were purchased
   from the animal center of Harbin Medical University and housed under
   temperature- and humidity-controlled animal room, with 12 h light-dark
   cycles and free access to food and water. All animal experiments were
   performed by National Institutes of Health guidelines and approved by
   the Animal Ethics Committee of the First Affiliated Hospital of Harbin
   Medical University.

   After 1 week of acclimation, all animals were randomly divided into the
   five groups (n = 8 for each group) and treated as follows: Control
   group (CON), rats only received carboxymethyl cellulose solution (1%)
   daily; CON + HAS-IV group, rats in the CON group received a high dose
   of AS-IV (40 mg/kg) daily; L-NAME group (LN), rats received 50 mg/kg of
   L-NAME in carboxymethyl cellulose solution (1%) daily for 5 weeks to
   evoke hypertension ([71]Berkban et al., 2015); LN + LAS-IV group, rats
   in the LN group received a low dose of AS-IV (20 mg/kg) daily; LN +
   HAS-IV group, rats in the LN group received a high dose of AS-IV
   (40 mg/kg) daily. LN + fosinopril group, rats in the LN group received
   fosinopril (4.67 mg/kg) daily. The doses of AS-IV and fosinopril were
   selected according to the previous report ([72]Jiang et al., 2018;
   [73]Wang et al., 2020). Drugs were suspended in carboxymethyl cellulose
   solution (1%). Carboxymethyl cellulose solution (1%) or drugs were
   intragastrically administered daily for 5 weeks.

Measurement of Cardiovascular Parameters

   After 5 weeks of continuous administration, a non-invasive blood
   pressure measurement and analysis system (ALC-NIBP, ALCBIO, China) were
   used to measure the systolic blood pressure (SBP), diastolic blood
   pressure (DBP), mean arterial pressure (MAP), and heart rates (HR) in
   conscious rats based on manufacturer’s instruction.

Assessment of Cardiac Function

   At the end of the experiment, the rats were fasted for 18 h and
   anesthetized with 40 mg/kg of sodium pentobarbital by intraperitoneal
   injection. The Doppler echocardiography (Agilent Sonos5500) was used to
   measure the left ventricular end-diastolic dimension (LVEDd), left
   ventricular end-systolic dimension (LVEDs), and fractional shortening
   (FS) of each group.

Collection of Tissue and Blood Samples

   At the end of the experiment, rats were anesthetized with 40 mg/kg of
   sodium pentobarbital by intraperitoneal injection and euthanized by
   inhaling CO[2]. Then, the blood samples were rapidly collected from the
   abdominal aorta. Hearts and thoracic aortas were rapidly harvested and
   stored at −20°C for further analysis.

Measurement of Cardiac Marker Enzymes Activities

   Collected blood samples were centrifuged at 3,000 r/min for 15 min at
   4°C and the serum was obtained. The activities of creatine kinase (CK),
   creatine kinase-MB (CK-MB), and lactate dehydrogenase (LDH) were
   measured using commercially available kits (Jiancheng Bioengineering,
   Nanjing, China) based on the manufacturer’s instruction.

Assay of Endothelial Function Biomarkers

   The plasma, aortic and cardiac NO levels and the eNOS activity were
   measured by commercially available kits (Jiancheng Bioengineering,
   Nanjing, China) based on the manufacturer’s protocol.

Measurement of Oxidative Stress Biomarkers

   The heart and aorta tissues were homogenized in ice physiological
   saline using a homogenizer and then centrifuged at 5,000 r/min for
   10 min. The supernatant was collected and the protein concentration was
   measured by the BCA method. The activities of SOD, GSH, and MDA levels
   in the aortic and cardiac homogenate were measured by corresponding kit
   (Jiancheng Bioengineering, Nanjing, China) based on the manufacturer’s
   protocols.

Histopathological Analysis

   The cardiac tissue was collected and washed by ice physiological
   saline, and then fixed by 10% formalin and embedded in paraffin. The
   pathological changes of the heart were examined using hematoxylin and
   eosin staining reagent. Cardiomyocyte injury and interstitial edema
   were evaluated for cardiac pathological score, in which the score was 0
   for normal, 1 for mild, 2 for moderate, and 3 for severe damage.

Cell Experiment

   Rat H9C2 cells were purchased from China Infrastructure of Cell Line
   Resources (Chinese Academy of Medical Sciences) and cultured in
   Dulbecco’s modified Eagle’s medium, containing antibiotics and 10%
   fetal bovine serum at 37°C, 95% air, and 5% CO[2]. We changed the
   medium daily until the H9C2 cells were at 80–90% confluence. H9C2 cells
   (3 × 10^4 cells/ml) were inoculated into a 96-well plate, and then
   added different concentrations of AS-IV (20, 40, and 80 μg/ml) in the
   absence or presence of L-NAME (1 mM) for 24 h. Then, cell Counting
   Kit-8 (CCK-8, Dojindo, Japan) was applied for measuring cell viability.

Quantitative Real-Time Polymerase Chain Reaction

   The whole RNA of H9C2 cells, heart, and aorta tissues was extracted by
   the TRIzol reagent (Invitrogen, United States ) following the
   manufacturer’s protocol. Then, the extracted total RNA was used for
   cDNA synthesis by the Prime Script RT reagent kit (Takara BioInc,
   Japan) according to the manufacturer’s protocol. qRT-PCR was carried
   out in the ABI StepOnePlus system (Applied Biosystems, United States )
   using the Sybergreen™ reactions. The primers used in the present study
   were listed in supplementary file [74]Supplementary Table S1. The
   results of mRNA were quantified using the delta Ct method and
   normalized to glyceraldehyde 3-phosphate dehydrogenase (GAPDH).

Data Analysis

   GraphPad Prism Version (version 7.0) software was used for all data
   analyses and all results were given as mean ± standard deviation (SD).
   Significant differences were analyzed by one-way analysis of variance
   (ANOVA) followed by the Mann Whitney test. A value of p < 0.05 was
   considered statistically significant.

Results

Targets Screening of AS-IV and Hypertensive Heart Disease

   As shown in [75]Figure 1A, we collected potential genes of AS-IV from
   CTD, PubChem, and Swiss Target Prediction databases. Those genes were
   combined and we removed the overlap genes. Then, 54 genes associated
   with AS-IV were obtained. Besides, potential targets of hypertensive
   heart disease were predicted from the Genecards and CTD databases. We
   combined those potential genes and removed the overlap ones. Then,
   7,900 hypertensive heart disease-associated genes were collected.
   Finally, 51 common genes were obtained as potential genes in the
   therapeutic effect of AS-IV against hypertensive heart disease.

FIGURE 1.

   [76]FIGURE 1
   [77]Open in a new tab

   Analysis of the potential genes of AS-IV for the treatment of
   hypertensive heart disease. Venn diagram of 51 potential common genes
   (A). Construction of a PPI network of those common genes. (B) PPI
   network construction of hub genes (C), the darker color means the more
   important in the network.

PPI Network Construction and Hub Genes Screening

   As shown in [78]Figure 1B, the string database was used to construct
   the PPI network of 51 common targets. Then, Cytoscape software was used
   to rearrange those 51 common genes based on the degree value, and the
   top 20 genes of high-node degree were selected as the hub genes
   ([79]Figure 1C). Interestingly, we found the inflammation-related (TNF,
   IL-1β, and IL6) and oxidative stress-related (SOD1 and SOD2) genes in
   the hub genes.

Enrichment Analysis of Common Genes

   The KEGG enrichment analysis indicated how AS-IV acts on this pathway,
   thus exerting a therapeutic effect in hypertensive heart disease. In
   the present study, the top 20 hub genes were selected for enrichment
   analysis, and the top 20 signaling pathways were screened for further
   analysis based on p-Value. These signaling pathways were shown in
   [80]Figure 2A. Among them, the IL-17 signaling pathway, TNF signaling
   pathway, NOD-like receptor signaling pathway, and Toll-like receptor
   signaling pathway as the top ones.

FIGURE 2.

   [81]FIGURE 2
   [82]Open in a new tab

   KEGG pathway enrichment analysis of top 20 genes. (A)
   Compound-targets-pathways-disease network: blue node is AS-IV. (B)
   Yellow node is hypertensive heart disease; green nodes represent 20
   genes; red nodes represent seven potential signaling pathways; these
   lines indicate the interactions between them.

Compound-Targets-Pathways-Disease Network

   As shown in [83]Figure 2B, a multidimensional network of
   “compound-targets-pathways-disease” was constructed by Cytoscape
   software, which included 29 nodes (1 compound, 20 genes, 7 signaling
   pathways, and 1 disease). The purple node is AS-IV; the yellow node is
   hypertensive heart disease; the green nodes represent 20 genes; the
   pink to red nodes represent 7 potential signaling pathways; these lines
   indicate the interactions between them. These findings indicated that
   the AS-IV could alleviate hypertension heart disease via regulating
   multi-targets and multi-signaling pathways.

Effects of AS-IV on Cardiovascular Parameters and Cardiac Function in
L-NAME-Treated Rats

   As shown in [84]Figure 3, administration of L-NAME for 5 weeks evoked a
   significant increase in SBP, DBP, MAP, HR, and HW/BW compared to the
   CON group. Treatment with a high dose of AS-IV (40 mg/kg) significantly
   decreased all these cardiovascular parameters in hypertensive heart
   disease rats. Fosinopril exerted similar effects as AS-IV (40 mg/kg).
   It’s worth noting that, a low dose of AS-IV (20 mg/kg) also decreased
   SBP, DBP, and HR in LN + LAS-IV group. Besides, our results indicated
   that the values of LVEDd and LVEDs in the LN group increased when
   compared with the CON group, accompanied by a significant decrease of
   FS ([85]Figure 4). After AS-IV (40 mg/kg) or fosinopril treatment,
   LVEDd and LVEDs were decreased, while FS was increased, implying
   remarkable cardioprotection of AS-IV against L-NAME-induced
   hypertensive heart disease.

FIGURE 3.

   [86]FIGURE 3
   [87]Open in a new tab

   Effects of Astragaloside IV (AS-IV) on cardiovascular parameters in
   L-NAME-treated rats. The SBP (A), DBP (B), MAP (C), HR (D), body weight
   (C), and HW/BW (F) was measured at the end of experiment. Results were
   showed as mean ± SD (n = 6 for each group). ##p < 0.001 versus CON
   group; *p < 0.01 versus LN group; **p < 0.01 versus LN group; ***p <
   0.001 versus LN group.

FIGURE 4.

   [88]FIGURE 4
   [89]Open in a new tab

   Effects of Astragaloside IV (AS-IV) on LVEDd (A), LVEDs (B), FS (C)
   values in L-NAME-treated rats. Results were showed as mean ± SD (n = 6
   for each group). ##p < 0.001 versus CON group; *p < 0.01 versus LN
   group; **p < 0.01 versus LN group; ***p < 0.001 versus LN group.

Effects of AS-IV on Cardiac Marker Enzymes and Cardiac Hypertrophy in
L-NAME-Treated Rats

   As shown in [90]Figures 5A–C, after administration of L-NAME for
   5 weeks, the activities of CK, CK-MB, and LDH were significantly
   increased in the LN group compared to the CON group. Treatment with a
   high dose of AS-IV (40 mg/kg) or fosinopril significantly reduced these
   cardiac marker enzymes activities in the model group. ANP and BNP are
   natriuretic peptides, play a major role in the regulation of
   cardiovascular, and as markers of myocyte hypertrophy ([91]Gardner,
   2003; [92]Kohno et al., 1995). After administration of L-NAME for
   5 weeks, the mRNA expression of ANP and BNP were significantly
   up-regulated in the LN group compared to the CON group ([93]Figures
   5D,E). Treatment with AS-IV (40 mg/kg) or fosinopril significantly
   reduced these cardiac hypertrophy markers expressions in the model
   group. Besides, hematoxylin-eosin (HE) staining was used to evaluate
   the pathologic features of cardiac tissue. [94]Figure 6 showed an
   enlarged cross-sectional area of cardiomyocytes, cardiomyocyte injury,
   and interstitial edema in the cardiac sections of the LN group.
   Treatment with AS-IV (40 mg/kg) or fosinopril significantly alleviated
   these histopathological changes and decreased heart pathology score in
   the model group ([95]Figures 6A,B). However, the low dose of AS-IV
   (20 mg/kg) has no effect on cardiac hypertrophy parameters and cardiac
   damage in L-NAME-treated rats. These findings revealed that the
   potential protective effects of AS-IV in the treatment of cardiac
   hypertrophy.

FIGURE 5.

   [96]FIGURE 5
   [97]Open in a new tab

   Effects of Astragaloside IV (AS-IV) on cardiac marker enzymes and
   cardiac hypertrophy in L-NAME-treated rats. The serum CK (A), CK-MB
   (B), LDH (C) activities were measured by commercially available kits.
   The cardiac tissue ANP (D), BNP (E) mRNA expression levels were
   measured by qRT-PCR. Results were shown as mean ± SD (n = 6 for each
   group). ##p < 0.001 versus CON group; *p < 0.01 versus LN group; **p <
   0.01 versus LN group; ***p < 0.001 versus LN group.

FIGURE 6.

   [98]FIGURE 6
   [99]Open in a new tab

   Effects of Astragaloside IV (AS-IV) on cardiac tissue histopathological
   changes in hypertensive rats. Results were shown as mean ± SD (n = 6
   for each group). ##p < 0.001 versus CON group, **p < 0.01 versus LN
   group. Hematoxylin and eosin staining method was used to evaluate the
   cardiac tissue histopathological changes. (A) Cardiac pathological
   score (B). Scale bar = 100 µm.

Effects of AS-IV on Endothelial Function Biomarkers in L-NAME-Treated Rats

   Chronic administration of L-NAME for 5 weeks, the levels of NO[X] and
   eNOS in plasma, heart, and aorta were significantly decreased in the LN
   group compared to the CON group ([100]Figure 7). Treatment with AS-IV
   (40 mg/kg) or fosinopril significantly increased these endothelial
   function biomarkers in the hypertensive heart disease group.

FIGURE 7.

   [101]FIGURE 7
   [102]Open in a new tab

   Effects of Astragaloside IV (AS-IV) on endothelial dysfunction in
   hypertensive rats evoked by L-NAME. The plasma NO[X] (A), aortic NO[X]
   (B), cardiac NO[X] (C), cardiac eNOS activity (D), aortic eNOS mRNA
   expression (E), cardiac eNOS mRNA expression (F) in CON, CON + HAS-IV,
   LN, LN + LAS-IV, LN + HAS-IV and LN + fosinopril groups. Results were
   showed as mean ± SD (n = 6 for each group). ##p < 0.001 versus CON
   group, *p < 0.01 versus LN group; **p < 0.01 versus LN group; ***p <
   0.001 versus LN group.

Effects of AS-IV on Inflammation Biomarkers in L-NAME-Treated Rats

   Chronic administration of L-NAME for 5 weeks, the expression levels of
   IL-6 and TNF-α in the heart and aorta were significantly increased in
   the LN group compared to the CON group ([103]Figure 8). Hypertensive
   heart disease rats treated with AS-IV (40 mg/kg) or fosinopril had
   significantly down-regulated IL-6 and TNF-α expression levels compared
   with the LN group.

FIGURE 8.

   [104]FIGURE 8
   [105]Open in a new tab

   Effects of Astragaloside IV (AS-IV) on inflammation biomarkers in
   hypertensive rats evoked by L-NAME. The mRNA expression levels of IL-6
   (A) and TNF-α (B) in CON, CON + HAS-IV, LN, LN + LAS-IV, LN + HAS-IV
   and LN + fosinopril groups. Results were shown as mean ± SD (n = 6 for
   each group). ##p < 0.001 versus CON group, *p < 0.01 versus LN group;
   **p < 0.01 versus LN group; ***p < 0.001 versus LN group.

Effects of AS-IV on Oxidative Stress Biomarkers in L-NAME-Treated Rats

   Chronic administration of L-NAME for 5 weeks, the activities of SOD and
   GSH in the heart and aorta were significantly decreased in the LN group
   compared to the CON group ([106]Figures 9A,B). Besides, the levels of
   MDA were significantly increased in the LN group compared to the CON
   group ([107]Figure 9C). Hypertensive heart disease rats treated with
   AS-IV (40 mg/kg) or fosinopril had significantly increased SOD and GSH
   activities and a significantly decreased MDA level compared with the LN
   group. It’s worth noting that, a low dose of AS-IV (20 mg/kg) also
   decreased MDA levels in LN + LAS-IV group. In addition, the qRT-PCR
   results indicated that AS-IV or fosinopril inhibited the
   down-regulation levels of SOD1 and SOD2 induced by L-NAME ([108]Figure
   9D,E).

FIGURE 9.

   [109]FIGURE 9
   [110]Open in a new tab

   Effects of Astragaloside IV (AS-IV) on oxidative stress biomarkers in
   hypertensive rats evoked by L-NAME. The SOD activity (A), GSH content
   (B), MDA content (C), SOD1 mRNA expression (D), and SOD2 mRNA
   expression (E) in CON, CON + HAS-IV, LN, LN + LAS-IV, LN + HAS-IV and
   LN + fosinopril groups. Results were shown as mean ± SD (n = 6 for each
   group). ##p < 0.001 versus CON group, *p < 0.01 versus LN group; **p <
   0.01 versus LN group; ***p < 0.001 versus LN group.

AS-IV Increased Cell Viability in L-NAME-Stimulated H9C2 Cells

   As shown in [111]Figure 10A, treatment with AS-IV (20–80 μg/ml) alone
   did not cause a decrease in cell viability of H9C2 cells. The H9C2
   cells were exposed to L-NAME caused the decline of cell viability
   ([112]Figure 10B). Treatment with AS-IV (40–80 μg/ml) improved cell
   viability in L-NAME-stimulated H9C2 cells.

FIGURE 10.

   [113]FIGURE 10
   [114]Open in a new tab

   Effect of Astragaloside IV (AS-IV) on the cell viability in
   L-NAME-stimulated H9C2 cells. H9C2 cells were stimulated with different
   concentrations of AS-IV (20, 40, and 80 μg/ml) in the absence or
   presence of L-NAME (1 mM) for 24 h. Results were presented as mean ±
   standard deviation (SD). n = 4. **p < 0.01, ***p < 0.001.

The Protective Effect of AS-IV Against L-NAME-Induced Inflammation and
Oxidative Stress in H9C2 Cells

   The cells experiment was performed to further validate the results of
   network pharmacology and animal experiment. As shown in [115]Figure 11,
   the expression of ANP, BNP, and IL-6 was up-regulated in
   L-NAME-stimulated H9C2 cells. AS-IV significantly down-regulated the
   expressions of ANP, BNP, and IL-6. The expressions of eNOS and SOD1
   were down-regulated in L-NAME-stimulated H9C2 cells. AS-IV
   significantly up-regulated the expressions of eNOS and SOD1.

FIGURE 11.

   [116]FIGURE 11
   [117]Open in a new tab

   The protection effect of Astragaloside IV (AS-IV) against
   L-NAME-induced inflammation and oxidative stress in H9C2 cells. H9C2
   cells were stimulated with different concentrations of AS-IV (40 and
   80 μg/ml) in the absence or presence of L-NAME (1 mM) for 24 h mRNA
   expression of ANP (A), ABP (B), eNOS (C), IL-6 (D), and SOD1 (E) were
   measured by qRT-PCR. Results were presented as mean ± standard
   deviation (SD). n = 4. *p < 0.05, **p < 0.01, ***p < 0.001.

Discussion

   Hypertensive heart disease is induced by chronic pressure overload and
   multiple mechanisms are involved in the progression of this disease
   ([118]Slivnick and Lampert, 2019). Hyperphosphorylation of titin
   protein, microtubule disarray, and abnormal calcium handing are
   involved in this pathological process ([119]Borbély et al., 2009;
   [120]Shah et al., 2014). Therefore, the detailed etiology of
   hypertensive heart disease is not fully understood. Because there is no
   specific drug to treat hypertensive heart disease; therefore,
   developing novel agents is very necessary.

   Over the past few decades, natural compounds extracted from herbs or
   Chinese herbal medicine have been one of the most primary resources for
   drug research and development, especially in the treatment and
   prevention of cardiovascular disease. AS-IV is the primary active
   compound extracted from Astragalus membranaceus. Previous reports have
   indicated that AS-IV exerts various protective activities in the brain,
   kidney, lung, and cardiovascular, and these pharmacological activities
   are related to multiple signaling pathways, such as Nrf2 antioxidant
   signaling pathways, NF-κB signaling pathway, and EGFR-Nrf2 signaling
   pathway (J. [121]Zhang et al., 2020a). However, the underlying
   mechanism actions of AS-IV against hypertensive heart disease have not
   been fully understood. Network pharmacology integrates omics, network
   visualization, and other methods to establish the system network model
   based on the theory of systems biology ([122]Li and Zhang, 2013). The
   complex relationship among drugs, diseases, targets, and pathways was
   revealed via network pharmacology analysis, which is of great
   significance for understanding the mechanism of action of traditional
   Chinese medicine, the basis of the medicinal substance, and the
   research and development of new drugs ([123]Hao da and Xiao, 2014). In
   the present study, the network pharmacology was used to investigate the
   therapeutic effect of AS-IV in the treatment of hypertensive heart
   disease and an animal experiment was performed to verify our
   speculation.

   First, we used network pharmacology and bioinformatics methods to
   construct a “compound-targets-pathways-disease” network. Our findings
   indicated that the targeted genes of AS-IV against hypertensive heart
   disease are involved in oxidative stress and inflammation, including
   SOD2, SOD1, IL-6, TNF, and IL-1β. Next, we investigated the
   antihypertensive and cardioprotective effects of AS-IV in
   L-NAME-induced hypertensive heart disease models. And our results
   revealed that these beneficial effects of AS-IV may be related to the
   down-regulation of IL-6 and TNF-α and up-regulation of SOD1 and SOD2.

   At present, multiple mechanisms have been proposed for the etiology of
   hypertensive heart disease. One of the most important is the reduction
   of NO bioavailability or NO synthesis ([124]Davel et al., 2011;
   [125]Lee et al., 2016). Previous reports have indicated that the
   suppression of eNOS resulted in hypertension and high vascular
   resistance ([126]Nyadjeu et al., 2013; [127]Selamoglu Talas, 2014).
   Blockade NO synthesis in L-NAME-induced high blood pressure is related
   to the down-regulation of eNOS expression and elevation of MDA level
   ([128]Fu et al., 2011; [129]Jan-On et al., 2020). The L-NAME-induced
   hypertension heart disease model is similar to that of patients with
   vascular endothelial dysfunction, who also experience structural and
   functional cardiac dysfunction due to the loss of bioavailability of NO
   ([130]Ndisang et al., 2014). In vivo, our findings showed that
   hypertension heart disease was induced by a decrease of NO[X] level and
   an increase of oxidative stress. Administration of AS-IV decreased high
   blood pressure, increased eNOS expression as well as attenuated cardiac
   dysfunction in the hypertensive heart disease model. Meanwhile, MDA
   level and oxidative stress markers were also alleviated in the model
   group after AS-IV administration. Thus, antioxidant properties may be
   one of the mechanisms by which AS-IV inhibited the progression of
   hypertensive heart disease. Moreover, the administration of L-NAME also
   leads to cardiomyocytes injury that induces the secretion of cardiac
   enzymes (LDH, CK-MB, and CK). The previous report showed that chronic
   depletion of NO by L-NAME resulted in a significant increase in CK,
   CK-MB, and LDH levels ([131]Kumar et al., 2014). Consistent with the
   previous report, our findings of cardiac enzyme measuring and H&E
   staining showed that the L-NAME administration caused the increase of
   cardiac enzymes (CK, CK-MB, and LDH) and cardiac structural
   abnormalities. In the present study, administration of AS-IV reversed
   those abnormalities induced by L-NAME, implying that AS-IV alleviated
   symptoms of hypertensive heart disease in L-NAME-treated rats.

   Inflammation is a primary factor in the occurrence of disease
   ([132]Nathan and Ding, 2010). The findings of the PPI network indicated
   that IL-6, TNF, IL-1β, and NFKBIA were predicted as the main genes of
   AS-IV against hypertensive heart disease and the KEGG pathways analysis
   showed that TNF signaling pathway was its key pathway. It has been
   reported that hypertensive caused chronic systemic inflammation, which
   stimulates the secretion of pro-inflammatory factors that lead to
   cardiac damage ([133]Mouton et al., 2020). Chronic cardiac inflammation
   is involved in the aggravation of cardiac remodeling in hypertensive
   heart disease ([134]Kai et al., 2009). AS-IV alleviates mechanical
   stress-induced myocardial hypertrophy via decreasing inflammation (T.
   [135]Zhang et al., 2020b). AS-IV prevents lipopolysaccharide-induced
   gestational hypertension via the suppression of inflammatory responses
   ([136]Tuerxun et al., 2021). Therefore, we speculated that AS-IV may
   alleviate systemic inflammation in hypertensive heart disease. In the
   present study, AS-IV down-regulated the expression of IL-6 and TNF and
   inhibited the progression of inflammation. This finding explained that
   AS-IV alleviated cardiac inflammation in hypertensive rats evoked by
   L-NAME.

   Oxidative stress is also one of the pathogenic factors of hypertensive
   heart disease ([137]Mi et al., 2019). Cardiac oxidative stress induced
   by excess reactive oxygen species has been indicated to be implicated
   in the occurrence and development of high blood pressure and pressure
   overload-induced cardiac damage (W. [138]Zhao et al., 2008). It has
   been reported that oxidative injury was present in hypertensive
   myocardial tissue ([139]Worou et al., 2011). Besides, the expression of
   SOD2 significantly changed in the hypertension-induced cardiac
   dysfunction ([140]Koyanagi et al., 2008). Therefore, inhibition of
   oxidative stress is a novel method to treat hypertensive heart disease.
   AS-IV alleviates myocardial ischemia injury via suppressing reactive
   oxygen species burst and improving antioxidant potential ([141]Luo et
   al., 2019). The findings of the PPI network indicated that SOD1 and
   SOD2 were predicted as the potential genes of AS-IV in the treatment of
   hypertensive heart disease. And the network pharmacological results
   were verified by in vivo and in vitro experiments. In the present
   study, we found that AS-IV up-regulated the expression of SOD1 and
   SOD2, improved the activities of SOD and GSH, and decreased the MDA
   level, indicating that AS-IV inhibited the progression of hypertensive
   heart disease via inhibition of oxidative stress.

Conclusion

   In the present report, the network pharmacology and experimental
   validation were performed to investigate the therapeutic effects of
   AS-IV against hypertensive heart disease. Our findings demonstrated
   that AS-IV prevents the progression of hypertensive heart disease via
   activation of eNOS and inhibition of oxidative stress. Our results not
   only provide a theoretical foundation for exploring the mechanism
   actions of AS-IV against hypertensive heart disease but also develop a
   promising treatment for hypertensive heart disease.

Data Availability Statement

   The original contributions presented in the study are included in the
   article/[142]Supplementary Files, further inquiries can be directed to
   the corresponding author.

Ethics Statement

   The animal study was reviewed and approved by the First Affiliated
   Hospital of Harbin Medical University. Written informed consent was
   obtained from the owners for the participation of their animals in this
   study.

Author Contributions

   HJ and RX designed the experiments and wrote the manuscript. YB, YD,
   CS, WY, and RC carried out the experiments and analyzed the data. ZL
   and XQ supervised and corrected the manuscript.

Conflict of Interest

   The authors declare that the research was conducted in the absence of
   any commercial or financial relationships that could be construed as a
   potential conflict of interest.

Publisher’s Note

   All claims expressed in this article are solely those of the authors
   and do not necessarily represent those of their affiliated
   organizations, or those of the publisher, the editors, and the
   reviewers. Any product that may be evaluated in this article, or claim
   that may be made by its manufacturer, is not guaranteed or endorsed by
   the publisher.

Supplementary Material

   The Supplementary Material for this article can be found online at:
   [143]https://www.frontiersin.org/articles/10.3389/fphar.2021.755653/ful
   l#supplementary-material
   [144]Click here for additional data file.^ (17KB, DOCX)

References