Abstract

   Trifolin exhibits anti-tumor activities; however, its effect on
   hypertension remains unknown. This study was performed to investigate
   trifolin’s potential therapeutic effects and underlying mechanisms of
   action on angiotensin II (Ang II)-induced hypertension in mice and Ang
   II stimulated A7R5 cells. Mice were randomly allocated into six groups:
   control, Ang II, Ang II + Trifolin (0.1 mg/kg), Ang II + Trifolin
   (1 mg/kg), Ang II + Trifolin (10 mg/kg), and Ang II + Valsartan
   (10 mg/kg). The hypertensive mouse model was constructed by infusing
   Ang II via a micro-osmotic pump (500 ng/kg/min), and trifolin,
   valsartan, or double distilled water was administered intragastrically
   once daily for 4 weeks. Blood pressure, vascular function, pathological
   morphology, and collagen deposition in Ang II infused mice and cell
   viability of Ang II stimulated A7R5 cells were assessed. A networking
   pharmacology analysis was performed to identify potential targets,
   pathways, and processes. These were verified by determining
   proliferating cell nuclear antigen (PCNA) expression, cell migration,
   collagen protein expression and related pathway activation in vivo and
   in vitro using masson, immunohistochemistry, cell counting Kit-8
   assays, phalloidin staining, wound healing assays, and
   western-blotting. Different concentrations of trifolin effectively
   mitigated the rise in systolic blood pressure, diastolic blood
   pressure, mean arterial pressure, pulse wave velocity, abdominal aorta
   wall thickness, and collagen deposition of Ang II infused mice.
   Notably, higher concentrations of trifolin exhibited greater
   attenuation which was similar to the effects of valsartan (a positive
   control). Networking pharmacology analysis identified 105 common
   targets and various gene ontology processes. The Kyoto Encyclopedia of
   Genes and Genomes pathways analysis identified multiple enriched
   signaling pathways, including responses to wounding,
   phosphatidylinositol 3-kinase complex, oxidoreductase, PI3K/AKT, and
   FoxO signaling pathways. Consistently, trifolin treatment significantly
   down-regulated the expression of PCNA and the ratio of p-PI3K/PI3K and
   p-AKT/AKT in the abdominal aorta tissues. In vitro study indicated that
   trifolin consistently reduced the cell viability, down-regulated the
   expression of PCNA, collagen I and collagen III, and reduced the cell
   migration, as well as reduced the ratio of p-PI3K/PI3K and p-AKT/AKT
   (similar with the effect of PI3K inhibitor: LY294002) in Ang II
   stimulated A7R5 cells. Trifolin treatment attenuated the elevation of
   blood pressure, the proliferation and collagen deposition of VSMCs, and
   modulated multiple signaling pathways, including PI3K/Akt pathway.
   These results suggest that trifolin could be a potential therapeutic
   approach for treating hypertension.

Supplementary Information

   The online version contains supplementary material available at
   10.1038/s41598-025-01022-1.

   Keywords: Hypertension, Networking pharmacology, Trifolin, Vascular
   smooth muscle cells, PI3K/AKT

   Subject terms: Cardiovascular biology, Cardiology

Introduction

   Hypertension is closely linked to cardiovascular disease and is
   considered a significant risk factor for global morbidity and
   mortality^[44]1. In a study involving 1,100,507 participants in low-
   and middle-income countries, 192,441 individuals were found to have
   hypertension. Of these, only a mere 10.3% had achieved control of their
   hypertension^[45]2. The prevalence of measures to control hypertension
   in China is relatively higher (13.0%) when compared to low-income
   countries, but the diagnosis, treatment, and control rates are still
   significantly lower than those of high-income countries
   (25%)^[46]3,[47]4. Therefore, identification of additional therapeutic
   approaches is urgently needed for hypertensive patients, particularly
   asymptomatic hypertensive patients, who are at an increased risk of the
   cardiovascular complications of damage to vital organs^[48]5.

   Due to a noticeable improvement in the effectiveness of treating
   hypertension when combined with western medicine, TCM has received
   growing interest^[49]6. Many formulas have been found to be effective
   in reducing blood pressure and alleviating hypertension-associated
   symptoms, as well as reducing hypertension-induced cardiovascular
   remodeling^[50]7,[51]8. Moreover, many natural compounds, such as
   quercetin and isoliensinine, have also been proven to have a good
   antihypertensive effect^[52]9–[53]12. Our previous studies have shown
   that Qingda Granule (QDG) play an antihypertensive role and reduce
   multiple target organs damage by regulating multiple signaling
   pathways^[54]13–[55]19. Further research revealed that many compounds
   in QDG exhibit antihypertensive effects, including alkaloids from
   nelumbinis plumula^[56]20, baicalin^[57]21, gastrodin^[58]22,
   neferine^[59]23, and Uncaria rhynchophylla^[60]24 and so on. However,
   the antihypertensive activity of many compounds is still unknown.
   Therefore, we thoroughly tested the antihypertensive activity of
   multiple compounds.

   Studies have shown that consuming flavonoids may lower the risk of
   cardiovascular-related deaths^[61]25,[62]26. Kaempferol
   3-O-galactosyltransferase catalyzes the formation of trifolin by adding
   a galactoside molecule to kaempferol^[63]27. The flavonoid trifolin
   (kaempferol-3-O-galactoside) has been isolated from the aerial portions
   of Plumula nelumbinis^[64]28. Trifolin exhibits anticancer effects by
   inducing lung cancer cell apoptosis and inhibits tumor angiogenesis by
   modulating multiple pathways, including death receptor-dependent and
   mitochondria-dependent pathways^[65]29,[66]30. Moreover, upon network
   pharmacology analysis, trifolin was identified as a potential active
   component of QDG, even though its potential targets and pharmacological
   mechanisms of antihypertension remain unknown.

   To further explore the effects and underling mechanisms of trifolin on
   hypertension, we established both in vivo and in vitro models of Ang
   II-induced hypertension to investigate the impact of trifolin on blood
   pressure and vascular dysfunction, as well as pathological changes of
   the abdominal aorta. Network pharmacology was employed to predict the
   potential molecular targets, pathways, and processes involved in the
   treatment of hypertension by trifolin. These were further validated,
   both in vivo and in vitro, by determining pathway activation and
   related functional changes.

Materials and methods

Reagents

   Ang II and antibodies against PI3 kinase p85 alpha (phospho Y607;
   p-PI3K; catalog # ab182651) and PCNA (catalog # ab29) were obtained
   from Abcam (Cambridge, MA, USA). Eosin Y stain solution (catalog #
   G1100) and Cole’s hematoxylin solution (catalog no.# G1140) were
   provided by Beijing Solarbio Science & Technology Co., Ltd. (Beijing,
   China). Antigen repair solution (catalog # MVS-0066), Ultra-Sensitive™
   SP (Mouse/Rabbit) immunohistochemistry (IHC) kit (catalog # KIT-9720),
   and 3,3’-Diaminobenzidine (DAB) substrate kit (catalog # DAB-0031) were
   provided by Maixin Biotechnology (Fuzhou, Fujian, China). Antibodies
   against PI3 kinase p85 (catalog # 4257), phospho-AKT (Ser473; catalog #
   4060), AKT (catalog # 4691), and rabbit secondary antibody (catalog #
   7074) were provided by Cell Signaling Technology (Danvers, MA, USA).
   Antibodies against p-PI3K (catalog # 41339) and p-AKT (catalog # 11054)
   for IHC staining were procured from Signalway Antibody (College Park,
   MD, USA). Dulbecco’s Modified Eagle Medium (DMEM), fetal bovine serum
   (FBS), trypsin-ethylenediaminetetraacetic acid, and bicinchoninic acid
   (BCA) protein assay reagent kits were provided by Thermo Fisher
   Scientific (Waltham, MA, USA). LY294002 was purchased from
   MedChemExpress (NJ, USA).

Preparation of trifolin

   Trifolin (catalog # ABL2391), with a purity of ≥ 98%, was acquired from
   Chengdu Alfa Biotechnology Co., Ltd. (Chengdu, Sichuan, China). For the
   animal experiments, trifolin was dissolved in double distilled water
   (ddH2O) to the appropriate concentrations, and fresh solutions were
   prepared daily. For cell culture experiments, trifolin was dissolved in
   DMEM medium at a concentration of 100 mM.

Animals and experimental protocols

   Male C57BL/6 mice aged 8 weeks were procured from Hangzhou Medical
   College (Certificate ID: SCXK 2019-0002; Hangzhou, China) and housed at
   the Animal Experiment Center of Fujian University of Traditional
   Chinese Medicine. The mice were kept in a pathogen-free environment
   under standard conditions at 24 °C ± 2 °C with a 12-hour light/dark
   cycle, and they were provided ad libitum access to food and water. To
   establish a hypertensive model, the mice were treated with Ang II via a
   micro-osmotic pump (500 ng/kg/min) and randomly allocated into six
   groups: control, Ang II, Ang II + Trifolin (0.1 mg/kg), Ang
   II + Trifolin (1 mg/kg), Ang II + Trifolin (10 mg/kg), and Ang
   II + Valsartan (10 mg/kg). The trifolin, dissolved in ddH2O, valsartan,
   dissolved in ddH2O, or plain ddH2O was infused intragastrically once
   daily for 4 weeks. Animal welfare and experimental protocols were
   performed in accordance with the ARRIVE guidelines and were applied in
   strict accordance with the guidelines of the Guide for the Care and Use
   of Laboratory Animals of the US National Institutes of Health. The
   animal experimental protocol for this study was approved by the Animal
   Care and Use Committee of Fujian University of Traditional Chinese
   Medicine (approval no. FJTCM IACUC 2022176) .

Assessment of blood pressure

   Blood pressure was monitored using the CODA™ non-invasive blood
   pressure system (Kent Scientific; Torrington, CT, USA) by connecting a
   catheter to a pressure transducer. The systolic, diastolic, and MAP
   were measured while the mice were secured in a holder under dark and
   quiet conditions at 37 °C. Blood pressure was recorded prior to
   micro-osmotic pump implantation and on days 7, 14, 21, and 28 after
   implantation.

Ultrasound imaging

   The Vevo 2100 high-resolution ultrasound instrument (FUJIFILM
   VisualSonics; Toronto, On, Canada) was used to conduct ultrasonic
   vascular assessments following the methodology described
   previously^[67]16. A 30 MHz probe was utilized to perform ultrasound
   scans while the mice were anesthetized with 1.5% isoflurane and
   positioned in a supine position on a heated table. B-mode and M-mode
   recordings were taken from below the xiphoid process, and measurements
   of the PWV and abdominal aorta thickness were measured and documented.

H&E staining

   Abdominal aortic tissue was collected, immersed in a 4%
   paraformaldehyde solution for 48 h at room temperature, and then
   embedded in paraffin. Coronal sections with a thickness of 4 μm were
   prepared and subjected to H&E staining. Three images at 400×
   magnification were obtained from random locations of each tissue by
   using an intelligent automated optical microscope (Leica; Weztlar,
   Germany).

Masson staining

   Paraffin sections were subjected to Masson trichrome staining.
   Representative images were captured using a light microscope (Leica) at
   a magnification of ×400 and collagen deposition was determined using
   Image J software.

IHC staining

   After dewaxing, endogenous peroxidase activity was inhibited using an
   Ultra-Sensitive™ SP (Mouse/Rabbit) IHC kit, then washed with
   phosphate‑buffered saline. After blocking, the sections were incubated
   for 24 h at 4 °C with antibodies to PCNA (1:100), PI3K (1:400), p-PI3K
   (1:200), AKT (1:400) or p-AKT (1:200) overnight. These sections were
   then incubated with a biotinylated secondary rabbit/mouse antibody and
   horseradish peroxidase-labeled streptavidin. After the sections were
   examined by an intelligent automated optical microscope (Leica),
   protein expression was visualized using DAB and quantitatively analyzed
   by positive rate via Image J software (National Institutes of Health;
   Bethesda, MD, USA).

Obtaining the targets for trifolin and hypertension

   Targets associated with hypertension were obtained via GeneCards
   ([68]https://www.genecards.org/). Additionally, DisGeNET
   ([69]https://disgenet.com/) was used to extract targets via the search
   term “hypertension.” Trifolin-related Targets were collected via
   PharmMapper ([70]https://www.lilab-ecust.cn/pharmmapper/) and
   Swiss-target prediction ([71]http://www.swisstargetprediction.ch/)
   databases.

Constructing and analyzing PPIs

   PPI network data for common targets of trifolin and hypertension were
   retrieved using the STRING search tool ([72]https://cn.string-db.org/,
   Version 11.5). Cytoscape (Version 3.8.2) software was used to construct
   the “trifolin-hypertension-targets” interaction network.

Exploration of functional annotations and pathways enrichment

   To determine the enriched GO functions and KEGG pathways, the DAVID
   tool was used to identify targets shared between trifolin and
   hypertension.

Cell culture

   A7R5 cells were purchased from Procell Life Science & Technology Co.,
   Ltd (Wuhan, Hubei, China; catalog # CL0316). All cells were maintained
   in DMEM with 10% FBS and 1% penicillin/streptomycin, kept in a
   humidified incubator at 37 °C with 5% CO[2]/95% air, and the medium was
   replaced every 48 h.

Cell viability analysis

   A7R5 cells were suspended and added to a 96-well plate at a density of
   0.3 × 10^5 cells/mL. Once the cells reached 40–50% confluence, they
   were deprived of serum for 6–8 h before being exposed to the different
   concentrations of trifolin for 24 h. Trifolin was administered at
   concentrations of 0, 25 µM, 50 µM, 100 µM, 200 µM, and 400 µM.
   Additionally, the A7R5 cells were treated with 1 µM Ang II and trifolin
   at concentrations of 0, 25 µM, 50 µM and 100 µM for 24 h. After the
   treatment period, 10 µL of Cell Counting Kit-8 was added to each well
   and incubated at 37 °C for an additional 2 h. Absorbance was measured
   at 450 nm using a microplate reader (Thermo Fisher Scientific; Waltham,
   MA, USA).

Western blotting

   Total proteins were extracted from cells using western and
   immunoprecipitation cell lysis buffer. The cells were subjected to this
   buffer for 30 min on ice and then centrifuged at 12,000 g for 20 min at
   4 °C. The supernatant, containing the extracted proteins, was
   collected, and protein concentration was measured using a BCA protein
   assay kit. An equal amount of protein was then subjected to sodium
   dodecyl sulfate polyacrylamide gel electrophoresis (10%) and
   transferred onto polyvinylidene difluoride (PVDF) membranes. To block
   any nonspecific binding, the PVDF membranes were incubated with
   blocking buffer at room temperature for 2 h. The membranes were then
   incubated with primary antibodies to p-PI3K, PI3K, p-AKT, AKT, PCNA, or
   GAPDH, all at a 1:1000 dilution, overnight at 4 °C. After washing with
   tris-buffered saline and tween 20 buffer to remove any unbound primary
   antibodies, the membranes were incubated with secondary rabbit/mouse
   antibody conjugated with horseradish peroxidase and diluted to a 1:5000
   concentration. Protein bands were detected using a chemiluminescence
   kit, and the resulting data were analyzed using Image J software.

Wound healing

   A7R5 cells were reseeded into 24-well plates (1 × 10^5 cells/well), and
   washed with PBS. A straight line was drawn through the cell monolayer
   using 10-µL pipette tip. Plates were incubated another 12 h, then
   photographed at magnification of 200× under a light microscope (Leica).

Phalloidin staining

   A7R5 cells were treated and fixed with 4% paraformaldehyde, stained for
   20 min with a conjugate of phalloidin and tetramethylrhodamine (AAT
   Bioquest, Sunnyvale, CA, USA), counterstained for 10 min with Hoechst
   solution, and photographed at a magnification of 200× under an
   UltraVIEW^® Vox fluorescence microscope (PerkinElmer, Santa Clara, CA,
   USA).

Statistical analysis

   Statistical analyses were performed using SPSS 26.0 software (SPSS/PC+;
   Chicago, IL, USA). The Shapiro-Wilk test was utilized to determine
   normality. Mean ± standard deviation was used when the data were
   normally distributed, or median ± quartile was used to present the
   data. One-way analysis of variance was performed, followed by
   Bonferroni post hoc analysis, if the data were normally distributed and
   had equal variance. If the data were normally distributed but did not
   have equal variance, Games Howell post hoc analysis was used. The
   nonparametric Kruskal-Wallis test was applied when the data were not
   normally distributed. Repeated analysis of measurement variance was
   performed to analyze statistical significance of blood pressure.
   Statistical significance was set at p < 0.05.

Results

Trifolin attenuated blood pressure in Ang II-infused hypertensive mice

   A significant increase in systolic blood pressure (SBP; Fig. [73]1A),
   diastolic blood pressure (DBP; Fig. [74]1B), and mean arterial pressure
   (MAP; Fig. [75]1C) was observed in an Ang II-induced mouse model.
   However, administration of trifolin or valsartan effectively mitigated
   the rise in SBP (Fig. [76]1A), DBP (Fig. [77]1B), and MAP
   (Fig. [78]1C). The most substantial reduction in blood pressure was
   observed in the group that received 10 mg/kg of trifolin and valsartan.
   Additionally, there was no notable difference in weight among the six
   groups (Fig. [79]1D).

Fig. 1.

   [80]Fig. 1
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   Impact of trifolin on blood pressure and body weight of angiotensin II
   (Ang II) infused mice. A tail-cuff plethysmograph method was used to
   measure blood pressure from each group: (A) systolic blood pressure,
   (B) diastolic blood pressure, (C) mean arterial pressure. (D) Body
   weight. Values are presented as mean ± standard deviation. Statistical
   significance is indicated as follows: ^ap < 0.05 vs. control group, ^bp
   < 0.05 vs. Ang II group, ^cp < 0.05 vs. Ang II + Trifolin (0.1 mg/kg)
   group, and ^dp < 0.05 vs. Ang II + Trifolin (1 mg/kg) group. N = 6 for
   each group.

Trifolin alleviated vascular dysfunction and pathological changes in the
abdominal aorta of Ang II-infused hypertensive mice

   Ultrasound was utilized to evaluate vascular function of the mice, and
   infusion of Ang II resulted in elevation of abdominal aorta pulse wave
   velocity (PWV), which was attenuated after trifolin and valsartan
   treatment (Fig. [82]2A–B). Furthermore, assessments using ultrasound
   (Fig. [83]2C) and hematoxylin and eosin (H&E) staining (Fig. [84]2D–E)
   demonstrated an increase in the thickness of the abdominal aortic wall
   of Ang II infused mice that was attenuated with the administration of
   trifolin and valsartan. Additionally, there was no notable difference
   in heart rate among the six groups (Supplementary Fig. [85]S1). Masson
   staining was revealed increased collagen content in Ang II infused mice
   that was attenuated with the administration of trifolin and valsartan
   (Fig. [86]2F-G).These above findings suggest that trifolin treatment
   could reduce Ang II-induced elavted blood pressure, vascular
   dysfunction and pathological changes.

Fig. 2.

   [87]Fig. 2
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   Effects of trifolin on vascular function and pathological changes of
   the abdominal aorta in Ang II infused mice. Ultrasound and
   hematoxylin-eosin (H&E) staining was used to assess pulse wave velocity
   (PWV) and the thickness of the abdominal aortic wall in each group. (A)
   Representative ultrasound measurements of the abdominal aorta. (B) PWV
   in the abdominal aorta from each group as determined by ultrasound. (C)
   Abdominal aortic wall thickness as measured by ultrasound. (D)
   Abdominal aortic wall thickness as measured by H&E. (E) Representative
   abdominal aorta cross-sections as determined by H&E staining at a
   magnification of 400×. (F–G) Masson staining was performed to observe
   collagen deposition. The collagen content was calculated as the
   percentage of positive area relative to the total area. The scale bar
   is set at 50 μm. Values represented as mean ± SD. Statistical
   significance is indicated as follows: ^ap < 0.05 vs. control group, ^bp
   < 0.05 vs. Ang II group, and ^cp < 0.05 vs. Ang II + Trifolin
   (0.1 mg/kg) group.

Analysis of protein-protein interaction (PPI) network highlights commonly
shared targets between trifolin and hypertension

   In this study, a Circos diagram (Fig. [89]3A) was used to visualize the
   intersection of targets from multiple databases. Gene searches were
   executed using the terms “hypertension” and “trifolin” on the
   PharmMapper and Swiss databases. An overlapping gene assessment was
   conducted, and 105 common genes were identified (Fig. [90]3B). These
   105 common targets were submitted to the Search Tool for Recurring
   Instances of Neighboring Genes (STRING) database to acquire data on
   target interactions. Network relationship data was obtained using a
   minimum required interaction score > 0.4 as a threshold. The resulting
   network consisted of 105 nodes and 1506 edges (Fig. [91]3C). To analyze
   the most potential genes, also known as hub genes, in the network, the
   Cytoscape integrated Cytohubba program was used. The top 15 hub genes
   were identified based on the Maximal Clique Centrality score, and a
   network with 15 nodes and 97 edges was constructed (Fig. [92]3D). These
   findings provide insight into the potential mechanisms underlying the
   effects of trifolin on antihypertension and may serve as a novel
   potential therapeutic approach for hypertension.

Fig. 3.

   [93]Fig. 3
   [94]Open in a new tab

   Networking pharmacology of common targets. (A) Circos plots displaying
   common targets between trifolin predicted targets and
   hypertension-related targets. Outer arcs represent the identity of each
   target, while inner arcs depict each target’s presence in various
   databases. Dark orange signifies targets found in multiple databases,
   while light orange represents those found in only one database. Purple
   lines connect identical targets found in specific databases, and blue
   lines connect targets with similar ontologies. (B) Depiction of common
   targets between trifolin predicted targets and hypertension-associated
   targets. (C) A network diagram presenting genes shared between trifolin
   predicted targets and hypertension-associated targets. (D) The top 15
   hub genes with their corresponding Matthew’s Correlation Coefficient
   scores represented by a color gradient, with red representing high
   interaction and yellow representing lower interaction in the network.

Clustering of common targets constructed network

   To gain insight into the structure and organization of complex
   networks, cauterization of the common network was performed, and five
   clusters were identified. Each cluster was characterized by a set of
   nodes, edges, and a score. The score is a measure of the strength of
   the clustering, with higher scores indicating more tightly connected
   nodes within the cluster. Cluster 1 consisted of 27 nodes and 372
   edges, with a score of 14.308. This cluster contained the genes PLAU,
   APP, PIK3CA, and MMP9, which are involved in cell signaling,
   proliferation, and differentiation (Fig. [95]4A). Cluster 2 consisted
   of 15 nodes and 98 edges, with a score of 7.000. This cluster contained
   the genes PRKCD, EGFR, and TNF, which are involved in cell growth,
   differentiation, and apoptosis (Fig. [96]4B). Cluster 3 consisted of 5
   nodes and 18 edges, with a score of 4.500. This cluster contained the
   genes COMT, ALDH2, and MAOA, which are involved in neurotransmitter
   metabolism and alcohol degradation (Fig. [97]4C). Cluster 4 consisted
   of 3 nodes and 6 edges, with a score of 3.000. This cluster contained
   the genes ADRA2A, PRKCB, and ADRA2C, which are involved in
   vasoconstriction, platelet aggregation, and protein kinase signaling
   (Fig. [98]4D). Cluster 5 consisted of 3 nodes and 6 edges, with a score
   of 3.000. This cluster contained the genes SHBG, GC, and F7, which are
   involved in hormone binding, vitamin D metabolism, and blood
   coagulation (Fig. [99]4E).

Fig. 4.

   [100]Fig. 4
   [101]Open in a new tab

   Cauterization of the network clusters highlighting common targets
   between proteins associated with hypertension and trifolin. The
   clusters are denoted as (A, B, C, D, and E), which stand for clusters
   1, 2, 3, 4, and 5 respectively. The nodes with the highest level of
   interaction in clusters (A, B, and C) are highlighted in the center
   (KDR, PI3KR1, and MAOB) with middle and outer layer nodes.

Assessment of GO and KEGG pathways

   Genetic ontology (GO) analysis using the Database for Annotation,
   Visualization, and Integrated Discovery (DAVID) database revealed that
   105 common genes were enriched in numerous GO processes (false
   discovery rate cutoff 0.05). The top 30 processes of each GO category
   were displayed. These targets were enriched into multiple biological
   processes including the response to wounding and phosphorus metabolic
   regulation. Additionally, these were involved in the negative
   regulation of programmed cell death, positive regulation of
   intracellular signal transduction, cellular response to
   oxygen-containing compounds, and response to organonitrogen compounds
   (Fig. [102]5A). Further, the cellular processes with which the targets
   were associated included the phosphatidylinositol 3-kinase complex,
   endocytic vesicle lumen secretory granule lumen, transferring
   phosphorus-containing groups, and receptor complex (Fig. [103]5B). The
   molecular functions that these targets exhibited included insulin
   receptor substrate binding, calcium-dependent protein kinase C
   activity, oxidoreductase, ATP binding, and transmembrane signaling
   receptor activity (Fig. [104]5C). Moreover, these key target genes
   showed a close association with various Kyoto Encyclopedia of Genes and
   Genomes (KEGG) pathways, including the vascular endothelial growth
   factor signaling pathway and the advanced glycation end-product
   (AGE)-receptor for AGE signaling pathway in diabetic complications,
   relaxin signaling pathway, FoxO, Rap1, and PI3K/AKT signaling pathways,
   and cancer and metabolic pathways (Fig. [105]5D).

Fig. 5.

   [106]Fig. 5
   [107]Open in a new tab

   Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG)
   pathways enrichment analysis. GO function and KEGG pathway enrichment
   were performed based on the common targets. (A) Biological processes,
   (B) Cellular processes, (C) Molecular function, and (D) KEGG pathways.
   The y-axis depicts terms that are significantly enriched, while the
   enrichment score of these terms is shown on the x-axis.

Trifolin attenuates up-regulated PCNA expression in vivo and in vitro, cell
viability, and proliferation in Ang II-induced A7R5 cells

   Compared with the control group, the protein expression level of
   proliferating cell nuclear antigen (PCNA), a biomarker of
   proliferation, was significantly up-regulated in abdominal aortic
   tissue of Ang II infused mice; however, the increase was attenuated
   after trifolin and valsartan treatment (Fig. [108]6A). In vitro, CCK-8
   analysis showed that trifolin did not affect cell viability at any of
   the concentrations tested (Supplementary Fig. [109]S2). Moreover,
   treatment with 25, 50, and 100 µM of trifolin significantly reduced the
   Ang II induced increase in cell viability (Fig. [110]6B–C). Similarly,
   Ang II stimulated expression of PCNA was reduced after trifolin
   treatment (Fig. [111]6D).

Fig. 6.

   [112]Fig. 6
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   Effects of trifolin on the protein expression of proliferating cell
   nuclear antigens (PCNA) in vivo and in vitro, and proliferation of Ang
   II-stimulated A7R5 cells. (A) Immunohistochemistry (IHC) staining for
   protein expression of PCNA in the abdominal aorta of Ang II infused
   mice. Images were taken at a magnification of 400×. The scale bar is
   set at 50 μm. Statistical significance is indicated by the following
   symbols: ^ap < 0.05 vs. control group, ^bp < 0.05 vs. Ang II group.
   N = 6 for each group. (B) Cell growth was observed using microscopy to
   determine cell confluence in Ang II stimulated A7R5 cells with or
   without trifolin treatment. Images were taken at a magnification of
   200×. Statistical significance is indicated by the following symbols:
   ^ap < 0.05 vs. control group, and ^bp < 0.05 vs. Ang II group. N = 3
   for each group. (C) Cell viability was determined by Cell Counting
   Kit-8 assay, and the viability of untreated A7R5 cells was defined as
   100%. (D–E) Protein expression of PCNA was examined by Western-blot,
   and protein expression was analyzed using Image J software.
   Glyceraldehyde 3-phosphate dehydrogenase expression was used as an
   internal control. Statistical significance is indicated by the
   following symbols: ^ap < 0.05 vs. control group, and ^bp < 0.05 vs. Ang
   II group. N = 3 for each group. All values in this figure are presented
   as mean ± SD.

Trifolin inhibits the formation of actin stress fibers and migration,
attenuates up-regulated collagen I and collagen III in Ang II-induced A7R5
cells

   Treatment of cultured A7R5 cells with Ang II also induced the formation
   of actin stress fibers and migration (Fig. [114]7A–C), while also
   upregulating collagens I and III at the level of protein
   (Fig. [115]7D–F). However, trifolin partially reversed all these
   effects (Fig. [116]7A–F). These results suggest that trifolin reverses
   the fibrosis of actin cytoskeleton in aortic smooth muscle, the
   migration of smooth muscle cells, and the accumulation of collagen
   associated with hypertensive vascular dysfunction.

Fig. 7.

   [117]Fig. 7
   [118]Open in a new tab

   Effects of trifolin on actin stress fibers, migration and fibrosis in
   Ang II-induced A7R5 cells. The A7R5 cells were treated with 1 µM Ang II
   and trifolin at concentrations of 0, 25 µM, 50 µM and 100 µM for 24 h.
   (A) Phalloidin staining was used to detected the expression of actin
   stress fibers. A7R5 cells were stained with Hoechst (blue) and
   Phalloidin (red). Representative images were taken at a magnification
   of ×200. (B–C) Wound-healing assay was used to detect the migration of
   A7R5 cells. Representative images were taken at a magnification of
   ×200. n = 3 for each group. Protein expression of (D) collagen I and
   collagen III was examined by Western-blot. Protein expression was
   analyzed using Image J software (E–F). GAPDH expression was used as an
   internal control. Statistical significance is indicated by the
   following symbols: ^ap < 0.05 vs. control group, and ^bp < 0.05 vs. Ang
   II group. N = 3 for each group. All values in this figure are presented
   as mean ± SD.

Trifolin attenuated PI3K/AKT pathway activation in abdominal aortic tissue of
Ang II infused mice

   The PI3K/AKT signaling pathway was explored to further explore the
   molecular underlying mechanism. Compared with the control group, the
   p-PI3K expression level, as well as the ratio of p-PI3K/PI3K, were
   significantly increased in the abdominal aortic tissue of Ang II
   infused mice; however, this increase was attenuated after trifolin and
   valsartan treatment (Fig. [119]8A–B). Moreover, trifolin treatment
   significantly attenuated increased expression of p-AKT and the
   p-AKT/AKT ratio in vivo (Fig. [120]8C–D).

Fig. 8.

   [121]Fig. 8
   [122]Open in a new tab

   Effects of trifolin on PI3K/AKT pathway activation in abdominal aortic
   tissue. Protein expression of (A) p-PI3K, (B) PI3K, (C) p-AKT and (D)
   AKT was examined by immunohistochemistry staining. Images were taken at
   a magnification of 200×. Protein expression is presented as mean ± SD.
   Statistical significance is indicated by the following symbols: ^ap <
   0.05 vs. control group, and ^bp < 0.05 vs. Ang II group.

Trifolin attenuated PI3K/AKT pathway activation in Ang II-induced A7R5 cells

   PI3K/AKT pathway activation in vitro demonstrated that trifolin
   treatment significantly attenuated the increase of p-PI3K/PI3K and the
   p-AKT/AKT ratio in Ang II-stimulated A7R5 cells (Fig. [123]9A–D).
   Moreover, treatment of trifolin, LY294002 alone or combination of
   Trifolin and LY294002 significantly reduced the ratio of p-PI3K/PI3K
   and the p-AKT/AKT in Ang II stimulated A7R5 cells, while didn’t
   exhibits significantly difference among the above groups
   (Fig. [124]9E–H). The results confirmed that trifolin plays a
   protective role in hypertension dependent on the activation of PI3K/AKT
   signaling pathway.

Fig. 9.

   [125]Fig. 9
   [126]Open in a new tab

   Effects of trifolin on protein expression of the PI3K/AKT pathway in
   A7R5 cells stimulated by Ang II. The A7R5 cells were treated with 1 µM
   Ang II and trifolin at concentrations of 0, 25 µM, 50 µM and 100 µM for
   24 h. (A–D) Protein expression of p-PI3K and PI3K and p-AKT and AKT was
   examined by Western-blot. Protein expression was analyzed using Image J
   software. GAPDH expression was used as an internal control. The A7R5
   cells were treated with 1 µM Ang II and/or 100 µM trifolin, 25 µM
   LY294002 for 24 h. (E–H) Protein expression of p-PI3K and PI3K and
   p-AKT and AKT was examined by Western-blot. Protein expression was
   analyzed using Image J software. GAPDH expression was used as an
   internal control. Values in this figure are represented as mean ± SD.
   Statistical significance is indicated by the following symbols: ^ap <
   0.05 vs. control group, and ^bp < 0.05 vs. Ang II group.

Discussion

   The current status of the prevention and control of hypertension
   indicates the need to explore new therapeutic strategies. Therefore, it
   is necessary to further explore the role of TCM on cardiovascular
   disease. QDG is a well-known TCM formula designed by academician Keji
   Chen, which functions as an antihypertensive^[127]15–[128]18. We
   previously demonstrated that multiple components (including baicalin,
   gastrodin, neferine, uncaria rhynchophylla) of QDG have
   antihypertensive effects. However, the antihypertensive activity of
   many compounds is still unclear. Previously we performed a network
   pharmacology analysis, and trifolin was identified as a potential
   active component from QDG. However, its active potential targets and
   the pharmacological mechanism of its antihypertensive effects remain
   unknown.

   Hypertension is linked to structural and functional alterations in the
   vascular system and is a significant health concern^[129]31,[130]32.
   Trifolin, a flavone belonging to the flavonoid family, is commonly
   referred to as kaempferol-3-O-galactoside^[131]33, and it is found in
   multiple plants including Vicia faba^[132]33, Camptotheca
   acuminate^[133]34, and Consolida oliveriana^[134]35. It possesses
   antioxidative and anticancer properties, and it was found to reduce the
   levels of atherosclerosis and cardiovascular risk
   index^[135]30,[136]36,[137]37. To the best of our knowledge, this study
   is the first to investigate the therapeutic potential of trifolin for
   managing hypertension using an Ang II infusion mouse model, and it
   found that trifolin effectively attenuated the Ang II-induced increase
   in blood pressure, which indicates its potential as an antihypertensive
   agent. Since vascular tone plays a crucial role in regulating blood
   pressure and blood flow^[138]38, we further evaluated functional
   changes of the abdominal aorta in vivo using ultrasound, and
   demonstrated that trifolin treatment significantly reduced Ang
   II-induced PWV and attenuated pathological changes in the abdominal
   aortic vessel wall. Overall, our findings suggest that trifolin
   treatment can alleviate both functional and pathological changes in the
   abdominal aorta of Ang II-infused mice, providing further evidence for
   its therapeutic potential in the treatment of hypertension. The current
   study further confirmed that trifolin treatment attenuated Ang
   II-induced the elevation of blood pressure, which was similar to the
   positive control valsartan (a kind of ARBs using as a positive
   control), suggesting the potential of trifolin to serve as an
   anti-hypertension medicine. However, the anti-hypertension effect of
   trifolin should be further verified in different animal models,
   including two-kidneys-one-clip and deoxycorticosterone acetate, and its
   underlying mechanisms should be further explored in depth.

   Network pharmacology is a novel field that builds upon the principles
   of systems biology. This involves the analysis of networks within
   biological systems and the identification of specific signaling nodes
   that can be targeted to develop a multi-target drug molecule
   network^[139]39. To comprehensively investigate the potential mechanism
   by which trifolin attenuates blood pressure elevation using network
   pharmacology analysis, this study identified 1036 trifolin-related
   target genes, 1947 hypertension-related genes, as well as 105 related
   common genes. These genes were significantly enriched in several GO
   processes, including intracellular signal transduction, programmed cell
   death, and others. Moreover, analysis of the topological variables of
   the network revealed that genes from the PI3K and protein serine kinase
   genes were highly ranked throughout the network, especially PIK3CA.
   PIK3CA encodes the p110α catalytic subunit of PI3K which through its
   role in the PI3K/AKT pathway is important for the regulation of
   important cellular functions such as proliferation, metabolism and
   protein synthesis, angiogenesis and apoptosis^[140]40. On the other
   hand, PIK3Rl, which encodes the regulatory subunit alpha of
   PI3Kmodulates PI3K’s catalytic activity and facilitates downstream
   activation of the AKT signaling pathway^[141]41,[142]42. This pathway
   is crucial for vascular smooth survival muscle cell proliferation,
   migration, and dysregulated expression or activity of PIK3Rl has been
   implicated in vascular pathologies, including endothelial dysfunction,
   oxidative stress, and vascular fibrosis^[143]43,[144]44. In addition,
   KEGG analysis analysis indicated that vascular endothelial growth
   factor signaling pathway, FoxO, Rap1, and PI3K/AKT signaling pathways
   were enriched, which have been widely reported to play vital functional
   roles in hypertensive vascular diseases^[145]17,[146]45–[147]47. Based
   on the target genes and potential pathway analysis that trifolin may
   exert its beneficial effects on blood pressure regulation through
   multiple signaling pathways, notably including the PI3K/AKT signaling
   pathway.

   Vascular smooth muscle cells (VSMC) proliferation, migration and
   fibrosis that results in vascular remodeling is a hallmark of vascular
   pathology including hypertension. To validate the network pharmacology
   results, we conducted both in vivo and in vitro experiments, which
   confirmed the antihypertensive effect of trifolin. Activated PI3K helps
   guide AKT to the plasma membrane where it is phosphorylated, and this
   contributes to the development of hypertension^[148]48. Therefore,
   targeting PI3K/AKT pathway activation significantly inhibits the
   proliferation and phenotypic conversion of VSMC, thereby inhibiting
   vascular remodeling and controlling blood pressure changes, which
   suggests a promising potential therapeutic strategy for managing
   hypertension^[149]20,[150]49–[151]53. In our current study demonstrated
   that trifolin consistently reduced Ang II stimulated A7R5 cell
   viability, down-regulated PCNA, collagen I and collagen III, reduced
   the cell migration and phenotypic transformation. However, we will
   further explore the effect of trifolin in adhesion, immunity,
   connection and diastole function of endothelial cells induced by
   hypertension. This study demonstrated that trifolin inhibited the
   proliferation and collagen deposition of Ang II-induced A7R5 cells and
   significantly decreased the expression of p-PI3K and p-AKT in Ang
   II-stimulated A7R5 cells and the abdominal aorta of Ang II infused
   mice. These results suggest that the antihypertensive properties of
   trifolin may be attributed to the inhibition of the PI3K/AKT pathway
   activation. These results justify further investigation into this
   traditional Chinese medicine compound as a treatment against
   hypertension, and they identify several molecules and pathways worth
   exploring in order to optimize this medicine and develop other
   anti-hypertensive drugs. Our results suggest that trifolin is a
   potential agent for treating hypertension patients and a promising drug
   for PI3K/AKT research. Based on network pharmacology analysis, more
   work is needed to further explore the underlying mechanisms of trifolin
   in sequenced potential target genes and other signaling pathways
   including redox enzymes, the FoxO and ERK signaling pathway, which
   indicated that the regulation and mechanism of vascular dysfunction. In
   addition, primary vascular smooth muscle cells in C57BL/6 mice of
   abdominal aorta should be used to investigate the effect of the
   treatment of trifolin on phenotypic switch (α-SMA, OPN), fibrosis
   (mmp2, mmp9) of smooth muscle cells after Ang II stimulation in future
   study. Our results should be verified and extended in other animal
   models of hypertension, and the mechanism of action of trifolin should
   be elucidated in detail to allow its optimization as an
   anti-hypertensive compound and inspire the design of similar compounds.

Conclusion

   Trifolin treatment attenuated the elevation of blood pressure, the
   proliferation and collagen deposition of VSMCs, and modulated multiple
   signaling pathways, including PI3K/Akt pathway. These results provide
   experimental evidence for the therapeutic potential of antihypertensive
   effects of trifolin.

Electronic supplementary material

   Below is the link to the electronic supplementary material.
   [152]Supplementary Material 1^ (115.7KB, docx)
   [153]Supplementary Material 2^ (1.8MB, pdf)

Abbreviations

   Ang II
          Angiotensin II

   BCA
          Bicinchoninic acid

   BSA
          Bovine serum albumin

   CVDs
          Cardiovascular diseases

   CCK-8
          Cell Counting Kit-8

   DBP
          Diastolic blood pressure

   DAVID
          Database for annotation visualization and integrated discovery

   DAB
          Diaminobenzidine

   ddH[2]O
          Double distilled water

   DMEM
          Dulbecco’s modified eagle medium

   FBS
          Fetal bovine serum

   GO
          Gene ontology

   GAPDH
          Glyceraldehyde 3-phosphate dehydrogenase

   H&E
          Hematoxylin and eosin

   IF
          Immunofluorescence

   KEGG
          Kyoto encyclopedia of genes and genomes

   MAP
          Mean arterial pressure

   PBS
          Phosphate-buffered saline

   PVDF
          Polyvinylidene fluoride membrane

   PWV
          Pulse wave velocity

   PPI
          Protein-protein interaction

   QDG
          Qingda granule

   STRING
          Search tool for recurring instances of neighboring genes

   SBP
          Systolic blood pressure

   TSV
          Tab-separated value

   TCM
          Traditional Chinese medicine

   VSMCs
          Vascular smooth muscle cells

Author contributions

   All authors contributed to the study conception and design. Jun Peng,
   Min Yu, and Keji Chen conceived and designed the experiments. Aling
   Shen, Meizhu Wu, Zhi Guo, and Jinkong Wu conducted the animal
   experiments and analyses. Farman Ali and Aling Shen performed network
   pharmacology analysis. Meizhu Wu, Zhi Guo, and Ying Cheng conducted H&E
   and IHC staining. Aling Shen, Hong Chen, and Meizhu Wu conducted the
   cell experiments and Western blotting. Aling Shen, Dawei Lian, and
   Meizhu Wu analyzed the data. Aling Shen, Meizhu Wu, and Farman Ali
   drafted the manuscript, and all authors commented on preliminary
   versions of the manuscript. Jun Peng, Min Yu, and Keji Chen provided
   the materials and revised the manuscript. All authors read and approved
   the final manuscript.

Funding

   This study was sponsored by the National Natural Science Foundation of
   China (U22A20372 and 82074363), Fujian Research and Training Grants for
   Young and Middle-aged Leaders in Healthcare, the Science and Technology
   Major Project of Fujian Province (2019YZ014004), the Development Fund
   of Chen Keji Integrative Medicine (CKJ2020003), the Young Elite
   Scientists Sponsorship Program of the China Association of Chinese
   Medicine (2021-QNRC2-B19), and the Youth Talent Support Program from
   Fujian University of Traditional Chinese Medicine (XQB202202).

Data availability

   The datasets used and/or analysed during the current study available
   from the corresponding author on reasonable request.

Declarations

Competing interests

   The authors declare no competing interests.

Ethical approval

   Animal welfare and experimental protocols were applied in strict
   accordance with the guidelines of the Animal Care and Use Committee of
   Fujian University of Traditional Chinese Medicine (Approval No. FJTCM
   IACUC 2022176).

Footnotes

   Publisher’s note

   Springer Nature remains neutral with regard to jurisdictional claims in
   published maps and institutional affiliations.

   Aling Shen and Meizhu Wu these have contributed equally to this work
   and share first authorship.

Contributor Information

   Jun Peng, Email: pjunlab@hotmail.com.

   Min Yu, Email: yumin@sinopharm.com.

   Keji Chen, Email: Kjchenvip@163.com.

References