Liensinine can improve vascular remodeling in hypertension through the ferroptosis-related TLR4 inflammatory pathway

Abstract

Abstract

Vascular remodeling, which results in aortic sclerosis, is one of the main risk factors for hypertension-related cardiovascular events. In the process of early intervention and clinical improvement, the inhibition of this process is very important. Previous studies have shown that liensinine (Lien) alleviates hypertension-induced vascular remodeling, but its mechanism of action remains unclear. This study aimed to investigate the therapeutic effects of Lien on hypertension, explore its molecular targets, and elucidate its anti-inflammatory mechanisms related to ferroptosis in preventing vascular remodeling. In vivo, Lien significantly reduced blood pressure and PWV and improved vascular pathology by reducing aortic wall thickness in hypertensive mice. The results of the WB experiment indicated that Lien can promote the expression of GPX4 protein and inhibit the expression of TFRC protein, which are ferroptosis markers. RNA-seq results suggested that Lien targets TLR4-mediated inflammation in vascular remodeling. The serum levels of IL-6, IL-1β, and TNF-α were decreased by Lien, according to ELISA results. IHC demonstrated decreased expression of inflammatory markers (p-IκB-α, IκB-α, p-P65, P65) in vascular tissues. In vitro, the results of the WB experiment indicated that Lien can promote the expression of GPX4 protein and inhibit the expression of TFRC protein; the ELISA results indicate that Lien can reduce the secretion of IL-6, IL-1β, and TNF-α induced by Ang II. Molecular docking and COIP experiments showed that Lien inhibits the formation of the Ang II-TLR4-MD2 complex and reduces MyD88 expression. Western blotting further confirmed that Lien inhibited the MAPK and TGF-β1/Smad2/3 pathways, both in the presence of Ang II and inflammatory stimuli (LPS, CKs). Subsequently, using the TLR4 inhibitor TAK242 and the NF-κB inhibitor PDTC, further evidence was provided that Lien effectively manages hypertension by lowering blood pressure, enhancing vascular health, and providing anti-inflammatory effects, mainly by inhibiting the TLR4/MD2 complex and suppressing the MAPK and TGF-β1/Smad2/3 pathways. These results underscore the promise of Lien as a therapeutic agent for preventing vascular remodeling caused by hypertension.

Key messages

• Liensinine significantly reduces blood pressure and improves vascular inflammatory in hypertensive mice.

• Liensinine inhibits vascular inflammatory and remodeling by suppressing the ferroptosis related GPX4 and TLR4/MD2 complex and its downstream NF-κB, MAPK, and TGF-β1/Smad2/3 signaling pathways.

• Liensinine demonstrates potential as a therapeutic agent and offers new insights into inflammation-driven vascular dysfunction and its intervention.

Supplementary Information

The online version contains supplementary material available at 10.1007/s00109-025-02624-y.

Introduction

Hypertension, defined by elevated arterial blood pressure (systolic ≥ 140 mmHg and/or diastolic ≥ 90 mmHg), is a major contributor to cardiovascular diseases (CVD) and premature mortality worldwide [1]. It frequently occurs alongside functional or structural damage to organs like the heart, brain, and kidneys [2–4]. In the process of hypertension, vascular remodeling is a major pathological process that contributes to target organ damage and raises the risk of aortic sclerosis and cardiovascular events. It is defined by inflammation, thickening of the elastic layer, and fibrosis. Inhibiting vascular remodeling is therefore critical for improving clinical outcomes in hypertensive patients [5]. Studies have shown that ferroptosis-related Toll-like receptor 4 (TLR4) is upregulated in hypertension and is linked to vascular inflammation and remodeling [6–8]. Angiotensin II (Ang II)-induced hypertension further exacerbates these processes through the MyD88/NF-κB/JNK signaling pathway [9] Our previous research confirms Ang II can activate TGF-β1/MAPK signaling pathway, promoting vascular remodeling [10]. However, whether the TLR4 signal is an upstream component of the MAPK/TGF-β1 signaling pathway still requires further exploration.

However, despite these insights into the molecular mechanisms of hypertension, current antihypertensive treatments remain largely focused on controlling blood pressure rather than addressing the underlying vascular remodeling. The main categories of medications utilized in clinical practice—diuretics, β-blockers, calcium channel blockers, angiotensin-converting enzyme (ACE) inhibitors, and angiotensin II receptor blockers (ARBs)—are effective in managing blood pressure and minimizing complications such as left ventricular failure and stroke. Nevertheless, these treatments offer limited protection against the progressive remodeling of blood vessels. Additionally, long-term use of these medications is often associated with adverse effects, underscoring the need for more comprehensive therapeutic approaches that can simultaneously target both hypertension and its impact on vascular structure [11].

In recent years, the modernization and internationalization of Traditional Chinese Medicine (TCM) have gained momentum, with extensive research being conducted on various herbal remedies and their active compounds. A well-known Chinese pharmacopoeia herb known as Nelumbo nucifera Gaertn has historically been used to treat hypertension [12]. Modern pharmacological studies have demonstrated that the alkaloid components of lotus plumule possess antihypertensive, anti-arrhythmic, antioxidant, and anti-inflammatory properties [13]. Among these alkaloids, liensinine (Lien, C₃₇H₄₂N₂O₆) stands out as a potent bioactive compound with well-documented cardiovascular protective effects [14–18]. Previous studies have shown that Lien can lower blood pressure and improve vascular remodeling [10]. However, there is still much to learn about the specific pathways that underlie its antihypertensive and vasoprotective properties.

In this study, we aim to investigate the therapeutic potential of Lien in hypertension by examining its effects on vascular inflammation and remodeling. Through in vivo and in vitro experiments, we demonstrate that Lien can inhibit ferroptosis-related TLR4-mediated inflammation, improve vascular remodeling, and modulate related signaling pathways, including mitogen-activated protein kinase (MAPK) and transforming growth factor-β1 (TGF-β1)/Smad2/3. More importantly, we show that Lien can competitively bind to the key TLR4 target myeloid differentiation protein-2 (MD2), thereby directly inhibiting Ang II-induced vascular remodeling. These findings provide new insights into the mechanism of hypertension and highlight Lien as a promising therapeutic candidate for the clinical management of hypertensive vascular remodeling.

Materials and methods

Liensinine preparation

Liensinine (Lien, purity 99.97%) was purchased from MedChemExpress (2586-96-1). For animal experiments, Lien was dissolved in NaCl solution to prepare a 5mg/mL working solution with ultrasonic assistance. After preliminary experiments, the final dose for animal studies was set at 5 mg/kg, administered via intraperitoneal injection every other day. For cell experiments, Lien was dissolved in a sterile PBS and DMSO mixture to a final concentration of 700µg/mL and kept at −20 °C, protected from light until further use.

Animal experiments

Male C57BL/6 mice were housed in the Animal Experimental Center of Fujian University of Traditional Chinese Medicine under controlled conditions (temperature, 20.8–24.3 °C; 12-h light/dark cycle). All mice had free access to food and water. Animal experiments were authorized by the Animal Management and Use Committee of Fujian University of Traditional Chinese Medicine (Ethics No FJTCM IACUC2021140) and complied with China’s “Guiding Opinions on Treating Experimental Animals.” The Ang II group, Ang II + Va l (valsartan) group, and Ang II + Lien group were continuously infused with Ang II (500 ng/kg/min) using an Alzet osmotic pump, while the control group received an equivalent volume of saline. Mice were anesthetized using isoflurane, and a longitudinal incision was performed in the scapular area to implant the osmotic pump. After surgery, the wound was sutured, and mice were provided with thermal support to enhance the success rate of the hypertensive model. Blood pressure measurements were taken to confirm the establishment of the hypertension model, and the Lien intervention group received intraperitoneal injections of Lien every other day.

Blood pressure measurement

The blood pressure of the mouse tail artery was assessed weekly with a Kent non-invasive blood pressure monitoring system, in conjunction with body weight measurements. The process involved placing a hollow occlusive sleeve at the base of the mouse tail, inflating the pressure sensor, and monitoring blood flow as it returned to the tail artery. Twenty rounds were used as the software set, and the average of the readings was used to calculate the mean arterial pressure, systolic, and diastolic values.

Small animal ultrasound

Small animal ultrasonography was done following the conclusion of hypertensive modeling. Mice were continuously anesthetized with isoflurane and fixed on the ultrasound examination table. Using depilatory cream, chest and abdominal hair were taken out. A color Doppler ultrasound system was used to assess aortic pulsation and pulse wave velocity (PWV).

Tissue processing

Following the ultrasound examination, tissues were collected. The mice were anesthetized with isoflurane and euthanized by cervical dislocation. Aortas were collected, with some portions stored in RNA preservation solution for RNA sequencing, others fixed in 4% paraformaldehyde for histopathological analysis (HE staining) and immunohistochemical (IHC) examination, and the remainder snap-frozen in liquid nitrogen and stored at −80 °C for Western blotting. Blood was also collected for ELISA cytokine analysis.

RNA-seq analysis

Trizol was used to extract total RNA from vascular tissues, and an Agilent 2100 Bioanalyzer and Qubit Fluorometer were used to measure the integrity of the RNA. RNA-seq libraries were created and sequenced by CapitalBio Technology (Beijing, China) with the NeoB Next Ultra RNA Library Prep Kit for Illumina sequencing. Differentially expressed genes were identified based on a fold-change threshold of |log2FC|≥ 1 and p ≤ 0.05, and pathway analysis was conducted using GO and KEGG databases.

HE staining and IHC analysis

HE staining was used to perform theological analysis on paraffin-embedded aortic sections. For IHC, sections were dewaxed, antigen retrieval was conducted using sodium citrate, and primary antibodies were incubated overnight at 4 °C. On the following day, secondary antibodies were used; DAB was used to visualize the staining, and hematoxylin was used as a counterstain. For each animal, the continuous cross-section of the aorta was taken, and three fields of view were selected for photography and statistics. Images were taken at a 400 × magnification with a light microscope, and protein expression was measured using ImageJ software. The DAB histochemical kit (DAB-0031), antigen retrieval solution (MVS-0066), and Immunohistochemistry kit (KIT-9720) were supplied by Maixin Biotechnology (Fuzhou, China).

ELISA

Using commercially available ELISA kits, the amounts of cytokines (IL-6, IL-1β, TNF-α) [Rat (EK301B/3–96 T, EK306/3–96 T, EK382/3–96 T) and mouse (EK206-96 T, EK201B/3–96 T, EK282/4–96 T) cytokine ELISA kits (IL-6, IL-1β, TNF-α) were purchased from Multisciences (Hangzhou, China)] in serum were determined in accordance with the manufacturer’s instructions. At 450 nm, absorbance was measured; standard curves were used to determine the amounts of cytokines.

Cell culture

Rat arterial smooth muscle cells (A7R5) were acquired from Wuhan Procell Life Science & Technology Co., Ltd. A7R5 cells were cultivated in DMEM full medium containing 10% fetal bovine serum (FBS) and 1% penicillin–streptomycin in a 37 °C, 5% CO₂ incubator. DMEM (25,200,072), fetal bovine serum (FBS) (10,099), Trypsin–EDTA (25,200,072), and BCA (DAB-0031) were procured from Thermo Fisher Scientific (MA, USA). The culture medium was changed every 2 days, and the cells were used for experiments once they reached the logarithmic growth phase (the cells grow to the 3rd to 10th generations).

Molecular docking experiment

The 3D structure of Lien was downloaded from the PubChem database in SDF format based on its CAS number. The structure was imported into ChemBio3D Ultra 14.0 for energy minimization, with the minimum RMS gradient set to 0.001, and saved in mol2 format. The optimized small molecule was then imported into AutoDockTools-1.5.6, where hydrogen atoms were added, charges calculated, and rotatable bonds set before saving the file in “pdbqt” format. The protein structure of mouse MD2 (PDB ID: 3VQ2) was downloaded from the PDB database. Using PyMOL2.3.0, crystal water and original ligands were removed, and the protein structure was processed with AutoDockTools-1.5.6 by adding hydrogen atoms, calculating charges, assigning atom types, and saving in “pdbqt” format. The protein binding site was predicted using POCASA 1.1, and molecular docking was performed using AutoDock Vina1.1.2. Parameters associated with ITPR1 and PLCB1 were established, and PyMOL2.3.0 was employed to analyze the interaction patterns from the docking results.

Molecular dynamics simulation experiment

Based on the molecular docking results, AMBER 22 was used to perform molecular dynamics (MD) simulations to investigate key interactions between Lien and MD2. The Lien pdb file was converted to mol2 format using Antechamber, and the electrostatic potential (ESP) of the small molecule was calculated using the Gaussian 16 software package. RESP was used to analyze the ESP and obtain the atomic charges of the small molecule. The MD2 protein was processed using pdb4amber to obtain the final formatted pdb file. After loading the MD simulation parameters, topology files (.prmtop) and input trajectory files (.inpcrd) were created, completing the system construction. Prior to the MD simulation, the system underwent energy optimization; the steepest descent method was employed for constrained backbone minimization, while unconstrained minimization was achieved using the conjugate gradient method, leading to an initial equilibrium structure. The system was then heated to 300 K using a 300-ps constant-volume NVT simulation, followed by a 20-ns NVT equilibration. The formal simulation was performed in the NVT ensemble, utilizing the PME method for long-range electrostatic interactions, applying hydrogen bond constraints with the SHAKE algorithm, and using a time step of 2 fs for the simulation. At 1 nm, there were truncated non-bonded contacts, and every 5000 steps were recorded in coordinates. A 200-ns molecular dynamics simulation was performed on the compound system of Lien and MD2, and the stability of the system was assessed by calculating the root-mean-square deviation (RMSD).

Western blot

Tissues or cells were gathered, lysed, and proteins extracted. For electrophoresis, the proteins were separated and placed onto a PAGE gel; then, they were transferred onto PVDF membranes. Following the blocking of nonspecific proteins with a blocking buffer, the membranes were incubated with primary and secondary antibodies. Antibodies against MyD88 (67969-1-Ig), tubulin (D20G3), GAPDH (D16H11), β-actin (13E5), phospho-P38 (4511s), phospho-ERK (4370S), phospho-P65 (3036S), IκB-α (4814S), and phospho-IκB-α (2859S) were obtained from Cell Signaling Technology (Boston, USA). Vinculin (26520-1-AP), P38 (14064-1-AP), ERK (11257-1-AP), JNK (66210-1-Ig), PCNA (10205-2-AP), P65 (66535-1-Ig), goat anti-rabbit IgG (SA00001-2), goat anti-mouse IgG (SA00001-1) antibodies, and GPX4 (14432-1-AP) were purchased from Proteintech (Wuhan, China). TFRC (PT0360R) was purchased from Immunoway. Antibodies for TGF-β1 (ABP52598), Smad2/3 (ABP57568), phospho-Smad2/3 (8828S), and phospho-JNK (ABP50351) were from Abbkine (Wuhan, China). The improved chemiluminescence (ECL) was used to visualize the protein bands, which were then measured using ImageJ software.

Co-immunoprecipitation (COIP)

Cells were plated at a density of 1.5 × 10⁵ cells in 10-cm dishes. After stimulation, two dishes were pooled for each condition. Cellular proteins were extracted and quantified using BCA. The proteins were divided into two parts: one for positive control (input) and the other for IP (immunoprecipitation with Flag antibody). IP required 800–1000 µg of protein. Magnetic beads were washed with lysis buffer containing PMSF (phenylmethylsulfonyl fluoride) to remove storage buffer. For each tube, 20 µL of protein A/G magnetic beads was washed three times and then resuspended in lysis buffer. Antibodies were incubated with the magnetic beads for 2h at 4 °C on a shaker. The beads were then incubated with 1 µg of protein in lysis buffer overnight at 4 °C. After incubation, the beads were washed with lysis buffer containing PMSF three times. Finally, the beads were resuspended in 2 × loading buffer and heated in a metal bath for 10 min at 100 °C. Protein A + G sepharose beads (P001-2) were acquired from Shanghai Qihai Futai Biotechnology Co., Ltd. (Shanghai, China). MD2 (ab24182) was from Abcam (Cambridge, UK). The immunoprecipitated proteins were then analyzed by Western blot.

Statistical analysis

Statistical analyses were performed using SPSS 26.0 (IBM Corp, Armonk, NY). Data were expressed as mean ± standard deviation. If the variance was not uniform, the Games Howell method was used for pairwise comparison. One-way analysis of variance was used. If the variance was equal, the T-TEST method was used for pairwise comparison between groups. A p-value of < 0.05 was considered statistically significant. No formal power calculation was conducted. All experiments were independently repeated at least three times (biological repetition). The sample size for the animal experiments was determined based on the effect size from the previous pre-experiment and previous literature.

Results

Lien reduces hypertension and mitigates vascular pathology in Ang II-induced hypertensive mice

The Ang II-induced hypertension model via subcutaneous pump implantation is a useful tool for studying the coordinated regulation of multiple cardiovascular factors in hypertension [19]. In this study, the hypertension model was established by administering Ang II (500 ng/kg/min) for 4 weeks, followed by Lien intervention. By day 7 post-implantation, the systolic, diastolic, and mean arterial pressures in the Ang II model group were significantly higher than in the control group, confirming successful model induction. Blood pressure measurements on days 7, 14, 21, and 28 revealed that blood pressure remained higher in the Ang II group compared to the control group. Treatment with valsartan (Val) or low, medium, and high doses of Lien significantly reduced systolic, diastolic, and mean arterial pressures compared to the Ang II group. The low-dose Lien group had a lower diastolic pressure than the Ang II group by day 28, although the difference was not statistically significant (Fig. 1A-C). Monitoring body weight during the intervention showed that Lien had no impact on mouse body weight (Fig. 1D). Small animal ultrasound revealed increased aortic medial thickness and pulse wave velocity (PWV) in the Ang II group compared to controls, while both the Val and Lien groups showed reductions in medial thickness and PWV (Fig. 1E and F). HE staining of aortic sections showed disorganized cells and increased vessel wall thickness in the Ang II group compared to controls. Treatment with Lien and Val reduced the disorganization and vessel wall thickening seen in the Ang II group (Fig. 1G). These results suggest that Lien can reduce Ang II-induced hypertension without affecting body weight and can alleviate pathological damage in the abdominal aorta of hypertensive mice.

Fig. 1.

Lien reduces hypertension and mitigates vascular pathology in Ang II-induced hypertensive mice. A–C Changes in systolic, diastolic, and mean arterial pressures in each group of mice (n = 6). D Changes in body weight of mice in each group (n = 6). E Representative small animal ultrasound images showing aortic medial thickness (right), and corresponding statistical graph of medial thickness (left) (n = 3). F Representative small animal ultrasound images showing abdominal aortic pulse wave velocity (PWV) (right) and statistical graph of PWV (left) (n = 3). G Representative HE-stained sections of abdominal aorta (right) and statistical graph of vascular wall thickness (n = 3). The data are expressed as the mean ± SD. ^***p < 0.001 Ang II vs. control. ^%%%p < 0.001 Ang II + Val (10.4 mg/kg) vs. Ang II ^&&p < 0.01; ^&&&p < 0.001 Ang II + Lien (2.5 mg/kg) vs. Ang II ^##p < 0.01; ^###p < 0.001 Ang II + Lien (5 mg/kg) vs. Ang II ^$$$p < 0.001 Ang II + Lien (10 mg/kg) vs. Ang II

Lien inhibits Ang II-induced ferroptosis-related inflammatory responses in hypertensive mice

The WB results indicated that, compared with the control group, the expression of ferroptosis-related protein GPX4 decreased and the expression of TFRC increased in the Ang II group. Compared with the Ang II group, after Lien intervention, the expression of GPX4 increased while the expression of TFRC decreased (Fig. 2A). We used GO and KEGG databases on differentially expressed genes (DEGs) to perform pathway enrichment analyses in order to investigate the signaling pathways that are regulated by Lien. GO analysis revealed enrichment of 1398 genes in the Ang II group compared to controls and 939 genes in the Lien group compared to the Ang II group, with 375 overlapping genes between the two comparisons (Fig. 2B). Among these overlapping genes, 26 inflammation-related pathways were enriched (Fig. 2C). KEGG analysis identified 25 Toll-like receptor (TLR)-related inflammation pathways that were enriched in the Ang II versus control and Lien versus Ang II comparisons (Fig. 2D). These data point to the possibility that Lien might help reduce vascular inflammation brought on by hypertension. RNA sequencing data further confirmed that Ang II induces vascular inflammation in hypertension [20], while Lien suppresses this inflammatory response. When compared to controls, the Ang II-induced inflammation in hypertensive mice in the Ang II group was shown in comparison to the serum levels of pro-inflammatory cytokines IL-6, IL-1β, and TNF-α in the ELISA. The release of certain cytokines was markedly decreased by Lien treatment (Fig. 2E). IHC analysis of abdominal aortic tissues revealed increased p-P65/P65 and p-IκB-α/IκB-α ratios in the Ang II group compared to controls, while Lien intervention reduced these ratios (Fig. 2F). Based on these data, Lien may prevent vascular inflammation caused by Ang II in people with hypertension.

Fig. 2.

Lien inhibits Ang II-induced ferroptosis-related inflammatory responses in hypertensive mice. A Western blot analysis of the effects of GPX4 and TFRC (n = 3). B, C GO functional enrichment analysis of the effect of Lien treatment on vascular inflammation (n = 5). D KEGG pathway analysis of the effect of Lien treatment on vascular inflammation (n = 5). E ELISA results showing the secretion of inflammatory cytokines IL-1β, IL-6, and TNF-α in the serum of each group of mice (n = 5). F IHC detection of p-P65, P65, p-IκB-α, and IκB-α expression in the abdominal aorta (n = 3). The data are expressed as the mean ± SD. ^*p < 0.05, ^**p < 0.01, ^***p < 0.001, compared with the control group; ^#p < 0.05, ^##p < 0.01, ^###p < 0.001 compared with the Ang II group

Lien inhibits inflammation-induced vascular remodeling in hypertension

In vitro experiments using Ang II to simulate hypertension showed activation of the MAPK and TGF-β1/Smad2/3 pathways compared to the control group. To further determine whether inflammation activates these pathways, we used lipopolysaccharide (LPS) and a cocktail of cytokines (CKs) to stimulate inflammatory signaling and observed that both LPS-induced TLR4 and CKs-induced NF-κB signaling activated the MAPK and TGF-β1/Smad2/3 pathways (Fig. 3A). In vivo experiments revealed increased PCNA expression and elevated p-JNK/JNK ratios in the Ang II group compared to controls. Lien treatment reduced PCNA expression and p-JNK/JNK ratios (Fig. 3B). These findings suggest that inflammatory responses activate vascular remodeling pathways. Given previous research indicating that Lien improves vascular remodeling [10], this study further explores the effects of Lien on vascular inflammation and remodeling in hypertension.

Fig. 3.

Lien suppresses inflammatory activation of MAPK and TGF-β1/Smad2/3 pathways in hypertension. A Western blot analysis of the effects of Ang II, CKs, and LPS stimulation on MAPK and TGF-β1/Smad2/3 signaling pathway-related proteins (n = 3). B IHC detection of PCNA, p-JNK, and JNK expression in the abdominal aorta of each group of mice (n = 3). The data are expressed as the mean ± SD. ^*p < 0.05, ^**p < 0.01, ^***p < 0.001, compared with the control group; ^#p < 0.05, ^##p < 0.01, ^###p < 0.001 compared with the Ang II group

Lien inhibits the formation of the Ang II-TLR4-MD2 complex to reduce hypertension

It was reported that angiogenesis results in TLR4 and MD2, which are complicated by the activation of downstream pathways [21, 22] . We hypothesized that Lien might exert its therapeutic effects by inhibiting the formation of the Ang II-TLR4-MD2 complex. The WB results indicated that, compared with the control group, Lien alone had no significant effect on the expression of ferroptosis-related protein GPX4 and TFRC in A7R5 cells, but the expression of ferroptosis-related protein GPX4 decreased and the expression of TFRC increased in the Ang II group. Compared with the Ang II group, after Lien intervention, the expression of GPX4 increased while the expression of TFRC decreased (Fig. 4A). ELISA results showed that Lien alone had no significant effect on inflammatory cytokine secretion in A7R5 cells, while Ang II stimulation significantly increased TNF-α, IL-6, and IL-1β levels. The Lien treatment decreased the levels of these cytokines in the cell supernatant (Fig. 4B). A stable contact between Lien and MD2 was suggested by molecular docking and molecular dynamics simulations (Fig. 4C). In vivo experiments confirmed that Lien reduced the expression of MyD88, a downstream protein of TLR4, in the abdominal aortas of Ang II-induced hypertensive mice (Fig. 4D). COIP experiments showed that Ang II stimulation enhanced the interaction between MD2 and TLR4 in A7R5 cells, while Lien intervention weakened this interaction (Fig. 4E). Remarkably, the MD2-TLR4 interaction was not impacted by Val (Fig. 4F), providing insights into the mechanism by which Val requires lifelong use and the development of resistance due to its inability to directly inhibit inflammation-induced vascular remodeling. These results suggest that Lien exerts its therapeutic effects by inhibiting the formation of the Ang II-TLR4-MD2 complex.

Fig. 4.

Lien inhibits the formation of the Ang II-TLR4-MD2 complex to reduce hypertension. A Western blot analysis of the effects of GPX4 and TFRC (n = 3). B ELISA analysis of Lien’s effects on the secretion of inflammatory cytokines IL-1β, IL-6, and TNF-α in the supernatant of A7R5 cells (n = 6). C Molecular docking diagram between MD2 protein and Lien (left upper); prediction of the protein binding site on MD2 (right upper); molecular dynamics simulation between Lien and MD2 (below). D Western blot detection of MyD88 expression in the abdominal aorta of each group of mice (n = 3). E, F COIP analysis of the effects of Lien and valsartan on the Ang II-TLR4-MD2 complex. The data are expressed as the mean ± SD. ^*p < 0.05, ^**p < 0.01, ^***p < 0.001, compared with the control group; ^#p < 0.05, ^##p < 0.01, ^###p < 0.001 compared with the Ang Ⅱ group

Lien suppresses TLR4-induced MAPK and TGF-β1/Smad2/3 activation in hypertensive mice

To verify the inhibitory effect of Lien on the TLR4 inflammatory response in A7R5 cells, we conducted Western blot analysis of TLR4 signaling activation. The expression of TLR4, MyD88, p-IκB-α, and p-P65 was dramatically increased by Ang II stimulation, whereas the expression of IκB-α was decreased. Lien treatment reversed these effects, suppressing the Ang II-induced TLR4 inflammatory signaling (Fig. 5A). Further experiments using LPS to activate TLR4 inflammation showed that LPS stimulation activated the MAPK and TGF-β1/Smad2/3 pathways, which were inhibited by Lien (Fig. 5B). These findings suggest that Lien inhibits the activation of these pathways induced by the TLR4 inflammatory response.

Fig. 5.

Lien suppresses TLR4-induced MAPK and TGF-β1/Smad2/3 activation in hypertensive mice. A The effects of Lien on the expression of TLR4, MyD88, p-P65, P65, p-IκB-α, and IκB-α in A7R5 cells stimulated by Ang II. B The effects of Lien on the expression of p-ERK, ERK, p-P38, P38, p-JNK, JNK, TGF-β1, p-Smad2/3, and Smad2/3 in A7R5 cells stimulated by LPS. C The effects of Lien and TAK242 on the expression of p-ERK, ERK, p-P38, P38, p-JNK, JNK, TGF-β1, p-Smad2/3, and Smad2/3 in A7R5 cells stimulated by Ang II (n = 3 per group). The data are expressed as the mean ± SD. ^*p < 0.05, ^**p < 0.01, ^***p < 0.001, compared with the control group; ^#p < 0.05, ^##p < 0.01, ^###p < 0.001 compared with the model group

To further validate the hypothesis that “Ang II-induced TLR4 inflammatory signaling activation in A7R5 can promote the activation of MAPK and TGF-β1/Smad2/3 signaling pathways related to vascular remodeling,” we intervened with the TLR4 inhibitor TAK242 in Ang II-stimulated A7R5 cells to observe its effects on the vascular remodeling-related pathways MAPK and TGF-β1/Smad2/3. The results showed that compared to the blank control group, Ang II stimulation led to the activation of the MAPK and TGF-β1/Smad2/3 signaling pathways. In comparison to the Ang II modeling group, the intervention with TAK242 notably decreased the levels of p-ERK, p-JNK, p-P38, TGF-β1, and p-Smad2/3, thereby inhibiting the activation of the MAPK and TGF-β1/Smad2/3 signaling pathways. Similarly, the Lien intervention also suppressed the activation of these pathways. Furthermore, the combination of TAK242 and Lien resulted in a significant reduction in the expression of the aforementioned proteins and also inhibited the activation of both the MAPK and TGF-β1/Smad2/3 signaling pathways (Fig. 5C). The results presented above suggest that Ang II-induced TLR4 inflammatory responses can enhance the activation of proliferation pathways associated with vascular remodeling, namely MAPK and TGF-β1/Smad2/3. Furthermore, Lien’s inhibitory effects on MAPK and TGF-β1/Smad2/3 may not solely occur through the suppression of TLR4 inflammatory responses; there could also be additional targets that warrant further exploration.

Lien inhibits NF-κB-induced activation of MAPK and TGF-β1/Smad2/3 in vascular remodeling

To investigate whether activation of the NF-κB inflammatory signaling downstream of TLR4 could promote the activation of MAPK and TGF-β1/Smad2/3 pathways, and to assess the effect of Lien, we used NF-κB activators (CKs) to stimulate A7R5 cells in combination with Lien treatment. Western blot analysis was employed to identify the activation of the MAPK and TGF-β1/Smad2/3 pathways. According to the data, in contrast to the control group, CKs stimulation raised the expression of p-ERK, p-JNK, p-P38, TGF-β1, and p-Smad2/3, which suggested that CKs stimulation activated the MAPK and TGF-β1/Smad2/3 pathways. In comparison to the model group, the CKs + Lien treatment significantly decreased the expression of these proteins, indicating that Lien may inhibit CKs-induced MAPK proliferative signaling and the activation of the TGF-β1/Smad2/3 pro-fibrotic pathway (Fig. 6A). These findings suggest that the NF-κB inflammatory response induced by CKs can activate vascular remodeling-related pathways, MAPK, and TGF-β1/Smad2/3, and this activation can be inhibited by Lien. To further validate the hypothesis that “Ang II-induced NF-κB inflammatory signaling in A7R5 cells promotes the activation of MAPK and TGF-β1/Smad2/3 pathways,” we used the NF-κB inhibitor PDTC to treat Ang II-stimulated A7R5 cells and observed its effects on the MAPK and TGF-β1/Smad2/3 pathways. The results showed that, compared to the control group, Ang II stimulation activated the MAPK and TGF-β1/Smad2/3 pathways. In comparison to the Ang II model group, PDTC treatment substantially decreased the levels of p-ERK, p-JNK, p-P38, TGF-β1, and p-Smad2/3, thereby inhibiting the activation of the MAPK and TGF-β1/Smad2/3 pathways. Additionally, Lien therapy decreased the activation of these pathways. Furthermore, the combined treatment of PDTC and Lien significantly reduced the expression of these proteins, similarly inhibiting the activation of MAPK and TGF-β1/Smad2/3 pathways (Fig. 6B). These results indicate that the NF-κB inflammatory response induced by Ang II can promote the activation of MAPK and TGF-β1/Smad2/3 pathways, and Lien’s inhibitory effect on these pathways may not be solely due to its suppression of NF-κB inflammation but may involve other targets, which warrant further investigation.

Fig. 6.

Lien inhibits NF-κB-induced activation of MAPK and TGF-β1/Smad2/3 in vascular remodeling. A The effects of Lien on the expression of p-ERK, ERK, p-P38, P38, p-JNK, JNK, TGF-β1, p-Smad2/3, and Smad2/3 in A7R5 cells stimulated by CKs. B The effects of Lien and PDTC on the expression of p-P65, P65, ERK, p-P38, P38, p-JNK, JNK, TGF-β1, p-Smad2/3, and Smad2/3 in A7R5 cells stimulated by Ang II (n = 3 per group). The data are expressed as the mean ± SD. ^*p < 0.05, ^**p < 0.01, ^***p < 0.001, compared with the control group; ^#p < 0.05, ^##p < 0.01, ^###p < 0.001 compared with the model group

Discussion

Chronic cardiovascular disease called hypertension seriously impedes the human body [23]. It is well recognized that ferroptosis-related inflammation is a critical factor in the development of hypertension and its related complications [24, 25] Our previous research showed that Lien, a natural compound, exhibits significant antihypertensive effects by markedly lowering systolic, diastolic, and mean arterial pressures in Ang II-induced hypertensive mice, all while having no impact on body weight. Beyond lowering blood pressure, Lien effectively reduced PWV and inhibited the thickening of the aortic medial layer, indicating its potential to prevent both hypertension and hypertension-induced vascular dysfunction. Furthermore, we discovered that Lien can inhibit vascular remodeling through the MAPK/TGF-β1/Smad2/3 signaling pathway [10]. The results of this study once again demonstrate that Lien has the effect of treating hypertension-induced vascular remodeling. However, we wonder what the upstream factor causing vascular remodeling is. By consulting the literature, vascular inflammation is closely connected to the process of vascular remodeling, particularly in cases of hypertension. Inflammatory responses are known to play a critical role in the progression of remodeling, which is characterized by structural changes such as cell proliferation, migration, collagen deposition, phenotypic changes, and apoptosis. These changes lead to increased vascular stiffness and progressive dysfunction [7, 26]. The association between inflammation and vascular remodeling in hypertension is further highlighted by the sustained activation of the renin–aAngiotensin–aldosterone system (RAAS). Ang II, a key factor in hypertension, not only induces continuous vasoconstriction but also triggers TLR4-mediated inflammatory responses, exacerbating pathological changes over time and contributing to vascular damage. Thus, in hypertensive circumstances, inflammation becomes a major factor in the vascular remodeling process. It has been reported that ferroptosis can trigger the TLR4 inflammatory response. In this process, GPX4 uses glutathione (GSH) to reduce lipid peroxides into non-toxic lipids, thereby blocking the ferroptosis process. Transferrin receptor 1 (TFRC) is a transferrin receptor located on the cell membrane. An increase in its expression leads to an increase in intracellular iron levels, which promotes the accumulation of lipid peroxides and triggers ferroptosis, thereby causing the TLR4 inflammatory response [27, 28]. The results of the WB assay indicated that Lien can promote the expression of the ferroptosis negative regulatory factor GPX4 and also inhibit the expression of the iron ion transporter receptor TFRC in vascular. At the same time, RNA-seq analysis revealed that Lien can inhibit Ang II-induced vascular inflammation. Our study shows that Ang II activates the TLR4 pathway, leading to increased levels of pro-inflammatory cytokines such as TNF-α, IL-6, and IL-1β in hypertensive mice. ELISA showed that Lien treatment dramatically decreased these cytokine levels, which suggests that Lien has strong anti-inflammatory properties. Additionally, IHC analysis revealed that Lien decreased the expression of p-IκB-α/IκB-α and p-P65/P65 in the abdominal aorta of hypertensive mice. These findings confirm that Lien inhibits the TLR4/NF-κB inflammatory signaling pathway, effectively curbing the inflammatory responses driven by Ang II. As a result, Lien’s anti-inflammatory properties provide a promising approach to prevent vascular remodeling by targeting the underlying inflammation in hypertension. Therefore, we speculate that Lien may exert its therapeutic effect on hypertension by inhibiting TLR4-induced vascular inflammation and thereby suppressing vascular remodeling. The Ang II infusion model provides a reliable and reproducible approach to study RAAS-dependent hypertension and vascular remodeling, making it suitable for mechanistic investigations. Nevertheless, this model mainly reflects RAAS-driven pathology, whereas human essential hypertension is multifactorial. Thus, while our findings are mechanistically informative, extrapolation to clinical patients should be cautious, and validation in other hypertension models will be valuable.

To further investigate the relationship between vascular inflammation and remodeling, we employed classical inflammatory stimuli, such as lipopolysaccharide (LPS) and cytokine cocktails (CKs), in addition to Ang II. Although hypertension is not directly caused by LPS or CKs, these well-established inflammatory stimulators are widely used in research to model inflammatory responses. LPS, derived from Gram-negative bacteria like E. coli, is a potent activator of the TLR4 signaling pathway, and LPS-induced inflammation has been shown to promote vascular dysfunction in animal models [29]. Similarly, CKs, which contain inflammatory mediators such as IL-1β, TNF-α, and GM-CSF, induce widespread inflammatory responses through NF-κB signaling activation [30–33]. The present study was conducted by simulating the inflammatory conditions that contribute to vascular remodeling using both LPS and CKs. Our findings demonstrated that the inflammatory response—whether driven by Ang II, LPS, or CKs—can activate key pathways associated with vascular remodeling, including MAPK and TGF-β1/Smad2/3. This supports the hypothesis that targeting inflammation could be a therapeutic strategy for addressing vascular remodeling in hypertension.

To delve deeper into how Lien produces its anti-inflammatory and anti-remodeling effects, we examined the TLR4 signaling pathway, which is crucial in mediating vascular inflammation in conditions like atherosclerosis, diabetes, and hypertension. TLR4, a key member of the pattern recognition receptor family, is known to initiate a signaling cascade upon activation by recruiting MyD88. This cascade involves the activation of IRAK4, TRAF6, and the IKK complex, leading to the phosphorylation and nuclear translocation of NF-κB. The result is the increase in pro-inflammatory cytokines, including TNF-α, IL-6, and IL-1β, which also worsen vascular inflammation [34–36]. Given the involvement of this pathway in inflammation-driven vascular remodeling, TLR4 was chosen as a potential therapeutic target. Previous studies suggest that Ang II forms a complex with TLR4 and MD2 [21, 22], further activating downstream inflammatory pathways. Our research demonstrated that Lien prevents the formation of the Ang II-TLR4-MD2 complex and lowers MyD88 expression, which in turn inhibits downstream signaling and reduces the secretion of pro-inflammatory cytokines in A7R5 cells stimulated by Ang II.

To validate the role of TLR4 in the anti-inflammatory effects of Lien, we used the TLR4 activator LPS and TLR4 inhibitor TAK242 [37]. The results showed that Lien could significantly inhibit the activation of MAPK and TGF-β1/Smad2/3 signaling pathways induced by LPS. Furthermore, TAK242 significantly decreased the activation of the MAPK and TGF-β1/Smad2/3 pathways in A7R5 cells stimulated by Ang II. When combined with Lien, this inhibition was even more pronounced, suggesting that suppressing TLR4-mediated inflammation is critical for preventing vascular remodeling. Additionally, we used the NF-κB activator CKs and NF-κB inhibitor PDTC to investigate whether NF-κB-mediated inflammation contributed to the activation of these pathways. The results showed that Lien could significantly inhibit the activation of MAPK and TGF-β1/Smad2/3 signaling pathways induced by CKs. Compared with TAK242, PDTC showed an even stronger inhibitory effect on the MAPK and TGF-β1/Smad2/3 pathways, probably because of the central role of NF-κB in mediating different inflammatory responses. The combination of PDTC and Lien resulted in the most significant inhibition of these pathways, further confirming the key role of NF-κB in hypertension-induced vascular remodeling. Lien treatment effectively inhibited the activation of these pathways, confirming its role in preventing vascular remodeling by suppressing inflammation. Additionally, Lien inhibited vascular cell proliferation, further demonstrating its ability to counteract hypertensive vascular remodeling.

Taken together, these findings suggest that decreasing both ferroptosis-related TLR4- and NF-κB-mediated inflammation successfully decreases vascular remodeling in hypertension. Lien, through its ability to competitively inhibit TLR4/MD2 binding, represents a promising therapeutic strategy for treating hypertension and preventing vascular remodeling. Furthermore, Lien’s long-term protective effects on vascular tissues may provide additional benefits beyond standard antihypertensive treatments, which primarily focus on blood pressure control without directly targeting the underlying inflammatory pathways. Lien lowers blood pressure and attenuates vascular remodeling in Ang II-induced models at low doses while inhibiting MAPK and TGF-β1/Smad2/3 pathways [10]. Rat PK studies show a half-life of 6–8 h, but human data are lacking [38]. Limited evidence suggests short-term tolerability [39], though potential cardiotoxicity via hERG interaction requires evaluation. Compared with standard antihypertensives, Lien may offer added benefit by targeting inflammatory and remodeling pathways, supporting its role as a candidate adjunct pending dose-finding and safety studies. However, this study has several limitations. We employed only a single hypertensive model; future work should validate the findings in spontaneously hypertensive rats (SHR) or DOCA-salt hypertensive models. In terms of mechanism research, in the future, we will still need to use VSMC-specific TLR4⁻/⁻ or GPX4⁻/⁻ mice. In addition, long-term toxicity and pharmacokinetic data for Lien are absent; dose–response curves and comprehensive toxicological studies are planned to provide a basis for clinical-trial design. Although we demonstrate that Lien markedly reduces phosphorylation of MAPKs (ERK, JNK, p38) and suppresses the TGF-β1/Smad2/3 pathway, we cannot yet determine whether this suppression arises from a direct action on the kinase machinery or receptor/Smad phosphorylation or is secondary to upstream anti-inflammatory effects; distinguishing these possibilities will require mechanistic follow-up studies such as isolated kinase assays, receptor phosphorylation analyses, and experimental systems in which inflammatory drivers are minimized.

In conclusion, our study confirms that inflammation plays a critical role in promoting vascular remodeling, and that Lien can improve hypertensive vascular remodeling by inhibiting ferroptosis-related TLR4-mediated inflammatory responses (Fig. 7). Lien, however, has similar antihypertensive properties to valsartan; however, the former may be able to directly inhibit the activation of inflammatory pathways in vascular tissues, which could be a long-term therapeutic benefit. These findings offer new insights into the treatment of hypertension and support the potential clinical application of Lien as a therapeutic agent.

Fig. 7.

Molecular mechanism of Lien to improve vascular remodeling through ferroptosis-related TLR4 inflammatory pathway

Supplementary Information

Below is the link to the electronic supplementary material.

Author contribution statement

Daxin Chen: methodology, visualization, writing—original draft. Peizhi Jia: methodology, investigation, writing—original draft. Meiling Wang: methodology, visualization. Baojie Du: investigation, verification. Haitao Du: visualization, validation. Xi Huang and Jianwei Zeng: conceptualization. Qiaoyan Cai: validation. Ling Zhang: methodology. Ying Zhu: validation. Shan Lin: manuscript preparation—review and editing, supervision, conceptualization, writing—review and editing, funding acquisition. Dawei Lian: funding acquisition, supervision. Jun Peng: funding acquisition. All data were generated in-house, and no paper mill was used. All authors agree to be accountable for all aspects of the work, ensuring integrity and accuracy.

Funding

This work was supported by the National Natural Science Foundation of China (No. U22A20372; No. 82204662; No. 82374359; No. 82074363), the Fujian Province Natural Science Foundation (2022J01369, 2024Y4009, 2025J01944), the Fujian Natural Science Foundation for Distinguished Young Scholars (No. 2024J010032), the Fundamental Research Program of Shanxi Province (No. 202103021223405), the Research project of the Health Commission of Shanxi Province (No. 2022137), and the School Management Project of Fujian University of Traditional Chinese Medicine (No. X2024036).

Data availability

The article presents original contributions, and any further inquiries regarding the data should be directed to the corresponding authors.

Declarations

Ethics approval

All procedures adhered to the Declaration of Helsinki. Ethics approval and animal experimental protocols were approved by the Ethics Committee of Fujian University of Traditional Chinese Medicine (Ethics No FJTCM IACUC2021140).

Competing interests

The authors declare no competing interests.