Kaempferol Protects Pulmonary Vascular Endothelial Function in Rats with High Altitude Pulmonary Hypertension by Regulating RAS System and AMPK/Arg2/eNOS Signaling Pathway

Abstract

Background

Previous studies have found that kaempferol can relieve pulmonary hypertension (PH).

Objective

Explore the protective impact of kaempferol on pulmonary vascular endothelium in rats with high altitude pulmonary hypertension (HAPH).

Materials and methods

In a simulated altitude of 5000 m environment, rats were induced to develop HAPH after continuous intragastric administration of kaempferol (25, 50 and 100 mg·kg⁻¹) and Sildenafil (30 mg·kg⁻¹) for 28 days. Assessment of isolated pulmonary arterial rings in rats and relevant indicators in lung tissue was performed, with the mechanism of action investigated using Western blotting.

Results

Kaempferol effectively dilates rat pulmonary arterial rings, with an EC₅₀ of 55.75 μmol/L. L-NAME can effectively counteract the vasodilatory effect of kaempferol. Acetylcholine demonstrated better relaxation of pulmonary arterial rings in HAPH rats after kaempferol intervention. Elastic Van Gieson staining (EVG) and immunohistochemistry (CD31) results indicate that kaempferol can partially protect pulmonary vascular endothelial function in HAPH rats. Western blotting reveals that kaempferol has the ability to regulate the Renin-Angiotensin System (RAS). This leads to a compensatory increase in eNOS expression, upregulation of AMPK activity, and downregulation of eNOS monomer/dimer levels.

Conclusions

Kaempferol can improve pulmonary vascular endothelial dysfunction caused by chronic hypoxia by upregulating the phosphorylation level of AMPK, regulating the RAS system, and inhibiting eNOS uncoupling, thereby achieving vasodilation and endothelial protection.

Keywords

Kaempferol high altitude pulmonary hypertension vascular rings AMPK/Arg2/eNOS signaling pathway RAS system

Introduction

HAPH is a prevalent condition in elevated regions, distinguished mainly by a mean pulmonary artery pressure exceeding 30 mm Hg or a systolic pulmonary artery pressure surpassing 50 mm Hg. It is linked to right ventricular hypertrophy, moderate hypoxemia, and the no polycythemia.^1,2 The condition involves two key components: pulmonary vascular constriction induced by early-stage hypoxia and pulmonary vascular remodeling resulting from prolonged hypoxia. This process includes thickening of the intimal layer and endothelial cell damage.³ Individuals with heightened sensitivity to hypoxia may experience exacerbated pulmonary vascular constriction, leading to the development of HAPH.⁴ Currently, sildenafil has been approved for the treatment of pulmonary arterial hypertension in clinical practice. It can inhibit phosphodiesterase-5 from degrading cGMP, thereby inducing vasodilation in the pulmonary circulation.^5,6 Therefore, this study selected sildenafil as a positive control drug.

The renin-angiotensin system, also known as the RAS system, maintains a dynamic balance within the organism through the interplay of its two axes, the ACE2/Mas axis and the ACE/Ang Ⅱ/AT1R axis.⁷ Research indicates that imbalances in the RAS system may contribute to the development of pulmonary arterial hypertension.^8,9 Additionally, activation of the ACE2/Mas axis and suppression of the ACE/Ang II/AT1R axis in pulmonary tissue are important mechanisms in mitigating chronic hypoxia-induced pulmonary arterial hypertension and play a significant role in the pathogenesis of pulmonary arterial hypertension.¹⁰ Moreover, the functional peptide hormones produced by this system are crucial for the physiological and pathological regulation of cardiovascular and respiratory system function.¹¹

Endothelial cells, situated in the innermost layer of blood vessels, release vascular active factors like nitric oxide (NO) and endothelin-1 upon stimulation.¹² NO is vital for regulating vascular tone, influencing immune responses, and modulating oxidative stress-sensitive processes.¹³ Generated from L-arginine by nitric oxide synthase, only the dimeric form of endothelial nitric oxide synthase (eNOS) produces NO, while its monomeric form generates O²⁻.¹⁴ Research has highlighted the close correlation between endothelial eNOS and human hypertension.¹⁵ Multiple signaling pathways, including phosphorylation, play a crucial role in regulating eNOS activity.¹⁶ AMPK is a key player among these kinases, playing a significant role in eNOS phosphorylation and serving as a primary pathway for eNOS activation.¹⁷ Additionally, via the Ca²⁺/CaM-dependent protein kinase II (CaMKII) pathway, Ca²⁺ binding to calmodulin activates CaMKII, promoting eNOS phosphorylation.¹⁸ Furthermore, protein kinase G (PKG) regulates eNOS activity by binding to and phosphorylating specific sites,¹⁹ while protein kinase C (PKC) can directly or indirectly phosphorylate eNOS, modulating its activity.^20,21 Thus, eNOS activity and NO release are pivotal for safeguarding vascular structure and function.

Kaempferol is a flavonoid compound, and its monomeric standard form is a yellow crystalline powder. Moreover, kaempferol has the ability to suppress the proliferation of primary rat pulmonary arterial smooth muscle cells induced by hypoxia via the Akt/GSK3β/Cyclin pathway.²²

Therefore, this study focuses on the vasodilatory effect of kaempferol on isolated rat pulmonary arterial rings and evaluates its regulatory effects on the RAS and AMPK/Arg2/eNOS signaling pathway in lung tissues of rats with HAPH. For the provide scientific evidence for understanding the protective mechanisms of kaempferol on the pulmonary arterial endothelium in rats with HAPH.

Materials and Methods

Animals

Healthy male Sprague-Dawley (SD) rats weighing (150 ± 20) g were obtained from the Animal Experiment Center of Xi'an Jiao Tong University, with the animal experiment license number: SCXK (Shan) 2018-001. Animals were kept in Plateau Medical Center, Qinghai University, without convection, room temperature: 23 °C ± 3 °C, relative humidity: 55%-60%, 12 h day and night. The experimental design involving these animals was reviewed and approved by the Ethics Committee of the Medical School, Qinghai University.

Reagents and Instruments

Kaempferol (Ka) and phosphodiesterase-5 inhibitor Sildenafil (Silde) were purchased from Shanghai YuanYe Bio-Technology Co., Ltd Norepinephrine bitartrate (NE), acetylcholine (Ach), and N-nitro-L-arginine methyl ester hydrochloride (L-NAME) were also obtained from Shanghai YuanYe Bio-Technology Co., Ltd Xylene and neutral gum were sourced from China National Pharmaceutical Group Chemical Reagent Co., Ltd (Shanghai, China). Citrate buffer was purchased from Beijing Zhongshan Golden Bridge Biotechnology Co., Ltd (Beijing, China), and hematoxylin staining solution was obtained from Beijing Baolingwei Technology Co., Ltd (Beijing, China). DAB Reagent Kit was purchased from Beijing Zhongshan Golden Bridge Biotechnology Co., Ltd (Beijing, China). BCA Protein Assay Kits were obtained from Thermo Fisher Technology Corporation (California, USA). EVG dye set was purchased from Servicebio Corporation (Wuhan, China).

The antibodies, including anti-AMPK and anti-p-AMPK^Thr172, were purchased from Cell Signaling Technology (Boston, USA); anti-eNOS was obtained from Abclonal (Wuhan, China); anti-p-eNOS^Ser1177 and anti-Ang II were acquired from Affbiotech (Jiangsu, China); anti-Arg2 was purchased from Boster (Wuhan, China); anti-ACE, anti-ACE2, and anti-AT1R were all obtained from Proteintech (Wuhan, China); anti-Mas was sourced from Novus (NBP1-78444, Littleton, USA); anti-CD31 was purchased from Abcam (Cambridge, USA); anti-β-actin and anti-β-Tubulin were procured from Abways (AB0035, AB0039, Shanghai, China), while goat anti-mouse IgG (H + L) and goat anti-rabbit IgG (H + L) were both from Proteintech (Wuhan, China).

The multi-channel isolated vascular tension measurement system, DMT 620 M, was purchased from Ed Instruments International Trading (Shanghai) Co., Ltd The cryostat for frozen sectioning was obtained from Thermo Fisher Technology Corporation (CRYOSTAR NX50, California, USA).

Establishment of HAPH Rats Model

Sixty healthy Sprague-Dawley (SD) rats, with a weight range of (150.0 ± 20.0) g, were randomly distributed into six groups, each consisting of 10 rats: control, hypoxia, Hox + Ka (25 mg·kg⁻¹·d⁻¹), Hox + Ka (50 mg·kg⁻¹·d⁻¹), Hox + Ka (100 mg·kg⁻¹·d⁻¹), and Hox + Silde (30 mg·kg⁻¹·d⁻¹). Except for the Ctrl group, the remaining five groups were exposed to a simulated high-altitude environment of 5000 m in a low-pressure oxygen chamber (DYC-300, Guizhou Feng Lei Oxygen Chamber Co., Ltd, Guizhou, China). The settings in the chamber included oxygen at 19.8%, pressure at 52.9 KPa, CO₂ concentration at 1298 ppm, temperature at 18 °C, and relative humidity at 46.9%.²³ The kaempferol low, medium, and high-dose groups, as well as the Sildenafil group, received daily oral gavage of the corresponding drug doses dissolved in 0.5% sodium carboxymethyl cellulose for a total of 28 days. The rats in the blank Ctrl group were kept under normal atmospheric conditions. The laboratory is located in Xining, Qinghai Province, at an altitude of approximately 2261 meters.

The Relaxant Effect of Kaempferol on Rat Pulmonary Arterial Rings

Rats were euthanized by cervical dislocation, and the pulmonary artery ring (2-3 mm) was dissected and mounted on a tension sensor. A tension of 6 mN was applied to the vascular ring for approximately 90 min (It is worth noting that the optimal initial tension can be designed based on different experimental designs and actual conditions, such as body weight of experimental animals, experimental animal model. In this experiment, the initial tension was optimized to 6 mN in order to achieve an appropriate balance time and stable contraction in response to norepinephrine, considering the changes in pulmonary vascular compliance and remodeling). Pulmonary artery contraction was stimulated using high potassium physiological saline solution (KPSS, 60 mM). Vascular rings that exhibited contractions exceeding 5 mN were selected for subsequent experiments. Drugs were cumulatively added at 15 min intervals using a cumulative dosing method. The final concentrations of the drug (Ka) were successively increased to 1.0, 3.2, 10.0, 32.0, 100.0, and 320.0 × 10⁻⁶ M., while the Ctrl group received cumulative additions of an equal volume of physiological saline solution (PSS). The impact of Ka on the pre-contracted pulmonary arterial tension induced by NE was evaluated.²⁴

The tension of the vascular rings was recorded using LabChart 7.2 software, and the percentage of vasodilation was calculated. Using GraphPad Prism 10 software, the concentration for 50% of maximal effect (EC₅₀) for Ka-induced vasodilation was calculated based on the relaxation rate. The formula for calculating the percentage of vasodilation (%) is as follows:

Vasodilation Percentage (%) = (M) aximum contraction tension of pulmonary arterial rings induced by NE stimulation−Baseline tension of pulmonary arterial rings)/(Maximum contraction tension of pulmonary arterial rings induced by NE stimulation−Tension of pulmonary arterial rings after drug administration) × 100%.

The Effect of L-NAME on the Relaxation of Pulmonary Arterial Rings Induced by Kaempferol

Take the complete pulmonary arterial ring experiment prepared according to the instructions in previous methods. The experimental groups include the Control group and the L-NAME group. The L-NAME group is pre-incubated with L-NAME (10⁻⁴ M) for 25 min, while the Control group is incubated without any pharmacological agent. After incubation, both groups are simultaneously stimulated with NE (10⁻⁶ M) to induce vasoconstriction until reaching a plateau. Kaempferol is then added to achieve a final concentration (EC₅₀) of 55.75 μM. The tension of the vascular rings is recorded using LabChart 7.2 software, and the vasodilation rates induced by kaempferol in each group are calculated. The impact of L-NAME on the vasodilatory effect of kaempferol in the pulmonary arterial rings is analyzed.

EVG Staining and Immunohistochemical (CD31) Staining

EVG Staining: Rat lung tissues were fixed in 4% paraformaldehyde for 48 h, paraffin-embedded, sectioned, deparaffinized to water, frozen-sectioned, thawed, and fixed. The sections were immersed in EVG staining solution for 5 min, followed by background differentiation and re-staining with VG for 1–3 min. After clearing in xylene, the sections were mounted with neutral gum, sealed with a coverslip, and examined under a microscope. Images were captured and analyzed.²⁵

Immunohistochemistry (CD31) Staining: Paraffin-embedded sections were deparaffinized to water, underwent antigen retrieval in citrate buffer (pH 6.0), and had endogenous peroxidase blocked. A blocking solution of goat serum (1:9) was applied at room temperature for 20 min. Subsequently, the sections were incubated with the primary antibody anti-CD31 (1:200) overnight at 4 °C, followed by incubation with the secondary antibody at 37 °C for 30 min. After DAB staining and counterstaining with hematoxylin for 3 min., the sections were dehydrated, mounted, and sealed with neutral gum. The average integrated optical density (IOD) at 400× magnification was calculated using Image-Pro Plus 6.0 software.²⁴

The Vasodilatory Effect of Ach on Isolated Pulmonary Arterial Rings from Kaempferol-Treated HAPH Rats

Fifteen Sprague-Dawley (SD) rats were randomly allocated into three groups, with each group comprising 5 rats: the Control group, the Hypoxia group, and the Hox + Ka group (50 mg·kg⁻¹·d⁻¹). The Control group was kept under normal atmospheric conditions, while the other two groups were treated according to the conditions described in previous methods. Pulmonary arterial rings prepared as per previous methods were used for the experiment after confirming good vascular reactivity. After inducing contraction with NE (10⁻⁶ M) until reaching a plateau, Ach was added using a cumulative dosing method at 5 min intervals. The final concentrations of Ach were set as 1 × 10⁻⁹, 3 × 10⁻⁹, 1 × 10⁻⁸, 3 × 10⁻⁸, 1 × 10⁻⁷, 3 × 10⁻⁷, 1 × 10⁻⁶, 3 × 10⁻⁶, 1 × 10⁻⁵ M. The same concentrations of Ach solution were cumulatively added to each group, evaluating the impact of Ach on the tension of pulmonary arterial rings in kaempferol-treated HAPH rats.²⁴ LabChart 7.2 software was used to record the tension of vascular rings, and the vascular relaxation rates in each group were calculated to analyze the protective effect of Ach on the pulmonary arteries of HAPH rats.

Western Blot Analysis

Extract total protein from lung tissues of HAPH rats using a total protein extraction kit. Determine the protein concentration using the BCA method. Conduct SDS-PAGE electrophoresis, transfer, and blocking. Incubate with primary antibodies.²⁶ It is worth noting that some samples are used for low-temperature western blotting analysis.²⁷ For this portion of protein samples, they only need to be mixed with non-denaturing loading buffer (without mercaptoethanol) and do not need to be heated for denaturation. This is done for the detection of eNOS monomer and dimer expression. Then include the following anti-AMPK (1:1000), anti-p-AMPK^Thr172 (1:1000), anti-eNOS (1:800), anti-p-eNOS^Ser1177 (1:1000), anti-Arg2 (1:1000), anti-ACE (1:1000), anti-ACE2 (1:1000), anti-Ang II (1:1000), anti-AT1R (1:700), and anti-Mas (1:1000), overnight at 4 °C. After membrane washing, add goat anti-rabbit IgG (H + L) (1:5000) and incubate for 1 h at room temperature. Apply ECL for chemiluminescent detection. Analyze the images using Image J software. Calculate the relative protein expression based on the grayscale values of bands with β-actin and β-tubulin as internal references.

Statistical Analysis

All data were analyzed using GraphPad Prism 10 software, and the results are expressed as mean ± standard deviation (SD). A t-test was used for comparisons between the normoxia and hypoxia groups. For comparisons among the drug group and the hypoxia group, one-way ANOVA followed by Tukey's or Dunnett's test for multiple comparisons was employed. Additionally, linear trend tests were conducted to assess the dose dependency of kaempferol. Dose-response curves for different concentrations of acetylcholine were analyzed using multiple tests (including nonparametric tests) with single-parameter row analysis. A P value of ≤ 0.05 was deemed statistically significant.

Results

The Effect of Kaempferol on Relaxation in Pulmonary Arterial Rings of Rats

The research results indicate that kaempferol can dilate the pulmonary arterial rings of rats precontracted with NE (10⁻⁶ M, Figure 1a). Trend analysis shows that the vasodilation rate increases with the dosage of kaempferol (F = 51.04, P < 0.01). The calculated EC₅₀ for kaempferol -induced vasodilation is 55.75 μM (Figure 1d). Therefore, subsequent studies were conducted at this concentration.

Figure 1.

Effects of kaempferol on pulmonary artery ring relaxation in rats. (a) Representative figure of effect of kaempferol (1.0∼320.0 × 10⁻⁶ M) on tension of NE (10⁻⁶ Μ) precontracted pulmonary vascular ring. (b, c) Representative figure of the effect of L-NAME (10⁻⁴ M) on vasodilation of kaempferol (55.75 μΜ), b: Control group; c: L-NAME group. (d) Dose-effect curve of the influence of kaempferol on the tension of pulmonary vascular ring in NE pre-constricted rats, kaempferol EC₅₀: 55.75 μΜ (n = 5). (e) Quantification of the percentage vasodilator effect of L-NAME (10⁻⁴ M) on kaempferol (55.75 μM). Data were expressed as means ± SD (n = 5. *P < 0.05 vs the Control group. Figure 1e: *P by t-test).

The Effect of L-NAME on Kaempferol-Induced Relaxation in Rats Pulmonary Arterial Rings

Following preincubation of complete pulmonary arterial rings with L-NAME, the relaxation rate induced by kaempferol in pulmonary arterial rings was 36.53 ± 3.68%, in contrast to 61.79 ± 1.31% in the Control group (Figure 1b, c, and e, P < 0.05).

The Effects of Kaempferol on the Histology of Lung Tissue in HAPH Rats

The results of EVG staining showed that, compared to the Ctrl group, rats in the Hox group exhibited increased black elastic fibers and pink collagen fibers in the pulmonary arteries (Figure 2a). Following intervention with kaempferol and Sildenafil, fibrosis in the pulmonary arteries of HAPH rats was reduced compared to the hypoxia group. According to the calculations, the WT % in the control group significantly increased compared to the hypoxia group, and after intervention with resveratrol and sildenafil, it showed a decrease (Figure 2b, P < 0.05), indicating effective improvement in pulmonary vascular remodeling.

Figure 2.

Kaempferol improves pulmonary vascular remodeling in HAPH rats. (a) Pulmonary arterioles were stained with EVG in Control, Hypoxia, Hox + Ka (25, 50, and 100 mg·kg⁻¹), and Hox + silde (30 mg·kg⁻¹, Positive Ctrl) rats (magnification, 400×) (n = 3). (b) Percentage of vascular wall thickness (WT %) of pulmonary arteries (5 Pulmonary arterioles per sample, outer diameter: 30-100 μm). (c) Immunohistochemistry images show the expression of CD31 protein (brown) in the media of small pulmonary arteries from rat lungs in each group (magnification, 400×) (n = 3). (d) Quantification of immunohistochemistry reveals CD31 (brown) expression in the endothelium of distal pulmonary arteries from HAPH rats. Measurements were taken from 5 distal pulmonary arteries, each < 100 μm in diameter (n = 3). Data are presented as means ± SD (*P < 0.05 vs the Ctrl group, ^#P < 0.05 vs the Hox group. Figure 2b and d: *P by t-test, ^#P by one-way ANOVA with Tukey's or Dunnett's multiple comparisons test).

Figure 3.

Dose-response curves depicting the impact of various concentrations of ach (10⁻⁹∼10⁻⁵ M) on pulmonary arteries in HAPH rats. (a, b, c) Representative plot of the effect of Ach on vasorelaxation in HAPH rats treated with kaempferol (50 mg·kg⁻¹), a: Control group b: Hox group c: Hox + Ka group. (d) Percentage of pulmonary vasodilator effect of Ach on kaempferol (50 mg·kg⁻¹) intervention in HAPH rats. Data were expressed as means ± SD (n = 5. *P < 0.05 vs the Hox group, ^#P < 0.05 vs the Control group. Figure 3d: *P and ^#P by multiple tests with single-parameter row analysis).

CD31 is primarily used to demonstrate the presence of endothelial cell tissue. Immunohistochemical results revealed that, except for the Hox group, all other groups exhibited expression of the CD31 protein, with immunopositive products appearing as brown granules. Following 28 days of hypoxia exposure, there was a significant decrease in the protein expression of CD31 compared to the Ctrl group (P < 0.05). However, after treatment with kaempferol and Sildenafil, the protein expression of CD31 showed a significant increase (Figure 2c and d, P < 0.05).

The Influence of Ach on the Vasodilatory Response of Isolated Pulmonary Arterial Vascular Rings in HAPH Rats Subjected to Kaempferol Treatment

The study revealed that Ach has the ability to dilate the isolated pulmonary arterial vascular rings pre-contracted with NE (10⁻⁶ M) in kaempferol-treated HAPH rats (Figure 3a, b, and c). Analysis revealed that, compared to the control group, acetylcholine did not induce relaxation in the pulmonary arteries of rats in the hypoxia group, suggesting that the vasodilation function of pulmonary arteries is impaired in HAPH rats. Results from varying concentrations of Ach (10⁻⁹∼10⁻⁵ M) indicate that kaempferol improves the response of pulmonary arterial vascular rings in hypoxic rats to Ach to different degrees. In comparison to the Hox group, kaempferol demonstrated better dilation of HAPH rat pulmonary arterial vascular rings at all concentrations of Ach except for Ach (3 × 10⁻⁹ M, Figure 3d, P < 0.05).

The Modulatory Effect of Kaempferol on the RAS Within the Pulmonary Tissue of Rats Experiencing HAPH

The results indicate a comparison with the Ctrl group, the expression of AT1R protein significantly increased in the lung tissue of hypoxic rats. However, its expression showed a significant decrease after kaempferol intervention (Figure 4a, b, c, and d, P < 0.05). The protein levels of ACE and Ang II exhibited a decrease with kaempferol intervention compared to the Hox group, although the difference was not statistically significant. Findings related to the counter-regulatory axis protein expression demonstrated a notable reduction in ACE2 and Mas protein levels in the Hox group. However, kaempferol intervention led to a significant increase in ACE2 and Mas protein expression (Figure 4a, e, and f, P < 0.05).

Figure 4.

Effects of kaempferol on the RAS system in pulmonary tissue of HAPH rats. (a) Representative western blots illustrating the protein levels of ACE, Ang II, AT1R, ACE2, and Mas in lung tissues from each rat group. (b-f) For quantitative analyzed of ACE, Ang II, AT1R, ACE2 and Mas protein expression, β-actin and β-tubulin were used as reference genes. Data were expressed as means ± SD (n = 5, *P < 0.05 vs the Ctrl group, ^#P < 0.05 vs the Hox group. Figure 4b-f: *P by t-test, ^#P by one-way ANOVA with Tukey's or Dunnett's multiple comparisons test).

The Regulatory Effect of Kaempferol on the AMPK / Arg2 / eNOS Signaling Pathway in HAPH Rats

The effect of kaempferol on the AMPK/Arg2/eNOS signaling pathway indicates (Figure 5a) that in the Hox group, there was no significant alteration in the expression of AMPK protein in lung tissue compared to the Ctrl group. However, the protein expression of p-AMPK^Thr172 increased with kaempferol intervention. The Hox + Ka (25 mg·kg⁻¹) group and Hox + Ka (50 mg·kg⁻¹) group exhibited statistically significant differences compared to the Hox group, the trend of p-AMPK^Thr172/AMPK is consistent with p-AMPK^Thr172 (Figure 5b, c and d, P < 0.05). The expression level of Arg2 protein in the lung tissue of rats in the Hox group significantly increased compared to the Ctrl group (Figure 5e, P < 0.05), while there was no significant difference in Arg2 protein expression in lung tissue among the other groups. The total protein expression of eNOS in the Hox group was significantly higher compared to the Ctrl group, and after kaempferol intervention, there was a slight increase in expression (Figure 5f, P < 0.05). The protein expression of p-eNOS^Ser1177 in the Hox group was notably elevated, while after kaempferol intervention, there was a significant decrease in expression (Figure 5g, P < 0.05). In p-eNOS^S1177/eNOS, there was no statistically significant difference between the Hox group and Ctrl group, but the ratio decreased after intervention with kaempferol and sildenafil (Figure 5h, P < 0.05). Regarding the expression levels of eNOS monomer/dimer proteins, there was a significant increase in the Hox group compared to the Ctrl group, and after intervention with kaempferol and Sildenafil (Figure 5i, P < 0.05), there was a significant decrease.

Figure 5.

Effect of kaempferol on the AMPK/Arg2/eNOS signaling in HAPH rats. (a) Representative western blots illustrating the protein levels of AMPK, p-AMPK^Thr172, Arg2, eNOS, p-eNOS^Ser1177, eNOS monomer, and eNOS dimer in lung tissues from each rat group. (b-i) Quantitative analysis of AMPK, p-AMPK^Thr172, p-AMPK/AMPK, Arg2, eNOS, p-eNOS^Ser1177, p-eNOS/eNOS, eNOS monomer, and eNOS dimer protein expression using β-actin as a reference gene. Data are presented as means ± SD (n = 5, *P < 0.05 vs the Ctrl group, ^#P < 0.05 vs the Hox group, NS indicates no significance. Figure 5b-i: *P by t-test, ^#P by one-way ANOVA with Tukey's or Dunnett's multiple comparisons test).

Discussion

We found that kaempferol effectively dilated the rats isolated pulmonary artery vascular rings pre-contracted with NE (10⁻⁶ M). Based on this, we pre-incubated L-NAME on rat isolated pulmonary artery vascular rings. Compared to the Ctrl group, L-NAME partially blocked the vasodilatory effect of kaempferol on rats isolated pulmonary artery vascular rings (Figure 1e). This suggests that the vasodilatory effect of kaempferol on vascular rings may be mediated by the activation of eNOS, leading to increased release of NO and modulation of endothelin contraction factors to regulate pulmonary artery vascular dilation. Additionally, studies had found that apart from endothelium-dependent vasodilation pathways, kaempferol had induced vasodilation in rat pulmonary arteries through non-endothelium-dependent mechanisms involving BK_Ca, L-type Ca⁺⁺ channels, sGC/cGMP, and PKA signaling pathways.²⁸

Recent years have seen notable progress in the treatment of PH; nevertheless, the prognosis for PH remains unfavorable, with an estimated five-year survival rate of less than 70%.²⁹ In the pathogenesis of PH, a substantial amount of data indicates that endothelial injury can lead to endothelial dysfunction.³⁰ High-altitude chronic hypoxic conditions can also affect pulmonary vascular endothelial function.³¹ Consequently, after having subjected the rats to chronic hypoxia for 28 days, we observed the effects of kaempferol on pulmonary vascular endothelium in rats with HAPH, sildenafil was chosen as the positive Ctrl drug for in vivo experiments.⁵ The HAPH rat model was established by simulating an altitude of 5000 m in a low-pressure oxygen chamber and raising the rats for 28 days. Previous studies have indicated that kaempferol can effectively reduce the mean pulmonary artery pressure and alleviate pulmonary vascular remodeling in HPH rats.³ In this study, EVG staining and immunohistochemistry (CD31) results suggest the successful establishment of the HAPH rat model and demonstrate that kaempferol can effectively preserve the integrity of the pulmonary vascular endothelium in HAPH rats.

Accumulated Ach can facilitate endothelium-dependent relaxation in the pulmonary arteries of HAPH rats following kaempferol treatment. Ach is recognized as an endothelium-dependent vasodilator capable of inducing relaxation in pre-constricted pulmonary arteries. Our results indicate that, compared to the Ctrl group, the high-altitude chronic hypoxic environment impairs the vascular dilation response of pulmonary small arteries to acetylcholine. This suggests the presence of endothelial dysfunction in the HAPH rat model. Based on preliminary work and current histological data, following a comprehensive assessment, we selected the kaempferol (50 mg·kg⁻¹) group for vascular function experiments. Compared to the hypoxic group, kaempferol enhanced the Ach-induced relaxation of pulmonary arteries, suggesting a specific protective effect, kaempferol on the pulmonary arterial endothelium may be linked to the release of vasodilators. Nonetheless, further exploration is required to determine whether the dose-dependent nature of kaempferol influences the vasodilatory function of pulmonary arteries in HAPH rats.

The ACE/Ang II/AT1R axis and the ACE2/Mas axis together constitute the RAS system. It is a cascading regulatory system that control cardiovascular function, maintains electrolyte balance, and regulates cellular proliferation and inflammatory responses.³² This study found that high-altitude chronic hypoxia upregulates the protein expression levels of ACE and AT1R while downregulating ACE2 and Mas. However, after intervention with kaempferol, there was a notable reduction in the expression levels of AT1R protein and a rise in the expression levels of ACE2 and Mas. This is consistent with the notion that inhibiting the ACE/Ang II/AT1R axis and activating the ACE2/Mas axis can alleviate pulmonary vascular constriction in HPAH rats,³³ leading to increased production of NO to reduce pulmonary arterial pressure and improve vascular remodeling outcomes.³⁴ In this study, sildenafil intervention did not decrease the expression level of AT1R protein. This is because unlike other drugs such as ACE inhibitors and ARBs (including AT1R antagonists), sildenafil does not directly intervene in the RAS system. AT1R (angiotensin II type 1 receptor) is the main receptor for angiotensin II and is closely related to blood pressure regulation and kidney function. ACE inhibitors and ARBs reduce AT1R levels through different mechanisms (inhibiting angiotensin II synthesis and blocking its action, respectively).^35,36 Sildenafil primarily acts on vasodilation, and its mechanism of action is not directly related to RAS, so it does not decrease AT1R levels like ACE inhibitors or ARBs. Furthermore, relevant studies suggest that similar to rats with ACE2 expression deficiency, Ang II can increase oxidative stress and downregulate NO levels by binding to its receptor AT1R, thereby mediating damage to endothelium-dependent vascular relaxation function.³⁷ On the other hand, the activation of ACE2 in endothelial cells may contribute to mitigating inflammatory responses and vascular dysfunction mediated by Ang II.³⁸ Within endothelial cells, an enzyme facilitating the conversion promotes the generation of Ang (1-7) from Ang I and Ang II.³⁹ Additionally, there exists a G protein-coupled receptor, Mas, specifically designed for Ang (1-7). When Ang (1-7) binds to Mas, it triggers the expression of eNOS in endothelial cells. Ang (1-7) has the capability to modulate the phosphorylation levels of eNOS at Thr495/Ser1177, leading to an elevation in eNOS activity and the production of NO.⁴⁰ Moreover, through the NO/cGMP signaling pathway, Ang (1-7) can suppression the pathological remodeling mediated by Ang II.³⁴ This indicates that kaempferol may promote the stability of pulmonary vascular endothelial function by suppressing the ACE/Ang II/AT1R axis and enhancing the ACE2-Mas axis.

Under high-altitude chronic hypoxia, there is a compensatory increase in eNOS protein expression compared to the Ctrl group. Following intervention with kaempferol and sildenafil, there is no alteration in the protein expression of eNOS compared to the Hox group. It is noteworthy that the expression of p-eNOS^S1177 shows different results, with the highest level in the Hox group. In the kaempferol and sildenafil groups, the levels of p-eNOS^S1177 significantly decrease, with the sildenafil group showing the most significant reduction. Numerous studies have demonstrated that hypoxia can increase the expression and/or activity of eNOS.⁴¹ In hypoxia-induced pulmonary arterial hypertension, it is possible to induce the expression of eNOS by increasing blood flow. However, an increase in the quantity of eNOS does not always coincide with an increase in eNOS activity. The expression and activity of eNOS can be modulated at the levels of transcription, post-transcription, and post-translation.⁴² Any interference in these complex regulatory processes can be reflected in changes in the biological utilization of NO. Some studies propose that in PH, the reduction in the biological utilization and activity of NO is a consequence of decreased utilization of eNOS cofactors or uncoupling of eNOS, rather than a decrease in the levels of eNOS itself.⁴³ Hypoxia has the potential to induce eNOS uncoupling by modifying the availability of cofactor BH₄ and substrate L-Arg.⁴⁴ Due to the competition for the same substrate between arginase and nitric oxide synthase, the upregulation or activation of arginase can weaken the ability of eNOS to produce NO. Elevated expression or heightened activity of arginase can result in the uncoupling of eNOS.⁴⁵ Our results are consistent. Where under high-altitude chronic hypoxia, the protein expression of Arg2 significantly increased compared to the Ctrl group. After intervention with kaempferol and sildenafil, the protein expression of Arg2 decreased compared to the Hox group.

Typically, eNOS exhibits its activity after phosphorylation, and it was anticipated that under high altitude chronic hypoxia, the phosphorylation level of eNOS should increase after intervention with kaempferol compared to the Hox group. However, the experimental results were contrary to this expectation. Therefore, in this study, the uncoupling status of eNOS in the lung tissue of HAPH rats was assessed. The results indicate a significant increase in the expression of eNOS monomer/dimer proteins under hypoxia compared to the Ctrl group. After intervention with kaempferol and sildenafil, the expression of eNOS monomer/dimer proteins significantly decreased compared to the Hox group. In conclusion, although the phosphorylation level of eNOS decreased after intervention with kaempferol, the total protein level of eNOS increased. We speculate that the decreased expression of phosphorylated eNOS is of the dimeric type. Overall, kaempferol to enhance dimeric eNOS activity by inhibiting eNOS uncoupling and attenuating the overexpression or activity of arginase. This, in turn, functions to safeguard the pulmonary vascular endothelial function in HAPH rats. For the above speculations, we are conducting further experiments to validate them.

At the same time, compared to the Ctrl group, the phosphorylation level of AMPK in the lung tissue of HAPH rats decreased, while kaempferol increased the decreased protein of hypoxia induced p-AMPK^T172. Studies have reported a decrease in endothelial AMPK protein expression in PH patients or HPH animals.^46,47 Endothelial cell AMPK, by phosphorylating eNOS and consequently upregulating NO generation, has become a therapeutic target for PH,⁴⁷ which aligns with our results. Additionally, there is evidence suggesting that AMPK and the ACE2 in the RAS system, acting as upstream regulators, sequentially modulate the function of eNOS. Within functional pulmonary endothelial cells, AMPK phosphorylates ACE2 at S680, enhancing its stability and thereby sustaining the biological utilization of NO derived from eNOS.⁴⁸ It is noteworthy that eNOS is primarily expressed in vascular endothelial cells,⁴⁹ while AMPK is widely distributed across various vascular types, including endothelial cells, muscle cells, adipocytes, and hepatocytes, playing crucial roles in cellular metabolism and survival.⁵⁰ Activation of AMPK may exert broad biological effects in the vasculature, not limited to endothelial cells. Differences in increased AMPK activity and decreased eNOS activity may stem from the complexity of intracellular signaling pathways. Although AMPK is expressed in various vascular types, its activation may have varying effects on eNOS phosphorylation and activity, particularly with more significant effects observed in endothelial cells. Additionally, while AMPK activation can promote eNOS phosphorylation, other regulatory mechanisms may exist, such as subsequent signaling pathways or protein-protein interactions, leading to reduced eNOS activity. Therefore, although increased AMPK phosphorylation may enhance the potential activity of eNOS, its specific effects may be modulated by cell type and other signaling pathways. Further research is needed to elucidate this complexity and gain a deeper understanding of the roles of these molecules in vascular function regulation. Additionally, another limitation is that we did not assess changes in other phosphorylation sites of eNOS. Further research is needed to elucidate the precise molecular mechanisms of kaempferol.

Conclusions

This study indicates that kaempferol can relax rat pulmonary artery vascular rings pre-constricted with norepinephrine, and this effect is mediated through the endothelium. Simultaneously, kaempferol regulates the RAS system, activates the AMPK/Arg2/eNOS signaling pathway, and inhibits eNOS uncoupling. This results in the improvement of pulmonary endothelial cell function and the alleviation of pulmonary arterial hypertension. The findings of this study provide initial insights into the mechanism by which kaempferol protects the pulmonary vascular endothelium in HAPH rats, suggesting that kaempferol may be a potential natural remedy for treating HAPH.

Supplemental Material

sj-docx-1-npx-10.1177_1934578X241274896 - Supplemental material for Kaempferol Protects Pulmonary Vascular Endothelial Function in Rats with High Altitude Pulmonary Hypertension by Regulating RAS System and AMPK/Arg2/eNOS Signaling Pathway

Supplemental material, sj-docx-1-npx-10.1177_1934578X241274896 for Kaempferol Protects Pulmonary Vascular Endothelial Function in Rats with High Altitude Pulmonary Hypertension by Regulating RAS System and AMPK/Arg2/eNOS Signaling Pathway by Xin Xie, Huiru Li, Liangqi Wang, Xiaonan Zhang, Dianxiang Lu and Zhanqiang Li in Natural Product Communications

Footnotes

Declaration of Conflicting Interests

The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: West Light Foundation of The Chinese Academy of Sciences”, “Qinghai Province Kunlun Talents, High-end Innovative and Entrepreneurial Talents Project”, Science and technology innovation project of Sanjiangyuan first-class discipline, School of ecological and environmental engineering, Qinghai University, National Natural Science Foundation of China, (grant number 2022-stxy-Y15, 82360831).

Statement of Informed Consent

There are no human subjects in this article and informed consent is not applicable.

ORCID iD

Zhanqiang Li

Supplemental Material

Supplemental material for this article is available online.

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