The present study was approved by the Clinical Trial Ethics Committee of the Affiliated Hospital of Southwest Medical University (approval no. KY2019039). All work was performed in accordance with the provisions of the Declaration of Helsinki and its later amendments. All patients provided written informed consent for specimen collection. A total of 10 pregnant women with a singleton pregnancy diagnosed with severe preeclampsia were selected as the experimental group (SP group), whose average maternal age ranged from 23 to 41 years, with a mean age of 32.20±5.226 years. In addition, 10 pregnant women with a singleton pregnancy with normotensive pregnancies were selected as the control group (NP group), whose average maternal age ranged from 22 to 44 years, with a mean age of 30.40±5.475 years (Table I). All patients were hospitalized and delivered by either vaginal delivery or cesarean section at the Department of Obstetrics, The Affiliated Hospital of Southwest Medical University (no. 8, Section 2, Kangcheng Road, Luzhou, Sichuan, China) between May 2020 and May 2021. The inclusion criteria for the SP group were based on the American College of Obstetricians and Gynecologists Guidelines (14). Subjects were excluded if they had chronic hypertension, kidney disease, cardio-cerebrovascular disease, severe liver and kidney function impairments, other primary diseases, systemic diseases or other pregnancy complications. Patients with a history of smoking, alcohol abuse, syphilis, hepatitis virus or human immunodeficiency virus were also excluded. Blood samples were acquired from the mother by peripheric venous puncture before any medical treatments were administered. Samples were then placed in anticoagulant tubes, centrifuged at 3,000 x g for 15 min at room temperature and stored in a -20˚C refrigerator until they were used for extraction. After the placenta was delivered, the fetal membrane was immediately stripped under sterile conditions, and 3-5 pieces of placental tissues with a small placental artery near the edge of the placenta, ~4x1x1 cm³ in size, were extracted and placed in the prepared specimen box, which was immediately transferred to the laboratory under low-temperature conditions. Next, the perivascular connective tissue was rapidly and gently removed under a microscope, taking care to minimize the damage to the blood vessels, and vessels with diameters of 0.1-0.2 cm and lengths of 2-3 cm were separated. Some of the isolated blood vessels were frozen in liquid nitrogen and then quickly transferred to a -80˚C freezer for cryopreservation, and these samples were used for subsequent PCR and western blot analyses, and the nitrate reductase method. The remainder were fixed in 4% paraformaldehyde for >24 h at room temperature, and the specimens were used for subsequent hematoxylin-eosin staining and immunohistochemical analysis.

Hematoxylin and eosin (H&E) staining was performed as previously described (15,16). To deparaffinize the paraffin sections, slides were submerged for 5 min in a slide jar filled with xylene (Sinopharm Chemical Reagent Co., Ltd.), repeating this procedure twice and using fresh xylene each time. Subsequently, sections were rehydrated by running slides through a series of decreasing concentrations of EtOH (Sinopharm Chemical Reagent Co., Ltd.). Hematoxylin (Shanghai Jingke Chemical Technology Co., Ltd.) was used to stain the tissues for 5 min at room temperature, and after washing with tap water, differentiation in staining was visualized by submerging for 30 sec in 1% acetic acid. To visualize the blue staining, the sections were rinsed under running tap water for 5 min, after which the slices were dyed in eosin solution (Shanghai Jingke Chemical Technology Co., Ltd.) for 2 min at room temperature. After dehydration with EtOH and transparency with xylene, the slices were sealed with neutral balsam (G8590-100; Solarbio). Finally, tissues were imaged under an electron microscope (400x magnification; OLYMPUS CX-21; Shanghai Xinyu Biotechnology Co., Ltd.).

HUVECs (cat. no. CL-0675; Procell Life Science & Technology Co., Ltd.) were cultured in high-glucose DMEM (AMEKO; cat. no. FB8025; Shanghai Lianshuobaowei Biological Technology Co., Ltd.) supplemented with 10% FBS (Guangzhou Ruite Biological Technology Co., Ltd.) and 1% penicillin and streptomycin (Shanghai Biyuntian Biotechnology Co., Ltd.) at 5% CO₂ and 37˚C in a humidified chamber. HUVECs in the logarithmic growth stage were subcultured and inoculated in six-well plates. The cells were divided into a control group, a capsaicin group (cat. no. HY-10448; MedChemExpress) and a capsazepine group (cat. no. HY-15640MCE; MedChemExpress). After reaching ~80% confluency, high-glucose DMEM containing capsaicin and capsazepine, prepared in DMSO to a final concentration of 1 µmol/l, was added. This concentration was based on a previous study by Yang et al (6), in which NO production was highest with 1 µM capsaicin in cultured endothelial cells from Wistar rats. In the control group, high-sugar DMEM with an equivalent volume of DMSO was used. After 24 h of incubation, total RNA and protein were extracted from the cells.

Total RNA was extracted from tissues and cells using TRIzol reagent (cat. no. 15596018; Thermo Fisher Scientific, Inc.), the absorbance of the extracted RNA was measured using a spectrophotometer on a Bio-Rad 3000 UV-vis spectrophotometer (Bio-Rad Laboratories, Inc.), and the integrity was examined by gel electrophoresis (Bio-Rad Laboratories, Inc.). RNA extracts with an A260/A280 ratio between 1.8-2.0 and three complete bands of 28S, 18S and 5S, after 2% agarose gel electrophoresis, were chosen for reverse transcription (Fig. 1A and B). A cDNA Reverse Transcription kit (Fuji Biological Co., Ltd.) was used to perform the reverse transcription procedure using the following reverse transcription temperature protocol: 42˚C for 15 min, 85˚C for 5 sec and cooling at 4˚C. A SYBR Green MasterMix kit (Toyobo Co., Ltd.) was used for qPCR in a StepOnePlus PCR System (Applied Biosystems; Thermo Fisher Scientific, Inc.), in a 20-µl reaction system with the following amplification temperature protocol: 95˚C for 10 min, followed by 40 cycles of 95˚C for 5 sec, 58˚C for 20 sec and 72˚C for 10 sec. GAPDH was used as the internal reference, and the internal reference and target gene primers were designed and synthesized by Shanghai Sangon Biological Co., Ltd. (Table II). Finally, the relative expression of each target gene was calculated using the 2^-∆∆Cq method (17). The experiments were repeated three times.

Total protein was extracted from tissues and cells using RIPA lysis buffer (Biyuntian Biotechnology Co., Ltd.) containing the protease inhibitor phenylmethanesulfonylfluoride (Biyuntian Biotechnology Co., Ltd.), and protein concentration was measured using a BCA protein assay kit (Biyuntian Biotechnology Co., Ltd.). The protein samples were denatured, and 25 µg protein per sample was loaded per lane on a 10% SDS gel, resolved using SDS-PAGE and transferred to a PVDF membrane. The membrane was blocked in 5% skimmed milk powder dissolved in 0.5% Tween-TBS for 2 h at room temperature. After washing the membranes, they were incubated overnight at 4˚C on an agitator with one of the following primary antibodies: Mouse anti-GAPDH (1:10,000; cat. no. MB001H; Bioworld), rabbit anti-TRPV1 (1:1,000; cat. no. KL-KT-008292; Abcam), rabbit anti-ABCC9 (also known as SUR2B; 1:500; cat. no. ab84299; Abcam), rabbit anti-Kir6.1 (1:1,000; cat. no. TF6468R; Crystal Wind Biological Co., Ltd.) or rabbit anti-eNOS (1:1,000; cat. no. ab15280-eNOS; Abcam). The following day, after the membranes were washed, the secondary HRP-conjugated Goat Anti-Rabbit IgG antibody (1:1,000; cat. no. PF01924; Pufei Bio Co., Ltd.) was added and incubated at 37˚C for 2 h. Densitometry analysis was performed using ImageJ version 1.49 (National Institutes of Health). Experiments were repeated three times.

Immunohistochemistry was performed using an HRP Streptavidin Conjugate kit according to the manufacturer's protocol (DBA cat. no. Im-05818B; Shanghai Caiyou Industrial Co., Ltd.). After baking at 60˚C for 2 h, the paraffin sections were dewaxed and hydrated in turn, washed with PBS and incubated with 3% hydrogen peroxide (TITABIO; cat. no. SY2622; Beijing Ita Biological Co., Ltd.) for 5-10 min at room temperature to quench endogenous peroxidase activity. The paraffin sections were subjected to antigen retrieval in a microwave in EDTA buffer (cat. no. AS1016; Wuhan Aspen Biotechnology Co., Ltd.). Subsequently, blocking solution (solution B included in the kit) was added and samples were incubated at room temperature for 30 min. The primary antibody working solution containing anti-TRPV1 (1:200 dilution; cat. no. KL-KT-008292; Abcam), anti-ABCC9 (1:200 dilution; cat. no. ab84299; Abcam), anti-Kir6.1 (1:200 dilution; cat. no. TF6468R; Crystal Wind Biological Co., Ltd.) or anti-eNOS (1:2,000 dilution; cat. no. ab15280-eNOS; Abcam) was added to cover the sections, which were incubated at 4˚C overnight. The following day, the samples were rinsed, and to stain the sections, the streptavidin-biotin-peroxidase complex was used after the sections had been treated with biotinylated goat anti-rabbit immunoglobulin G secondary antibody for 30 min at room temperature. A DBA solution was used to develop the stain. Next, the samples were stained again with hematoxylin for 5 min at room temperature and differentiated by hydrochloric acid alcohol, dehydrated, made transparent and sealed with neutral glue. Finally, the samples were observed and imaged under a light microscope (400x magnification; Leica Aperio AT2; Leica Microsystems, Co., Ltd.). The expression levels of various proteins in the endothelial cells were observed. Brown-yellow staining indicated positive expression, and the optical density value represented the relative expression level. The images were analyzed using Q-IMAGING software (MicroPublisher 6 CCD; Nano Hai Bioscientific Instruments Co., Ltd.).

An NO assay kit (Nanjing Jiancheng Biological Co., Ltd.) was used in the present study according to the manufacturer's protocol. Prior to the experiment, solutions III, IV and V were prepared into chromogenic agents according to the ratio of 2.5:1:1. A total of 300 µl homogenate supernatant and 100 µl plasma was deproteinized by adding 200 µl reagent I and stirring, after which 100 µl reagent II was added, the solution was stirred and centrifuged at 3,500-4,500 x g for 15 min at 4˚C, and the supernatant was recovered. A preheated microplate reader (BMG LABTECH, CLARIOstar PLUS) (preheated for >30 min) was used and the wavelength was adjusted to 550 nm. The samples to be tested were added to the 96-well plate, mixed, and left for 15 min, after which the OD value of absorbance in each well was measured using a microplate reader.

All data were analyzed using SPSS version 20.0 (IBM Corp.) and GraphPad Prism 5.0 (GraphPad Software, Inc.). Normally distributed data are presented as the mean ± SD. An independent samples t-test was used to compare the NP and SP groups, and a one-way ANOVA followed by a Newman-Keuls multiple comparison post-hoc test was used for multiple comparisons. P<0.05 was considered to indicate a statistically significant difference.

Table I presents the demographic and clinical characteristics of the included patients when they were admitted to hospital, including maternal age, gestational age and blood pressure. There was no significant difference in maternal age between the NP and SP groups (P>0.05), but the gestational age difference was statistically significant in the two groups (P<0.01). In addition, systolic and diastolic blood pressure were significantly higher in the SP group than in the NP group (P<0.01).

As shown in Fig. 2A endothelial cells in the NP group were uniformly and regularly arranged, and the nucleus was oblong, bluish in color and protruded slightly from the official cavity. In the SP group, the walls of the placental artery were thickened, the number of collagen fibers had increased, the artery was hyalinized, the endothelial cells were arranged in a disorderly manner and the endodermis was notably damaged (Fig. 2B).

Immunohistochemistry was used to evaluate the localization of the TRPV1, Kir6.1/SUR2B and eNOS channels in the placental artery (Figs. 3 and 4). The results showed that each channel was primarily distributed in the vascular endothelial layer. It was noted that the number of counterstained nuclei in the endothelial cell layer was significantly decreased, as was the membrane staining of each channel in the SP group compared with the NP group. The OD values of TRPV1, Kir6.1/SUR2B and eNOS in the SP group were 11,660.0±1,721.0, 2,975.0±505.5, 10,236.0±1,355.0 and 14,663.0±2,320.0, respectively, which were lower than those in the NP group (24,917.0±2,044.0, 4,495.0±775.0, 14,663.0±2,320.0, and 20,988.0±2,289.0; all P<0.05) (Fig. 5). RT-qPCR was used to examine the relative expression of the four-channel genes. The relative expression of TRPV1, Kir6.1, SUR2B and eNOS in the SP group was 0.559±0.609, 0.419±0.281, 0.166±0.087 and 0.383±0.110, respectively, which was significantly lower than that in the NP group (1.098±0.341, 1.024±0.085, 1.243±0.335 and 1.219±0.247; all P<0.01) (Fig. 6). Since gene expression does not fully reflect protein expression, western blotting was used to quantitatively examine whether this difference was also present at the protein level (Fig. 7). The relative quantitative analysis results showed that the ratio of the gray value of each target protein to GAPDH in the SP group was 0.282±0.058, 0.058±0.005, 0.132±0.007 and 0.059±0.023, respectively, which was lower than that in the NP group (0.688±0.145, 0.196±0.010, 0.514±0.018 and 0.327±0.063; all P<0.01) (Fig. 8), suggesting that the relative protein expression of TRPV1, KATP subtype Kir6.1/SUR2B and eNOS in the SP group was also significantly downregulated.

To explore the interactions of the four channels, further cytological experiments were performed. The relative gene expression of TRPV1, Kir6.1, SUR2B and eNOS in the control group was 0.986±0.129, 1.439±0.358, 1.479±0.403 and 1.162±0.090, respectively, while in the capsaicin group, it was 4.568±0.810, 4.014±0.781, 5.505±0.287 and 2.821±0.377, respectively, and in the capsazepine group, it was 0.077±0.010, 0.036±0.014, 0.046±0.010 and 0.083±0.005, respectively; the differences between the control group and capsaicin groups, and the capsazepine group were statistically significant (all P<0.01; Fig. 9). The results of western blotting showed that the relative protein expression of TRPV1, Kir6.1, SUR2B and eNOS in the control group was 0.266±0.026, 0.226±0.014, 0.166±0.013 and 0.277±0.025, respectively, while the expression in the capsaicin group was 0.763±0.044, 0.687±0.046, 0.493±0.035 and 0.498±0.022, respectively, and expression in the capsazepine group was 0.157±0.040, 0.057±0.005, 0.065±0.005 and 0.058±0.008, respectively (all P<0.01), and these differences between the control group and capsaicin groups, and the capsazepine group were statistically significant (P<0.01; Figs. 10 and 11).

The nitrate reduction method indicated that the mean levels of total nitrites in the placental artery and plasma were 33.31±11.73 and 45.12±14.92, respectively, in severe preeclampsia patients, which were significantly lower than those in the control group (47.65±9.81 and 66.01±13.81, respectively; both P<0.01) (Figs. 12 and 13).