Animals and reagents

Male Sprague-Dawley rats weighing 200–250 g were acquired from Beijing Vital River Laboratory Animal Technology Co., Ltd. in China. Approval for the experimental procedure was granted by the Laboratory Animal Ethics Committee at Wenzhou Medical University (No. wydw2019-0858). The study followed the National Institutes of Health Guide for Laboratory Animals (NIH Publications No. 80-23, revised 1978).

Endothelin-1 (ET-1) and sarafotoxin 6c (S6c) were acquired from China Peptides and then dissolved in a solution composed of 0.9% saline and 0.1% bovine serum albumin. MAPK pathway inhibitor SB203580 (for p38 pathway), SP600125 (for JNK pathway), U0126 (for ERK1/2 pathway), and ET_B receptor antagonist BQ 788 were obtained from Selleck (Houston, Texas, USA) and Sigma (St. Louis, Missouri, USA). These compounds were dissolved in dimethyl sulfoxide (DMSO) for experimental use. Each inhibitor was added in a volume of 1 µl to the corresponding culture wells, while an equal amount of DMSO was used as a control. Final molar concentrations in the tissue baths were displayed.

Artery culture

Rats were anaesthetised with CO₂ and sacrificed. Middle cerebral arteries were carefully separated, sliced into ring segments measuring 1~2 mm, and then transferred to the wells of a 24-well plate containing Dulbecco’s modified Eagle’s medium without serum. Artery segments were divided into various experimental categories including a Fresh group, Control group (using DMSO as the solvent), UA group (200, 400, and 800 µmol/l), UA 400 µmol/l + inhibitor groups, and DMSO + inhibitor groups (n = 12 experiments per group, and only one sample was taken from each rat). The plates were incubated at 37°C in humidified 5% CO₂ and 95% air for 24 h.

Contractile function studies

A wire myograph was used to record artery contractile function. Two thin wires (40 mm in diameter) were used to thread each artery segment, which was then mounted in myograph baths containing 5 ml of Krebs solution. The segments were permitted to reach a stable state at 1.5 mN for a minimum of 1 h. Arterial activity was tested by K⁺-rich Krebs solution (containing 60 mmol/l KCl). Only segments that elicited reproducible responses > 1 mN to K⁺ were used. The selective ET_B receptor agonist, S6c, was gradually introduced at concentrations ranging from 10^–7 to 10^–11 M into the tissue baths. This allowed for a concentration-response curve specific to the ET_B receptors. Following the inhibition of ET_B receptors with BQ788, the contractions elicited by ET-1 (10^–7–10^–11 M), which acts as an agonist for both ET_B and ET_A receptors, were assessed. These response curves were specifically attributed to the activation of ET_A receptors.

RT-PCR

Total RNA was isolated from samples of cerebral arteries by using an E.Z.N.A. ^@MicroElute Total RNA Kit (R6831-02, Omega Biotech, Co. Ltd.). Reverse transcription-PCR Kits (Bioer Technology Co., Ltd., China) were utilised to synthesise cDNA from mRNA. RT-PCR was conducted with SYBRs Premix Ex TaqTM Kits from Takara, Japan, using the iQTM5 Multicolor Real-Time PCR Detection System by Bio-Rad, USA. Specific primer sequences for ET receptor genes were designed as follows: ET_A receptor, forward: 50-GTC GAG AGG TGG CAA AGA CC-30; ET_A receptor, reverse: 50-ACA GGG CGA AGA TGA CAA CC-30; ET_B receptor, forward: 50-TGACGCCACCCACTAAGACC-30; ET_B receptor, reverse: 50-GGCACGGAGGAGGGAAGG-30. ET receptor gene expression levels were compared using β-actin mRNA as an internal reference during the measurement process and were expressed as a fold-change relative to the fresh group (n = 6 rat for each group).

Western blot

The protein content in cerebral artery (n = 3 rat for each group) extracts was determined using a BSA assay kit. Equal portions of 40 µg protein were electrophoresed and transferred to a nitrocellulose membrane. Following a 2-h period of incubation at ambient temperature with 5% non-fat milk to prevent non-specific binding, the membranes were exposed to primary antibodies overnight at 4°C: rabbit anti-ET_A receptor (Abcam, ab85163) at 1 : 300, rabbit anti-ET_B receptor (Abcam, ab262700) at 1 : 500, and mouse anti-β-actin receptor (Santa Cruz Biotechnology, sc-47778) at 1 : 2000. Following this, the membranes were exposed to secondary antibodies at room temperature for 1 h. Finally, protein bands were detected by chemiluminescent HRP substrate and captured using a computerised chemiluminescent image analysis system (Bio-Rad, Hercules, CA). Band intensity was analysed using the Imaging J System (Media Cybernetics, Bethesda, MD).

Immunohistochemistry

Cerebral arterial tissues (n = 3 rat for each group) were prepared by fixation and dehydration, followed by cutting into slices of 10 µm thickness. Next, these sections were incubated in a 10% goat serum solution for 30 min at 37°C. Afterward, they were permitted to engage with primary antibodies at a temperature of 4°C for the duration of the night: mouse anti-ET_A receptor (Santa Cruz Biotechnology, sc-518060) diluted at 1 : 500; sheep anti-ET_B receptor (Abbexa, abx411769) diluted at 1 : 300. Ultimately, the tissue sections were exposed to secondary antibodies – either goat anti-mouse IgG H&L conjugated with FITC (Bioss, bs-0296G-FITC) or donkey anti-sheep IgG H&L (Santa Cruz Biotechnology, ab150177) – for 1 h under dark conditions. Immunoreactivity was visualised using an epifluorescence microscope (Olympus BX53, Tokyo, Japan) and photographed with a digital camera. Data analysis was performed using Image J software. Omission of primary antibodies served as a negative control.

Statistical analysis

Data are presented as the average value with the standard error of the mean (SEM). Statistical analyses were conducted using GraphPad Prism software version 8.2. E_max refers to the highest level of contraction observed, whereas pEC₅₀ represents the logarithm of the concentration of the agonist that produces half of the maximum response. Statistical comparisons were conducted using unpaired t-tests with Welch’s correction for 2-group comparisons, or 2-way ANOVA with Bonferroni’s post-test for multiple comparisons. For the analysis of multiple data sets, one-way ANOVA was conducted, followed by Dunnett’s post-test. A p-value below 0.05 was deemed to show statistical significance.

Elevated UA levels increased the contraction of cerebral arteries mediated by ET receptors

In Figure 1 A, it is shown that UA at concentrations of 400 µmol/l and 800 µmol/l notably increased the contractions induced by S6c in cerebral arteries when compared to the control group (0 µmol/l UA). The E_max values were also notably higher in the UA-treated groups. In Figure 1 B, it can be observed that being exposed to 400 µmol/l UA for 24 h led to more intense ET_B receptor-induced contractions in cerebral arteries when compared to exposure durations of 12 h or 48 h. In Figure 1 C, it is shown that 400 µmol/l UA notably increased the contractions to ET-1 in cerebral arteries when compared to the control group (0 µmol/l UA), resulting in a significant rise in the E_max values. Figure 1 D shows that exposure to UA for 24 h or 48 h induced an increase in ET_A receptor-mediated contractions. Therefore, a concentration of 400 µmol/l and an exposure time of 24 h were selected as the UA culture conditions for further studies.

Figure 1

The effects of high levels of UA on concentration-contractile curves for rat cerebral artery segments, as meditated by ET receptors. A – Dose-dependent curve of cerebral artery constriction induced by S6c. B – Time-dependent curve of cerebral artery constriction induced by S6c. C – Dose-dependent curve of cerebral artery constriction induced by ET-1. D – Time-dependent curve of cerebral artery constriction induced by ET-1. Data are presented as mean ± SEM. n = 12. *p < 0.05, **p < 0.01 vs. UA 0 μmol/l (A, C); #p < 0.05, ##p < 0.01 vs. UA 200 μmol/l (A, C); $p < 0.05 vs. UA 400 μmol/l (A) or *p < 0.05, **p < 0.01 vs. Exposure to UA 400 μmol/l for 0 h (B, D)

High levels of UA-enhanced contraction are associated with the MAPK pathways

Inhibitors were utilised to study whether MAPK pathways are involved in UA-enhanced contractions. For ET_B receptors, SB203580, SP600125, and U0126 significantly attenuated the UA-enhanced contractions mediated by ET_B receptors, with a significant reduction in E_max values (Figures 2 A–C, p < 0.05). For ET_A receptors, U0126 and SP600125 did not alter the UA-enhanced contractions significantly. However, SB203580 suppressed the contractions, causing a rightward shift in the concentration-response curves, which is indicative of a notably diminished E_max value (Figures 2 D–F, p < 0.05).

Figure 2

Effects of MAPK pathway inhibitors on UA-induced enhancement of ET_B (A, B, C) and ET_A (D, E, F) receptor-mediated concentration-contraction curves for rat cerebral arteries. The cerebral artery segments were cultured with or without UA 400 μmol/l for 24 h. In addition, the segments were cultured with UA 400 μmol/l and different MAPK inhibitors and DMSO for 24 h. S6c-induced and ET-1-induced contractions were recorded, and concentration- contraction curves for p38 inhibitor SB203580 (A, D), JNK inhibitor SP600125 (B, E), and ERK1/2 inhibitor U0126 (C, F) were constructed. Data are presented as mean ± SEM. n = 12. *p < 0.05, **p < 0.01 vs. Control (cultured with DMSO); #p < 0.05, ##p < 0.01 vs. UA 400 μmol/l

High levels of UA upregulate the expression of ET receptor mRNA and proteins, and are involved in the MAPK pathway

Figure 3 demonstrates that UA at concentrations of 400 µmol/l and 800 µmol/l stimulated the de novo transcription of ET_B receptor mRNA. We found that exposure to UA 400 µmol/l significantly elevated the expression of ET_A receptor mRNA (p < 0.05, Figure 3). However, when the UA concentration was increased to 800 µmol/l, there was a non-significant downward trend in the ET_A receptor mRNA level.

Figure 3

Effects of UA 400 μmol/l on ET_B and ET_A receptor mRNA expression in cerebral arteries. Rat cerebral arterial segments were cultured with or without UA 200, 400, and 800 μmol/l or DMSO for 24 h. The mRNA expression levels of ET_B and ET_A were measured using RT-PCR and were expressed as a fold-change relative to the fresh group. Data are presented as mean ± SEM. n = 6. *p < 0.05, **p < 0.01 vs. Control (cultured with DMSO)

Furthermore, as shown in Figure 4, exposure to UA at a concentration of 400 µmol/l resulted in a notable enhancement in the protein levels of ET_B receptor in comparison to the control group, displaying a statistically significant variance (p < 0.01). The increase in ET_B receptor protein levels could be significantly reduced by using inhibitors of the MAPK pathway such as SB203580, SP600125, or U0126 (p < 0.01, Figure 4). Meanwhile, the protein expression of ET_A receptor was observed to be higher relative to the control group (p < 0.01, Figure 4). This enhancement was notably mitigated solely by the treatment with SB203580 (p < 0.01, Figure 4). Neither SP600125 nor U0126 treatments had any significant effect on the ET_A receptor protein levels in both UA-exposed and control groups.

Figure 4

Effects of UA 400 μmol/l on protein expression in cerebral arteries with the influence of MAPK inhibitors. Rat cerebral arterial segments were cultured with UA 400 μmol/l and p38 inhibitor SB203580 (A), JNK inhibitor SP600125 (B), ERK1/2 inhibitor U0126 (C), and DMSO for 24 h. The ET receptor protein expression levels were expressed relative to β-actin. Data are presented as mean ± SEM. n = 3. **p < 0.01 vs. Control (cultured with DMSO). ##p < 0.01 vs. UA 400 μmol/l

Figure 5 illustrates the application of immunohistochemistry to detect ET receptor proteins in cerebral arterial tissues, which were staining green. The negative control group were without any detectable staining for both ET_B and ET_A receptors (data not shown). The brightness level of ET_B receptors in cerebral arteries was discovered to be notably higher in the UA-treated group than in the control group (p < 0.01), and it was decreased by the MAPK pathway blockers SB203580, SP600125, and U0126 (p < 0.01). Likewise, the UA-treated group exhibited higher fluorescence intensity of ET_A receptors compared to the control group (p < 0.01). While SP600125 and U0126 did not significantly alter the fluorescence intensity in the UA group, SB203580 notably suppressed the UA-enhanced in ET_A receptor fluorescence intensity in cerebral arteries (p < 0.01).

Figure 5

Immunohistology analysis of the effects of high levels of UA and MAPK inhibitors on ET receptor protein expression in cerebral arteries. Rat cerebral arterial segments were cultured with UA 400 μmol/l and MAPK inhibitors or DMSO for 24 h. Representative images for each group (A – ET_B receptor, B – ET_A receptor). Scale bar 100 μm, magnification × 20. Data are presented as mean ± SEM. n = 3. **p < 0.01 vs. Control. ##p < 0.01 vs. UA 400 μmol/l Representative images for each group the results of analyses (C – ET_B receptor, D – ET_A receptor). Scale bar 100 μm, magnification. 20. Data are presented as mean ± SEM. n = 3. **p < 0.01 vs. Control. ##p < 0.01 vs. UA 400 μmol/l