The experimental procedures in the present study were in accordance with the ARRIVE (Animals in Research: Reporting In Vivo Experiments) guidelines and were approved (approval no. 20210304037) by the Animal Care and Use Committee of Guangzhou University of Traditional Chinese Medicine (Guangzhou, China). Male Sprague-Dawley rats (8–10 weeks old; 250–300 g; Experimental Animal Center affiliated with Guangzhou University of Traditional Chinese Medicine, Guangzhou, China) were kept in a clean animal room, with free access to food and water (12 h:12 h light/dark cycle, 22±1°C, 50% humidity). The rats were randomly assigned to 5 groups: Sham, the group of rats with sham operation; CIR, the group of rats with MCAO; Saline, the group of rats with MCAO and saline pretreatment; Ast, the group of rats with MCAO and astragaloside IV pretreatment; Bum, the group of rats with MCAO and bumetanide pretreatment. The animals with tissue sampling were anesthetized by sodium pentobarbital (20 mg/ml, i.p.), and the rest were sacrificed by carbon dioxide asphyxiation (flow rate=70% of the cage volume per min). Finally, the valid data was produced from a total of 86 rats. All possible efforts were made to minimize the number of animals and their suffering.

Rats were first subjected to 5% isoflurane (RWD Life Science) in 30% O₂ balanced with N₂O, then with 1.5% isoflurane for anesthesia maintenance. Cerebral blood flow was monitored in the territory of the middle cerebral artery using a laser doppler flowmetry (RWD Life Science). Thereafter, the right common carotid artery, internal carotid artery and external carotid artery of individual rats were exposed. Focal ischemia was induced by inserting a monofilament nylon suture with a round tip (Yuyan Instruments Co., Ltd.) inserted into the internal carotid artery through the external carotid artery stump and gently advanced to the middle cerebral artery. After 30 min of ischemia, the filament was removed to restore blood flow (reperfusion) and the skin incision was sutured. Sham-operated control rats received the same surgical procedure without insertion of a filament. Rats with intracranial hemorrhage and those that did not show a reduction in cerebral blood flow >80% during MCAO were excluded.

Astragaloside IV (Shanghai Yuanye Bio-Technology Co., Ltd.) was diluted in corn oil. Rats were treated with astragaloside IV (7.5 mg/ml) or saline via intraperitoneal injection 15 min before surgery. Bumetanide (0.18 mg/ml, 2 µl, 0.25 µl/min; cat. no. B3023; MilliporeSigma) was microinjected into bilateral paraventricular [AP 1.5 mm, ML 0.4 mm, DV 7.7 mm (16)] with a microinjection pump (Yuyan Instruments Co. Ltd.).

Tail cuff blood pressure system (IITC Life Science Inc.) was used for the measurement of MAP. First, the rats were sufficiently acclimated to the restraint holder, and each rat was separated by an opaque partition. Then the tail was warmed with a warming pad for 15–20 min (until the rat was no longer irritable) before each cycle of blood pressure measurements. The final data was obtained from the average of 10 valid data. The data of animals those could not cooperate well in the experiment were eliminated.

To estimate the degree of neurological impairment, a 48-point scoring system was used. This scoring system composes of general status (spontaneous activity, body symmetry, gait; 0–12), simple motor deficit (forelimb asymmetry, circling, hind-limb placement; 0–14), complex motor deficit (vertical screen climbing, beam walking; 0–8), and sensory deficit (hind limb, trunk, vibrissae and face touch; 0–14). The total score was the sum of the 4 individual scores, with 0=no deficit and 48=maximal deficit.

Rats were euthanized on indicated time point. The brain sections were placed in 1% TTC (cat. no. 298-96-4; MilliporeSigma) at 37°C for 30 min. The slices were flipped once every 5 min and then washed 3 times with ddH₂O. Images of the sections were captured with a digital camera. The area of infarct of each section (1 mm) was measured by subtracting the non-infarcted area in the ipsilateral hemisphere from the total area of the contralateral hemisphere, and then the final infarct volume was calculated by summing the infarct areas in all sections and multiplying by the section thickness (ImageJ v1.53e; National Institutes of Health).

To detect the level of arginine vasopressin (AVP), rat pituitary tissue was isolated, and dissolved with homogenizing medium (1:9). The sample was centrifuged at 3,500 × g for 10 min, and the supernatant was harvested. Diluted supernatant was added to the AVP ELISA kit (Rat) (cat. no. OKCD08532; Aviva Systems Biology, Corp.). Then, the plate was sealed with sealing film and incubated at 37°C for 30 min. The sealing plate film was carefully removed and the liquid was discarded. Τhe plate was washed for 5 times. Then, labeling reagents (50 µl) were added into each well (except blank). The incubation and washing process were repeated. Chromogenic agent A and B (50 µl) was added into each well in sequence. After 10 min coloration at 37°C, 50 µl termination solution was added to each well. The absorbance of each well was measured at the wavelength of 450 nm.

The paraventricular nucleus (PVN) tissue was homogenized in the lysis buffer (10 mM Tris-HCl and 320 mM sucrose) containing 1% protease inhibitor mixture (cat. no. 36978; Thermo Fisher Scientific) and then centrifuged (Eppendorf 5810) at 8,000 × g for 5 min. Then the supernatant was centrifuged (Eppendorf 5810) at 40,000 × g for 30 min to obtain crude membrane in the pellet. The cellular membranes were resuspended in lysis buffer with protease inhibitor mixture. Protein concentrations were determined using Bio-Rad protein reagent (Bio-Rad Laboratories, Inc.). Samples were heated at 95°C for 10 min in loading buffer before 20 µg protein was loaded for 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis, electrophoretically separated, and then transferred to polyvinylidene difluoride membranes at 4°C. After blocking with 5% non-fat milk for 2 h at room temperature, membranes were incubated overnight at 4°C with a primary antibody [rabbit anti-Na⁺-K⁺−2Cl⁻ cotransporter isoform 1 (NKCC1) (1:1,000; cat. no. 4828S; Cell Signaling Technology, Inc.)]. Membranes were washed with PBS buffer, incubated with horseradish peroxidase-conjugated secondary anti-rabbit IgG (1:2,000; cat. no. ab6721; Abcam) for 2 h at room temperature, and the bands were visualized using Immobilon Western Chemiluminescent HRP Substrate (cat. no. WBKLS0050; MilliporeSigma). The relative levels of the target protein were determined by performing a densitometry analysis using ImageJ v1.53e software (National Institutes of Health).

SigmaStat 3.5 (Jandel Scientific Software) was used to perform the statistical analyses. The data were expressed as the mean ± SD. Student's t-test (independent t-test) was used when two groups were compared. The MAP data were analyzed by using one-way repeated-measures ANOVA or two-way repeated-measures ANOVA followed by the Bonferroni post hoc test. P≤0.05 was considered to indicate a statistically significant difference.

To reduce the mortality of the animals, the middle cerebral artery of the rat was occluded for only 30 min, and the subsequent reperfusion lasted 210 min in the present study (Fig. 1). The results showed significant neurologic dysfunction (Sham vs. CIR=0 vs. 22.60±5.07, n=5 per group, Student's t-test, P<0.001, Fig. 2A) and increased infarct volume (Sham vs. CIR=0 mm³ vs. 130.13±27.08 mm³, n=5 per group, Student's t-test, P<0.001, Fig. 2B and C) after CIR. Meanwhile, the MAP significantly elevated, and prolonged at least for 210 min (n=10 per group, two-way repeated-measures ANOVA followed by the Bonferroni post hoc test, P<0.001, Fig. 2D, Table I). These results indicated that CIR induces a long-term MAP elevation.

It is consistent with most of previous findings that astragaloside IV pretreatment relieved the neurologic dysfunction of rats induced by CIR (Saline vs. Ast=23.67±4.55 vs. 14.33±4.46, n=6 per group, Student's t-test, P=0.005, Fig. 3A) and reduced infarct volume (Saline vs. Ast=173.46±60.97 mm³ vs. 74.97±35.05 mm³, n=6 per group, Student's t-test, P=0.006, Fig. 3B and C). Moreover, the rats treated with astragaloside IV showed a milder fluctuation of MAP (n=10, one-way repeated-measures ANOVA followed by the Holm-Sidak method, P<0.001 compared with the baseline before modeling, Fig. 3D, Table I). These results indicated that astragaloside IV relieves CIR-induced MAP elevation.

AVP, also called antidiuretic hormone, is synthesized in the PVN of the hypothalamus and stored in the pituitary gland. It is released into the blood circulatory system to regulate systemic blood pressure when needed (17). It has been demonstrated that the upregulation of NKCC1 in AVP neurons contributes to the generation of sodium-dependent hypertension by increasing AVP release (18). One of the main functions of NKCC1 is to transport Cl⁻ into cells, increasing the concentration of intracellular Cl⁻, thereby weakening GABAergic inhibition and even turning it into depolarization (19,20). Therefore, the upregulation of NKCC1 in hypothalamus may promote the excitability of hypothalamic neurons, thus increasing the synthesis and secretion of AVP.

The present results showed that CIR significantly increased the AVP level (ng) in the pituitary gland (g) (Sham vs. CIR=18.12±2.25 ng/g vs. 48.64±9.06 ng/g, n=6 per group, Student's t-test, P<0.001, Fig. 4A) and the expression level of NKCC1 in the PVN (Sham vs. CIR=1.00±0.19 vs. 3.61±1.30, n=6 per group, Student's t-test, P<0.001, Fig. 4B). To investigate the contribution of NKCC1 to generating MAP elevation, bumetanide, an NKCC1 inhibitor, was delivered by paraventricular microinjection. It was found that bumetanide pretreatment significantly reduced the MAP during CIR (n=10, one-way repeated-measures ANOVA followed by the Holm-Sidak method, P<0.001 compared with the baseline before modeling, Fig. 4C, Table I). These results indicated that upregulation of NKCC1 in hypothalamus mediates MAP elevation during CIR.

The results of the present study showed that astragaloside IV intraperitoneal injection significantly inhibited the NKCC1 expression in PVN of MCAO rats (Saline vs. Ast=1.00±0.32 vs. 0.43±0.15, n=6 per group, Student's t-test, P=0.002, Fig. 5A). Moreover, the AVP level in pituitary gland was also reduced. (Saline vs. Ast=49.05±9.59 ng/g vs. 31.89±6.45 ng/g, n=6 per group, Student's t-test, P=0.005, Fig. 5B). These results indicated that astragaloside IV inhibits NKCC1 expression in hypothalamus of MCAO rats.