Contributed equally
Stroke is one of the leading causes of death from diseases. When the blood supply to the brain tissue is interrupted, neuronal core death occurs due to the lack of glucose and oxygen in min. Blood pressure lowering after ischemic stroke was proven to be an effective strategy to achieve neurovascular protection and reduce the risk of recurrent stroke. Astragaloside IV is a pure small molecular compound isolated from
Stroke is one of the leading causes of death from diseases. According to the Heart Disease and Stroke Statistics report from the American Heart Association (2019 updated), more than 10 million people are affected by stroke every year (
Thrombolytic therapy is a standard strategy for clinical treatment for ischemic stroke (
Astragaloside IV is a pure small molecular compound isolated from
The experimental procedures in the present study were in accordance with the ARRIVE (Animals in Research: Reporting
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 (
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 (
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,
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 (
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,
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,
The human brain accounts for 15–20% of the cardiac output and is extremely sensitive to ischemia. Blood pressure is one of the important vital signs of the human body. As a driving force to promote the blood flow, blood pressure ensures the blood supply of important organs. When ischemia occurs in tissues or organs, the human body may activate the autoregulation system (heart rate, cardiac output, blood pressure) to maintain normal hemodynamics (
AVP elevates blood pressure mainly by promoting water reabsorption, blood volume and the contraction of vascular smooth muscle (
In traditional Chinese medicine, the stroke is described as a series of symptoms (abrupt coma, paraesthesia, hemiplegia, facial paralysis, speech disorder) induced by obstruction of cerebral meridians (ischemic stroke) or blood spill over from cerebral meridians (hemorrhagic stroke). The most important pathogenesis of ischemic stroke is the decline of vital Qi (energy) with aging or prolonged illness.
It is consistent with previous findings that astragaloside IV had an obvious preventive effect on CIR injury. Meanwhile, the rats treated with astragaloside IV showed a milder fluctuation of MAP. Those results indicated that the inhibitory effect of astragaloside IV on blood pressure may contribute to alleviate ischemia reperfusion injury to a certain extent. In addition, pretreatment with astragaloside IV inhibited the expression of NKCC1 in PVN nuclei and the level of AVP in pituitary of CIR rats. However, in the present study, it was not clear whether the antihypertensive effect of astragaloside IV directly acts on the central system or peripheral.
It was considered that there are two possible mechanisms for astragaloside IV regulating blood pressure during CIR. According to the data provided by Traditional Chinese Medicine Systems Pharmacology database and analysis platform, astragaloside IV is BBB non-penetrable, which may have limited effect on the central nervous system. It has been reported that astragaloside IV relieved hypertension via improving inflammation, pulmonary artery remodelling and oxidative stress (
Not applicable.
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.
FS, JH and LW designed the research. FS, YM and YH conducted the experiments. FS, YM and BH analyzed the data. JH and LW confirm the authenticity of all the raw data. FS, JH and LW prepared the manuscript. All authors read and approved the final version of the manuscript.
The experimental procedures in the present study were in accordance with the ARRIVE (Animals in Research: Reporting
Not applicable.
The authors declare that they have no competing interests.
Schematic of experimental process. MCAO, middle cerebral artery occlusion; MAP, mean arterial pressure; 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. TTC, 2,3,5-triphenyltetrazolium chloride; ELISA, enzyme linked immunosorbent assay.
CIR-induced brain damage and hypertension. (A) Statistical chart of neurologic score (n=5 per group, Student's t-test, ***P<0.001). (B) 2,3,5-triphenyltetrazolium chloride-stained brain tissue specimens. (C) Statistical chart of infarct volume (n=5 per group, Student's t-test, ***P<0.001). (D) Statistical chart of MAP (n=10 per group, two-way repeated-measures ANOVA followed by the Bonferroni post hoc test, ***P<0.001, Sham vs. CIR at the same time point). Sham, the group of rats with sham operation; CIR, the group of rats with CIR. Triangles indicate the individual data obtained from each group. CIR, cerebral ischemia reperfusion; MAP, mean arterial pressure.
Astragaloside IV relieves CIR-induced brain damage and hypertension. (A) Statistical chart of neurologic score (n=6 per group, Student's t-test, **P<0.01). (B) 2,3,5-triphenyltetrazolium chloride-stained brain tissue specimens. (C) Statistical chart of infarct volume (n=6 per group, Student's t-test, **P<0.01). (D) Statistical chart of MAP (n=10, one-way repeated-measures ANOVA followed by the Holm-Sidak method, ***P<0.001, compared with the baseline before modeling). Saline, the group of rats with CIR and saline pretreatment; Ast, the group of rats with CIR and Astragaloside IV pretreatment. Triangles indicate the individual data obtained from each group. CIR, cerebral ischemia reperfusion; MAP, mean arterial pressure.
Upregulation of NKCC1 in hypothalamus-mediated hypertension during CIR. (A) Statistical chart of AVP level in pituitary (n=6 per group, Student's t-test, ***P<0.001). (B) Statistical chart of NKCC1 level in hypothalamus. Upper panel represents three pairs of western blot samples (n=6 per group, Student's t-test, ***P<0.001). (C) Statistical chart of MAP with bumetanide pretreatment (n=10, one-way repeated-measures ANOVA followed by the Holm-Sidak method, ***P<0.001, compared with the baseline before modeling). Sham, the group of rats with sham operation; CIR, the group of rats with CIR. Triangles indicate the individual data obtained from each group. NKCC1, Na+-K+−2Cl− cotransporter isoform 1; CIR, cerebral ischemia reperfusion; AVP, arginine vasopressin; MAP, mean arterial pressure.
Astragaloside IV inhibits NKCC1 expression in hypothalamus during CIR. (A) Statistical chart of NKCC1 level in hypothalamus. Upper panel represents three pairs of western blot samples (n=6 per group, Student's t-test, **P<0.01). (B) Statistical chart of AVP level in pituitary (n=6 per group, Student's t-test, **P<0.01). Saline, the group of rats with CIR and saline pretreatment; Ast, the group of rats with CIR and Astragaloside IV pretreatment. Triangles indicate the individual data obtained from each group. NKCC1, Na+-K+−2Cl− cotransporter isoform 1; CIR, cerebral ischemia reperfusion; AVP, arginine vasopressin.
Mean arterial pressure (Sham vs. CIR).
Group | −30 min | 30 min | 60 min | 120 min | 240 min |
---|---|---|---|---|---|
Sham (mmHg) | 85.90±2.60 | 87.40±2.17 | 87.80±2.70 | 85.80±3.36 | 86.70±3.68 |
CIR (mmHg) | 86.40±3.24 | 111.40±4.50 | 105.00±4.45 | 105.60±7.00 | 102.40±4.53 |
Ast (mmHg) | 86.80±3.08 | 102.10±6.23 | 88.60±4.60 | 87.80±4.59 | 89.30±3.40 |
Bum (mmHg) | 86.10±3.48 | 98.50±6.29 | 88.50±4.12 | 85.70±4.50 | 87.30±6.00 |
CIR, cerebral ischemia reperfusion; Ast, the group of rats with MCAO and Astragaloside IV pretreatment; Bum, the group of rats with MCAO and bumetanide pretreatment.