BuyangHuanwu Decoction attenuates cerebral vasospasm caused by subarachnoid hemorrhage in rats via PI3K/AKT/eNOS axis

2.1 Main materials and regents

The Second Affiliated Hospital of Shaanxi University of Chinese Medicine Pharmacy created the BHD. Radix Astragali (Huangqi), Radix angelicaesinensis (Danggui), Radix paeoniaerubra (Chishao), Rhizoma ligustici chuanxiong (Chuanxiong), Semen persicae (Taoren), Flos carthami (Honghua), and Lumbricus (Dilong) were prepared by decoction in water and alcohol, as 5.0 g/mL extracts. Primary antibodies targeting phosphoinositide 3-kinase (PI3K) (cat. No. AF3241), p-Akt (cat. No. AF0908), and endothelial nitric oxide synthase (eNOS) (cat. No. GB11086) and secondary antibodies horseradish peroxidase (HRP)-labeled goat anti-mouse IgG (cat. No. GB23301) and HRP-labeled goat anti-rabbit IgG (cat. No. GB23303) were obtained from Wuhan Servicebio Biotechnology Co., Ltd. The hematoxylin and eosin (HE) staining kit (cat. No. G1120) was purchased from Beijing Soleibao Biotechnology Co., Ltd. The bicinchoninic acid (BCA) protein quantitative detection kit and the SDS-PAGE gel preparation kit were purchased from Google Biotechnology Co., Ltd. The nitric oxide (NO) enzyme-linked immunosorbent assay (ELISA) Test Kit (cat. No. A013-2) was purchased from Shanghai Qiaodu Biotechnology Co., Ltd.

2.2 Animals and experimental design

Our investigations used Sprague–Dawley rats (bought from the Laboratory Animal Center, Xi’an Jiaotong University, Medical Experimental Animal License Number: SCXK 2012-003). They were kept in a facility with a 12-h light–dark cycle and free access to food and water. During our investigation, 60 male Sprague Dawley rats weighing between 300 and 400 g were used.

Rats were divided into four groups: (1) control group, (2) SAH group, (3) SAH + BHD [13 g/(kg day)] group, and (4) the SAH + BHD [26 g/(kg day)] group. The dose of BHD administered was converted from that used in humans based on the rat body surface area; this dose (0.06 mL/kg) was administered twice per day for 7 days. A double-blind method was used to evaluate the behavior of the animals from all groups using the Garcia neurological scoring scale [13]. After Garcia neurological scores were measured, the animals were given a 10% chloral hydrate (350 mg/kg) anesthetic. Thoracotomy was performed on the animals, followed by perfusion. The right atrium was opened by blocking the descending aorta, and the ventricular chamber was mounted with an NO18 catheter. The animal was then perfused with 100 mL of 0.01 M phosphate-buffered saline (pH 7.4), which was followed by 100 mL of 2% paraformaldehyde in phosphate-buffered saline at 36°C and 80 mmHg pressure. The extracted brain was immersed in 2% paraformaldehyde overnight at 4°C. In all SAH animals, visual assessment confirmed that the clump had clogged the basilar artery. CSF was collected and retained for further analysis.

1. Ethical approval: The research related to animal use has been complied with all the relevant national regulations and institutional policies for the care and use of animals and was approved by the Ethics Committee of Shaanxi University of Chinese Medicine (license no: sucmdl20210305017).

2.3 SAH animal model and BHD treatment

Rats were anesthetized with 350 mg/kg of 10% chloral hydrate intraperitoneally. We used the upgraded double injection SAH model [14]. For the first induction of SAH, fresh arterial blood (0.1 mL/100 g) was collected from the caudal artery and administered into the cisterna magna. The mice were placed in a central position for 30 min after induction. Rats with respiratory distress were observed and, if necessary, ventilated. When they were completely awake, they were reintroduced to the veterinary house. For the second induction of SAH, animals were again injected with blood 48 h after the first SAH to delay vasoconstriction. After the model was established, the neurological function score of the SAH mice was lower than that of the control group, indicating that the model was successful.

On the second day after the model was established, two experimental groups were handled as follows: BHD was administered by continuous enema for 7 days, twice per day in the morning and evening. The equivalent dose was converted in accordance with the surface area of the experimental animals and humans using the conversion formula of the relative ratio of the body mass to the body surface area per kilogram.

2.4 The Garcia neurological score

The Garcia neurological scoring scale evaluated the extent of neurological impairment in six categories: rat autonomy, body symmetry, forelimb extension function, screen test, bilateral body touch, and bilateral beard reflex. A score of 18 points indicates normal function [13]. The most severe injury scored the lowest score of 3 points.

2.5 Basilar artery diameter analysis (HE staining)

The basilar artery was sliced into five cross-sections, and paraffin-embedded brain tissue was sliced into 25-µm-thick sections. HE staining was performed, and the tissue was observed with a video-assisted microscope. The diameter of the basilar artery was measured and analyzed with ImageJ 1.50i [15].

2.6 Immunohistochemical analysis

Endothelial cells were identified using video-assisted microscopy (400×). Separated rat basilar arteries were perfused and preserved with 4% paraformaldehyde. Paraffin-embedded slices of the basilar artery were treated with polyclonal anti-rabbit eNOS antibody (1:200; Servicebio, Wuhan, China) at 25°C for 40 min. Five consecutive sections of each specimen were collected, and the morphology of the lumen cross-section was analyzed.

2.7 ELISA analysis

An ELISA kit was used to measure the NO content of the CSF. The testing was carried out as directed by the manufacturer. As indicated in the supplier’s instructions, the upper limit of NO detected was 40 µmol/L.

2.8 RNA extraction and quantitative real-time PCR

A TRIzol reagent (Invitrogen, Carlsbad, CA, USA) was used to extract total RNA from brain tissue, which was then measured using a UV spectrophotometer. A SuperScript reverse transcriptase kit was used for reverse transcription (Invitrogen, Carlsbad, CA, USA). Gene expression was measured using a quantitative real-time PCR with SYBR^® dye (LightCycler^® 96 Real-Time PCR System) after the cDNA was generated. The transcription level was calculated relative to the GAPDH signal and normalized to the mean of the control group data.

The primers for PI3K, AKT and GAPDH were as follows. PI3K: forward, 5′-GGTGAGGAACGAAGAATGGC-3′ and reverse, 5′-TCCGAGGCAAGACAGGGATA-3′; AKT: forward, 5′-TGAGACCGACACCAGGTATTTTG-3′ and reverse, 5′-GCTGAGTAGGAGAACTG GGGAAA-3′; GAPDH: forward, 5′-CTGGAGAAACCTGCCAAGTATG-3′ and reverse, 5′ GGTG GAAGAATGGGAGTTGCT-3′.

2.9 Western blot analysis

Samples from the basilar artery (6 animals per group) were homogenized with a protease inhibitor in RIPA lysate (Servicebio) and centrifuged for 12 min at 12,000 rpm. Protein was then quantified by BCA protein quantitation (Google Biotechnology Co., Ltd.), and then, sample buffer (4×; Servicebio) was added. The samples were loaded and run on 8% SDS-PAGE gels and transferred to polyvinylidene difluoride membranes. At room temperature, membranes were blocked in 5% skim milk powder in Tris-saline with 0.2% Tween 20 (TBST). Rabbit polyclonal anti-rat PI3K (1:1000; Wuhan Servicebio Biotechnology Co., Ltd.), Akt, and p-AKT antibodies were used and anti-actin (monoclonal antibody, 1:1,000; Servicebio) served as a control. HRP-conjugated secondary antibody in TBST was added for 1 h at room temperature. SuperSignal™ West Femto Maximum Sensitivity Substrate (Thermo Fisher Scientific, Inc.) was used as a visualization substrate. Optical density was measured using a Pierce chemiluminescence image analyzer.

2.10 Statistical analysis

GraphPad Prism software and SPSS 25.0 (IBM Corp.) were used to analyze all of the data (version 8.2). The results were provided as mean with standard deviation. To reduce error, the trials were performed three times. To examine the differences between the groups, one-way analysis of variance was performed, followed by Tukey’s post hoc analysis. P < 0.05 was considered statistically significant.

3.1 Establishment of the SAH animal model

To investigate the protective effect of BHD on CVS caused by SAH in rats, we established an SAH animal model using the improved double injection SAH model. Garcia neurological scores were measured after the model was completed. The neurological function (Figure 1a) score of SAH mice was lower than that (Figure 1b) of control group mice; when the two groups were compared, there was a significant difference (P < 0.01). This demonstrates that the model was successfully established.

Figure 1

Establishment of the SAH animal model. (a) The procedure for a catheter-based double blood injection into the cisterna magna via a parieto-occipital burr hole in an experimental animal. (b) The neurological function score of the SAH group was lower than that of the control group. There was a statistically significant difference between the two groups. The data are shown as mean ± standard deviation (n = 5); ^* P < 0.05 is used to indicate significance.

3.2 BHD improved CVS-induced neurological deficits after SAH (the Garcia neurological score and the diameter of basilar artery)

Rats in the experimental group were treated with BHD for 1, 3, and 7 days, and neurological function scores were recorded. As demonstrated in Figure 2a, BHD considerably improved the neurological score after SAH; after 3 days of BHD treatment after SAH, the neurological function score was 14.3% higher in the BHD group than in the SAH group. After 7 days of BHD treatment, the neurological function score was 18.8% (17.0 ± 0.71 points) higher in the BHD group than in the SAH group. These results indicate that BHD improved CVS-induced neurological deficits after SAH.

Figure 2

(a) BHD significantly improved the neurological score after SAH. (b) Cross-sections of the basilar artery in the different groups. (c) The diameter of the basilar artery in the SAH group was significantly reduced compared with that in the control group (^* P < 0.05 was used to indicate significance, compared with the control group). The basilar artery diameter in the SAH + BHD group was substantially larger than that in the SAH group (^# P < 0.05 was used to indicate significance, compared with the SAH group). The data were shown as mean ± standard deviation (n = 3).

Basilar artery cross-sections were stained with HE (Figure 2b). The cross-sectional area of the basilar artery (which is regarded as a useful marker for CVS following SAH) in the SAH group was reduced compared with that in the control group. The cross-sectional area of the basilar artery in the SAH + BHD group was increased after BHD treatment; the basilar artery diameter in the SAH group was 273.548 ± 4.333 µm, whereas in the control group, the SAH + BHD [13 g/(kg day)] group, and the SAH + BHD [26 g/(kg day)] group, the diameter was 291.394 ± 6.367, 303.336 ± 4.376, and 317.306 ± 7.725 µm (mean ± SD), respectively. The basilar artery diameter in the SAH + BHD group was substantially larger than that in the control group and in the SAH group. These results suggest that BHD plays a protective role in regulating vasomotor tone in CVS after SAH (Figure 2c).

3.3 eNOS and NO play an important role in regulating vasomotor tone in CVS after SAH

Immunohistochemistry analysis showed higher protein levels of eNOS in the control group (Figure 3a). Compared with the SAH group, the SAH + BHD group had increased expression of eNOS proteins (Figure 3b). NO levels in the CSF were measured using an ELISA kit that was purchased commercially, and it was found that the concentration of NO in the CSF of the SAH group was considerably lower than that in the control group (Figure 3c). In the SAH rats, BHD raised NO levels in the CSF. In the SAH group, the baseline NO level was 20.32 ± 1.13 mol/L. After 7 days of treatment with BHD, the level of NO was significantly increased compared with the SAH group. These results suggest that BHD reverses the NO decrease in CSF.

Figure 3

Expression of eNOS using immunohistochemistry and of NO using ELISA. (a) Immunostaining for eNOS in basilar artery cross-sections from different groups. (b) The eNOS level in the SAH group was significantly lower than that in the control group (^* P < 0.05 was used to indicate significance, compared with the control group). BHD significantly increased the expression of eNOSafter SAH (^## P < 0.01 was used to indicate significance, compared with the SAH group). The data were shown as mean ± standard deviation (n = 3). (c) The concentration of NO in the CSF in the SAH group was considerably lower than that in the control group (^* P < 0.05 was used to indicate significance, compared with the control group). BHD significantly increased the expression of NO after SAH (^# P < 0.05 and ^## P < 0.01 were used to indicate significance, compared with the SAH group). The data were shown as mean ± standard deviation (n = 3).

3.4 BHD significantly upregulated the PI3K/AKT signaling pathway

We found a reduction in PI3K, AKT, and p-AKT in the SAH group compared with the control group at both the protein level (Figure 4a) and transcription level (Figure 4c). The levels of PI3K, AKT, and p-AKT in the SAH + BHD group were substantially higher than those in the SAH group (P < 0.01) (Figure 4). These findings indicate that BHD induced the expression of PI3K, AKT, and p-AKT and suggest that BHD alleviated CVS and reduced the recurrence of CVS after SAH by significantly upregulating the PI3K/AKT signaling pathway.

Figure 4

Analysis of PI3K, AKT, and p-AKT expression levels by western blot and quantitative real-time PCR. (a) The blots showed that the SAH group had lower levels of PI3K, AKT, and p-AKT than the control group, and the graphs (b) show the ratio of expression compared with actin. BHD increased the expression of PI3K, AKT, and p-AKT as compared with the SAH group. (c) Quantitative real-time PCR analysis of PI3K, AKT, and p-AKT. Quantitative real-time PCR analysis of data was adjusted for GAPDH mRNA content (n = 3) and examined in three separate quadruplicate experiments; data were shown as mean ± standard deviation (n = 3); ^* P < 0.05, compared with the control group and ^# P < 0.05, compared with the SAH group, were used to indicate significance.