Chinese Journal of Physiology

ORIGINAL ARTICLE
Year
: 2022  |  Volume : 65  |  Issue : 2  |  Page : 72--79

Connexin 43 mediated the angiogenesis of buyang huanwu decoction via vascular endothelial growth factor and angiopoietin-1 after ischemic stroke


Ying Zhou1, Ya-Xing Zhang1, Kai-Ling Yang2, Yu-Lian Liu1, Fang-Hua Wu1, Yu-Rong Gao1, Wei Liu1,  
1 Department of Physiology, School of Basic Medical Science, Guangzhou University of Chinese Medicine, Guangzhou, China
2 Medical School, South China University of Technology, Guangzhou, China

Correspondence Address:
Prof. Wei Liu
Department of Physiology, School of Basic Medical Science, Guangzhou University of Chinese Medicine, No. 232, East Waihuan Road, Guangzhou Higher Education Mega Centre, Panyu District, Guangzhou, Guangdong
China

Abstract

Buyang Huanwu decoction (BYHWD), a classical prescription for ischemic stroke, has been reported to promote angiogenesis after focal ischemia. However, the mechanisms of the contribution of BYHWD on angiogenesis are still unclear. Connexin 43 (Cx43) played important roles in the functions of neurogliovascular unit. Therefore, the aim of this study was to explore the potential role of Cx43 in angiogenesis of the ischemic brain after BYHWD treatment. Middle cerebral artery occlusion (MCAO) was used to establish the model of focal ischemia. BYHWD was administrated intragastrically twice a day after MCAO with or without Gap26 (a specific Cx43 inhibitor). Western blot, neurological deficits, immunofluorescent staining, and Evans blue dye were used to confirm the role of Cx43 in angiogenesis after BYHWD treatment. The expression levels of total Cx43 and phosphorylated Cx43 were upregulated by BYHWD and peaked at 7 days post MCAO. Inhibition of Cx43 with Gap26 significantly attenuated the protective role of BYHWD in neurological behavior. BYHWD treatment promoted angiogenesis demonstrated by increased microvascular density, upregulated vascular endothelial growth factor (VEGF), and angiopoietin-1 (Ang-1), while inhibition of Cx43 with Gap26 attenuated these effects of BYHWD. In addition, Gap26 inhibited the beneficial effect of BYHWD on blood-brain barrier (BBB) integrity. These results suggested that Cx43 mediated the angiogenesis of BYHWD via VEGF and Ang-1 after focal ischemic stroke.



How to cite this article:
Zhou Y, Zhang YX, Yang KL, Liu YL, Wu FH, Gao YR, Liu W. Connexin 43 mediated the angiogenesis of buyang huanwu decoction via vascular endothelial growth factor and angiopoietin-1 after ischemic stroke.Chin J Physiol 2022;65:72-79


How to cite this URL:
Zhou Y, Zhang YX, Yang KL, Liu YL, Wu FH, Gao YR, Liu W. Connexin 43 mediated the angiogenesis of buyang huanwu decoction via vascular endothelial growth factor and angiopoietin-1 after ischemic stroke. Chin J Physiol [serial online] 2022 [cited 2022 Jul 1 ];65:72-79
Available from: https://www.cjphysiology.org/text.asp?2022/65/2/72/344169


Full Text



 Introduction



Stroke is the most common cause of death and disability in the world, among which 80% is ischemic stroke.[1] Apart from great sufferings for patients, it brings mental and economic pressure to families and society. Ischemic stroke is generally caused by thrombosis of the cerebral vessels, which deprives the brain cells of oxygen and nutrients, thus leading to cell injury or death. The mechanisms of cerebral ischemic injury are very complicated, including mitochondrial dysfunction, oxidative stress, excitotoxicity, calcium overload, neuroinflammation, and disruption of blood-brain barrier (BBB). However, the applications of these corresponding interventions are limited.[2] Moreover, tissue plasminogen activator, the most effective intervention for ischemic stroke, requires treatments within 6 h. Numerous patients are beyond the treatment window. Therefore, it is necessary to discover and confirm novel strategies for treating ischemic stroke beyond acute phase.[3]

Buyang Huanwu decoction (BYHWD), which was first recorded in Yilin Gaicuo (Correction on Errors in Medical Classics, written by Wang Qingren), is a well-known classical Chinese formula for treating ischemic stroke. Both basic and clinical trials have demonstrated that BYHWD improved neurological behavior after focal ischemia.[4],[5] Its protective effects on focal ischemia were closely related to its promotion of angiogenesis.[6] Angiogenesis is a key factor for promoting restoration after ischemia, which includes the growth of collateral blood vessels and formation of new vessels.[7] However, the mechanisms of BYHWD on angiogenesis after ischemic stroke remain unclear.

Connexin 43 (Cx43), the main gap junction protein subtype, existed in astrocytic processes along blood vessels and was essential for neurogliovascular unit.[8] It has been reported that Cx43 improved brain blood flow recovery by mediating reparative angiogenesis after chronic cerebral hypoperfusion.[9] Conversely, Cx43 downregulation attenuated angiogenic potential of smooth muscle progenitor cells.[10] The protective role of erythropoietin against ischemia was mediated by Cx43.[11] The present study aimed to investigate whether Cx43 mediated the angiogenesis of BYHWD after ischemic stroke.

 Materials and Methods



Buyang Huanwu decoction quality control

All the herbal components of BYHWD [Table 1] were obtained from The Second Affiliated Hospital of Guangzhou University of Chinese Medicine. Herbs were identified and then decocted in distilled water twice (each time 100°C for 30 min). The drug solution was condensed into final concentration of 2 g/ml (equivalent to dry weight of raw materials).{Table 1}

The solution of BYHWD (0.1 ml) was added into 10 ml 50% methanol and filtered by 0.22 μM membrane. The six standards of amygdalin, hydroxysafflor yellow A, paeoniflorin, calycosin glycoside, formononetin, and calycosin were dissolved in methanol at concentration of 1 mg/ml, 300 μg/ml, 300 μg/ml, 5 μg/ml, 50 μg/ml, and 25 μg/ml, respectively. Chromatographic analysis was performed by ultra high-performance liquid chromatography (UHPLC, Shimadzu, Japan). Chromatographic separation was operated on a Shim-pack GISS-C18 column (2.1 × 100 mm, 1.9 μm, Shimadzu) at 30°C. Formic acid aqueous solution and acetonitrile were used for gradient elution: 0–10 min, 5%–10% acetonitrile; 10–13 min, 10%–15% acetonitrile, 13–25 min, 15%–25% acetonitrile, and 25–43 min, 25%–55% acetonitrile. The sample volume was 2 μL and the flow velocity was 0.3 mL/min. The detection wavelength was 264 nm.

Drug administration

BYHWD was administrated intragastrically twice a day (32 g/kg/d).[12] Gap26 (a specific Cx43 inhibitor) was dissolved in saline and injected intraperitoneally (25 μg/kg/d).[13],[14] Notably, Gap26 was treated at 3 days after middle cerebral artery occlusion (MCAO) to avoid its influence during acute phase.[14],[15] Saline was used as a control in sham and MCAO groups.

Animal experiments

Adult male Sprague-Dawley rats (300–350 g) were obtained from Experimental Animal Center of Guangzhou University of Chinese Medicine (Guangzhou, China). All procedures conducted in this study were approved by the Animal Care and Use Committee of Guangzhou University of Chinese Medicine (2016014) and performed in accordance with the National Institutes of Health guidelines for the Care and Use of Laboratory Animals.

Rats were anesthetized with 3.5% isoflurane and maintained with 2% isoflurane via vaporizer for isoflurane (RWD Life Science, Shenzhen, China). Under anesthesia, the right carotid bifurcation was exposed and the external carotid artery was coagulated distal to the bifurcation. A 4.0-s nylon monofilament suture was inserted into the right internal carotid artery through the stump of external carotid artery and gently advanced to block the blood flow. After occlusion for 2 h, the filament was withdrawn for reperfusion. The rats of sham group underwent the same surgical procedure without a suture insertion. According to Longa's assessment, the neurological deficit was scored: 0, no neurological deficit; 1, failure to fully extend left forepaw; 2, circling to the left; 3, falling to the left; 4, loss of spontaneous walking with a depressed level of consciousness, and 5, dead. The rats scored less than or equal to 3 were randomized by table of random number into three groups: MCAO, BYHWD, and BYHWD + Gap26 (n = 12 for each group). The following experiments were under double-blinded methods.

Western blotting

After rats were deeply anesthetized, brains were quickly taken out. The hippocampus were collected and frozen in liquid nitrogen. Samples were homogenized in RIPA buffer (Sigma, USA) that contained inhibitors of protease and phosphatase. After standing on ice for 30 min, samples were centrifuged at 12,000 rpm for 20 min at 4°C. The supernatant was collected and protein concentration was quantified using a BCA Protein Assay Kit (Thermo, USA). Proteins (30 μg per well) were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred onto polyvinylidene difluoride membranes (Millipore, USA). Membranes were blocked with 5% BSA (Roche, Germany) and incubated with primary antibodies (Cx43, 1:1000, Abcam, USA; phosphorylated Cx43 (p-Cx43), 1:1000, Abcam, USA, and glyceraldehyde-3-phosphate dehydrogenase, 1:5000, Abcam, USA) overnight at 4°C. After incubation with secondary antibodies (anti-mouse, 1:5000, Abcam, USA, and anti-rabbit, 1:5000, Abcam, USA) for 1 h at room temperature, the membranes were developed with ECL solution (Millipore, USA) and exposed to an X-ray film. For quantitative calculation, the mean intensity of bands was measured using the ImageJ software (National Institutes of Health, USA).

Behavioral tests

Coordination and integration of motor movements were evaluated by beam-walking test at 1 day, 3 days, and 7 days after MCAO. The beam (90 cm long, 3 cm wide) was placed at the height of 50 cm above the ground.[14] Rats were trained before the operation to ensure that they could walk smoothly on the beam. The scoring criteria developed by Ohlsson was referenced: 0, the rat traversed the beam with no foot slips; 1, the rat traversed the beam with less than 50% foot slips; 2, the rat traversed the beam with more than or equal to 50% foot slips; 3, the rat could cross the beam, but the affected limbs could not aid in forward locomotion; 4, the rat fell down while walking; 5, the rats were unable to cross the beam but remained sitting on the beam, and 6, the rat fell down.[16] The mean value was obtained from three repeated measurements. There were two rats excluded from testing because of death. Extra rats were supplemented.

Spontaneous alteration behavior test was performed with Y-maze at 1 day, 3 days, and 7 days after operation. Arms of the “Y” maze (80 cm long, 16 cm wide) were converged at angles of 120°. Each rat was placed in the center of the maze and allowed to move freely for 8 min. During this period, the total number and sequence of arm entries were recorded. An alternation was defined if the rat entered different arms in succession. The relative alternation in percentage of total arm entries was calculated as follows: relative alternation % = (number of alternations/numbers of total arm entries –2) ×100%.[17]

Immunofluorescent staining

At 7 days, after being deeply anesthetized, the rats were transcardially perfused with 0.9% saline and then 4% paraformaldehyde. Brains were removed and immersed in sucrose solution for dehydration (sucrose of 15% and 30% successively). Coronal sections were cut at the thickness of 10 μm by freezing microtome (Leica, Germany). The sections were permeabilized with 0.3% Triton X-100, blocked by goat serum, and incubated at 4°C overnight with primary antibodies (CD31, 1:200, Abcam, USA; vascular endothelial growth factor (VEGF), 1:400, Millipore, USA; and angiopoietin-1 (Ang-1), 1:100, Abcam, USA). After being washed with Tris-buffered saline, sections were incubated with goat anti-mouse/rabbit IgG H&L secondary antibody (1:200, Abcam, USA) for 2 h at room temperature. After being mounted with DAPI (Solarbio, Shanghai, China), the stained sections were photographed with a fluorescent laser scanning confocal microscope (ZEISS, Germany) and analyzed with Image-Pro Plus 6.0. Microvascular density (MVD) was counted by quantity of CD31 positive cells per mm2. Expressions of VEGF and Ang-1 were calculated by integrated optical density per image vision.

Evans blue dye

Rats were injected intravenously with 2% Evans blue (EB, 4 ml/kg) 2 h before sacrifice. Brain sections were cut coronally into 2-mm slices and photographed. Then, the whole brains were homogenized in 50% trichloroacetic acid and centrifuged at 12,000 rpm for 20 min. The supernatant was collected and mixed with ethanol (1:3). The concentration of EB in each sample was measured at 610 nm and quantified by the linear standard curve.

Statistical analysis

All values were expressed as mean ± SEM. Statistical analysis was performed by SPSS17.0. Multiple groups were compared using one-way analysis of variance and followed by least significant difference for pairwise comparison. P < 0.05 was considered a statistically significant difference.

 Results



Quality control of Buyang Huanwu decoction

To identify the active ingredients contained in BYHWD extracts, quality control was performed by UHPLC. The representative chromatograms of BYHWD and six standards are shown in [Figure 1]. The time peaks of amygdalin, hydroxysafflor yellow A, paeoniflorin, calycosin glycoside, formononetin, and calycosin as standards or in BYHWD appeared at 8 min, 9 min, 13.5 min, 17 min, 23.5 min, and 25 min, respectively.{Figure 1}

Buyang Huanwu decoction increased the expressions of connexin 43 and phosphorylated connexin 43 in hippocampus

To investigate the role of Cx43 in BYHWD treatment after focal ischemia, we firstly detected the effect of BYHWD on the expression of Cx43. As shown in [Figure 2], compared with the sham group, the expressions of Cx43 in hippocampus of the MCAO group were obviously upregulated at 3 days and 7 days [P < 0.05, [Figure 2]a and [Figure 2]b]. BYHWD further increased the expressions of Cx43 at 3 days, 7 days, and 14 days when compared with the MCAO group [P < 0.05, [Figure 2]a and [Figure 2]b]. Moreover, p-Cx43, the activated form of Cx43, was measured. Compared with the MCAO group, p-Cx43 was significantly increased by BYHWD at 7 days and 14 days [P < 0.05, [Figure 2]a, [Figure 2]c and [Figure 2]d]. Notably, both upregulations of Cx43 and p-Cx43 peaked at 7 days, therefore, the subsequent experiments were conducted at 7 days after MCAO.{Figure 2}

Gap26 inhibited the protective effects of Buyang Huanwu decoction on neurological behavior

To confirm the role of Cx43, Gap26 (a specific inhibitor of Cx43) was administrated and neurological behavior was examined. For the beam-walking test, from 1 day to 7 days, scores were dramatically higher due to the MCAO operation when compared with the sham group (P < 0.05). However, BYHWD significantly decreased the score at 3 days and 7 days. At 7 days, the score of the BYHWD group was significantly lower than that of the MCAO group (P < 0.05); however, Gap26 attenuated this protective effect of BYHWD (P < 0.05) [Figure 3]a.{Figure 3}

As to the Y-maze test, rats with MCAO operations showed remarkably decreased alteration percentage from 1 day to 7 days (P < 0.05). The alteration percentage of the BYHWD group was significantly higher than that of the MCAO group (P < 0.05), while Gap26 attenuated the protective effect of BYHWD (P < 0.05) [Figure 3]b.

Gap26 attenuated the beneficial effects of Buyang Huanwu decoction on microvascular density and blood-brain barrier integrity

To explore whether Cx43 mediated the angiogenesis of BYHWD, MVD and BBB integrity were detected. MVD was increased in hippocampal CA1, CA3, and dentate gyrus (DG) after MCAO (P < 0.05). It was further significantly increased by BYHWD treatment (P < 0.05); however, this effect of BYHWD was remarkably reversed by Gap26 (P < 0.05) [Figure 4]a, [Figure 4]b, [Figure 4]c, [Figure 4]d, [Figure 4]e.{Figure 4}

MCAO induced serious extravasation of EB compared to the sham group at 7 days (P < 0.05), indicating higher permeability. BYHWD reduced the permeability of BBB compared with the MCAO group (P < 0.05), while co-administration with Gap26 blocked this effect (P < 0.05) [Figure 4]f, [Figure 4]g.

Gap26 reversed the beneficial effects of Buyang Huanwu decoction on vascular endothelial growth factor and angiopoietin-1

To further investigate the mechanism of Cx43 mediating angiogenesis after BYHWD treatment, levels of representative factors (VEGF and Ang-1) were measured. The expressions of VEGF were dramatically increased in hippocampal CA1, CA3, and DG in the MCAO group (P < 0.05). Compared with the MCAO group, BYHWD treatment significantly augmented its expression in CA1, CA3, and DG (P < 0.05), while Gap26 reversed the effects of BYHWD on VEGF in CA1 and DG areas (P < 0.05) [Figure 5]a, [Figure 5]b, [Figure 5]c, [Figure 5]d.{Figure 5}

Ang-1 was another widely accepted angiogenic factor. As shown in [Figure 5], BYHWD upregulated the expression of Ang-1 in hippocampal CA1, CA3, and DG compared with the MCAO group (P < 0.05), while co-administration with Gap26 attenuated these increase (P < 0.05) [Figure 5]e, [Figure 5]f, [Figure 5]g, [Figure 5]h.

 Discussion



In the present study, we demonstrated that expressions of Cx43 and p-Cx43 were both upregulated by BYHWD. Inhibition of Cx43 by Gap26 abolished the protective role of BYHWD, demonstrated by aggravated neurological behaviors. These effects were probably related to angiogenesis, as Gap26 attenuated the effect of BYHWD on increasing MVD and promoting the integrity of BBB. Furthermore, upregulations of VEGF and Ang-1 after BYHWD were reversed by Gap26. Therefore, Cx43 mediated the protective role of BYHWD in angiogenesis.

Cx43, the most widely expressed connexin subtype, was distributed in perivascular end feet of astrocytes and vascular cells. Cx43 could form either intercellular channels (permeable for regulatory molecules and ions) or hemichannels open to the extracellular space to provide the release of cell metabolites.[18],[19] In cultured human endothelial cells, endothelial tube and sprout formation were minimized after Cx43 knockdown, while these were significantly enhanced after Cx43 overexpression.[20] p-Cx43 (activated form) mediated the protective effects of erythropoietin against ischemic neurovascular unit injuries.[9] BYHWD has been reported to facilitate angiogenesis and improve neurological behavior after focal cerebral ischemia.[21],[22] However, the mechanisms have not been fully explored. Here, whether Cx43 mediated the angiogenesis of BYHWD was investigated.

In our study, the temporal expressions of Cx43 were firstly evaluated after BYHWD treatment for focal ischemia. The data showed that both Cx43 and p-Cx43 (activated form) were upregulated by BYHWD from 3 days and peaked at 7 days, suggesting that Cx43 probably mediated the protective role of BYHWD in subacute period. To further demonstrate the role of Cx43 in the protective effects of BYHWD after focal ischemia, Gap26 (a specific inhibitor of Cx43) was administrated. We found that the effects of BYHWD on improving neurological function were abolished when Gap26 was co-administrated with BYHWD. Moreover, our data showed that BYHWD increased MVD (identified by CD31 staining, a platelet endothelial adhesion factor, often used as an indicator of angiogenesis), while co-administration with Gap26 reversed this effect. This was comparable with recent studies that tanshinone IIA and astragaloside IV promoted angiogenesis via upregulation of Cx43.[23] Our study was the first to show the beneficial role of Cx43 in angiogenesis after BYHWD treatment. Just as previously reported that increased MVD was positively correlated with functional recovery following focal ischemia,[24],[25] consistently in the present study, Gap26 decreased MVD and attenuated the protective effects of BYHWD on neurological behavior.

Moreover, we observed that BYHWD increased the expression of VEGF and Ang-1, while Gap26 abolished this effect. VEGF and Ang-1, secreted by endothelial cells, smooth muscle cells, or pericytes, are soluble factors, which promoted angiogenesis.[26],[27],[28] These findings were comparable with previous studies where VEGF production was increased in mesenchymal stem cells with elevated levels of Cx43.[29] VEGF expression was decreased by Cx43 siRNA in endothelial progenitor cells.[30] Moreover, expressions of Cx43 and Ang-1 were reported to be decreased concurrently.[31] Notably, Ang-1 was considered a well-suited complement angiogenic factor for VEGF: VEGF induced vascular budding while Ang-1 promoted maturation of vessels.[32],[33] A considerable amount of literature has been published about the function of BYHWD in increasing VEGF and Ang-1.[21],[34] In our study, the effects of Gap26 on decreased VEGF and Ang-1 indicated that Cx43 mediated the angiogenesis of BYHWD via VEGF and Ang-1. Due to the increased Cx43, the communications among extracellular environment, cytoplasm, and neighboring cells were enhanced, which probably contributed to the upregulation of VEGF and Ang-1. However, further explorations are needed for identifying specific molecules for the upregulation of VEGF and Ang-1.

Within the first few hours of ischemic stroke, BBB was disrupted.[35] During subacute period after ischemic stroke, BBB restoration started and continued to be a selective barrier that protected brain from various insults. BYHWD could preserve the integrity of BBB after cerebral ischemia.[36],[37] Comparable with previous studies, our data showed that BYHWD reduced the leakage of EB, and co-administration of Gap26 attenuated this effect, indicating that Cx43 was beneficial for BBB integrity. Similarly, it has been reported that channels formed by Cx43 on both astrocytes and capillary endothelial cells were critical to the function of BBB,[38] while Cx43 deletion weakened it.[39] A point that needs to be noted: in our study, the reduced permeability of BBB was coexisted with increased expression of VEGF. However, there have been studies that VEGF increased vascular permeability and BBB disruption,[40],[41] which seemed to be paradoxical with our study. This discrepancy may be explained due to different phases after ischemic stroke. For instance, rhVEGF165, which was found to increase BBB leakage, was administrated during early postischemic (1 h).[40] In our study, the increased VEGF was found in sub-acute phase after ischemic stroke. In addition, our results were comparable with previous studies where BYHWD promoted VEGF expression and BBB integrity after cerebral ischemia.[22]

 Conclusion



The present study showed that total Cx43 and p-Cx43 were increased by BYHWD treatment after focal ischemic stroke, concomitant with improved neurological behavior, increased MVD, enhanced integrity of BBB, and upregulated expression of VEGF and Ang-1, while co-administration with Gap26 (a specific Cx43 inhibitor) abolished these effects of BYHWD. Taken together, our results showed that BYHWD promoted angiogenesis via Cx43, therefore playing protective role after focal ischemia.

Financial support and sponsorship

This work was supported by grants from the National Natural Science Foundation of China (81673772) and Elite Youth Education Program of Guangzhou University of Chinese Medicine (QNYC20170102).

Conflicts of interest

There are no conflicts of interest.

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