Modified Renshen Yangrong decoction enhances angiogenesis in ischemic stroke through promotion of MicroRNA‐210 expression by regulating the HIF/VEGF/Notch signaling pathway

Abstract Objective This study aims to investigate the efficacy of modified Ginseng Yangrong decoction (GSYRD) promoting angiogenesis after ischemic stroke. Methods In an in vivo study, rats that survived surgery were allocated into four groups: the control group and model group were treated with normal saline, the GSYRD group was treated with 18.9 mg/kg of GSYRD daily, and the positive control group was treated with Tongxinluo (TXL) (1 g/kg/d). At the end of the seven‐day treatment, the area of cerebral infarction, the expression changes of miRNA‐210 and ephrin A3 were determined. In an in vitro study, HUVECs were divided into a normal control serum group (NC group), normal control serum OGD group (Oxygen Glucose Deprivation group) (OGD group), OGD + drug‐containing serum group (OGD+GSYRD group), and OGD + drug‐containing serum + ES group (Endostatin group) (OGD+GSYRD+ES group). The cells in all groups except the NC group were cultured in a sugar‐free DMEM medium under hypoxia for 48 h. Cell proliferation, angiogenic structure formation ability, the expression changes of miRNA‐210, ephrin A3, and the HIF/VEGF/Notch signaling pathway‐related molecules were determined. Results In vivo, GSYRD significantly reduced infarct size (p < .01), the expression of miRNA‐210 and ephrin A3 were decreased in the GSYRD group (p < .05). In vitro, the cell proliferation and tube formation ability were significantly increased in the GSYRD group (p < .05), and the expression of miRNA‐210 and ephrin A3 was decreased (p < .05). In addition, in the GSYRD group, the expression of the HIF/VEGF/Notch signaling pathway‐related molecules was significantly increased (p < .01 or p < .05). Conclusion GSYRD promotes cerebral protection following angiogenesis and ischemic brain injury. The specific mechanism was activating the HIF/VEGF/Notch signaling pathway via miRNA‐210.


INTRODUCTION
Ischemic stroke is a common clinical disease with high mortality (D. Chen et al., 2015;Donnan et al., 2008;Lapergue et al., 2017;Rodgers, 2013). Interruption of cerebral artery blood flow and hypoxic-ischemic necrosis of local brain tissue are the leading causes of ischemic stroke (Du et al., 2011). After the disease occurs, the blood flow in the ischemic center decreases rapidly, and some blood flow in the periphery penumbra still passes through. If the blood flow in this area is restored quickly, the functions of the patient could be improved (Sharp et al., 2000).
The recovery of blood flow includes the opening of the infarcted blood vessels and the compensation of the collateral circulation. As a critical measure of early blood flow recovery, thrombolysis is often not practical due to strict time windows and indications. Therefore, promoting the regeneration of blood vessels and actively establishing the collateral circulation becomes an urgent and new treatment direction for ischemic stroke. Studies have shown that the establishment of collateral circulation can effectively reduce the area of cerebral infarction and improve the survival rate of nerve cells and prognosis (Bang et al., 2011;Kao et al., 2017;Souza et al., 2012).
Angiogenesis refers to the formation of new vascular networks by existing vascular endothelial cells by proliferation, migration, and budding and the delivery of nutrients and oxygen to various organs and tissues (Carmeliet, 2005). It is a crucial process of local blood supply (Ruan et al., 2015) and is regulated by various vascular growth factors.
Studies have shown that miRNA regulation of tissue angiogenesis after ischemia has become one of the research hotspots at home and abroad. MicroRNAs-210 are hypoxia-specific microRNAs, which play an essential role in promoting angiogenesis by negatively regulating its target gene, ephrin A3 (Fasanaro et al., 2008). Ephrin A3 plays a vital role in the mechanism of VEGF (Vascular endothelial growth factor) signaling, promoting angiogenesis. HIF-1 (HyPoxia-inducible factor 1α) plays an important role as a core regulator of angiogenesis. When hypoxia occurs in tissues or cells, HIF-1 binds specifically with VEGF and promotes the recovery of blood supply in ischemic areas by regulating the downstream Notch signaling pathway. Therefore, finding effective drugs that can promote angiogenesis, alleviate brain damage after ischemia and hypoxia, and understand the molecular mechanism of its occurrence is of great significance for the treatment of ischemic stroke. promote the proliferation of endothelial cells, inhibit the synthesis of endothelin, and accelerate its decomposition (Xuejiang et al., 2001).
Our preliminary research results show that GSYRD can promote the proliferation and migration of HUVECs in a dose-dependent manner (Zhu et al., 2017). However, the mechanical function of GSYRD is not fully grasped in promoting angiogenesis. Therefore, we investigated whether GSYRD can improve ischemia by promoting angiogenesis and whether the miRNA-210 and HIF/VEGF/Notch signaling pathway is involved in the molecular mechanism of GSYRD.

Experimental animals
In this trial, we selected adult male Sprague Dawley (SD) rats

Establishment of the MCAO (Middle cerebral artery occlusion) model
Referring to the improved middle cerebral artery ischemia method in rats such as Longa, the specific steps were as follows: the SD rats were weighed, anesthetized with 20% uratan, and placed on the operating table in a supine position. The neck skin was cut, the neck fascia, platysma, sternocleidomastoid, and sternal hyoid muscles were bluntly separated. The right CCA (common carotid artery) bifurcation, internal carotid artery (ICA), and external carotid artery (ECA) were identified.
The distal end of the ECA was ligated, and the proximal end of the heart was ligated for a short time. The artery clamp was completely clamped at 0.5 cm from the proximal end of the CCA bifurcation. The artery clamp completely clamped the ICA, and a "V" was cut along the distal end of the ECA. The ECA stump was straightened along the ICA, a 0.2 mm diameter blunt nylon thread was inserted into the artery, the ICA was passed through the CCA bifurcation (releasing the arterial clip), and the beginning of the middle cerebral artery was entered to block the blood flow of the middle cerebral artery. The silk thread was slowly pushed until stopped by resistance, the ECA line was ligated, the CCA artery clamp was relaxed for hemostasis, the skin was sutured, and the rat was placed inside a cage to have free access to food and water. Rats were given an incandescent lamp (100 W) during the operation and before waking, maintaining an anal temperature of about 36 • C and room temperature of 25 • C. Animals operated in the control group only had blood vessels separated and exposed without any damage.

Preparation of drug-containing serum
Healthy adult male SD rats weighing 250-280 g were randomly divided into two groups: the normal control group and the GSYRD group. The GSYRD group was administered 18.9 g/kg twice a day for three days.
One hour after the last administration, the rats were routinely anesthetized. Blood was taken from the abdominal aorta, left at room temperature for 2 h, centrifuged for 20 min. The serum was collected, inactivated at 56 • C for 30 min, filtered through a 0.22 μm filter, and stored at −80 • C. The normal control group was given the same amount of normal saline, and the other groups were prepared with the medicinal serum of the GSYRD.
The whole experimental process conformed to the principles outlined in the Declaration of Helsinki. The cells were divided into the normal control serum group (NC group), the normal control serum OGD group (OGD group), the OGD + drug-containing serum group (OGD+GSYRD group), and the OGD + drug-containing serum + ES group (OGD+GSYRD+ES group). The cells in all groups except the NC group were cultured in a sugar-free DMEM medium under hypoxia for 48 h. The hypoxic culture conditions were 1% O 2 , 94% N 2 , and 5% CO 2 .

The calculation of the infarct area
After treatment, six animals from each group were sacrificed, and the whole brain was excised immediately and then stored at −20 • C for approximately 20 min after PBS washing to harden tissue. Then, the brain was cut into five pieces (each piece is about 2 mm thick), incubated with 2% TTC, covered with foil, and incubated in a 37 • C water bath for 30 min. The brain slices were intermittently turned over to ensure the section fully contacted the dye solution. Sections were photographed, and the size of the infarcted region was evaluated. The nonischemic area was red, while the infarcted tissue was white. All sections were fixed in 4% paraformaldehyde, and the area of infarct was calculated with ImageJ.

Immunofluorescence staining
The SD rats in each group were sacrificed, and the brain tissue of the ischemic cortex was taken and fixed with 4% paraformaldehyde All images were captured on a fluorescence microscope (OLUMPUS, Japan).  Table 1.
Endothelial cells were cultured in a 96-well plate and cultured under the above conditions. The cells were incubated for 4 h in a medium containing 0.5% MTT, the yellow mitochondrial dye. The reaction was terminated by adding 150 μl DMSO to the cells and incubating them for 10 min. Absorbance at 570 nm was recorded with an enzyme-linked immunosorbent assay plate reader.

Flow cytometry
Cells in each group were collected and digested with trypsin, then centrifuged for 5 min; the antibody was added in the dark. The cells were centrifuged and the supernatant discarded. Each cell sample had 500 μl of the staining buffer added for 15 min, after which flow cytometry was performed.

Western blotting
The total protein was isolated from HUVECs according to the standard protocols. Subsequently, the protein concentration was determined by Plus sensitization solution, and the gels were exposed to a scan. The mean gray values of the bands were analyzed by ImageJ.

Statistical analysis
We used the software program SPSS 22.0 (IBM, Armonk, NY, USA) to conduct the statistical analysis. Continuous variables were expressed as mean ± SD. Discontinuous variables were expressed as a percentage (%). For multiple comparisons, each value was compared by oneway ANOVA following the Dunnett test when each datum conformed to a normal distribution, while the non-normally distributed continuous data were compared using non-parametric tests. The counting data were tested by the chi-square test. A value of p < .05 was considered statistically significant.

GSYRD reduces infarct size in rats with cerebral ischemia
TTC staining showed that infarct size was lower in the GSYRD and TXL groups compared with the model group, and the infarct size of the GSYRD group was smaller than the TXL group ( Figure 1A,B). It shows that the treatment of the GSYRD group has a better therapeutic effect.

Changes of miRNA-210 and ephrin A3 after GSYRD administration
After cerebral ischemia, the expression of miRNA-210 and its target gene, ephrin A3, will change accordingly. RT-qPCR detected the expression of miRNA-210, and the change of ephrin A3 was observed by immunofluorescence staining. Compared with the control group, the expression of the model, the GSYRD, and the TXL groups miRNA-210 was increased ( Figure 2B), and the expression of ephrin A3 was decreased (Figure 2A,C). Among them, the expression of miRNA-210 in the GSYRD group was higher than in the model and TXL groups (Figure 2B), and the expression of ephrin A3 was lower than in the model and TXL groups (Figure 2A,C). It indicates that GSYRD has a regulatory effect on miRNA-210 and ephrin A3.

GSYRD augmented proliferation and tube formation in HUVECs
To explore the roles of the pro-angiogenesis effect induced by GSYRD, we investigated cell proliferation and tube formation in GSYRD treated HUVECs using MTT and Matrigel tube formation assays, respectively. Figure 3, compared with the OGD group, the GSYRD group has a significantly increased cell proliferation ability and can effectively form a slender capillary-like structure. After the addition of ES, the cell's proliferative capacity and angiogenic ability were significantly inhibited.

GSYRD stimulates angiogenesis through the HIF/VEGF/Notch pathway
The HIF/VEGF/Notch pathway has been shown to play an important role in the angiogenesis process. To clarify the effect of GSYRD on the HIF/VEGF/Notch pathway, we introduced inhibitor ES into cells.
The introduction of ES reduced the expression of the HIF/VEGF/Notch pathway-related molecular proteins and mRNA, and the expression of each protein and mRNA of the GSYRD group was higher than the OGD group, which explains that the addition of ES weakens the role of GSYRD (Figure 4). These results indicate that GSYRD can activate the HIF/VEGF/Notch pathway.

GSYRD regulated miRNA-210 and ephrin A3 expressions
To further verify the effect of GSYRD on miRNA-210, changes in miRNA-210 and ephrin A3 in GSYRD treated cells were measured using RT-qPCR and flow cytometry, respectively. The results showed that compared with the OGD group, the expression of miRNA-210 in the GSYRD group was significantly increased (Figure 5B), and the expression of ephrin A3 decreased considerably ( Figure 5A,B). With the addition of ES, the expression of miRNA-210 and ephrin A3 was affected. This indicates that GSYRD can indirectly regulate the expression of miRNA-210 and ephrin A3 in cells. The study has shown that under the guidance of traditional Chinese medicine theory, according to the symptoms and characteristics of patients, the Chinese herbal compound is intended to contain a combination of various plants and minerals, which can improve clinical efficacy (Liu et al., 2007). It is believed that, at least in certain formulations, multiple components can bind to multiple targets to exert a synergistic therapeutic effect. The synergism between component herbs can play an essential role in that the effects of a combination of two herbs can be significantly greater than that of either in isolation (Liu et al., 2007;Wang et al., 2008;Xuejiang et al., 2001;Zhu et al., 2017). GSYRD is a traditional ancient prescription of Chinese medicine for qi-supplementing, blood-nourishing, Xinnourishing, and mind-tranquilizing (Uehiyama, 1994). Modern studies show that it affects aging, enhances immunity, and improves anemia, and is frequently used to treat various diseases, including malignant tumors (Y. Z. Chen, Lin, F., Zhuang, G. B., et al., 2011).
HIF-1α is a specific marker of hypoxia, a key regulator upstream of the VEGF signaling pathway, and a critical transcription factor that triggers the expression of various hypoxic stress proteins . VEGF is an important factor regulating the promotion of angiogenesis, promoting the division and proliferation of vascular endothelial cells, and finally inducing the formation of new blood vessels (Ferrara & Davis-Smyth, 1997;Hicklin & Ellis, 2005). Moreover, it plays a key role in neovascular remodeling after ischemic stroke (Ma et al., 2012). The Notch signaling pathway is involved in forming the collateral network of ischemic stroke, and inhibition of the Notch signaling pathway can impair the repair of post-ischemic angiogenesis (Cristofaro et al., 2013). The expression of HIF-1α is significantly increased after hypoxia, which promotes the expression of VEGF, activates the Notch signaling pathway and transcription of downstream target genes, promotes cell proliferation, survival, vascular endothelial cell migration, and arteriovenous differentiation.
To verify whether GSYRD promotes angiogenesis through the HIF/VEGF/Notch signaling pathway, we used endostatin (ES), a signal transduction inhibitor of VEGF. We observed the level of the HIF/VEGF/Notch protein and molecular changes by western blot and RT-qPCR method and found that the expression level of related factors was significantly increased after the addition of GSYRD. When we added ES, the HIF/VEGF/Notch signaling pathway was blocked, and the pro-angiogenic function of GSYRD was greatly inhibited, even lower than the control group. The above experimental results indicate that GSYRD promotes angiogenesis by activating the HIF/VEGF/Notch signaling pathway.
MiRNAs are widely involved in various physiological and pathological metabolic processes in vivo, especially in angiogenesis under hypoxia (Chan et al., 2012). Among them, miRNA-210 is a hypoxiaspecific miRNA, which can be stably expressed in different concentrations of hypoxic environments and different cell types. In hypoxia, miRNA-210 promotes endothelial cells to form blood vessels by inhibiting the expression of the target gene ephrin A3 (Ivan et al., 2008). Liang et al. (2020) have reported that miRNA-210 can regulate the expression of the HIF/VEGF/Notch pathway-associated molecules and participate in the process of promoting angiogenesis after hypoxia.
In previous research, the expression of VEGF is regulated by many miRNAs, and it is predicted by computer software that miRNA-210 can regulate VEGF, which affects the process of angiogenesis (Hua et al., 2006). miRNA-210 can regulate the expression of ephrin A3, promote the differentiation of HUVECs into capillary-like structures, and promote the migration of HUVECs under the action of VEGF (Huang et al., 2010). Simultaneously, miRNA-210 can promote angiogenesis in normal brain tissue by up-regulating VEGF expression (Zeng et al., 2014). Kati et al (Pulkkinen et al., 2008)  There were some limitations in this study. The specific active ingredients of GSYRD still need further research.

CONCLUSION
GSYRD promotes cerebral protection following angiogenesis and ischemic brain injury. The specific mechanism was activating the HIF/VEGF/Notch signaling pathway via miRNA-210. GSYRD has good therapeutic ability as a treatment of ischemic diseases.

DATA AVAILABILITY STATEMENT
The datasets generated and/or analyzed during the current study are not publicly available due to the lack of an online platform but are available from the corresponding author on reasonable request.

CONFLICT OF INTEREST
Authors declare no conflicts of interest.

AUTHOR CONTRIBUTIONS
Ce Liang, Teng Zhang, Ya-Li Wang and Cui-Huan Yan were involved in drafting the manuscript and revising it critically for important intellectual content and conception and design; Xu-Liang Shi made substantial contributions to acquisition and interpretation of data; Lin Jia analyzed the data; All authors gave final approval of the version to be published.