The promoting effect of modified Dioscorea pills on vascular remodeling in chronic cerebral hypoperfusion via the Ang/Tie signaling pathway

2.1 Animals

Male Sprague–Dawley rats (40 in total) with specific-pathogen-free status were purchased from the Hubei Provincial Academy of Preventive Medicine (License number: SCXK(Hubei)2015-0018). The rats had an average body weight of approximately 280 ± 20 g and were housed in the Experimental Animal House of Guizhou Medical University under standard laboratory conditions, including an ambient temperature of 23 ± 1°C, relative humidity of 50 ± 10%, and a 12 h light/dark cycle. They were provided with free access to food and water. The rats were acclimated to the housing conditions for 1 week prior to the experimental procedures.

2.2 Preparation of the CCH model and intervention measures

A total of 40 rats were randomly assigned to three groups: control group (n = 10), model group (n = 15), and MDP group (n = 15), using a random number table. The CCH models, consisting of the model group and MDP group, were induced by bilateral common carotid artery occlusion (2-VO) wherein the rats’ left and right common carotid arteries were ligated separately, with a 1 week interval. Rats in the control group underwent the same surgical procedures as the model groups, except for the ligation and occlusion of the bilateral common carotid arteries. During surgery, the rectal temperature of the rats was maintained at 37.0–37.9°C using a heating pad to prevent brain injury due to low temperature. After approximately 2–4 min, all animals regained consciousness and were provided with free access to food and water. After 1 week of 2-VO surgery, rats in the MDP group were administered MDP condensed decoction intragastrically at a dose of 1 ml/100 g body weight (prepared by the Preparation Room of Hubei Provincial Hospital of Traditional Chinese Medicine) for 45 days, while rats in the other two groups were treated with normal saline intragastrically at the same dose and duration as the MDP group.

2.3 Sampling

After the gavage intervention, rats were euthanized, and brain tissues were obtained by perfusion sampling and fresh sampling. Perfusion sampling was conducted as follows: five rats from each group were deeply anesthetized with isoflurane inhalation, as previously described. Their hearts were exposed, and perfusion needles were inserted through the apex into the left ventricle and then into the aorta. The right atrial appendage was quickly cut, and 250 ml of normal saline was perfused through the perfusion needles. As clear perfusate flowed out, the rats’ tails straightened, trembling limbs ceased, and the livers gradually turned white. Subsequently, 250 ml of 4% paraformaldehyde was perfused successively. After approximately 40 min, the rats were decapitated, and the hippocampus was isolated and harvested. The harvested tissues were then fixed in 4% paraformaldehyde buffer solution for 12–24 h, followed by conventional dehydration, paraffin embedding, and staining with hematoxylin and eosin (HE) and immunohistochemical staining. Fresh sampling was performed for reverse transcription-polymerase chain reaction (RT-PCR) and western blot detection, involving five rats from each group. The steps for fresh sampling were as follows: rats were deeply anesthetized with isoflurane inhalation and then decapitated, and brain tissues were quickly stripped for RT-PCR detection.

2.4 Histopathological observation

Paraffin sections of the hippocampus were obtained for HE staining in order to observe the pathological characteristics of the CA1, CA3, and DG regions. The hippocampal tissue was dehydrated, rendered transparent, and embedded in paraffin. Subsequently, the tissue was cut into 4 µm slices, which were then baked, dewaxed, and stained with HE for subsequent microscopic observation using an Olympus biological microscope (Type BX53) at magnifications of 100× and 400×.

2.5 Immunohistochemistry and microvessel density (MVD) analysis

Immunohistochemistry and MVD detection were performed using the hippocampus of three rats from each group. Paraffin sections were prepared following the same procedure as for histopathological analysis. After antigen retrieval and blocking of endogenous peroxidase, the first antibodies (mouse anti-CD43) were added to the hippocampus paraffin sections. Subsequently, IgG polymers of horseradish peroxidase-labeled secondary antibody and freshly prepared DAB color solution were successively added to these sections. Finally, the sections were counterstained with Harris hematoxylin.

For the analysis of MVD, CD43-labeled images were collected. According to the corrected Weidner method, any single endothelial cell or cell clusters stained by anti-CD43, as long as they have a clear boundary with the surrounding microvessels or other connecting tissues, regardless of whether a lumen has formed, were counted as a microvessel, excluding vessels with smooth muscle walls or lumen diameters greater than eight times the diameter of erythrocytes. Each sample was first observed at 100× magnification to select three spots with the highest number of microvessels as “hot spots.” Then, the microvessels were counted at high magnification (400×) from the hot spots, and the average number of microvessels was calculated as MVD.

2.6 Western blot

To the hippocampus tissue was added RIPA lysate (Meilunbio, product no.: MA0151) to extract the total proteins of the hippocampus. By diluting and boiling, the extracted proteins were denatured prior to electrophoresis on the gel, and these proteins were transferred into the PVDF membrane (Genscript, product no. L00791C). They were sealed and reacted with primary antibodies (rabbit anti-GAPDH, 37 kD, Hangzhou Xianzhi Biological Co., Ltd., product no. AB-P-R 001, 1:10,000 dilution; rabbit anti-Ang-1 [70 kD], Wuhan Sanying, product no. 23302-1-AP, 1:10,000 dilution; rabbit anti-Ang-2, 75 kD, product number: ABclonal A0698, diluted 1:10,000; rabbit anti-tie2126 kD, ABclonal, product number A7222, diluted at 1:10,000; rabbit anti-VEGF, 45 kD, Wuhan Sanying, product number: 19003-1-AP, 1:10,000 dilution). Afterward, HRP-labeled secondary antibodies (HRP-labeled sheep antimouse secondary antibody, Wuhan Doctor Bioengineering Co., Ltd., product number: BA1051, 1:10,000 dilution) were added and the films were developed, fixed, and processed. Finally, the films were analyzed with ipp6.0 software to determine the film grayscale value using GAPDH as an inner reference.

2.7 RT-PCR

Hippocampal tissues weighing 100 mg were extracted to obtain RNA using the Trizol method. The extracted RNA was then reverse-transcribed into cDNA, followed by the addition of designed primers for real-time fluorescence quantitative reaction using the SYBR Green dye method. Amplification and melting curves were drawn, and the relative expression of target genes such as Ang-1, Ang-2, Tie-2, and VEGF was calculated using the QPCR algorithm (relative quantification, 2⁻^ΔΔCt), with GAPDH as an internal reference (Table 1).

2.8 Statistics

All data obtained in this experiment, except for HE staining, are presented as quantitative values expressed as mean ± standard deviation (mean ± SD). Statistical analysis was performed using SPSS 19.0 software. T-test was used for intergroup comparisons, and one-way ANOVA was used for comparisons among multiple groups. A significance level of P < 0.05 was considered statistically significant, while P < 0.01 was considered extremely statistically significant.

1. Ethical approval: The research related to animals’ use complied with all the relevant national regulations and institutional policies for the care and use of animals. All animal handling and experimental protocols were conducted in accordance with the Guidance Suggestions for the Care and Use of Laboratory Animals, formulated by the Ministry of Science and Technology of the People’s Republic of China. The study was approved by the Committee on Ethics of the First People’s Hospital of Guiyang (Guizhou, China), with ethical approval number 2022007.

3.1 Pathology observation

HE staining revealed that compared to the control group, the model group showed a significant loss of neurons with disordered arrangement in the CA1, CA3, and DG regions of the hippocampus. The radiation layer appeared loose with pale staining and showed multiple cracks in the CA1 region. Many disarranged cells infiltrated the white matter of the CA3 region, and a large blank gap was observed between the cortex and subcortical in the DG region. However, in the MDP group, these abnormal pathological findings were significantly restored, with evident regeneration of neurons in the CA1, CA3, and DG regions (Figure 1).

Figure 1

The pathology of CA1, CA3, and DG zones in the hippocampus among the three groups. CCH induced neurons loss, white matters loosen and disarrange in CA1, CA3, and DG zones in the hippocampus. MDP significantly restored them. Using Image J software, we calculated that the total neurons in the CA1 zone are, respectively, 155 ± 21, 110 ± 18, 182 ± 36 per slice (400 times magnetite). There are significant differences between the two groups (P < 0.05).

3.2 Immunohistochemistry

CCH induced by 2-VO resulted in evident neovascularization in the hippocampus. Compared with the control group, the MVD in the model group (14.93 ± 1.92) was significantly higher (P < 0.01) than that in the control group (5.78 ± 1.65). Furthermore, the MVD in the MDP group (21.19 ± 2.62) was significantly higher (P < 0.01, P < 0.01) than that of both the model group and control group, indicating that MDP further promotes microvascular regeneration (Figure 2).

Figure 2

Immunohistochemistry CD43 staining in the hippocampus and MVD analysis. MVD in the model group (14.93 ± 1.92), control group (5.78 ± 1.65), and the MDP group (21.19 ± 2.62). **P < 0.01 vs control group; ^## P < 0.01 vs the model group.

3.3 Western blot

The expression of Ang-1 in the model group was significantly increased compared to the control group (0.597 ± 0.090 vs 0.345 ± 0.058, P = 0.005), and MDP further upregulated the expression of Ang-1 (0.750 ± 0.060), with significant differences observed when compared to both the control group and the model group (P < 0.001, P = 0.039). Similarly, the expression of Ang-2 in the model group was significantly increased compared to the control group (0.592 ± 0.035 vs 0.442 ± 0.068, P = 0.007), and MDP further upregulated the expression of Ang-2 (0.771 ± 0.017), with significant differences observed when compared to both the control group and the model group (P < 0.001, P = 0.003). Furthermore, the expression of Tie-2 in the model group was significantly increased compared to the control group (0.703 ± 0.025 vs 0.524 ± 0.044, P < 0.001), and MDP further upregulated the expression of Tie-2 (0.909 ± 0.019), with significant differences observed when compared to both the control group and the model group (P < 0.001, P < 0.001). Finally, the expression of VEGF in the model group was significantly increased compared to the control group (0.706 ± 0.075 vs 0.486 ± 0.030, P = 0.002), and MDP further upregulated the expression of VEGF (0.844 ± 0.034), with significant differences observed when compared to both the control group and the model group (P < 0.001, P = 0.016) (Figure 3).

Figure 3

Expressions of Ang-1, Ang-2, Tie-2, and VEGF in the hippocampus among the three groups. Using GAPDH as the inner reference, the relative expression levels of Ang-1, Ang-2, Tie-2, and VEGF in the hippocampus form are calculated. The left image shows the immunoblotting of GAPDH, Ang-1, Ang-2, Tie-2, and VEGF, while the right bar graph, respectively, shows the relative expression levels of Ang-1, Ang-2, Tie-2, and VEGF in each group of rats. **P < 0.01 vs control group; ^# P < 0.05 vs model group; ^## P < 0.01 vs model group.

3.4 RT-PCR

The expression of Ang-1 mRNA in the model group was significantly increased compared to the control group (1.691 ± 0.488 vs 0.992 ± 0.034, P = 0.032), and MDP further upregulated it (3.358 ± 0.596), with significant differences observed compared to both the control group and the model group (P <0.001, P <0.001). The expression of Ang-2 mRNA in the model group showed a tendency to increase compared to the control group but did not reach statistical significance (1.560 ± 0.491 vs 1.011 ± 0.016, P = 0.203). However, MDP further upregulated the expression of Ang-2 mRNA (2.673 ± 0.804), with significant differences observed compared to both the control group and the model group (P < 0.001, P = 0.002). Similarly, the expression of Tie-2 mRNA in the model group showed an increasing trend compared to the control group but did not reach statistical significance (1.362 ± 0.297 vs 1.001 ± 0.002, P = 0.095). Nevertheless, MDP further upregulated the expression of Tie-2 mRNA (2.294 ± 0.443), with significant differences observed compared to both the control group and the model group (P < 0.001, P < 0.001). The expression of VEGF mRNA in the model group also showed a tendency to increase compared to the control group but did not reach statistical significance (1.447 ± 0.264 vs 1.012 ± 0.036, P = 0.219). However, MDP further upregulated the expression of VEGF mRNA (2.754 ± 0.797), with significant differences observed compared to both the control group and the model group (P < 0.001, P < 0.001) (Figure 4).

Figure 4

Expressions of Ang-1 mRNA, Ang-2 mRNA, Tie-2 mRNA, and VEGF mRNA in the hippocampus among the three groups. Using GAPDH as the inner reference, the relative expression levels of Ang-1 mRNA, Ang-2 mRNA, Tie-2 mRNA, and VEGF mRNA in the hippocampus form are calculated. The left image shows the amplification and fusion curves of GAPDH, Ang-1, Ang-2, Tie-2, and VEGF genes. The right bar graph shows the relative expression levels of Ang-1 mRNA, Ang-2 mRNA, Tie-2 mRNA, and VEGF mRNA in each group of rats. **P < 0.01 vs control group; *P < 0.05 vs control group; ^## P < 0.01 vs model group.

3.5 Ang-1/Ang-2 ratio

The Ang-1/Ang-2 ratio suggests that there are no significant differences in the gene expression ratio among the three groups (F = 2.576, P = 0.097) but MDP still significantly elevated the gene expression ratio of Ang-1/Ang-2 compared to the control group (P = 0.041). In terms of protein expression, in the physiological state (control group), the expression of Ang-1 is less than Ang-2, the ratio of Ang-1/Ang-2 is 0.779 ± 0.046, the interventions of CCH or MDP obviously increase the expression of Ang-1 much more than that of Ang-2, thus the ratios of Ang-1/Ang-2, respectively, reach 1.007 ± 0.113 and 0.972 ± 0.077. All reach significant differences when compared to that of the control group (P = 0.015, P = 0.029) but there is no significant difference between the model group and the MDP group (P = 0.627) (Table 2).