Scholarly article on topic 'Microvascular pathological features and changes in related injury factors in a rat acute blood stasis model'

Microvascular pathological features and changes in related injury factors in a rat acute blood stasis model Academic research paper on "Basic medicine"

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{"Blood stasis" / "Acute-phase reaction" / Microvessels / "Endothelial cells" / "Medicine / Chinese traditional"}

Abstract of research paper on Basic medicine, author of scientific article — Zhang Junxiu, Feng Yu, Li Shaodan, Liu Yi, Zhang Yin, et al.

Abstract OBJECTIVE To examine the microvascular pathological characteristics and changes in related injury factors in a rat model of acute blood stasis. METHODS A total of 75 Sprague-Dawley rats were divided randomly and equally into a control group and four experimental groups assessed at different times after the induction of stasis (0, 1, 3 or 6 h after stasis) (n = 15). The acute blood stasis model was established through rat tail-vein injection of high-molecular-weight dextran. After Electrocardiograph (ECG) detection at predetermined times (0, 1, 3 and 6 h after induction of stasis), the rats were sacrificed and blood and cardiac samples were harvested for analysis. Hematoxylin-eosin (HE) staining and transmission electron microscopy were used for histopathological detection; an enzyme linked immunosorbent assay (ELISA) was used to detect thromboxane B2 (TXB2) and 6-keto-prostaglandin F1α (6-Keto-PGF1α) concentrations; a real-time polymerase chain reaction (PCR) reaction system was used to detect intercellular adhesion molecule 1 (ICAM-1) and vascular cell adhesion molecule1 (VCAM-1) mRNA expression; western blotting was used to detect vascular endothelial cadherin (VE-cadherin) protein expression. RESULTS The ST segment in the ECG showed gradual elevation after induction of stasis and continued elevation at a high level at 3 and 6 h. The HE staining showed changes in myocardial cell necrosis and tissue dissociation after the induction of stasis, along with inflammatory infiltration. Results of transmission electron microscopy showed immediate changes in blood stasis and lumen occlusion in the microvasculature, along with endothelial cell swelling. After the induction of stasis, TXB2 concentrations gradually increased while 6-Keto-PGF1α concentrations were immediately significantly reduced. The TXB2/6-Keto-PGF1α ratio was maintained at a high level. ICAM-1 mRNA expression showed an unstable elevation while VCAM-1 mRNA expression was significantly reduced after the induction of stasis. Compared with the control group, VE-cadherin protein expression increased at 0 and 3 h after the induction of stasis, while no change occurred at 1 and 6 h. CONCLUSION The pathological manifestations of acute blood stasis are microvascular blood retention, lumen stenosis and even occlusion. The condition is also called “blood coagulation and weep” in Traditional Chinese Medicine. The blood stasis model resulted in the injury and necrosis of endothelial cells and cardiomyocytes, along with the presence of an imbalance of vasomotor factor levels, platelet activation, and increases in the expression of adhesion molecules and endothelial barrier dysfunction, which corresponds to “blood failed to nourish” in Traditional Chinese Medicine.

Academic research paper on topic "Microvascular pathological features and changes in related injury factors in a rat acute blood stasis model"

JTCM

Online Submissions: http://www.journaltcm.com JTradit Chin Med 2017 February 15; 37(1): 108-115

info@journaltcm.com ISSN 0255-2922

© 2016 JTCM. This is an Open Access article under the CC BY-NC-ND License (http://creativecommons.org/licenses/by-nc-nd/4.0/).

EXPERIMENTAL STUDY

Microvascular pathological features and changes in related injury factors in a rat acute blood stasis model

Zhang Junxiu, Feng Yu, Li Shaodan, Liu Yi, Zhang Yin, Guo Yunxia,Yang Minghui

Zhang Junxiu, Feng Yu, Li Shaodan, Liu Yi, Zhang Yin, Guo Yunxia, Yang Minghui, Institute of Traditional Chinese Medicine, Chinese People's Liberation Army General Hospital, Beijing 100853, China

Correspondence to: Prof. Yang Minghui, Institute of Traditional Chinese Medicine, Chinese People's Liberation Army General Hospital, Beijing 100853, China.ymh9651@sina.com Supported by the National Program on Key Basic Research Project (973 Program, No. 2012CB518601) Telephone: +86-15810998369 Accepted: October 12,2016

Abstract

OBJECTIVE: To examine the microvascular pathological characteristics and changes in related injury factors in a rat model of acute blood stasis.

METHODS: A total of 75 Sprague-Dawley rats were divided randomly and equally into a control group and four experimental groups assessed at different times after the induction of stasis (0, 1, 3 or 6 h after stasis) (n = 15). The acute blood stasis model was established through rat tail-vein injection of high-molecular-weight dextran. After Electrocardiograph (ECG) detection at predetermined times (0, 1, 3 and 6 h after induction of stasis), the rats were sacrificed and blood and cardiac samples were harvested for analysis. Hematoxylin-eosin (HE) staining and transmission electron microscopy were used for histopathological detection; an enzyme linked immunosorbent assay (ELISA) was used to detect thromboxane B2 (TXB2) and 6-keto-prostaglandin F1a (6-Keto-PGF1a) concentrations; a real-time polymerase chain reaction (PCR) reaction system was used to detect intercellular adhesion molecule 1 (ICAM-1) and vascular cell adhesion molecule!

(VCAM-1) mRNA expression; western blotting was used to detect vascular endothelial cadherin (VE-cadherin) protein expression.

RESULTS: The ST segment in the ECG showed gradual elevation after induction of stasis and continued elevation at a high level at 3 and 6 h. The HE staining showed changes in myocardial cell necrosis and tissue dissociation after the induction of stasis, along with inflammatory infiltration. Results of transmission electron microscopy showed immediate changes in blood stasis and lumen occlusion in the microvasculature, along with endothelial cell swelling. After the induction of stasis, TXB2 concentrations gradually increased while 6-Keto-PGFiaconcentrations were immediately significantly reduced. The TXB2/6-Keto-PGFio ratio was maintained at a high level. ICAM-1 mRNA expression showed an unstable elevation while VCAM-1 mRNA expression was significantly reduced after the induction of stasis. Compared with the control group, VE-cad-herin protein expression increased at 0 and 3 h after the induction of stasis, while no change occurred at 1 and 6 h.

CONCLUSION: The pathological manifestations of acute blood stasis are microvascular blood retention, lumen stenosis and even occlusion. The condition is also called "blood coagulation and weep" in Traditional Chinese Medicine. The blood stasis model resulted in the injury and necrosis of endothelial cells and cardiomyocytes, along with the presence of an imbalance of vasomotor factor levels, platelet activation, and increases in the expression of adhesion molecules and endothelial barrier dysfunction, which corresponds to "blood failed to nourish" in Traditional Chinese Medicine.

© 2016 JTCM. This is an Open Access article under the CC BY-NC-ND License (http://creativecommons.org/licenses/by-nc-nd/4.0/).

Key words: Blood stasis; Acute-phase reaction; Mi-crovessels; Endothelial cells; Medicine, Chinese traditional

INTRODUCTION

Blood stasis is a common pathological mechanism in Traditional Chinese Medicine (TCM), but is also the common pathological basis of cardiovascular disease. The concept of blood stasis includes two types: poor blood stasis and blood running outside meridians. This paper focuses on poor blood stasis. The TCM classic Huang Di Nei Jing does not propose the concept of "blood stasis",1 but repeatedly mentions "blood coagulation and weep" to highlight the pathological mechanisms of blood barriers. Around 220 AD, the Shang Han Lun officially proposed "blood stasis" as a disease.2 In the 14th century, Pu Ji Fang stressed that blood stasis plays a major role in the development of chronic diseases.3 TCM physicians Ye Tianshi in the 17th century and Wang Qingren in the 18 th century highlighted the significance of blood stasis in the vein in disease. Previous theories correlated the collateral vasculature and the microvasculature,4 in terms of the nature of blood stasis, and from a modern scientific point of view, blood stasis is closely related with the microcircula-tion.5 High-molecular-weight dextran (HMWD) features a high molecular weight and high viscosity, and can form a stable bridge between erythrocytes, for which the surface force is greater than the surface repulsion of the electric charge, thereby inducing erythro-cyte aggregation, platelet aggregation, and an increase in blood flow resistance. An infusion of HMWD for 5 min will result in microcirculatory dysfunction and a gradually worsening microcirculatory situation.6 Therefore, it is the ideal drug to model for poor blood stasis. In this study, we conducted a tail-vein injection of HMWD in rats, as an acute blood stasis model, to study the pathological characteristics of the microvascu-lature, and changes in related injury factors. The objective of this study was to assess the physiological changes of blood stasis, a classic TCM pathological state.

MATERIALS AND METHODOLOGY

Animals

A total of 75 male Sprague-Dawley rats of specific-pathogen-free grade (Beijing, Certificate No. SCXK 2012-0001), weighing 280-300 g, 10 weeks old, were provided by the Laboratory Animal Center, Chinese PLA General Hospital. The rats were permitted a 3 d acclimatization period with a normal feeding regime before the experiment. Rats were fed and treated in accordance with the guidelines of the animal experiments

committee of PLA General Hospital. Protocols and surgical procedures were approved by the PLA General Hospital Animal Ethics Committee.

Acute blood stasis model in rats

After 3 days of acclimatization, the rats were anesthetized using an intraperitoneal injection of 0.3% sodium pentobarbital (1 mL/100 g body weight; Sigma, St. Louis, MO, USA), and then fixed in a supine position. Electrodes were connected to the limbs to monitor the electrocardiogram (ECG). The acute blood stasis model was established through a rat tail-vein injection of 6% HMWD (0.8 mL/100 g body weight; HMWD 500, Amresco, OH, USA). After the induction of stasis, the rats curled and were agitated, and demonstrated a reduced response to external stimuli. ST segment elevation proved that the induction of stasis was successful.

Experimental groups

The rats (n = 75) were divided randomly and equally into a control group, and four experimental groups assessed at different times after the induction of stasis (0, 1, 3 or 6 h after stasis) (n = 15). The control group rats were injected with saline (0.8 mL/100 g body weight) through the tail vein, and the remaining procedures were consistent with the other experimental groups. ECG was assessed at predetermined times (0, 1, 3 and 6 h after the induction of stasis), and abdominal aortic blood and cardiac samples were harvested from each group.

Optical microscopy

Two rats demonstrating successful induction of stasis were selected randomly in each group. The rats underwent carotid artery intubation. The heart was perfused using 4% paraformaldehyde and was removed. The excised hearts underwent 10% formalin fixation and conventional dehydration until transparent. The hearts were embedded in paraffin wax, and sectioned at a thickness of 5 pm. Hematoxylin-eosin (HE) staining was then conducted. Briefly, the paraffin sections underwent conventional dewaxing followed by hematoxy-lin staining. The sections were then washed using water, and underwent 1% hydrochloric acid alcohol differentiation and anti-blue and eosin staining. Sections were dehydrated until transparent and then mounted using neutral gum and observed using a light microscope.

Transmission electron microscopy

Three rats were selected randomly in each group. Abdominal aortic blood samples were harvested and the heart was removed immediately and placed in an ice bath. A piece of tissue (1 mm x 1 mm x 2 mm) was removed from the left and right ventricle, respectively. The specimens were fixed using 2.5% glutaraldehyde for 2 h and were then washed using phosphate buffer

(pH 7.0), post-fixed using 1% osmium tetroxide for 1 h, and dehydrated through a graded series of acetone solutions. The samples were then embedded in epoxy resin, and sliced to a thickness of 70 nm, followed by citric acid and uranyl acetate staining. The samples were then viewed using a transmission electron microscope.

Determination ofTXB2 and 6-Keto-PGFla levels in the plasma

Ten rats were selected randomly from each group. The rats were anesthetized using an intraperitoneal injection of 0.3% (1 mL/100 g body weight) sodium pentobarbital (Sigma, St. Louis, MO, USA) and fixed in a supine position. A midline abdominal incision was performed to expose the abdominal aorta, and 10 mL of abdominal aortic blood was collected in a sterile EP tube (with the pre-addition of 0.2 mL of Ethylene Diamine Tetraacetic Acid Na2). The samples were mixed and centrifuged at 4 t and 3500 rpm for 15 min to obtain platelet-poor plasma. TXB2 and 6-Ke-to-PGF1a were detected using the enzyme linked immunosorbent assay (ELISA) method.

Determination of intercellular adhesion molecule 1 (ICAM-1) and vascular cell adhesion molecule 1 (VCAM-1) expression in myocardium

Ten rats were selected randomly in each group, and approximately 100 mg of left ventricular anterior cardiac tissue was harvested from each rat. The tissue samples were placed in liquid nitrogen, and stored at — 80 t until further use. Genomic RNA was extracted using a TRIzol Kit. RNA purity was determined using a microplate reader. cDNA was synthesized using reverse transcription. A real-time polymerase chain reaction (PCR) reaction system was employed to measure ICAM-1 and VCAM-1 mRNA expression. PCR was performed in a final volume of 20 pL, containing 2 pL of cDNA, 7 pL of nuclease-free water, 10 pL of FAST SYBR Green Master Mix, 0.5 pL of upstream primer, and 0.5 pL of downstream primer. The temperature profile was 20 s at 95 t, 30 s at 95 t, and 10 s at 60 t for 40 cycles. The amplification curve and melting curve of the real-time PCR were determined. The relative levels of mRNA were calculated using the 2-AAct method. (Primer sequence see Table 1).

Determination of VE-cadherin protein expression in myocardium

Ten rats were selected randomly in each group, and approximately 100 mg of left ventricular anterior cardiac tissue was harvested from each rat. The tissue samples were placed in liquid nitrogen, and stored at — 80 t until further use. The myocardial samples were crushed and 200 pL of protein lysis buffer was added. The samples were vortexed 8-10 times, were homogenized on ice for 35-40 min, and were centrifuged at 12000 rpm and 4 t for 15 min. Loading buffer was added to the supernatant, which was heated to 100 t for 5 min, and stored at — 80 t . Protein standards were mixed with loading buffer at 100 t for 5 min for prior to use. Western blotting was used to assess protein levels. A total of 100 pg of the cellular protein samples was loaded in each well of the sodium dodecyl sulfate poly-acrylamide gel electrophoresis gel. A constant current of 15 mA was used for the electrophoresis. Based on the relative position of the pre-stained marker and the molecular weight of the target protein, the optimal running time was determined to ensure that the target protein was located in the lower 1/3 of the separation surface. The polyvinylidene fluoride membrane was immersed in methanol for 2 min. The membrane was opened, a wet sponge pad was placed on the negative electrode and two more layers of wet filter paper were added. The SDS-PAGE gel was carefully placed on the filter paper and the surface covered with the appropriate PVDF membrane. The gel and membrane were then sandwiched with another wet sponge pad, the red positive plate was closed, clipped, and inserted into the corresponding location of the transfer rig. The process was conducted at 4 t, using 100 V/100 mA. Blocking solution (2 mL) and the appropriate amount of primary antibody were added to a 5 mL EP tube, and stored in a refrigerator at 4 t , overnight. The primary antibody was incubated with the membrane in 5% skim milk + tris buffered saline tween for 2 h on the shaker at room temperature. Membranes were placed in a washing box containing TBST, and were washed three times on a shaker for 10 min. The procedure for the application of the secondary antibody is similar to the pri-

able 1 Primer sequences

Sequence

ICAM-1

VCAM-1

Upstream primer 5'-AAG AAG GTG GTG AAC CAC GC-3' Downstream primer 5'-TTC ACC ACC CTG TTG CTG TA-3' Upstream primer 5'- GTC ACT GTT CAA GAATGT C -3' Downstream primer 5'- ACA AGA ACT TCT GCT GGG T -3' Upstream primer 5'- GAG AAC TAC AAG TCT ACA CC -3' Downstream primer 5'- CAC ACA CAT AGA TTC CAG -3'

Notes: ICAM-1: intercellular adhesion molecule 1; VCAM-1: vascular cell adhesion molecule 1.

mary antibody application procedure, except that it was performed at room temperate for 1 h. The dilution of the primary and secondary antibody was 1: 2000 and 1: 5000, respectively. Western blot chemilumines-cence reagent was added to the dry and flat membrane surface. After 5 min, the extra ECL reagent was absorbed using filter paper and then the membrane was placed in a chemiluminescence instrument to measure the signal.

Statistical analysis

All data are expressed as mean ± standard deviation ( x ± s). Statistical analysis of data was performed using the SPSS package (version 13.0, SPSS Inc., Armonk, NY, US). A one-way analysis of variance followed by the Least Significant Difference test was used for multiple comparisons. Qualitative data were analyzed using Fisher's exact probability method. A value of P < 0.05 was selected to denote statistical significance.

RESULTS

ECG changes

After the induction of stasis, the rats curled and were agitated, and demonstrated a reduced response to external stimuli. ST segment elevation proved that the induction of stasis was successful. The ST segment was elevated compared with control in the 0 h group, but the elevation was lower than the other treatment groups (Figure 1). The ST segment in the 1 h group continued to increase and the elevation amplitude increased significantly. The ST segment in the 3 h group continued to increase and part of the ECG demonstrated a T wave in a one-way curve. The ST segment in the 6 h group continued to increase but demonstrated no significant difference compared with that of the 3 h group. In the process of monitoring the ECG, some rats demonstrated atrioventricular block and ventricular premature beat-based arrhythmias, there was no group difference of this occurrence. Experimental groups assessed at different times after the induction of stasis (0, 1, 3 or 6 h after stasis); The control group rats were injected with saline (0.8 mL/

100 g body weight) through the tail vein, and the remaining procedures were consistent with the other experimental groups.

HE staining of the myocardium

In the control group myocardial fibers were arranged as expected. The cells were arranged clearly and the nucleus appeared in the center; the myocardial tissues were not dissociated or necrotic and inflammatory cell infiltration was not apparent. In the 0 and 1 h groups, myocardial cells were dissociated and necrotic and inflammatory cell infiltration was observed. In the 3 h group, myocardial cells were dissociated, flaky and ne-crotic and inflammatory cell infiltration was observed. In the 6 h group, a significant level of inflammatory cell infiltration and myocardial cell dissociation, necrosis and flaking was observed. The myocardial edge appeared to be damaged and fibrous tissue hyperplasia was apparent, while red blood cell retention was observed in the myocardial microvasculature (Figure 2).

Myocardial ultrastructure observed using electron microscopy

The electron microscopy observation showed that in the control group the vascular endothelial cells were flat and close to the vessel wall. Vascular lumen patency was apparent and the endothelial cells were not swollen. In the 0 h group, the cytoplasm of the vascular en-dothelial cells contained more absorptive vesicles. The vessel lumens demonstrated plasma retention and stenosis. In the 1 h group, the endothelial cells were swollen and the cytoplasm contained more absorptive vesicles. The vessel lumens demonstrated blood aggregation. In the 3 h group, the morphology of the endothe-lial cells was irregular. The vessel lumens demonstrated plasma retention and stenosis. In the 6 h group, the en-dothelial cells were swollen and demonstrated abnormal nuclei. The vessel lumens demonstrated blood aggregation and stenosis (Figure 3).

Expression ofTXB2 and 6-Keto-PGFM in plasma

The level of TXB2 in the plasma did not significantly change in the 0 h group. However, the level of TXB2 in the plasma was significantly increased at 1 h, 3 h, and

Figure 1 Electrocardiogram of rats in each group

A: control group; B: 0 h group; C: 1 h group; D: 3 h group; E: 6 h group.

Figure 2 HE staining in the myocardium in each group (x100)

A: control group; B: 0 h group; C: 1 h group; D: 3 h group; E: 6 h group. HE: Hematoxylin-eosin.

6 h after the induction of stasis (P < 0.05), and the level in the 6 h group was significantly higher than that in the 1 h and 3 h groups (P < 0.05), and reached the highest level. The level of 6-Keto-PGF1„ in the plasma in the 0 h, 1 h, 3 h and 6 h groups was significantly lower than that in the control group (P < 0.05). The level in the 0 h group was the lowest. The level of 6-Ke-to-PGF1a in the plasma in the 3 h and 6 h groups was significantly higher than that in the 0 h group (P < 0.05). The ratio of TXB2/6-Keto-PGF1„ in the plasma in the 0, 1, 3, and 6 h groups after the induction of stasis was significantly higher than that in the control group (P < 0.05) and the highest ratio was observed in the 1 h group (Table 2).

Expression ofICAM-1 and VCAM-1 mRNA in the myocardium

The expression of ICAM-1 in the myocardium in the 0 h group was significantly higher than that in the control group (P < 0.05). The expression of ICAM-1 in the myocardium decreased in the 1 h group and increased in the 3 h and 6 h groups. The expression of VCAM-1 mRNA in the myocardium in the 0, 1, 3 and 6 h groups was significantly lower than that in the control group (P < 0.05). The expression of VCAM-1 mRNA in the myocardium in the 6 h group was significantly increased compared with the 0 h group (P < 0.05).

Expression of VE-cadherin protein in the myocardium

The expression of VE-cadherin protein in the myocardium in the 0 h group was significantly higher than that in the control group (P < 0.05). The expression of VE-cadherin decreased in the 1 h group, but was not

significantly different compared with the control group (P > 0.05). The expression of VE-cadherin in the 3 h group was significantly higher than that in the control group (P < 0.05). The expression of VE-cadherin in the 6 h group didn't show any difference compared to control group. ( P > 0.05).

DISCUSSION

Blood stasis is a pathological state of blood retention and stagnation in the capillary lumen. Previous patho-physiological studies of blood stasis have mostly focused on atherosclerotic diseases, emphasizing the formation of large arterial plaques and thrombosis. Jin-zhou Tian et al. suggested that carotid plaques and blood stasis are highly correlated.7 In this research, we have studied pathological changes in the microvascula-ture, endothelial cells and cardiomyocytes and the variation in microvascular injury-related factors in a acute blood stasis rat model to elucidate the theory of the classic pathological state of blood stasis from a micro-pathological point of view.

This study found that erythrocyte aggregation, plasma retention, lumen stenosis and even occlusion were observed in microvascular lumens after the induction of stasis, and these phenomena were aggravated with increasing time, along with the development of further pathological changes such as endothelial swelling and abnormal nuclei. Therefore, the pathological state of blood stasis, as characterized by blood flow hysteresis and stagnation, was verified from an endothelial and microvascular perspective. Endothelial cells are also injured in blood stasis. We verifed the Traditional Chinese Medicine theory of "blood coagulation and weep" in modern pathology performance. A typical myocardi-

Figure 3 The ultrastructural changes in the myocardial tissue in each group (A, B: X5000; CDE: X2000) A: control group; B: 0 h group; C: 1 h group; D: 3 h group; E: 6 h group.

|Table 2 Expression of TXB2 and 6-Keto-PGF1a in plasma ( x ± s) |

Group TXB2 (pmol/L) 6-Keto-PGFi„ (pmol/L) TXB2/6-Keto-PGFi„ (ratio)

Control 78.29±15.77 1.74±0.16 45.05±9.71

0 h 74.33±18.31 0.l4±0.03a 54l.63±95.61a

1 h 298.99±26.71ab 0.34±0.09a 871.19±50.44ab

3 h 291.38±28.37ab 0.86±0.12ab 340.19±26.61a

6h 428.49±36.50ab 0.93±0.13ab 460.39±10.97a

Notes: the control group rats were injected with saline (0.8 mL/100 g body weight) through the tail vein; the four experimental groups were assessed at different times after the induction of stasis (0, 1, 3 or 6 h after stasis). TXB2: thromboxane B2; 6-Keto-PGF1a: 6-ke-to-prostaglandin F1a. Compared with control group, aP < 0.05; compared with the 0 h group, bP < 0.05.

Table 3 Expression of intercellular adhesion molecule 1 (ICAM-1) and vascular cell adhesion molecule 1 (VCAM-1) mRNA and VE-cadherin protein in the myocardium (x ± s)

ICAM-1 mRNA

VCAM-1 mRNA

VE-Cadherin (dots per inch )

Control 1.00±0.00 1.00±0.00 0.76±0.14

0 h 1.43±0.08a 0.39±0.05a 1.22±0.28a

1h 0.93±0.12b 0.34±0.05a 0.60±0.10b

3h 1.46±0.13a 0.53±0.07a 1.04±0.21a

6h 1.32±0.16a 0.62±0.10ab 0.74±0.19b

Notes: the control group rats were injected with saline (0.8 mL/100 g body weight) through the tail vein; the four experimental groups were assessed at different times after the induction of stasis (0, 1, 3 or 6 h after stasis). ICAM-1: intercellular adhesion molecule 1; VCAM-1: vascular cell adhesion molecule 1; VE-Cadherin: vascular endothelial cadherin. Compared with control group, aP < 0.05; compared with the 0 h group, bP < 0.05.

ß-actin

ß-actin

ß-actin

ß -actin

ß-actin M^M ••»- 42

Figure 4 Western blot images depicting VE-cadherin protein expression in the myocardium in each group 1: control group; 2: 1 h group; 3: 3 h group; 4: 6 h group; 5: 0 h group.

al ischemia changes showed in ECG after the induction of stasis, aggravated as time prolonged and been serious in group of 3 h, 6 h. . The HE staining showed increases over time in myocardial cell necrosis and dissociation after the induction of stasis, along with inflammatory infiltration. Therefore, we demonstrated that organs and tissues were injured and damaged by blood stasis, which corresponds to the "blood failed to nourish" aspect of blood stasis, as described inTCM. We studied the pathological features of the acute blood stasis model at the molecular level. Thromboxane a2 (TXA2) and prostaglandin I2 (PGI2) are thought to be the two of the most important prostaglandins that regulate the homeostasis of the cardiovascular system.8'9 TXA2 induces vasoconstriction, platelet aggregation, smooth cell proliferation and promotes atherosclerosis formation.10 PGI2 is a prostaglandin derived from endo-thelial cells and smooth muscle, and plays a role in relaxing blood vessels and inhibiting platelet aggregation,

and is anti-inflammatory and anti-thrombotic.11 An imbalance between TXA2 and PGI2 can damage vascular homeostasis. If the proportion of TXA2 increases, the effect of vasoconstriction and platelet aggregation can increase the risk of thrombosis, including cardiac thrombosis.12-14 Because PGI2 and TXA2 are very unstable in vivo, the levels of TXA2 and PGI2 in the blood were measured in the current study by analyzing the levels of the TXB2 and 6-keto-PGF^ metabolites, respectively. We found that the level of plasma TXB2 increased rapidly after the induction of stasis, and was continuously increased within a 6 h observation time, while the plasma levels of 6-keto-PGF^ decreased significantly. The lowest 6-keto-PGF^ concentration was observed immediately after the induction of stasis, and levels then recovered but were still lower than in the control group. The TXB2/6-Keto-PGF1a ratio increased rapidly after the induction of stasis, and remained at a high level for 6 h. TXB2, which promotes platelet aggregation and adhesion, rapidly increased, while 6-keto-PGF1a, which suppresses platelet aggregation, rapidly decreased. This resulted in a pathological state consisting of blood flow hysteresis and stagnation, and vasomotor dysfunction further aggravated blood stasis.

In addition, the mRNA expression of the intercellular adhesion molecule, ICAM-1, was significantly increased after the induction of stasis, which likely increased the interaction between leukocytes and endo-thelial cells, including capture, rolling, and adhesion.15 These pathological processes might be involved in the pathological process of blood flow hysteresis and stagnation. A previous study has indicated that the expression of ICAM-1 is increased in endothelial cells by activated platelets, demonstrating that the mechanism of blood stasis formation involves a variety of factors.16 VCAM-1 is expressed on the inner and outer sides of endothelial cells, mediating inflammatory cell rolling, adhesion and penetration of the vessel wall to reach inflammatory sites during inflammatory reactions.17'18 The mRNA expression of VCAM-1 was not signifi-

cantly increased after the induction of stasis, indicating that VCAM-1 is not involved in the high blood viscosity produced during acute blood stasis. In the acute blood stasis model, we examined levels of VE-cadherin, a vascular endothelial cell tight junction protein. VE-cadherin is not only a key molecule in the endothelial cell barrier,19 protecting the vessel wall from decomposition, but also acts as a regulator of tight junctions between endothelial cells, regulating the expression and distribution of other tight junction molecules such as claudin-5 and N-cadherin. It plays a key role in maintaining microvascular integrity and endo-thelial cell barrier function.20-22 In the current study, the expression of VE-cadherin protein was disordered after the induction of stasis, indicating that the endothelial cell barrier was unstable during acute blood stasis. Previous studies have shown that the aggregation of ICAM-1 could lead to tyrosine phosphorylation of VE-cadherin, which increases endothelial cell permea-bility.23-25 There was a trend towards increased expression of VE-cadherin after the induction of stasis. The disorganized expression of VE-cadherin during acute blood stasis could be related to the unstable increasing expression of ICAM-1.

In conclusion, the pathological manifestations of acute blood stasis are microvascular blood retention, lumen stenosis and even occlusion. Acute blood stasis is also called "blood coagulation and weep" in TCM theory. Blood stasis is also characterized by endothelial cell and cardiomyocyte injury and even necrosis, which corresponds to "blood failed to nourish" in TCM theory. Furthermore, blood stasis is characterized by vasomotor factor imbalance, platelet activation, adhesion molecule increases and endothelial barrier disorder. In summary, we have demonstrated the pathological features and related mechanisms of acute blood stasis from a micro-pathological perspective, which verifies the classic TCM theory using modern methods.

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