Effects of Hexueshengluo Method on Angiogenesis and Vasodilatation of Rats with Myocardial Infarction and Its Mechanisms
|School||Hebei Medical University|
|Course||Chinese and Western medicine combined with the basis|
|Keywords||Collaterals and blood born Myocardial infarction Angiogenesis Vascular endothelial growth factor Endothelial function Endothelium-dependent vasodilation of aortic ring Human umbilical vein endothelial cells|
Objective: Coronary heart disease is a kind of disease that seriously threatens the health of the human being. In our country, the morbidity and mortality of the disease is increasing in recent years. The coronary stenosis or obliteration in the disease can lead to myocardial ischemia and necrosis, thus influence the heart function. Collateral circulation of the coronary artery will have a protective action. Through the promotion of new vessels in the ischemic region, therapeutic angiogenesis has provided a new and hopeful treatment for serious CHD patients. It has a long history to treat the disease with traditional Chinese medicine. According to the traditional Chinese medicine theory, and combined with modern medicine research, our tutor think that the contradiction between blood and vessels is the important pathogenesis of CHD, so he raise the therapeutic method of Hexueshengluo to harmonize the blood and vessels. Through the copy of experimental myocardial infarction model, we observed the effects of Hexueshengluo method on the heart function, angiogenesis and vasodilatation of the model rats, and confirm the therapeutic action of the method to pro-angiogenesis. We also explored the mechanisms of pro-angiogenesis activity and endothelium protection with method of cell culture and isolated thoracic aorta rings.Part I Changes of Angiogenesis and Vasodilatation in Rats with Myocardial Infarction and the Intervention of Hexueshengluo MethodMethods: The model of experimental myocardial infarction was copied with the ligation of left anterior descending of coronary artery (LAD) in 96 healthy Wistar male rats. 48 successful model rats were randomly divided into 4 groups: the model group, the isosorbide dinitrate group, and the high and low doses of Hexueshengluo Method group, sham operated group was also prepared. They were treated with different therapies for 4 weeks, then we checked the left ventricular contraction function with colored Doppler, observed the infracted size with TTC staining, studied the expression of FVIII, VEGF at protein level with immune histochemical method, detected the serum VEGF, NO, and NOS with ELISA method, and studied the vasodilatation function with isolated thoracic aorta rings.Results: (1) Left ventricular contraction function: Compared with sham group, left ventricular internal dimension diastole (LVIDd) and left ventricular internal dimension systole (LVIDs) are significantly increased in model group (P<0.01), left ventricular fractional shortening (LVFS,%) and the left ventricular ejection fraction (LVEF,%) are significantly decreased in model group (P<0.01); Left ventricular contraction function in treatment groups are all significantly increased compared with model group(P<0.01), and the high dose group is the best one among them. (2) Myocardial infarction size: Myocardial infarction size in HXSL-L, HXSL-H and ID groups are significantly smaller compared with model group (P<0.05~0.01), and HXSL-H group is better than HXSL-L and ID groups (P<0.05); (3) The quantity of myocardial blood vessel and capillaries density (MVD): The MVD in model group is higher than the sham group (P<0.05); The MVD in HXSL-L, HXSL-H and ID groups are significantly higher than the model group (P<0.05); (4) Blood serum VEGF、NO and NOS content: There is no significant difference for serum VEGF between model group and sham group (P>0.05),the NO and NOS content in model group are significantly slower than sham group (P<0.01); Serum VEGF、NO and NOS in HXSL-L、HXSL-H and ID groups are significantly higher than the model group (P<0.05),and the HXSL-H group is the best one among treatment groups (P<0.05~0.01); (5) The expression of VEGF around the infarction region: Compared with sham group, the expression of VEGF around the infarction region in model group is significantly higher (P<0.05), the expression of VEGF in HXSL-L、HXSL-H and ID groups are significantly higher than the model group (P<0.05~0.01). (6) Vasodilatation of thoracic aorta rings: The endothelial-dependent vasodilatation in the model group is decreased compared with sham group (P<0.01), the endothelial-dependent vasodilatation in HXSL-L、HXSL-H and ID groups are significantly increased compared with model group (P<0.05~0.01). There is no significant difference in endothelial-independent vasodilatation among each group (P>0.05).Part II Effects of Hexueshengluo Method on the Proliferation and Migration of Human Umbilicus Vein Endothelial CellsMethods: HUVEC cell line ECV-304 was used as the model. Experiment group: (1) normal control group; (2) HXSL recipe group (after 24 hours adherent culture, ECV-304 cells were treated with RPMI-1640 culture fluid containing 0.1mg/mL、0.5mg/mL、1mg/mL、2mg/mL、4mg/mL Chinese medicine respectively). Every experiment was performed three times in each group. The proliferation was measured by MTT assay; FCM was used to detect the cell cycle; the cell migration was observed by scratch wound assay.Results: (1) Effects of HXSL recipe on the proliferation of ECV-304 cells: Compared with control group, HXSL groups significantly increased ECV-304 cell proliferation in a dose-dependent manner (P<0.05 or P<0.01); (2) Time action of HXSL recipe on the proliferation of ECV-304 cells: Certain dosage (2mg/mL) of HXSL recipe promoted the cell proliferation significantly in different time (P<0.01); (3) Cell cycle analysis by FCM: Cell cycle analysis showed a significant increase of cell percentage of synthetic period in HXSL groups compared with control group(P<0.01); (4) Effects of HXSL recipe on the migration of ECV-304 cells: Compared with control group, HXSL groups significantly promoted ECV-304 cell migration in a dose-dependent manner(P<0.05 or P<0.01). Part III Relaxation Effects of Hexueshengluo Method on Isolated Thoracic Aorta Rings of Rats and Its MechanismsMethods: The study was performed with the model of isolated rat thoracic aorta rings in organ bath. The effects of HXSL method on aorta rings in resting tension were observed with accumulated concentration. Aorta rings were pre-constricted with norepinephrine or KCl, then the tension of aorta rings with intact endothelium or endothelium denuded were observed by stimulating with HXSL at concentration of 0.0125、0.025、0.05、0.1、0.2g/L. Aorta rings were pre-treated with L-NAME, MB, indomethacin or propranolol, then the tension of aorta rings with endothelium denuded were observed by stimulating with HXSL.Results: (1) HXSL had no significant effects on aorta rings in resting tension or pre-constricted with KCl (P>0.05). (2) When the concentration of HXSL was 0.0125、0.025、0.05、0.1、0.2g/L respectively, it caused concentration-dependent relaxation while aorta rings were pre-constricted with NE (1×10- 5mol/L) compared with the control(P<0.05~0.01). (3) The relaxation effect of HXSL on aorta rings with endothelium denuded had no significant change when pre-treated with propranolol (P>0.05). (4) when pre-treated with L-NAME or MB,the relaxation effect of HXSL on aorta rings with endothelium intact was partly inhibited(P<0.05~0.01). The relaxation effect of HXSL on aorta rings with endothelium intact had no significant change when pre-treated with indomethacin (P>0.05).Conclusions: (1) HXSL method can improve the left ventricular contraction function, and decrease the size of myocardial infarction; it has therapeutic effects on myocardial infarction rats. (2) HXSL method can increase MVD and VEGF expression in the area around the myocardial infarction, and elevated the content of VEGF and NO in the blood; it has pro-angiogenesis activity for ischemic myocardium. (3) HXSL method can improve the endothelium-dependent vasodilatation of the AMI rats, and increase the content of NO and NOS in the blood; it can inhibit the endothelial dysfunction in AMI rats. (4) HXSL method has the action of endothelium-dependent vasodilatation and endothelium-independent vasodilatation on aorta rings, its endothelium-dependent vasodilatation is related to NOS in aorta rings, and the endothelium-dependent vasodilatation may be induced by the way of NO- sGC- cGMP.