| Objective: Under the guidance of the Ying-nutrient and Wei-defense theory of the Vessel-Collateral Doctrine, this study established the rabbits early atherosclerotic lesions model by giving high-fat diet, and founded vasa vasorum angiogenesis animal model via high-fat diet combined with right carotid artery cannula, discussed the effects of vasa vasorum angiogenesis in early atherosclerotic lesion through observing the morphological changes of the carotid artery, the changes of blood lipids and adventitia angiogenesis at 3 day, 1 week, 2 weeks and 4 weeks; and furtherexplored related mechanisms of vasa vasorum angiogenes and the effect of Tongluo drugs on the vasa vasorum angiogenesis in Early Atherosclerosis around oxidative stress and sympathetic / parasym-pathetic neurotransmitters effects by means of selecting the node of the fourth week after surgery.Methods: 1 The effects of vasa vasorum angiogenesis in early atherosclerotic lesion180 healthy New Zealand rabbits were fed adaptively for 2 weeks, then randomly divided into normal groups, high-fat group and complex model group. n=60. Normal group gives ordinary feed, while high-fat group were given a high-fat feed to establish AS early lesion animal model, and complex model group were given high-fat diet with positioning of a soft silicone collar around the rabbits right carotid artery established vasa vasorum angiogenesis animal model, the experimental period was 4 weeks. Fifteen rabbits were randomly taken from the three groups respectively at 3 days, 1 week, 2 weeks and 4 weeks after surgery to selecte for 3D imaging of Micro-CT, to detect the blood perfusion changes of the cartoid artery microvascular by injecting DYE-TRAK color microsphere through Left ventricular, to detect blood lipids by biochemistry, to detect morphological of the carotid artery by HE staining, and to detect CD34 protein’ s expression in the cartoid artery by Western Blot. 2 Sympathetic and parasympathetic nervous and Oxidative stress mechanism of vasa vasorum angiogenesis in carotid artery and intervention effects of Tongluo drugs90 healthy New Zealand rabbits were randomly divided into normal group, high-fat group, complex model group, complex model + GSTL-H group, complex model + GSTL-L group, complex model + ATO group. n=15. The treatment groups were given high-fat diet with positioning of a soft silicone collar around the rabbits right carotid artery, On the same day of surgery while giving the drug intervention. Drugs were compounded to corresponding concentration of mixture suspension liquid with 0.5% sodium carboxymethyl cellulose(CMC-Na). Dosage: Guishaotongluo high-dose 0.6g/kg/d, low-dose 0.3g/kg/d, Atorvastatin 2.5mg/kg/d. Oral dosing volume of 3ml/kg, 1 times a day. The experiment lasted for 4 weeks, Blood lipids, MDA, SOD, T-AOC levels, morphological of the carotid artery,NADPH subunits gp91 phox, p22 phox mRNA were detected, Micro-CT method to test the microvascular density of the right carotid artery, color microsphere method to detect the blood perfusion changes of the cartoid artery microvascular, immunohisto chemical staining method to detect the expression of NGF antibody, TH antibody in the carotid artery tissue. and NGF, TH, AchE, VEGF, VEGFR-2, ERK and p-ERK protein’ s expression in the cartoid artery were detected by Western Blot. 3 NGF/TrkA/ERK1/2 pathway of vasa vasorum angiogenesis in carotid artery and intervention effects of Tongluo drugs90 healthy New Zealand rabbits were randomly divided into normal group, high-fat group, complex model group, complex model + PD98059(ERK inhibitor) group, complex model + GSTL-H group, complex model + PD98059 + GSTL-H group. n=15. 100μl PD98059 dissolved solution was placed in 25 ml 25% Pluronic@ F-127 gel to prepare sustained release gel which contained ERK inhibitors(Final concentration of 200μmol/L). Injected the PD98059 sustained release gel into silicone tube, which wrapped in right carotid artery, to inhibiti the signal pathway of adventitia. It will be given drug intervention simultaneously on the surgery day,continuously to 4 weeks. The right carotid artery morphology were observed by optical microscope, color microsphere method to detect the blood perfusion changes of the cartoid artery microvascular, immunohisto chemical staining method to detect in the carotid artery tissue expression of NGF antibody, and NGF, TrkA, VEGF, ERK and p-ERK protein’s expression in the cartoid artery were detected by Western Blot.Results:1 The effects of vasa vasorum angiogenesis in early atherosclerotic lesion 1.1 The changes of blood lipid levels in every groups at different periodCompared with the normal group, the serum TC, TG and LDL-C levels in high-fat group gradually increased from 2 weeks to 4 weeks, and in complex model group significantly elevated from 1 week to 4 weeks, The differences are all statistically significant( P<0.01).Compared with the high-fat group,the blood lipid levels in complex model group increased at 1 week. The differences are statistically significant( P<0.01). 1.2 Morphological changes of carotid(HE staining)Normal group: The carotid intima smooth wavy, endothelial cells are complete and continuous, the tunica media consists mainly smooth muscle. High-fat group and complex model group: The carotid intima endothelial cells are complete, nucleus are flat,the tunica media line is clear in the first 1 week. The intima are slight thickening at 2 weeks. There is exfoliate of endothelial cells and small amount of foamy macrophages, the intima are obvious thickening, tunica media gap widening at 4 weeks. 1.3 The comparation of carotid artery wall MVD in each groupNormal group: vasa vasorum longitudinally aligned along the carotid artery, the vessel wall area and microvascular density of the wall in High-fat group and complex model group increased since 1 week, The differences are all statistically significant(P<0.05 or P<0.01).Compared with the High-fat group, the vessel wall area and microvascular density of the wall in the complex model group appeared to increase since 1 week, gradually increased at 2 weeks and 4 weeks, vasa vasorum showed a dense capillary plexus distribution. The differences are all statistically significant(P<0.05 or P<0.01). 1.4 Microvascular blood flow volume of the carotid artery wall in each groupsCompared with the normal group at the same period: the microvascular blood flow volume of the carotid artery wall in high-fat group increased from 2 weeks, and complex model group appeared increasing trend at 1 week, while gradually increased from 2 weeks.(P<0.01).The microvascular blood flow volume appeared increasing trend at 1 week and significante increasing at 4 weeks compared with high-fat group(P<0.05). 1.5 The comparison of CD34 protein levels in carotid artery tissueNormal group: There were less protein expression of the microvascular density of the wall(CD34 antibody staining positive), the protein expression of CD34 in high-fat group were presented progressively increased since 1 week to 4 weeks(P<0.05 or P<0.01); the protein expression of CD34 in complex model group were presented progressively increased since 3 day to 4 weeks(P<0.05 or P<0.01). 2 Sympathetic and parasympathetic nervous and Oxidative stress mechanism of vasa vasorum angiogenesis in carotid artery and intervention effects of Tongluo drugs 2.1 The changes of blood lipid levels in each groupThe serum TC, TG and LDL-C levels in high-fat group and complex group significantly increased compared with normal group( P<0.01). Compared with complex model group, TC, TG and LDL-C levels decreased significantly in complex group + GSTL-H, complex group + GSTL-L group and complex group + Atorvastatin group(P<0.01), and there is no statistical difference among the three groups(P>0.05). 2.2 Comparison of serum MDA, SOD, and T-AOC levels in different groupsCompared with normal group, serum MDA levels in high-fat group and complex model group show progressive increase, while serum SOD and T-AOC levels are all progressive decrease(P<0.01). Compared with complex model group, serum MDA levels decreased significantly(P<0.01), while serum SOD and T-AOC levels progressive increase in complex group + GSTL-H, complex group + GSTL-L group and complex group + Atorvastatin group(P<0.01), and there is no statistical difference among the three groups(P>0.05). 2.3 Histomorphology changes of artery in different groups 2.3.1 Histomorphology changes of carotid(HE staining)Normal group:The intima were smooth, endothelial cells were continuous and complete, nucleus fit closely with the inner elastic plate, smooth muscle were clear, and there were small amount of microvascular in the carotid adventitia. High-fat group: The intima are slight thickening, tunica media gap widened, microvascular quantities increased. Complex model group:The intima diffuse hyperplasia, small amount of foamy macrophages can be seen, smooth muscle and elastic fiber disordered arrangement, microvascular proliferation significantly. The pathological damages of carotid alleviated in different degrees in treatment groups. 2.3.2 Histomorphology changes of aorta abdominals(Oil red O staining)Aortas observation are milk white, intimal level off, almost no Oil red staining. While the aortas elasticity decreased and small amount of patchy red area can be seen in High-fat group. There were numerous of patchy red area(namely fatty streak formed),mainly located in the lower thoracic aortic segment and abdominal aorta. 2.4 Distribution and expression of NADPH subunits p22 phox, gp91 phox mRNA in carotid arteriesThere was small amount of p22 phox, gp91 phox hybridization signals in carotid tissues of normal group, while the hybridization signals were strongly positive in high-fat group and complex model group, mainly in neointima and adventitial area. The hybridization signals of carotid alleviated in different degrees in complex group + GSTL-H, complex group + GSTL-L group and complex group + Atorvastatin group compared with complex model group. 2.5 Microvascular blood flow volume of the carotid artery wall in each groupThe microvascular blood flow volume of the carotid artery wall in high-fat group have a progressive increase compared with normal group(P<0.01). Compared with high-fat group, the blood flow significantly increased in the complex model group(P<0.01). Compared with complex model group, microvascular blood flow volume decreased significantly in complex group + GSTL-H, complex group + GSTL-L group and complex group + Atorvastatin group(P<0.05, P<0.01), and there is no statistical difference among these treatment groups(P>0.05). 2.6 Distribution and expression of NGF, TH in carotid arteries in different groupsImmunohisto chemical staining showed,Compared with normal group, NGF,TH antibody staining positive in the high-fat group and complex model group presents a progressive expression, Compared with complex model group, the protein level of NGF, TH in every treatment groups reduced significantly. 2.7 The comparison of NGF, TH, AchE, VEGF, VEGFR-2, total ERK and p-ERK protein levels of the carotid artery tissue in different groupsThere is no apparent abnormality in the protein level of total ERK among normal group, high-fat group, complex model group and treatment intervention groups(P>0.05).Compared with normal group, the protein levels of NGF, TH, AchE, VEGF, VEGFR-2 and p-ERK increased in high-fat group(P<0.01). Compared with high-fat group, the protein levels of above indicators increased significantly in complex model group(P<0.01). Compared with complex model group, the protein level of NGF, TH, AchE,VEGF, VEGFR-2 and p-ERK in every treatment group reduced significantly(P<0.01). 3 NGF/TrkA/ERK1/2 pathway of vasa vasorum angiogenesis in carotid artery and intervention effects of Tongluo drugs 3.1 Histomorphology changes of carotid(HE staining)Normal group: The intima were smooth, endothelial cells were continuous and complete, nucleus fit closely with the inner elastic plate, smooth muscle were clear, and there were small amount of microvascular in the carotid adventitia. The intima are slight thickening, tunica media gap widened, microvascular quantities increased in high-fat group, complex model group and complex model+PD98059+Guishaotongluo high group. The pathological damages of carotid alleviated in different degrees in complex model + PD98059 and complex model + Guishaotongluo high group. 3.2 Microvascular blood flow volume of the carotid artery wall in each groupThe microvascular blood flow volume of the carotid artery wall in high-fat group have a progressive increase compared with normal group(P<0.01). Compared with high-fat group, the blood flow significantly increased in the complex model group(P<0.01). Compared with complex model group, microvascular blood flow volume decreased significantly in complex model + PD98059 and complex model + Guishaotongluo high group(P<0.01). The microvascular blood flow volume have a progressive increase in complex model + PD98059+Guishaotongluo high group compared with complex model + PD98059 group. 3.3 The comparison of NGF, TrkA, VEGF, total ERK and p-ERK protein levels of the carotid artery tissue in different groupsThere is no apparent abnormality in the protein level of total ERK among normal group, high-fat group, complex model group and treatment intervention groups(P>0.05).Compared with normal group, the protein levels of NGF, TrkA, VEGF and p-ERK increased in high-fat group(P<0.01). Compared with high-fat group, the protein levels of above indicators increased significantly in complex model group(P<0.01). Compared with complex model group, complex model + PD98059 group and complex model +Guishaotongluo high obviously lowered the protein level of NGF, TrkA, VEGF and p-ERK(P<0.01). Compared with PD98059 group, the intervention effect of complex model + PD98059+Guishaotongluo high group to the the protein levels of NGF, TrkA, VEGF and p-ERK showed significant statistical difference.Conclusions:1 Under the guidance of the Ying-nutrient and Wei-defense theory of the Vessel-Collateral Doctrine to discuss the effects of vasa vasorum angiogenesis in early atherosclerotic lesion. Based on the correlation between “Ying zai mai zhong”and endothelial, “Wei zai mai wai” and adventitia, identity on the anatomic between “collaterals-microvessels”, this study established the rabbits early atherosclerotic lesions model by giving high-fat diet, founded complex animal model via high-fat diet combined with right carotid artery cannula to discuss the effects of vasa vasorum angiogenesis in early atherosclerotic lesion. and Tongluo drug intervention, not only conducive to reveal pathophysiological basis of abnormal Ying-nutrient and Wei-defense “unblocking collaterals with subsequent convergence and substance-qi transformation”,and it is important for clarify AS “outside-in” pathogenesis and to find effective drug intervention.2 This study established the rabbits early atherosclerotic lesions model by giving high-fat diet,and founded complex animal model via high-fat diet combined with right carotid artery cannula, Compared with normal group, the protein expression of CD34 and microvessel density in high-fat group were presented progressively increased at 1 week, the microvascular blood flow volume of the carotid artery wall in high-fat group increased from 2 weeks; While protein expression of CD34 in complex model group were presented progressively increased since 3 day compared with normal group and presented progressively increased at 3 day, 1 week. The microvessel density appeared increasing trend at 1 week, and appeared significant differences at 2 weeks and 4 weeks compared with high-fat group. The microvascular blood flow volume appeared increasing trend at 1 week and significante increasing at 4 weeks compared with high-fat group; Carotid morphology showed slight thickening of the intima at 2 weeks and more obvious at 4 weeks in high-fat group and complex model group, while the degree of intima thickening showed more serious at 4 weeks in complex model group compared with high-fat group. These results suggest that hyperlipidemia can promote adventitia angiogenesis, which occurred early than intimal hyperplasia. The degree of vasa vasorum angiogenesis is heavier in complex model group, which affected and aggravated atherosclerosis early lesion.3 Compared with normal group, The expression of hybridization signals of gp91 phox and p22 phox appeared positive strongly in intima and adventitia, content of MDA increased and activity of SOD, T-AOC decreased in high-fat group and complex model group; the protein levels of NGF, TrkA, TH, AchE, VEGF, VEGFR-2 and p-ERK1/2 increased in high-fat group, the expression of NE increased, the carotid artery microvascular density and blood flow excessive perfusion; while apart from NE and microvascular density, above the rest indicators are elevated in complex model group compared with high-fat group. Above demonstrated that hyperlipidemia and complex model caused the vasa vasorum angiogenesis with oxidative stress and excessive sympathetic activation at the same time, the degree of sympathetic activation was heavier in complex model group compared with high-fat group; NGF/TrkA/ERK1/2 passageway involved in the pathological process of sympathetic overactivity promote vasa vasorum angiogenesis.4 Ying-nutrient and Wei-defense Tongluo drugs-Guishaotongluo can inhibit vasa vasorum angiogenesis, reduce carotid artery microvascular density and blood flow excessive perfusion. It may related to inhibited NGF/TrkA/ERK1/2 passageway and sympathetic overactivity, and improved the imbalance of sympathetic and parasympathetic nervous system activity, relieve adventitia and serum oxidative stress. |
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