| Objective: With the guidance of Ying-Wei Theory “pass through choroids, intersected biochemistry” in Choroids Theory, the angiogenesis rabbit model were established induced by combining a high-fat diet with collar placed around carotid artery in early AS. After the observation on the arterial angiogenesis, neuroendocrine and oxidative stress in carotid artery, we explored the effects of angiogenesis on early AS and the intervention effects of Tongxinluo. This provided the study model and experimental datas for application of Ying-Wei Theory “pass through choroids, intersected biochemistry” in Choroid Theory in vascular lesions, but also this helped elucidate AS pathogenesis channel that "from outside to in". It was of great significance to establish a novel way for effective prevention and treatment in early AS.Method:1 To establish the arterial angiogenesis model by combining a high-fat diet with collar placed around carotid artery in early AS and study the intervention effects of Tongluo drugsWe established the arterial angiogenesis models by combining a high-fat diet with collar placed around right common carotid artery in New Zealand rabbit, observed the pathomorphological changes and arterial angiogenesis, and further demonstrated the effects of angiogenesis on neointimal formation and the intervention effects of Tongxinluo on this model. 120 New Zealand rabbits were randomly divided into 8 groups: NC, HC, HC+high, HC+mod, HC+low, HC+ato, LY294002(PI3K inhibitor group) and PD98059(MEK inhibitor group), 15 rabbits in each group. Using 25% concentration Pluronic®F-127 gel as the carrier to prepare sustained release gel which contained PI3 K, MEK inhibitors, the final concentration was 200μmol/L. Injected the sustained release gel containing PI3 K, MEK inhibitors into silicone tube through a catheter, rabbits were received local drug delivery by vascular adventitial administration. For each rabbit, 0.5ml gel preparations for sustained release for local application. Experiment lasted for 4 weeks. NC group was fed with common diet all along; HC, HC+high, HC+mod, HC+low, HC+ato, LY294002, PD98059 groups were fed with a high-fat diet the next day after collar implantation. Each rabbit consumed about 120 g of food daily and corresponding drug treatment. HC+high, HC+mod and HC+low groups were given Tongxinluo ultra fine powder superfine powder at 0.60 g/(Kg·d), 0.30 g/(Kg·d) and 0.15 g/(Kg·d), respectively, and HC+ato was administered atorvastatin by gavage at 2.50 mg/(Kg·d). The serum lipid level was detected to reflect lipid metabolism; the changes of pathological morphology in vessels by HE staining, the vascular smooth muscle cell proliferation and formation of collagenous fiber in arterial wall by Masson staining, the intimal lipid deposit of abdominal aorta by Oil Red O staining was observed; CD31 and VEGF immunohistochemical staining reflected the angiogenesis in carotid artery. Blood flow through microvessel of carotid arteries was detected with color microspheres to reflect microvessel dysfunction.2 Neuroendocrine mechanism of angiogenesis in carotid artery wall and intervention effects of Tongluo drugsThis part was based on the successful model in former part, observed the possible neuroendocrine mechanism of angiogenesis in rabbit carotid artery and intervention effects of Tongluo drugs. 90 New Zealand rabbits were randomly divided into 6 groups: NC, HC, HC+high, HC+mod, HC+low and HC+ato, 15 rabbits in each group. The method for the rapid establishment of rabbit model and drug dose were the same as above. The plasma NE, Ach content were tested; location and expression of TH, Ach E, NGF, NPY in carotid artery tissues were detected by immunofluorescence technique; Western Blotting technique was used to determine α1-adrenergic receptor(α1-AR) and α7-nicotinic acetylcholine receptor(α7n Ach R) protein expression in carotid arteries.3 Oxidative stress mechanism of angiogenesis in carotid artery and intervention effects of Tongxinluo drugsThis part further discussed the possible oxidative stress mechanism of angiogenesis in carotid artery and intervention effects of Tongxinluo. 120 conventional New Zealand rabbits were randomly divided into 8 groups: NC, HC, HC+high, HC+mod, HC+low, HC+ato, LY294002 and PD98059, 15 rabbits in each group. The experimental period was 4 weeks. Using 25% concentration Pluronic®F-127 gel as the carrier to prepare sustained release gel which contained PI3 K, MEK inhibitors, the final concentration was 200μmol/L. Injected the sustained release gel containing PI3 K, MEK inhibitors into silicone tube through a catheter, rabbits were received local drug delivery by adventitia. 0.5ml sustained release gel inhibitor was locally applicated for each rabbit. The administration doses of Tongxinluo and atorvastatin were the same as above. After the specimen collection, serum MDA, SOD, T-AOC, CAT and GSH-px were detected; NADPH subunits p22 phox m RNA, gp91 phox m RNA in carotid arteries were located and semi-quantitated by fluorescence in situ hybridization and reverse transcription PCR; ROS production of carotid tissues were detected by DHE fluorescence probe; PI3K/AKT, ERK pathway protein and VEGF-A/ VEGFR-2 in carotid tissues were detected by Western Blotting technique; observed the microvessel density changes marked by CD31 in carotid artery wall and localization, expression of p-AKT, p-ERK proteins by immunohistochemical technique.Results:1 Model establishment of the angiogenesis model by combining a high-fat diet with collar placed around carotid artery in early AS and intervention effects of Tongluo drugs1.1 The serum lipid level changes in each groupSerum TC, TG, LDL-C and HDL-C levels of the animals in HC group were obviously higher than those in NC group and the differences were statistically significant(P<0.01). There was no statistical difference in serum lipid level between LY294002, PD98059 and HC groups(P>0.05). Serum TC, TG and LDL-C levels in HC+high, HC+mod and HC+ato groups were lower than HC group, with statistically significant differences(P<0.05, P<0.01). HDL-C levels in HC+high and HC+ato groups were higher than HC group, with statistically significant differences(P<0.01). There were no statistically significant differences in TC, TG, LDL-C and HDL-C levels between HC+high, HC+mod and HC+ato groups(P>0.05).1.2 Oil Red O staining of abdominal aortaThe normal abdominal aorta showed cream color by general observation, intima was flat and smooth, without lipid deposition. There were multiple scattered red lipid deposition in the abdominal aortic intima in HC group, LY294002 and PD98059 group, manifested spot or stripes of various lengths, which were fatty streak or lipid plaque formation, and early atherosclerotic lesions. Compared with HC group, aortic lipid deposition decreased at different degrees in HC+high, HC+mod, HC+low and HC+ato groups, and obviously decreased in HC+high and HC+ato groups.1.3 Masson staining of carotid artery tissuesThe structures of normal carotid arteries were clear; intima was flat and smooth and smooth muscle cell layers had regular arrangement. In HC group, the tunica intima was obviously thickened, smooth muscle cell had migration, the collagen fiber accumulated largely, medial smooth muscle cell increased, with irregular arrangement. Compared with HC group, the intimal hyperplasia were not prominent, with little collagen fiber accumulated, no obvious smooth muscle cell proliferation and migration, and little medial smooth muscle cell hyperplasia in HC+high, HC+mod, HC+ato, LY294002 and PD98059 groups.1.4 HE staining of carotid arteriesFor the common carotid artery, take the internal and external elastic membrane as the boundary, which includes intima, media and adventitia outward from the lumen in order. There were a smooth intima, intact internal elastic membrane and endothelial cell monolayers, clear medial smooth muscle cell, little microvessel and fibroblasts and nerve ending in the normal arterial adventitia. In HC group, neointima formation, internal elastic lamina continuation was interrupted, smooth muscle cell significantly increased, its branches were in disorder, some elastic fibers broke or disappeared, media layer gap was obviously widened, with connective tissue changes in adventitia and inflammatory cell infiltration, microvessel counts increased. Compared with HC group, the intima and adventitia hyperplasia were alleviated at different degrees, medial smooth muscle cell proliferation and migration, microvessel quantity decreased at different degrees and inflammatory cell infiltration were alleviated in HC+high, HC+mod, HC+low, HC+ato, LY294002 and PD98059 groups.1.5 Immunohistochemical staining of CD31 and VEGF in carotid arteryVEGF positive substance was yellow granular and mainly located in cytoplasm and extracellular matrix. Little VEGF positive expression can be observed in intima, media and adventitia in normal carotid artery. VEGF positive expression in carotid artery significantly increased, microvascular endothelial cells and adventitia showed positive expression in HC group. VEGF positive expression in adventitia decreased at different degrees after Tongxinluo and Atorvastatin intervention. Take VEGF positive rate as the assisted markers: adventitial VEGF positive rate of HC was significantly higher than NC group, and the difference was statistically significant(P<0.01). Compared with HC group, after intervention of Tongxinluo and Atorvastatin, adventitial VEGF positive rate significantly decreased(P<0.05, P<0.01), which had decreased the most significantly in HC+high groups and the difference was statistically significant(P<0.01). Compared with HC+ato group,the differences reached statistical significance between HC+high, HC+mod, LY294002 and PD98059 groups(P>0.05).Little microvessels marked by CD31 can be seen in carotid artery in NC group. Microvessel quantity in adventitia in HC group significantly increased, microvessels marked by CD31 can not be seen in tunica media. Compared with HC group, adventitial microvessel quantity in all Tongxinluo dose groups, HC+ato, LY294002 and PD98059 groups decreased at different degrees, which significantly decreased in HC+high, HC+mod, HC+ato, LY294002 and PD98059 groups. Quantitative analysis of microvessel density indicated that MVD labeling index in HC were significant higher than NC group, with the difference statistically significant(P<0.01). MVD labeling index in HC+high, HC+mod and HC+ato groups were significantly lower than HC and all the difference were statistically significant(P<0.01). MVD labeling index in HC+high group decreased more significantly than HC+ato group, and the difference was statistically significant(P<0.05). The differences were not statistically significant between LY294002, PD98059 and HC+ato groups(P>0.05).1.6 Changes of microvascular blood low MBF in carotid artery wallCompared with NC group, MBF in HC group increased significantly and the difference was statistically significant(P<0.01). Compared with HC group, MBF in all Tongxinluo dose groups and HC+ato groups decreased at different degrees, which obviously decreased in HC+high, HC+mod and HC+ato groups, and the differences were statistically significant(P<0.05, P<0.01). Compared with HC+ato group, MBF in HC+high and LY294002 groups significantly decreased, and the differences were statistically significant(P<0.05, P<0.01). The difference was not statistically significant between PD98059 and HC+ato groups(P>0.05).2 Neuroendocrine mechanism of angiogenesis in carotid artery and intervention effects of Tongluo drugs 2.1 Plasma NE and Ach levels of experimental rabbit Plasma NE and Ach levels in experimental rabbit were detected by Elisa detection. Result analysis indicated that there was no statistical difference between all groups(P>0.05).2.2 Location of TH and Ach E in arterial arteries by immunofluorescence Immunofluorescence showed, noradrenergic nerve marked by TH distributed in intima and media—adventitial boundary region of normal carotid artery and cholinergic nerve marked by Ach E mainly distributed in media layer/ adventitia region. In HC group, noradrenergic nerve which distributed in media—adventitial boundary disappeared, but obvious noradrenergic nerve hyperplasia occurred around microvessel in adventitia, with the noradrenergic nerve hyperplasia was more significant than cholinergic nerve in adventitia. All Tongxinluo dose groups can inhibit arterial neural remodeling at different degrees, showing that the noradrenergic nerve in media—adventitial boundary region decreased can dominate, and inhibited the noradrenergic nerve hyperplasia around adventitial microvessels.2.3 Location and and expression of NGF and NPY in arterial arteries by immunofluorescence NGF and NPY immunofluorescence indicated, there was no NGF expression in normal carotid wall, but NPY expression existed in media—adventitial boundary. In HC group, NGF and NPY expression around vasa vaorum in adventitia significantly increased, but NPY expression significantly decreased in media—adventitial boundary region. In all Tongxinluo dose groups, NGF and NPY positive expression decreased in adventitia, but NPY expression increased in media—adventitial boundary region.2.4 α1-AR and α7n Ach R protein expression in carotid tissuesThere were little α1-AR, α7n Ach R protein expression in NC group, α1-AR, α7n Ach R protein expression increased significantly in HC group; α1-AR, α7n Ach R protein expression in HC+high, HC+mod, HC+low and HC+ato groups were downregulated at different degrees. Gray analysis indicated, compared with NC group, α1-AR, α7n Ach R in HC group significantly increased and the differences were statistically significant(P<0.01). Compared with HC group, α1-AR, α7n Ach R of HC+high, HC+mod and HC+ato groups significantly decreased, and the differences were statistically significant(P<0.01); Compared with HC+ato group, α1-AR, α7n Ach R of HC+high group significantly decreased, the differences were all statistically significant(P<0.05).3 Oxidative stress mechanism of angiogenesis in carotid artery and intervention effects of Tongluo drugs 3.1 The level changes of serum MDA, SOD, T-AOC, CAT and GSH-pxCompared with NC group, the activity of serum SOD, CAT, T-AOC, GSH-px in HC significantly decreased, MDA content significantly increased, and the differences were all statistically significant(P<0.01). Compared with HC group, the serum SOD, GSH-px, CAT, T-AOC activity in HC+high, HC+mod and HC+ato groups obviously increased, MDA content obviously decreased, and the differences were all statistically significant(P<0.05, P<0.01). There were no statistical differences in SOD, CAT, T-AOC, GSH-px activity and MDA content between HC+high, HC+mod and HC+ato groups(P>0.05).3.2 ROS distribution in carotid artery and adventitial quantitative comparison There was low ROS content in intima, media and adventitia of NC group. Adventitial ROS production had a prominent increase in HC group, and the expression in microvessel and adjacent area was significant. Adventitial ROS production in HC+high, HC+mod and HC+ato groups decreased more significantly than HC group.Immunofluorescence analysis showed compared with NC group, adventitial ROS content in HC group increased relatively significantly, and the difference was statistically significant(P<0.01). Compared with HC group, adventitial ROS content in LY294002、PD98059 and HC+low groups didn’t decreased relatively significantly(P>0.05). Compared with HC group, adventitial ROS content in HC+high, HC+mod and HC+ato groups relatively significantly decreased, and the differences were statistically significant(P<0.01). Compared with HC+ato group, adventitial ROS content in HC+high group significantly decreased, and the difference was statistically significant(P<0.05).3.3 Correlation analysis of microvessel density in carotid artery with ROS content in adventitiaA Spearman correlation analysis of microvessel density in carotid wall with adventitial ROS content was made, and the statistical results indicated MVD in carotid wall had significant positive correlation with adventitial ROS content(r=0.893, P<0.01).3.4 Location and quantitative expression of NADPH oxidase subunit p22 phox and gp91 phox in carotid arteryIn situ hybridization results indicated, there were little gene p22 phox hybridization signals in carotid artery tissues of NC group, only limited in adventitia, and gp91 phox hybridization signals distributed in intima and adventitia, mainly in adventitia. Gp91 phox hybridization signals distributed more densely than p22 phox in adventitia. p22 phox and gp91 phox hybridization signals in HC group were strongly positive, mainly in neointima, adventitial microvessel and adjacent area. Compared with HC group, p22 phox and gp91 phox hybridization signals didn’t obviously changes in LY294002, PD98059 and HC+low groups. Positive hybridization signals of adventitial p22 phox and gp91 phox decreased at different degrees in HC+high, HC+mod and HC+ato groups,.In order to evaluate the effects of NADPH oxidase from ROS, we detected the expression of p22 phox and gp91 phox m RNA in carotid artery by reverse transcription PCR. The results showed expression of p22 phox and gp91 phox m RNA in HC were significantly higher than NC group, and the difference was statistically significant(P<0.01). Compared with HC group, the expression level of p22 phox m RNA and gp91 phox m RNA in LY294002, PD98059 and HC+low groups had no significant difference(P>0.05).Compared with HC group, the expression level of p22 phox m RNA and gp91 phox m RNA in HC+high, HC+mod and HC+ato groups significantly decreased, and the differences were statistically significant(P<0.01). The expression level of p22 phox m RNA and gp91 phox m RNA in HC+high groups were obviously lower than HC+ato group, and the differences were statistically significant(P<0.05).3.5 Expression of PI3K/AKT, ERK and VEGF-A/VEGFR-2 transduction associated proteins in carotid artery wallThere were no statistically differences between PI3 K protein relative expression in all groups(P>0.05). p-PI3 K protein relative expression: compared with NC group, p-PI3 K protein relative expression in HC group increased significantly(P<0.01); compared with HC group, p-PI3 K protein relative expression in HC+high, HC+mod, HC+ato, LY294002 groups significantly decreased, and the differences were statistically significant(P<0.01); compared with HC+ato group, p-PI3 K protein relative expression in HC+high and LY294002 groups significantly decreased, the differences were statistically significant(P<0.01). p-PI3K/PI3 K level: compared with NC group, p-PI3K/PI3 K level in HC group increased significantly, the difference was statistically significant(P<0.01); compared with HC group, p-PI3K/PI3 K level in HC+high, HC+mod, HC+ato and LY294002 groups significantly decreased, and the differences were statistically significant(P<0.05, P<0.01); compared with HC+ato group, p-PI3K/PI3 K level in HC+high and LY294002 groups obviously decreased, and the differences were statistically significant(P<0.05, P<0.01).There was no statistical difference in AKT protein relative expression between all groups(P>0.05). p-AKT protein relative expression: compared with NC group, p-AKT protein relative expression in HC groups significantly increased, and the difference was statistically significant(P<0.01); compared with HC group, p-AKT protein relative expression of HC+high, HC+mod, HC+ato and LY294002 groups significantly decreased, and the differences were statistically significant(P<0.01); compared with HC+ato group, p-AKT protein relative expression of HC+high and LY294002 groups significantly decreased, and the differences were statistically significant(P<0.01). p-AKT/ AKT level: compared with NC group, p-AKT/AKT level in HC group significantly increased, and the difference was statistically significant(P<0.01); compared with HC group, p-AKT/AK level in HC+high, HC+mod, HC+ato and LY294002 groups significantly decreased, and the differences were statistically significant(P<0.01); compared with HC+ato group, p-AKT/AKT level in HC+high and LY294002 groups obviously decreased, and the differences were statistically significant(P<0.05, P<0.01).There were no statistical differences between all groups in MEK protein relative expression(P>0.05). p-MEK protein relative expression: compared with NC group, p-MEK protein relative expression in HC group increased significantly(P<0.01), the difference was statistically significant; compared with HC group, p-MEK protein relative expression in HC+high, HC+mod and PD98059 groups significantly decreased, and the differences were statistically significant(P<0.01); p-MEK protein relative expression of HC+ato group significantly decreased(P<0.05); compared with HC+ato group, p-MEK protein relative expression in HC+high and PD98059 groups significantly decreased(P<0.05). p-MEK/MEK: compared with NC group, p-MEK/ MEK level in HC groups increased significantly, and the difference was statistically significant(P<0.01); compared with HC group, p-MEK/MEK level in HC+high, HC+mod and PD98059 groups significantly decreased, and the differences were statistically significant(P<0.05, P<0.01); compared with HC+ato group, p-MEK/MEK level in HC+high and PD98059 groups obviously decreased, and the differences were statistically significant(P<0.05, P<0.01).There was no statistical difference between all groups in ERK protein relative expression. p-ERK protein expression: compared with NC group, p-ERK protein relative expression significantly increased in HC group(P<0.01); compared with HC group, p-ERK protein relative expression decreased by a certain extent in HC+low, HC+mod and HC+ato groups, but had no statistical difference(P>0.05). p-ERK protein relative expression in HC+high and PD98059 groups were significant(P<0.01); compared with HC+ato group, p-ERK protein relative expression in HC+high and PD98059 groups obviously decreased(P<0.05). p-ERK/ERK level: compared with NC group, p-ERK/ERK level in HC group increased significantly(P<0.01); compared with HC group, p-ERK/ERK in HC+high and PD98059 groups obviously decreased, and the differences were statistically significant(P<0.05, P<0.01); compared with HC+ato group, p-ERK/ERK level in HC+high and PD98059 groups obviously decreased, and the differences were statistically significant(P<0.05, P<0.01).VEGF-A and VEGFR-2 protein expression: compared with NC group, the VEGF-A and VEGFR-2 protein expression in HC group increased significantly, and the differences were statistically significant(P<0.01); compared with HC group, VEGFR-A of HC+high, HC+mod, HC+ato, LY294002 and PD98059 groups significantly decreased, and the differences were statistically significant(P<0.01). VEGFR-2 expression obviously decreased in HC+mod group, LY294002 and PD98059, and the differences were statistically significant(P<0.05). VEGFR-2 expression significantly decreased in HC+high and HC+ato groups, and the differences were statistically significant(P<0.01); compared with HC+ato group, HC+high and LY294002 groups obviously decreased, and the differences were statistically significant(P<0.05, P<0.01).3.6 Localization and expression of p-AKT, p-ERK in carotid artery by immunohistochemistryp-AKT positive substance was yellow granular which mainly located in cell nuclear or cytoplasm. In NC group, p-AKT positive expression was visible in intima, media and adventitia of carotid artery. Adventitial p-AKT expressed little in NC group, and strongly positive in HC group, with microvascular endothelial cells and some fibroblasts expression were positive; Adventitial p-AKT positive expression in HC+high, HC+mod, HC+ato and LY294002 groups more obviously decreased than HC group.p-ERK positive substance was yellow granular which mainly located in cell nuclear or cytoplasm. In NC group, there was little p-ERK positive expression in intima, media player and adventitia; adventitial p-ERK expression was strongly positive in HC group, with microvascular endothelial cells and some fibroblasts expression positive. Adventitial p-ERK positive expression in HC+high, HC+mod, HC+ato and PD98059 groups more obviously decreased than HC group.Conclusion:1 Based on the identity of collaterals and microvessels in anatomic morphology with the guidance of choroid theory, this research pointed out arterial wall “collaterals- microvessels” is the important place for ying-qi(vascular endothelial) and wei-qi(vascular adventitia or neuroendocrine) “pass through choroids, intersected biochemistry”. Arterial angiogenesis caused by “collaterals- microvessels” ying-wei “pass through choroids, intersected biochemistry” dysfunction is the important pathological factor of AS disease. This research established carotid artery injury model by combining a high-fat diet with collar placed around right common carotid artery in New Zealand rabbit. As a result, microvessel obviously increased and neointima formation in carotid atrtery. After inhibitors’ application to inhibite arterial angiogenesis, neointimal hyperplasia reduced. It demonstrated that arterial angiogenesis contributed to early atherosclerostic lesions. The study provides the experimental evidences, effective treatment strategies and drug intervention to prevention and treatment for early AS.2 The angiogenesis in carotid artery may be related to adventitial neuroendocrine imbalance. In early AS, neural remodeling occurred in arterial wall, that the noradrenergic nerve in media—adventitial boundary region disappeared to some extent, but noradrenergic nerve had hyperplasia in adventitia, and finally local noradrenergic nerve was more dominant in adventitia than cholinergic nerve. During the process of angiogenesis, there was adventitial neuroendocrine regulation imbalance, with adventitial NGF, NPY and α1-AR, α7n Ach R expression significantly increased in carotid artery.3 In early AS, adventitial oxidative stress is the critical mechanism for arterial angiogenesis. During the angiogenesis, arterial p22 phox and gp91 phox gene expression, adventitial ROS production and VEGF-A, VEGF-R2 protein expression significantly increased. After blocking adventitial PI3K/Akt and ERK signal pathway in vivo, AKT and ERK phosphorylation level, VEGF-A, VEGF-R2 protein expression and angiogenes is effectively decreased. It is demonstrated that PI3K/Akt and ERK signal pathway are the critical signal pathways to be involved in arterial angiogenesis.4 Tongxinluo significantly inhibited arterial angiogenesis and neointima formation in early AS. Compared with atorvastatin, Tongxinluo moderate dose has similar effects as atorvastatin to reduce angiogenesis and neointimal hyperplasia, but Tongxinluo high dose is obviously better than atorvastatin. And the mechanisms were related to regulation of neuroendocrine imbalance, lowering the level of oxidative stress in adventitia. Tongxinluo significantly inhibited adventitial sympathetic remodeling, improveded neuroendocrine imbalance, with Adventitial NGF and NPY expression decreased, α1-AR, α7n Ach R protein expression downregulated; Tongxinluo obviously inhibited p22 phox and gp91 phox gene expression in carotid artery, and adventitial ROS production to downregulate PI3K/Akt, ERK signal pathway and VEGF-A,VEGFR-2 protein expression, which play the critical role for arterial angiogenesis. |
PDF Full Text Request