| Objective: Under the guidance of the Ying-nutrient and Wei-defense“unblocking collaterals with subsequent convergence and substance-qitransformation†theory of the Vessel-Collateral Doctrine, this studyestablished the rabbits early atherosclerotic lesions model by giving high-fatdiet, dynamic observed the changes of blood lipids, hypoxia condition of theaorta, microvascular density of the aorta wall, endothelium-dependentvasodilatation, intima lesion, and the correlation factors expressions ofinflammation and oxidative stress, discussed the effects and relatedmechanisms of arterial wall “collateral-microvascular†angiogenesis in earlyatherosclerotic lesion. Using high-fat diet with positioning of a soft siliconecollar around the rabbits left carotid artery established microvascularangiogenesis animal models, and intervened with drugs, further explored theeffect and mechanism of Tongxinluo on the microvascular angiogenesis of thearterial wall, and provided experimental basis for revealing the scientificconnotation of the Ying-nutrient and Wei-defense “unblocking collaterals withsubsequent convergence and substance-qi transformation†theory of theVessel-Collateral Doctrine.Methods: This section mainly discussed AS etiology and pathogenesisfrom Traditional Chinese Medicine (TCM) and guided by theVessel-Collateral Doctrine, and discussed the guidance value of ying-wei“unblocking collaterals with subsequent convergence and substance-qitransformation†theory in the study of microvascular lesions.1The role of arterial wall “collateral-microvascular†angiogenesis in earlyatherosclerotic lesionThis section experiment established AS early lesion animal model byfeeding New Zealand rabbits high-fat diet, and dynamically observe its hypoxia condition, microvascular density, endothelial dependent diastolicfunction and pathological changes of the aortic intima and so on. A total of110conventional grade New Zealand rabbits were randomly divided intonormal diet group (50) and high-fat diet group (60). Normal diet group givesordinary feed, while high-fat diet were given a high-fat feed (normal dietsupplemented with1.0%cholesterol,7.5%yolk powder,5%lard oil). All ofthe animals were given drinking water freely and were on a12h light and darkcircadian rhythm, and experimental period was6weeks. Ten to twelve rabbitswere taken from the two groups respectively at3days,1week,2weeks,4weeks and6weeks after high-fat feeding, two of them were randomly selectedfrom each group for whole aorta Oil-red O staining, and the other animalsabdominal aorta were removed quickly and used to check other indicators. Atthe nodes in each time, blood was withdrawn from the ear vein after overnightfasting, and all animals were sacrificed with overdose of sodium pentobarbital(100mg/Kg). Serum lipoprotein levels, endothelin-1, nitric oxide, MDA, SOD,T-AOC levels, and endothelium dependent vasodilatation function ofabdominal aorta were detected, morphological changes of each animalabdominal aorta were observed under light microscope, immunohistochemicalstaining method to detect in the abdominal aorta tissue expression of CD34antibody, HIF-1alpha antibody, and molecular biology method to detect thegene and protein expression of HIF-1α, NF-κB, TNF-α, IL-6, Nrf2, γ-GCSand HO-1in aorta tissue.2The effects of Tongxinluo on “collateral-microvascular†angiogenesis,inflammation and oxidative stress levelsThis section experiment using high-fat diet with positioning of a softsilicone collar around the rabbits left carotid artery established microvascularangiogenesis animal model, to observe the intervention effect of Tongxinluoon wall microvascular develop and explore its action mechanism. Operationmethod: animals were weighed and fixed on the operating table supine afteranesthesia, Layered cut and separated the skin, subcutaneous fascia andmuscle tissue, carefully remove the left total artery sheath membrane by ophthalmic forceps, about3cm of left carotid artery was isolated, siliconecollar disinfected (long28mm, diameter1.7mm, outside diameter3.2mm)opened longitudinally positioning around the rabbits carotid artery, and thecollars were sealed with silicone (Silastic dow corning732, RTV), then putback the carotid artery. Penicillin160000IU were local dripped to preventinfection. A total of290conventional grade New Zealand rabbits wererandomly divided into normal group (40), Atorvastatin group (50), andTongxinluo high-, medium-and low-dose group (50rabbits in each group).Normal group gives ordinary feed, while other groups were given a high-fatfeed (the same to above) and positioning of a soft silicone collar around therabbits carotid artery. The treatment groups were given drugs on the same dayof surgery. Dosage: Atorvastatin2.5mg/kg, Tongxinluo high-dose0.6g/kg,medium-dose0.3g/kg, low-dose0.15g/kg. Drugs were compounded tocorresponding concentration of mixture suspension liquid with0.5%sodiumcarboxymethyl cellulose (CMC-Na), Oral dosing volume of3ml/kg,1times aday. All of the animals were given drinking water freely and were on a12hlight and dark circadian rhythm, and experimental period was6weeks. Eightto Ten rabbits were taken from each groups respectively at3days,1week,2weeks,4weeks and6weeks after given drugs. Serum lipoprotein levels,endothelin-1, nitric oxide, MDA, SOD, T-AOC levels were detected as thesame as the second section, the left carotid artery morphology andmicrovascular endothelial ultrastructure on it were observed by opticalmicroscope and transmission electron microscopy (TEM),3D imaging ofMicro-CT and immunohistochemical staining to test the microvascular density(MVD) of the left carotid artery, color microsphere method to detect the bloodflow volume change of the microvascular, inflammation and oxidative stressrelated factors NF-κB, TNF-α, IL-6, Nrf2, and NQO1gene and proteinexpression were detected by Real-time qPCR and Western Blot, respectively.3The effects of Tongxinluo on DLL4/Notch signal pathway of artery wall“collateral-microvascular†angiogenesisMolding method and the experimental group were the same with the third section. VEGF-A, VEGF-R2, DLL4, and Notch1gene and protein expressionwere detected by Real-time qPCR and Western Blot, respectively.Results:1The role of arterial wall “collateral-microvascular†angiogenesis in earlyatherosclerotic lesion1.1The changes of blood lipid levels in every groups at different period:Compared with the normal diet group, the serum TC, TG, HDL-C andLDL-C levels in high-fat diet group significantly elevated in turn from2weeks to6weeks, and are higher than the normal diet group at the sameperiod. The differences are all statistically significant (P<0.05or P<0.01).1.2Morphological changes of aorta abdominals (Oil red O staining and HEstaining):Oil red O staining: Aortas general observation are milk white, goodelasticity, intimal smooth and level off, no Oil red staining. There are nosignificant changes between normal diet group and high-fat diet group after3days,1week,2weeks in the oil red O staining results, while the aortaselasticity decreased and small amount of patchy red area (namely fatty streakformed) can be seen in the intima after4and6weeks, and located in the lowerthoracic aortic segment, abdominal aorta and where its branches.Aortic intima-media changes (HE staining): The aortic intima endothelialcells are complete and continuous, nucleus are flat, fit closely with the innerelastic plate, smooth muscle in the tunica media line is clear in normal dietgroup and high-fat diet group in the first2weeks. Small amount of foamymacrophages can be seen in the intima, tunica media gap widening at4weeks.The intima are obvious thickening, a large number of foam cells accumulationin the subintima, parts of elastic fiber corrugated fracture or disappear, tunicamedia gap widening more obvious.Aortic adventitia changes (HE staining): Small amount of microvascularscan be seen in the abdominal aorta adventitia in normal diet group and high-fatdiet group in the first1week. The microvascular quantities begin to increaseafter high-fat2weeks. 1.3Comparison of microvascular density of the abdominal aorta wall andHIF-1α antibody expression (immunohistochemistry):The microvascular density of the wall (CD34antibody staining positive)in the high-fat diet group have a progressive increase from high-fat feeding2weeks to6weeks (P <0.05), and are higher than the normal group at the sameperiod (P<0.05or P<0.01). There are no obvious HIF-1α antibodies stainingpositive expression in either of the two groups from3days to4weeks, andonly a little weakly positive expression in the high-fat diet group at6weeks.1.4The changes of abdominal aorta endothelium dependent vasodilatationfunction at different times:The endothelium dependent vasodilatation functions of abdominal aortain normal diet group are all higher than90%from3days to6weeks, whilethere is a progressive decrease in high-fat diet group, from53.55%to49.95%,respectively, and lower than those of normal diet group. The differences areall significant (all P<0.01).1.5Correlation analysis of abdominal aorta endothelium dependentvasodilatation function and its microvascular density:MVD as the independent variable, endothelium-dependent vasodilationas the dependent variable, given a Spearman correlation analysis for them, Theresult shows that the correlation coefficient (r) is-0.824, P<0.01, Two of themwere significantly negative correlation relationship.1.6The changes of serum ET-1, NO, MDA, SOD, and T-AOC levels:Compared with normal diet group, serum ET-1and MDA levels inhigh-fat diet group show progressive increase, differences between groups in4weeks and6weeks are all significant (all P<0.01), while serum NO, SOD, andT-AOC levels are all progressive decrease, lower than the normal group at thesame period, and significant difference also appear in4weeks and6weeks(all P<0.01).1.7The comparison of HIF-1α, NF-κB, TNF-α, IL-6, Nrf2, γ-GCS and HO-1mRNA expressions in abdominal aorta tissue (4weeks):Compared with normal diet group, the mRNA expressions of HIF-1α, NF-κB, and Nrf2do not appear significant difference in high-fat diet group(all P>0.05), the mRNA expressions of TNF-α and IL-6increasedsignificantly (all P<0.01), while γ-GCS and HO-1mRNA expressionsdecreased significantly (all P<0.01).1.8The comparison of HIF-1α, NF-κB, TNF-α, IL-6, Nrf2, γ-GCS and HO-1protein levels in abdominal aorta tissue (4weeks):Compared with normal diet group, the protein level of HIF-1α does notappear significant difference in high-fat diet group yet (P>0.05), the proteinlevels of NF-κB, TNF-α and IL-6increased significantly (P<0.05or P<0.01),while Nrf2, γ-GCS and HO-1protein levels decreased significantly (allP<0.01).2The effects of TXL on “collateral-microvascular†angiogenesis,inflammation and oxidative stress levels2.1The changes of blood lipid levels in each group:The serum TC, TG, HDL-C and LDL-C levels in high-fat diet groupsignificantly increased in turn from2weeks to6weeks, and are higher thanthe normal group at the same period. The differences are all statisticallysignificant (P<0.05or P<0.01). Compared with the model group, TC andLDL-C levels began to decline in Atorvastatin group, TXL-H, and TXL-Lgroup from2weeks (P<0.05or P<0.01), and there is no statistical differenceamong the three groups (P>0.05). TG level began to decrease significantly inthe three groups from4weeks (P<0.05or P<0.01). Although the HDL-C levelis higher in the three group than that of model group, there is no statisticalsignificance (P>0.05). TXL-L group does not appear significant difference atdifferent times compared with model group (P>0.05).2.2The comparison of serum ET-1, NO, MDA, SOD, and T-AOC levels indifferent groups:Compared with normal group, serum ET-1and MDA levels of modelgroup show progressive increase, differences between groups in4weeks and6weeks are all significant (all P<0.01), while serum NO, SOD, and T-AOClevels are all progressive decrease, lower than the normal group at the same period, and significant difference also appear in4weeks and6weeks (allP<0.01).Compared with model group, serum ET-1and MDA levels inAtorvastatin group and TXL-H group from4weeks, TXL-M group from6weeks began to decline (P<0.05or P<0.01), serum NO, SOD, and T-AOClevels increased significantly (P<0.05or P<0.01), and there was no significantdifference among the three groups (P>0.05). TXL-L group showed nostatistically significant indicators at various time points with the model group(P>0.05).2.3Histomorphology changes of carotid artery in different groups:2.3.1Observe under optical microscope (HE staining):The intima were smooth, endothelial cells were complete and continuous,nucleus were flat, fit closely with the inner elastic plate, smooth muscle in thetunica media line were clear, and small amount of microvascular can be seenin the carotid adventitia in normal group and non-silicone positioned.Model group left carotid artery morphological changes markedly: a.Tunica intima: The intima were complete and no hyperplasia at3days, lightproliferation at1weeks, diffuse hyperplasia and progressive aggravating from2weeks to6weeks. b. Tunica media: The inner elastic plate curving andtunica media light thickening at3days and1week, inner elastic plate curveaggravated at2weeks, media inter space broadening, smooth muscle cells andelastic fiber disordered arrangement at4weeks and6weeks. c. Tunicaadventitia: Microvasculars began to proliferation at3days, inflammatory cellsinfiltration markedly at1week, the number of microvasculars increasedfurther from2weeks to6weeks.Compared with model group at the same period, the pathologicaldamages of left carotid artery alleviated in different degrees in Atorvastatingroup, TXL-H, and TXL-L groups.2.3.2The ultrastructure of microvascular endothelial cells observed bytransmission electron microscopeNormal group: Endothelial cells were complete, eumorphism, structure clear, intracytoplasmic mitochondria morphology normal, and pinocytosisvesicles were visible, intercellular connection tightness, extracellular matrixmembrane continuous and completed.Model group: Endothelial cells were atrophy, broken, the mitochondria incells of crest and membrane fusion and even disappeared, and vacuolarchanges were visible, intercellular space expansion, intercellular connectionbreakage, fall off partly, extracellular matrix were loosely discontinuous, andparts were dissolvedTreatment groups: The above ultrastructural changes can be alleviated indifferent degrees, the whole cellular structure were roughly normal.2.4Comparison of microvascular density of the carotid artery wall in differentgroups:Compared with normal group at the same period, the microvasculardensity of the wall in the model group have a progressive increase from3daysto6weeks (P<0.05or P<0.01).Compared with model group, the microvascular density reducedsignificantly in Atorvastatin and TXL-H groups from4weeks to6weeks (allP<0.05), and there was no significant difference between them (P>0.05).There was no statistical difference compared to TXL-M and TXL-L groups(P>0.05).2.5Comparison of microvascular blood flow volume of the carotid artery wallin different groups:Compared with normal group at the same period, the microvascular bloodflow volume of the carotid artery wall in the model group have a progressiveincrease from1week to6weeks (P<0.05or P<0.01). Compared with modelgroup, the microvascular blood flow volume reduced significantly in TXL-Hgroup from4weeks to6weeks, and TXL-M at6weeks (all P<0.05), andthere was no statistical difference compared to Atorvastatin and TXL-Lgroups (P>0.05).2.6The comparison of NF-κB, TNF-α, IL-6, Nrf2, and NQO1mRNAexpressions of the carotid artery tissue in different groups: Compared with normal group, the mRNA expressions of TNF-α and IL-6increased, while NQO1declined significantly in model group (all P<0.01),and NF-κB and Nrf2mRNA expressions did not appear significant difference(all P>0.05). Compared with model group, Atorvastatin group, TXL-H andTXL-L groups all can down-regulate TNF-α mRNA expression significantly(P<0.01). Simultaneously, every treatment groups can down-regulate IL-6andup-regulate NQO1mRNA expressions (P<0.05or P<0.01).2.7The comparison of total NF-κB, nucleus NF-κB, TNF-α, IL-6, total Nrf2,nucleus Nrf2, and NQO1protein levels of the carotid artery tissue in differentgroups:Compared with normal group, the protein level of total NF-κB and totalNrf2did not appear significant difference in model group (P>0.05). Comparedwith model group, the total NF-κB and total Nrf2protein levels were nosignificant difference in every treatment groups (P>0.05).Compared with normal group, the protein levels of nucleus NF-κB,TNF-α, and IL-6increased significantly (P<0.01). Compared with modelgroup, Atorvastatin and TXL-H groups can both decrease the nucleus NF-κBprotein level (P<0.01), TNF-α protein level in TXL-H group and IL-6proteinlevel in every treatment groups reduced significantly (P<0.05or P<0.01).Compared with normal group, the protein levels nucleus Nrf2and NQO1decreased significantly (P<0.05). Compared with model group, nucleus Nrf2protein levels rise significantly in Atorvastatin, TXL-H and TXL-M groups(P<0.05or P<0.01), and NQO1protein levels also rise significantly in everytreatment groups (P<0.05or P<0.01).3The effects of TXL on DLL4/Notch signal pathway3.1The comparison of VEGF-A, VEGF-R2, DLL4, and Notch1mRNAexpressions of the carotid artery tissue in different groups:Compared with normal group, the mRNA expressions of VEGF-A andVEGF-R2increased, while DLL4and Notch1declined significantly in modelgroup (all P<0.01). Compared with model group, every treatment groups canall down-regulate VEGF-A and VEGF-R2mRNA expressions significantly (P<0.05or P<0.01). Simultaneously, Atorvastatin, TXL-H and TXL-Mgroups all can up-regulate DLL4and Notch1mRNA expressions significantly(P<0.01). Compared with Atorvastatin group, there were no significantdifference on down-regulating VEGF-A/VEGF-R2and up-regulatingDLL4/Notch1mRNA expressions in TXL-H and TXL-M groups (all P>0.05).3.2The comparison of VEGF-A, VEGF-R2, DLL4, and Notch1protein levelsof the carotid artery tissue in different groups:Compared with normal group, the protein levels of VEGF-A andVEGF-R2increased, while DLL4and Notch1declined significantly in modelgroup (all P<0.01). Compared with model group, every treatment groups candown-regulate VEGF-A and VEGF-R2protein levels significantly (P<0.05orP<0.01). Simultaneously, Atorvastatin, TXL-H and TXL-M groups all canup-regulate DLL4and Notch1protein levels significantly (P<0.01). Comparedwith Atorvastatin group, there were no significant difference ondown-regulating VEGF-A/VEGF-R2and up-regulating DLL4/Notch1proteinlevels in TXL-H and TXL-M groups (all P>0.05).Conclusions:1This study established AS early lesion animal model by feeding NewZealand rabbits high-fat diet, found that microvascular angiogenesis of thearterial wall anterior to host vascular structure and function lesion, andanterior to intima fatty steak formation, namely the microvascular lesion isearlier than the host vascular lesion. Moreover, microvascular density had asignificantly negative correlation relationship to endothelium dependentvasodilatation function. This result suggested that microvascular angiogenesispromoted the development and progression of atherosclerosis early lesion.Inflammation level rising and antioxidative stress decline played an importantrole during angiogenesis.2Using high-fat diet with positioning of a soft silicone collar around therabbits left carotid artery established microvascular angiogenesis animalmodel. TXL can lower arterial wall microvascular density, reducemicrovascular blood flow excessive perfusion, and protect the ultrastructure of microvascular endothelial cells. Its mechanism may be related to inhibit theNF-κB nuclear transfer and promote Nrf2nuclear transfer, which lower theinflammation levels of the artery, increase and antioxidative stress function,and alleviate atherosclerosis early lesions.3Angiogenesis in atherosclerosis early lesions was related withDLL4/Notch signaling pathway restrained. TXL can activate this signalingpathway, and inhibit the gene and protein expressions of VEGF-A/VEGF-R2,regulate angiogenesis from two aspects of positive and negative, and alleviateatherosclerosis early lesions.4Under the guidance of the Vessel-Collateral Doctrine, Based on“collateral-microvascular†homoousia in anatomical morphology, this studypoints that “collateral-microvascular†of the arterial wall is an important wayand place of Ying-nutrient (vascular endothelium) and Wei-defense (vascularadventitia)“unblocking collaterals with subsequent convergence andsubstance-qi transformationâ€, which maintained the nutrition metabolicbalance of arteries. The wall microvascular angiogenesis caused by“collateral-microvascular†Ying-Wei “unblocking collaterals with subsequentconvergence and substance-qi transformation†is the important pathologicbasis of atherosclerosis. TXL alleviate atherosclerosis early lesions byinhibiting the microvascular angiogenesis of the artery wall, whichmechanisms are related to anti-inflammatory, antioxidation, inhibiting theNF-κB nuclear transfer and promoting Nrf2nuclear transfer, activatingDLL4/Notch signaling pathway, inhibiting the gene and protein expressions ofVEGF-A/VEGF-R2. This study provides a new approach and drug foratherosclerosis early prevention and treatment, which shows the guidancevalue of Ying-Wei “unblocking collaterals with subsequent convergence andsubstance-qi transformation†theory of the Vessel-Collateral Doctrine inpreventing and treating atherosclerosis,... |
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