| Objective:In this study, directed by the theory of"Chengzhitiaoping of Ying and Wei", together with Western medicine recent research of the vascular adventitia play an important role in vascular lesion, from the "Ying-qi" and the endometrium, "Wei qi" and the outer membrane as an entry point, through the establishment of internal and external memebrane cell model ,to explore the influence of internal and external membrane and the discipline of Chengzhitiaoping in angiopathy which was interposed by Tongluo drug. This is not only pass again the traditional Chinese medicine theory of Ying and Wei ,but also a more comprehensive and profound study of vascular lesions.Methods: There are two sections in this research:1 The influence of vascular adventitia-derived nitric oxide to AngⅡ-induced apoptosis of vascular endothelial cells1.1 The influence of AngⅡon endothelial cell viabilityNormal cultured human umbilical vein endothelial cells ECV-304, using good integration of endothelial cells in logarithmic growth phase, after digestion with trypsin, adjusting the cell density of 1×105 / ml, inoculated in 96 orifice plate. Cells were divided into(1) control group: using serum-free DMEM to culture; (2)AngⅡgroup: joining the AngⅡwhich the final concentrations were 10-9mol/L, 10-8mol/L, 10-7mol/L, 10-6mol/L, 10-5mol/L. After the cells were cultured 0h,6h,12h,24h,48h, we determine the cell viability of each group with SRB.1.2 The influence of AngⅡon adventitia-derived NOSelecting clean and healthy male SD rats,carefully separated and interception of thoracic aorta, peel outer membrane, cut the outer membrane organization to 2cm×2cm, dry and weigh. Using containtion 10% FBS in DMEM medium to culture, then using serum-free DMEM to continue to culture 24h. Joining the AngⅡ,which the final concentrations were 10-6)mol/L, then culture 0h,1h,2h,12h,24h,48h. When the test was end, we detection the NO value of each group and detection the iNOS protein of each group.1.3 The influence of vascular adventitia-derived nitric oxide to AngⅡ-induced apoptosis of ECV304.Cells are then divided into 5 groups:(1)pure endothelial cell group: Normal cultured human umbilical vein endothelial cells ECV-304; (2)endothelial cells + adventitia: endothelial cells and adventitia were incubated; (3)endothelial cells+ AngⅡ:jointing the AngⅡto endothelial cells(the final concentrations were 10-6)mol/L); (4)endothelial cells+ adventitia+ AngⅡ:jointing the AngⅡto adventitia, after 2 hours, the outer membrane and the supernatant were added to the endothelial cells which were incubated for 24h; (5)endothelial cells+ L-adventitia+ AngⅡ:using L-NNA pre-incubation with the membrane for 30 minutes, washing 3 times with PBS,then adding the AngⅡpre-incubation, finally, incubated with endothelial cells.When the experiment was end, we observe the morphological changes of vascular endothelial cells with HE, detection of cell viability with SRB, detection of apoptosis rate with FCM, detection of nitric oxide in the supernatantwith Nitrate reductase, detection of eNOS protein of each group with western blot.2 The influence of Tongluo durg to AngⅡ-induced membrane-derived nitric oxide.2.1 The influence of TXL durg to adventitia-derived NO.The preparation of outer membrane as the first part, then join the TXL, which the final concentrations were 0ug/ml, 25ug/ml, 50ug/ml, 100ug/ml, 200ug/ml,500ug/ml, culture 0h,1h,2h,4h,6h,8h。When the experiment was end, we collected supernatant and determination the NO value of each group.2.2 The influence of YWTJF to adventitia-derived NOThe preparation of outer membrane as the first part, then join the YWTJF, which the final concentrations were0%,0.01%,0.05%,0.1%,0.5%,1%, culture 0h,1h,2h,4h,6h,8h。When the experiment was end, we collected supernatant and determination the NO value of each group.2.3 The influence of Tongluo durg to AngⅡ-induced membrane-derived nitric oxide:The preparation of outer membrane as the first part. The test divided into 8 groups:(1)outer membrane group: Outer membrane was cultured in serum-free DMEM; (2)outer membrane+AngⅡ:joining the AngⅡto outer membrane; (3)outer membrane+TXL: joining the TXLto outer membrane; (4)outer membrane+AngⅡ+TXL: using AngⅡpre-incubation with the outer membrane, after 2 hours ,incubated with TXL for 24-hour; (5)outer membrane+L-NNA+AngⅡ+TXL: using L-NNA pre-incubation with the membrane for 30 minutes, washing 3 times with PBS,then adding the AngⅡpre-incubation, finally, incubated with TXL for 24 hours; (6)outer membrane+YWTJF: joining the YWTJF to outer membrane; (7)outer membrane+ AngⅡ+YWTJF: using AngⅡpre-incubation with the outer membrane, after 2 hours,incubated with YWTJF for 24 hours; (8)outer membrane+L-NNA + AngⅡ+YWTJF: using L-NNA pre-incubation with the membrane for 30 minutes, washing 3 times with PBS,then adding the AngⅡpre-incubation, finally, incubated with YWTJF for 24 hours.When the experiment was end, we detection of nitric oxide in the supernatant with nitrate reductase, detection of iNOS protein of each group with western blot.Results:1.The influence of vascular adventitia-derived nitric oxide to AngⅡ-induced apoptosis of vascular endothelial cells.1.1The influence of AngⅡon endothelial cell viability.AngⅡcan significantly reduce the vitality of ECV304,and have dose- effect relationship.Compared with normal control group, the different concentrations of AngⅡcan obviously cause apoptosis of ECV304. Compared with normal control group, AngⅡOD value of 10-9mol/L had reduce (P<0.05), AngⅡOD value of 10(-8,10(-7,10(-6,10-5 mol/L had more obvious reduce (P<0.01);Compared with 10-9mol/L group, AngⅡOD value of 10-6 mol/L had most obvious reduce (P<0.001). AngⅡcan significantly reduce the vitality of ECV304,and had aging relationship. Compared with 0h group,AngⅡOD value of 6h ,12h ,24h,48h had more obvious reduce (P<0.01), ang With the lengthen of the time, there is a decreased tendency, but no statistical significance(P>0.05).1.2The influence of AngⅡto adventitia-derived NO at different time: AngⅡcan induction NO level raised in supernatant of vascular adventitia, and to peak at 2h time point. Compared with 0h group,there had statistical significance(P<0.01).1.3The influence of AngⅡto iNOS protein at different time: AngⅡcan induction iNOS protein level raised in vascular adventitia, and to peak at 2h time point.1.4 The influence of vascular adventitia-derived nitric oxide to AngⅡ-induced apoptosis of ECV304.1.4.1 Observation the vascular endothelial cell morphologyPure endothelial cell group:Cells were flat and polygonal,cobblestone mosaic arrangement. The boundary was distinct. The cytoplasm was rich .The Nucleus was visible, round or oval-shaped.Endothelial cells+adventitia: Cells were flat and polygonal, cobblestone mosaic arrangement. The boundary was distinct. The cytoplasm was rich .The Nucleus was visible, round or oval-shaped.Endothelial cells+ AngⅡ:The cells change round, swelling. The edge of cells membrane were blurred, and some cells membrane were incomplete, even damage rupture.Endothelial cells+ adventitia+ AngⅡ:The cells shape were rule and the boundary was still clearly. The cells were still connected to each other. Endothelial cells+ L-adventitia+ AngⅡ: The cells change round, swelling.The edge of cells membrane were blurred, and some cells membrane were incomplete, even damage rupture.1.4.2 Detection of endothelial cell viability.Compared with normal control group,the OD value of model group was significantly lower (P<0.01); Compared with model group, the OD value of adventitia intervention group was significantly exaltation(P<0.01); Compared with adventitia intervention group, the OD value of L-NNA group was significantly lower (P<0.05). The results suggest that the Outer membrane have a certain protective effect to the endothelial cells.1.4.3 Detection of endothelial cell apoptosis rate.Compared with normal control group,the cell apoptosis rate of model group was significantly exaltation (P<0.05); Compared with model group, the cell apoptosis rate of adventitia intervention group was significantly lower (P<0.05), the cell apoptosis rate of L-NNA group had no difference(P>0.05).1.4.4 Detection of NO level in each groupCompared with normal control group,the NO OD value of model group was significantly lower (P<0.05); Compared with model group, the NO OD value of adventitia intervention group was significantly exaltation(P<0.01); Compared with adventitia intervention group, the NO OD value of L-NNA group was significantly lower (P<0.05).1.4.5 Detection of eNOS protein level in each groupAdventitia have antagonistic effects to the reduce of eNOS protein level,which induced by AngⅡ.When the L-NNA affect outer membrane, the protection effect of adventitia to endothelial cells was lower. Then the eNOS protein level was also lower, which had difference compared with adventitia intervention group (P<0.05) and had no difference compared with model group(P>0.05).2 The influence of Tongluo durg to AngⅡ-induced membrane-derived nitric oxide2.1 The effect of TXL to adventitia-derived NO.TXL can improve the level of adventitia-derived NO, and have dose- effect relationship. Compared with 0ug/ml group, TXIL of 25ug/ml,50ug/ml can Significantly promote the expression of adventitia-derived NO;The effects of 50ug/ml was most Significantly(P<0.01).TXL can improve the level of adventitia-derived NO, and have aging relationship. For 6 hours intention, the NO content in supernatant was most highest(P<0.001).2.2 The effect of YWTJF to adventitia-derived NO.YWTJF can improve the level of adventitia-derived NO, and have dose- effect relationship. Compared with 0ug/ml group, TXIL of 0.05%,0.01% can Significantly promote the expression of adventitia-derived NO;The effects of 0.05% was most Significantly(P<0.01).YWTJF can improve the level of adventitia-derived NO, and have aging relationship. For 2 hours intention, the NO content in supernatant was most highest(P<0.001).2.3 The influence of Tongluo durg to AngⅡ-induced membrane-derived nitric oxide2.3.1 The NO level of each groupCompared with pure outer membrane group, AngⅡ,TXL,YWTJF can advance the level of NO (P<0.01); Compared with AngⅡgroup and TXL group, the NO level of TXL+AngⅡgroup had Significantly exaltation (P<0.01); After the use of blocking agents(L-NNA),the level of NO had Significantly reducing, which compared with TXL+AngⅡ,there had significant difference(P<0.01); TXL+AngⅡgroup and TXL+AngⅡgroup had difference(P<0.05),which show that the affect of TXL was superiorer than the affect ofYWTJF.2.3.2 The iNOS protein level of each groupCompared with pure outer membrane group, AngⅡ,TXL,YWTJF can advance expression of iNOS protein(P<0.01);In the effect of TXL+AngⅡ, the expression of iNOS protein is highter than the effect of single factor; When the L-NNA affect the outer membrane, the expression of iNOS protein was significantly lower; Compared with TXL+AngⅡ,there had significant difference(P<0.01).TXL and YWTJF can advance expression of iNOS protein, and the effect of TXL was better than the effect of YWTJF.Conclusion:1 In this study, directed by the theory of"Chengzhitiaoping of Ying and Wei", based of""the ying-nutrient qi travels within the vessels,but wei-nutrient qi travels outside of the vessels". Ying and Wei are running together which are bounded by blood vessels, regulate vein systolic and diastolic function and blood running. Together with Western medicine recent research of the vascular adventitia play an important role in vascular lesion, point out that Ying-qi and vascular endothelium, Wei-qi and vascular adventitia have highly relevant, which have directive function for us explore pathogenesis of "context - the vascular system disease" form the interaction between the membrane and the endothelium, and reveal the scientific of the theory of"Chengzhitiaoping of Ying and Wei".2 Based of the relevant between Ying-qi and vascular endothelium, Wei-qi and vascular adventitia,this test build model of outside the membrane and the endothelial cells incubated. Discussion that the function of adventitia to AngⅡ-induced apoptosis in vascular endothelial cells. The result shows that Vascular adventitia could inhibit endothelial cell apoptosis. The role was by way of activating NOS/NO. This show that vascular adventitia play a role in vascular disease, which provide data support for the function of Ying-Wei estranged in Context - vascular system disease.3 TXL directed by collateral theory and YWTJF directed by Ying and Wei theory can protect the vascular endothelial by the way of improving the level of adventitia NO and increasing the expression of iNOS protein. The effect of TXL was superior than the effect of YWTJF. The mechanism was by way of NOS/NO in adventitia.4 These results suggest that: AngⅡcan injury endothelial cell ,reduce NO secretion; After join the membrane organization, NO secretion is compensatory increase , which can antagonistic endothelial injury by AngⅡinduced;Tongluo drug can obviously increase the secretion of adventitia-derived NO, which protection of endothelial cells. This reflect the discipline of"Chengzhitiaoping of Ying and Wei"and provide endorsement of the experimental data for using"Chengzhitiaoping of Ying and Wei"to direct angiopathy.
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