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The Influence of Vascular Adventitia-derived Nitric Oxide to Ang Ⅱ-induced Apoptosis of Vascular Endothelial Cells and the Effect of Tongluo Drug

Author: GeXiaoLi
Tutor: WuYiLing
School: Hebei Medical University
Course: Traditional Chinese Medicine
Keywords: Camp David is for the system leveling The vascular adventitia source NO Angiotensin II Vascular endothelial cells Apoptosis Tongluo drugs
CLC: R285
Type: Master's thesis
Year: 2010
Downloads: 64
Quote: 0
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Abstract


Objective: In this experiment, the theory of \outer membrane as the starting point, through the establishment of the inner and outer membranes were incubated with cell model to explore the outer membrane interaction and tongluo drug intervention vascular lesions Order of the system, adjusting the level law, both the Chinese camp guard doctrine heritage, but also more comprehensive and profound reveal the internal mechanism of the evolution of the development of vascular disease has guiding significance. Methods: vascular adventitia source of nitric oxide on vascular endothelial cell apoptosis induced by Ang Ⅱ 1.1 Ang II on cultured vascular endothelial cell viability routinely cultured human umbilical vein endothelial cells ECV-304, select the logarithmic growth phase fusion a good endothelial cells, a cell density of 1 × 10 5 a / mL adjusted after trypsinization and seeded in 96-well plates until the cell fusion induced by 80%, the cells were divided into: ( 1) control group: adding serum-free DMEM medium; (2) Ang II group: added to a final concentration of 10 -9 mol / L, 10 -8 mol / L , 10 -7 mol / L, 10 -6 mol / L, 10 -5 mol / L of Ang II. Each group were cultured for 0h, 6h, 12h, 24h, 48h after using the SRB assay to detect the expression dynamic changes. 1.2 Ang II the vascular adventitia source NO release of selected clean healthy male SD rats were carefully separated and interception of the thoracic aorta, peel the outer membrane and cut into 2mm × 2mm organizations flakes, dry weighing, with 10 ? S cultured in DMEM medium, 24h replaced with serum-free DMEM medium cultured for 24h, and then added to a final concentration of 10 -6 mol / L of Ang Ⅱ continue to cultivate 1h, 2H, 12h, 24h , 48h, while the establishment of the control group. Nitrate reductase method to detect the NO content in the culture supernatant, detected by Western blot outer membrane of iNOS protein expression levels. 1.3 outer membrane of NO ECV304 apoptosis induced by Ang Ⅱ. The experiment was divided into five groups: (1) endothelial cells alone group: ECV-304 cells were incubated with conventional culture; (2) endothelial cell the adventitia group: endothelial cells and adventitia were incubated; (3) endothelial cell Ang Ⅱ group: endothelial cells incubated Add to Ang II (final concentration of 10 -6 mol / L) were incubated; (4) endothelial cell outer membrane Ang Ⅱ group: to join Ang II in the outer membrane of incubation (final concentration of 10 -6 mol / L), 2h after the outer membrane and the supernatant join together to continue endothelial cells were incubated for 24 h; (5) endothelial cells in the outer membrane of L-NNA Ang Ⅱ group: outer membrane NOS blocker L-NNA (10 -5 mol / L) pre-incubated for 30 min, washed 3 times with PBS solution, and then added to a final concentration of 10 -6 mol / L Ang II pre-incubation, and finally with endothelial cells incubated. After the end of the experiment, HE staining of vascular endothelial cell morphological changes, SRB method cell viability was detected by flow cytometry apoptosis rate, nitrate reductase indirect detection of nitric oxide levels in the supernatant, western blot method to detect the expression eNOS protein expression levels tongluo drugs on the outer membrane of the source of the Ang II-induced nitric oxide 2.1 Tongxinluo the vascular adventitia source NO release of vascular outer membrane preparation and training with the first part, and then were added to a final concentration of for 0ug/ml 25ug/ml 50ug/ml, 100ug/ml, 200ug/ml 500ug/ml macaroni network to continue training 0h, 1h, 2h, 4H, 6h, 8h. The end of the experiment, the supernatant was collected after centrifugation to detect the level of NO. Vascular adventitia 2.2 business health regulator side of the vascular adventitia of NO release of preparation and training with the first part, and then were added to a final concentration of 0%, 0.01%, 0.05%, 0.1%, 0.5%, 1% of the camp Wei regulator parties to continue to foster 0h, 1h, 2h, 4h, 6h, 8h. The end of the experiment, the supernatant was collected after centrifugation to detect the level of NO. 2.3 tongluo the impact of drugs on the outer membrane of the Ang II-induced nitric oxide in the outer membrane preparation and training with the first part, and then divided into eight groups: (1) simple the adventitia group: normal complete medium were incubated outer membrane; ( 2) outer membrane Ang II: the outer membrane of the incubation Add to Ang II (final concentration of 10 -6 mol / L) were incubated; (3) the outer membrane of Tongxinluo: incubation adventitia added Tongxinluo (the final concentration 50ug/ml) were incubated 24h; (4) outer membrane Ang II Tongxinluo: Ang II and outer membrane were pre-incubated 2h after Tongxinluo continue to incubate for 24 hours; (5) the outer membrane L-NNA Ang II through tongxinluo: the outer membrane and the NOS blocker L-NNA (10 -5 mol / L) pre-incubated for 30 min, washed three times with PBS solution, and then added to a final concentration of 10 - 6 mol / L of Ang Ⅱ pre-incubation, 2h Add to Tongxinluo continue to incubate for 24 hours; (6) outer membrane business health regulator side: incubation outer membrane organization a party to the business health regulator added (final concentration of 0.05% ) were incubated; (7) outer membrane Ang II business health regulator side: Ang II and outer membrane pre-incubated for 2h after the business health regulators continue incubated for 24 h; (8) the outer membrane L-NNA Ang II business health regulator Group: the outer membrane NOS blocker L-NNA (10 -5 mol / L) pre-incubated for 30 min in PBS solution was washed 3 times, and then added to a final concentration of 10 -6 mol / L Ang II pre-incubated for 2h join business health regulators continue incubated for 24 h. End of the experiment, nitrate reductase indirect detection of the supernatant of nitric oxide (NO), western blot method to detect the expression of iNOS protein. Results: the vascular adventitia sources of nitric oxide on vascular endothelial cell apoptosis induced by Ang Ⅱ 1.1 Ang II ECV304 cell viability Ang Ⅱ can significantly reduce ECV304 vitality, and the dose-effect relationship, compared with the control group, various concentrations of Ang II could obviously cause ECV304 apoptosis, 10 -9 mol / L Ang II OD value of the normal group to reduce significantly (P lt; 0.05), 10 -8 10 -7 , 10 of -6 , 10 to 5 mol / L Ang II OD values ??decreased significantly (P lt; 0.01) ; compared with 10 -9 mol / L Ang Ⅱ, 10 -6 mol / L of Ang II OD value loss was the most significant (P lt; 0.001). Ang Ⅱ on reducing the vitality of ECV304 time effect, compared with 0h, 6h, 12h, 24h, the Ang Ⅱ OD value was significantly reduced (P lt; 0.01), and with the role of time extend have reduced trend, but not statistically significant (P gt; 0.05 ). 1.2 at different time points Ang Ⅱ outer membrane of NO: Ang II-induced vascular adventitia culture supernatant NO expression increased and peaked 2h time point was statistically significant (P lt; 0.01) 0h. 1.3 at different time points Ang II outer membrane of iNOS protein impact: Ang II can promote outer membrane source of NO (iNOS) protein expression increased peak 2h point in time. 1.4 vascular adventitial nitric oxide in vascular endothelial cell apoptosis induced by Ang Ⅱ 1.4.1 vascular endothelial cell morphology pure endothelial cells group: cells were flat polygonal, cobblestone mosaic arrangement, clear boundary, cell pulp rich. The nucleus is visible as a circular or oval. Endothelial cell outer membrane Group: cell flat polygonal, cobblestone mosaic arrangement, clear boundary, abundant cytoplasm. The nucleus is visible as a circular or oval. Endothelial cells Ang Ⅱ group: cells became round, swelling, blurred edge of the cell membrane, the part of the cell membrane is incomplete, or even damage the rupture. Endothelial cell outer membrane Ang Ⅱ group: regular shape, cell borders is still clear, the cells are still connected to each other. L-NNA outer membrane of endothelial cells Ang Ⅱ group: cells became round, swelling, cell membrane blurred edges, incomplete part of the cell membrane rupture or damage. 1.4.2 endothelial cell viability detection compared with normal control group, model group OD value significantly lower (P lt; 0.01); compared with the model group, the OD value of the outer membrane in the intervention group was significantly higher (P lt; 0.01) The; the outer membrane intervention group compared to the OD value of the L-NNA group were significantly lower (P lt; 0.05). Tip: The outer membrane on Ang II intervention endothelial cells have a protective effect. 1.4.3 detection of endothelial cell apoptosis rate compared with normal control group, model group, the apoptosis rate increased significantly (P lt; 0.05); outer membrane intervention group compared with the model group, the apoptosis rate lower (P lt; 0.05), L-NNA group of apoptosis was no difference (P gt; 0.05). 1.4.4 of NO level detection with the normal control group, model group NO OD values ??were significantly lower (P lt; 0.05); compared with the model group, NO OD value of the outer membrane in the intervention group was significantly higher (P lt ; 0.01); intervention group compared with the outer membrane, NO OD value of the L-NNA group were significantly lower (P lt; 0.05). 1.4.5 groups in eNOS protein expression of outer membrane induced by Ang Ⅱ ECV304 eNOS protein expression reduce antagonism; L-NNA role outer membrane, reducing the protective effect of the outer membrane of endothelial cells, thus ECV304 cells eNOS protein reduced expression difference (P lt; 0.05), compared with the outer membrane of the intervention group, no difference compared with the model group (P gt; 0.05). The 2 tongluo drugs induced by Ang Ⅱ outer membrane source of nitric oxide from 2.1 through Tongxinluo the outer membrane of NO impact Tongxinluo can promote the outer membrane of NO levels, and dose-effect relationship relative to 0ug/ml than 25ug/ml 50ug/ml Tongxinluo can significantly promote the expression 50ug/ml of the outer membrane of NO NO OD value increased most significantly (P lt; 0.01) Tongxinluo outer membrane of NO time effect, extend of Tongxinluo (50ug/ml) are induced by varying degrees of the outer membrane of the culture supernatant of NO increased expression over time from 2h, 6h time point peak, and then down, but still higher than the the 0h point in time. Compared with 0 h, 6h caused supernatant NO levels increased most significantly (P lt; 0.001). 2.2 business health regulator's outer membrane of NO impact on business health regulation may promote the outer membrane of NO levels, and dose-effect relationship, compared with 0%, 0.05%, 0.01% of the business health regulator side could significantly to promote the expression of outer membrane source of NO, NO OD value of 0.05% increased most significantly (P lt; 0.01). A party to the business health regulator outer membrane of NO time effect, with time 1h business health regulator side (0.05%) were induced by varying degrees of the outer membrane of the culture supernatant of NO increased expression and peak 2h point in time, and then decreased, but still higher than the 1h time point. 0h, 2h point in time caused supernatant NO content increased most significantly (P lt; 0.001). 2.3 tongluo the source of the drugs on the outer membrane of the Ang II-induced nitric oxide in 2.3.1 NO expression level changes compared with the outer membrane alone group, Ang Ⅱ group, TXL group, business health regulator side group NO OD value could significantly elevated (P lt; 0.01); compared with Ang Ⅱ group and Tongxinluo group, the TXL Ang II group NO OD value was significantly higher (P lt; 0.01); blockers (TXL Ang II L -NNA) NO OD value was significantly reduced compared to the significant difference (P lt; 0.01) TXL Ang Ⅱ; YWTJF Ang Ⅱ group TXL Ang Ⅱ group compared there is difference (P lt; 0.05), indicating better than Tongxinlou Camp David is adjust the side. 2.3.2 adventitia iNOS protein expression and outer membrane alone group, Ang II, macaroni network, the the camp guard regulator parties can promote the outer membrane expression of iNOS (P lt; 0.01); joint action in TXL Ang II of under, the expression of iNOS higher than the single factors, the L-NNA role of the outer membrane, the outer membrane of iNOS expression was significantly lower, compared with the TXL Ang Ⅱ were significantly different (P lt; 0.01); with Ang II, Tongxinluo group phase than decreased, but not statistically significant. Tongxinluo and business health regulator parties can promote outer membrane expression of iNOS and Tongxinluo better than a party to the business health regulator. Conclusion: a research business health for the system leveling theory as a guide, based on \function and blood running, combined with Western medicine reveals vascular adventitia and endothelial play an important role in vascular disease, pointed out that the the camp gas and vascular endothelium, Wei Qi and vascular adventitia highly correlated, for from the outer membrane Influence of context - vascular system disease pathogenesis, and the interaction between endothelial reveal the scientific value of \2 based on the the camp gas and endothelium, Wei Qi outer membrane correlation, the film outside the endothelial cells were incubated with the establishment of an in vitro model of the outer membrane of vascular endothelial cell apoptosis induced by Ang Ⅱ, the results showed vascular adventitia by activation of NOS / NO pathway inhibition of endothelial cell apoptosis induced by Ang Ⅱ, initially revealed not only the role of the outer membrane in vascular lesions, experimental data also explore the Chinese business health estranged context - vascular system disease \Support. 3 network disease theory the Tongxinluo guidance and vascular disease based on the theory of business health guidance developed business health regulator side can improve the the Ang Ⅱ role of outer membrane NO levels, and enhance the expression of iNOS protein protect endothelial and Tongxinluo better than a party to the business health regulator, the mechanism and regulation of the outer membrane NOS / NO system. 4 results suggest: Ang II allows endothelial cell damage, reduced NO secretion; join adventitia, NO the secretion compensatory increase can antagonize Ang Ⅱ induced endothelial injury; tongluo drug intervention of the outer membrane of NO secretion significantly increased, thereby protecting endothelial cells. This reflects the inner outer membrane were incubated business health for the system evolution of the leveling system, guidance on the use of business health for the system leveling theory vascular lesions experimental data support.

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