| Objective:Endothelial dysfunction is the basis of the development of cardiovascular disease.This research evaluated the therapeutic effect of Aspirin on endothelial injury in both theory of TCM and Western medicine through study the endothelial dysfunction model with Noxious Heat and Blood Stasis caused by intraperitoneal injection of LPS and the pathological changes after aspirin administration.We explain the pathogenesis of Noxious Heat and Blood Stasis by LPS-induced,and the treatment mechanism in the theory of TCM and Western medicine to guide the use of Aspirin,and provide experimental basis for the future study of Western medicine with the basic theory of TCM.Methods:(l)Clinical questionnaire were conducted to look for the biological indicators of Noxious Heat and Blood Stasis Syndrome,and the relevant biological indicators were verified by animal models:According to the literature review,a questionnaire was conducted to investigate the differences in clinical indicators of TCM syndromes related to cardiovascular diseases.Doctors and scholars in this field were invited to answer the questionnaire,the selection of biological indicators of cardiovascular diseases caused by Noxious Heat and Blood Stasis Syndrome was statistically analyzed and calculated by the answers of doctors and scholars.After determining the biological indicators of blood stasis,the corresponding indicators were verified by the animal model of Noxious Heat and Blood Stasis Syndrome.Intraperitoneal injection of LPS was used to induce vascular endothelial layer damage with Noxious Heat and Blood Stasis Syndrome.Twenty-four mice were randomly divided into three groups,including normal group,the low-dose LPS stimulation group and the high-dose LPS stimulation group,with 8 rats in each group.From the 1 to 7 days of the experiment,phosphate buffered sa.l ine,100 μg/kg LPS and 300 μg/kg LPS were given by intraperitoneal injection respectively.Observe and record the changes of animal status,diet and body weight.On the 8th day,the rats were sacrificed and their blood and the thoracic aorta were collected.Blood was collected from the abdominal aorta of rats,and one of them were used to detect hemorheological indexes,such as whole blood reduction viscosity,hemotocricrit,erythrocyte aggregation index,erythrocyte sedimentation rate and fibrinogen.Another part of the serum was used to detect various kits:TG,TC,Apo-A,Apo-B,SOD,LPO,TXB2,and 6-keto-PGF1α,etc.The thoracic aorta was taken for immunofluorescence detection of the integrity of vascular layer composed of VE-Cadherin,Z01 and other junction proteins in the vascular endothelium-(2)The holistic pharmacodynamies of Aspirin to endothelial dysfunction with Noxious Heat and Blood Stasis Syndrome in LPS-induce:LPS(2 μg/mL)was used to stimulate inflammation model of vascular endothelial cells,and then the cells were divided into control group,LPS model group,positive control group,0.lmmol/L,0.5mmol/L,lmmol/L,2mmol/L and 3mmol/L aspirin group.The endothelial cells were incubated with irritants and drugs for 24 hours before the experiments.MTT,LDH and cell proliferation assay were used to detect cell activity and determine the low toxicity and effectiveness.The expression of endothelial junction protein Z01/Z02 was detected by immunofluorescence.The endothelial cell membrane permeability and resistance were detected to determine the damage of endothelial cell membrane.And detected the release of nitric oxide to determine the change of endothelial function.C57BL/6 mice were randomly divided into Normal group,LPS group,positive drug dexamethasone group(Dexamethasone,0.0182mg/kg),low-dose aspirin group(12.5mg/kg),medium-dose aspirin group(62.5mg/kg)and high-dose aspirin group(125mg/kg),8 mice in each group.LPS was molded at a concentration of 100 μg/kg.The experiment lasted 7 days.Mice were killed and sampled on 8th day.The frozen heart sections of mice were cut in 8 μm thick slices and then were frozen and preserved.The integrity of the vascular endothelial layer in the heart sections of mice was detected by immunofluorescence colocalization method,such as the colocalization of the VWF/Z01,VWF/Z02.(3)Mechanism study of aspirin in treatment of endothelial damage:LPS(2 μg/mL)was used to stimulate inflammation model of vascular endothelial cells,and then the cells were divided into Control group,LPS model group,0.1mmol/L,0.5mmol/L,lmmol/L and 2mmol/L aspirin group.The endothelial cells were incubated with irritants and drugs for 24 hours before the experiments.Intracellular ROS were detected by fluorescence DCFH-DA probe and then detected by cell real-time detection microscopy,the expression of TXNIP protein detected by Western Blot,and immunofluorescent co-located TXNIP/NLRP3 confirmed that the formation of NLRP3 inflammasome was related to activation and ROS/TXNIP signaling pathway.The expressions of NLRP3,ASC,Caspas-1 and HMGB1 proteins were detected by Western Blot,and the formation process of NLRP3 inflammasome was determined by immunofluorescence co-localization about the recruitment of NLRP3/ASC and NLRP3/Caspase-1.The cell of shRNA transfection RAGE and gRNA transfection NLRP3 were measured the expression of endothelial junction protein Z01 to determined that the restore of endothelial dysfunction was closely related to the activation of HMGB1-RAGE axis activated by the activation NLRP3 inflammasome.The frozen sections of mice hearts that had been frozen and preserved in the early stage were used to detect the formation and activation of NLRP3 inflammasome:NLRP3/Caspase-1,VWF/HMGB1 in the sections of mice by immunofluorescence co-location method.Enzyme-linked immunosorbent assay was used to further determine the content of HMGB1 in serum of mice.Results:(1)Clinical questionnaire were conducted to look for the biological indicators of Noxious Heat and Blood Stasis Syndrome,and the relevant biological indicators were verified by animal models:According to the statistical analysis of the questionnaire,the biological indicators of cardiovascular diseases caused by Noxious Heat and Blood Stasis Syndrome were selected as the following:hemorheology indicators,blood lipid metabolism indicators TG and TC,apo-a and apo-b,txa2-pgi2 relative balance indicators TBX2 and 6-keto-PGF1,oxygen free radical system indicators SOD and LPO,etc.The results showed that the whole blood reduction viscosity,erythrocyte aggregation index and erythrocyte sedimentation rate were significantly increased in LPS stimulated.The changes of hemotocricrit was not obvious.The changes of fibrinogen detected also increased in a concentration-dependent increased by LPS stimulation.The contents of TG and TC in serum were increased in the stimulation of LPS,but the increase of high-dose LPS was less than of low-dose LPS stimulation.The ratio of Apo-B/Apo-A increased with the increase of LPS dose.The activity of SOD enzyme was activated in the stimulation of LPS,but the increased of high dose LPS was no statistical significance.LPO content increased in LPS-induce,but there was also no statistical significance.The expression of 6-keto-PGF1α and TXB2 increased with LPS dose.The expression level of TXB2 was highest when LPS stimulation was given at a low-dose LPS,while the content at a high-dose LPS was lower than the low-dose LPS.However,the ratio of TXB2/6-keto-PGF1α decreased in a concentration-dependent increased-With the increase of LPS dose,the integrity of endodermal connective junction proteins VE-Cadherin and Z01 were damaged in a concentration-dependent,and the damaged area was larger and larger,with more and more damaged serious.(2)The holistic pharmacodynamics of Aspirin to endothelial dysfunction with Noxious Heat and Blood Stasis Syndrome in LPS-induce:MTT and LDH assay showed that the selected LPS concentration,Aspirin concentration and dexamethasone were low toxic to the cells,except 3 mmol/L Aspirin.Cell proliferation was detected using a real-time cell detection microscope.LPS,2mmol/L Aspirin and 3mmol/L Aspirin inhibited cell proliferation,especially 3mmol/L Aspirin.While other concentrations restored cell proliferation.Then the changes of endothelial function were detected.It was found that junction protein was damaged,membrane permeability was exceeding,TEER and Nitro oxide release were decreased in LPS-induce.While Aspirin could improve the expression of junction protein and the cell membrane permeability,and increase the TEER and Nitro oxide release.However,the endothelial dysfunction was not improved in dexamethasone administration.Immunofluorescence detection was performed on the heart sections of mice,and it was found that LPS stimulation significantly reduced the expression of endothelial tight junction protein Z01/Z02.Dexamethasone intervention could restore the tight junction protein;The expression of Z01/Z02 also could repaired when intervention with low-concentration aspirin and high-concentration aspirin,but the medium-concentration aspirin did nothing to restore the tight junction proteins in the heart vascular endothelium.(3)Mechanism study of aspirin in treatment of endothelial damage:Cell real-time detection microscope was used to detect intracellular ROS.LPS stimulation would increase the release of intracellular ROS,and Aspirin intervention could inhibit the relea.se of ROS.Western Blot was used to detect the changes in the expressions of endothelial proteins such as TXNIP,NLRP3,Caspase-1,HMGB1,and it was found that LPS increased the expression of these proteins,while Aspirin intervention could inhibit the expression.However,the ASC expression remained unchanged both LPS stimulation and Aspirin intervention.LPS stimulation increased the co-localization of TXNIP/NLRP3,NLRP3/ASC,NLRP3/Caspase-1,while the co-localization area decreased after aspirin administration.Endothelial junction protein Z01 was detected in cells transfected with shRAGE and gNLRP3,there was no significant change in the connective junction protein both LPS stimulation and aspirin intervention.NLRP3/Caspase-1 in the frozen heart sections of the co-infected mice showed that LPS stimulation increased the co-localization area,and the intervention of dexamethasone and medium concentration Aspirin could not reduce the co-localization between the two fluorescence,while low and high concentration of both could reduce the co-localization between the two fluorescence.The same results were obtained for the co-stained VWE/HMGB1.Elisa kit was used to detect the HMGB1 content in the serum of mice.Low concentration of HMGB1 reduced the content of HMGB1 in the serum,while the other two concentrations showed no inhibitory effect.Dexamethasone effectively reduced the content of HMGB1 in the serum.Conclusion:1.LPS induced vascular endothelial damage with Noxious Heat and Blood Stasis Syndrome2.Aspirin has therapeutic effect on endothelial injury with Noxious Heat and Blood Stasis by restoring the function of endothelial gap3.Aspirin can improve endothelial dysfunction with Noxious Heat and Blood Stasis Syndrome in LPS-induced by inhibiting activation of NLRP3 inflammasome... |
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