| Guided by TCM Vessel-collateral Theory,and based on the identity of“Sun Luo-microvascular”,this study proposed that the dysfunction of pulmonary microvascular barrier plays an important role in mediating chronic obstructive pulmonary disease（COPD）on the effect of atherosclerosis（AS）.Based on the previous research,it is proved that treatment of“Yiqi,Huoxue,Tongluo”and representative drug Tongxinluo in the protection of microvascular of heart,brain and kidney,this method and representative drug were used for the first time to explore the intervention effect on pulmonary microvascular barrier function and its influence on the development of COPD aggravating AS.Through the establishment of COPD plus AS mouse model and cigarette smoke extract（CSE）induced human pulmonary microvascular endothelial cells（HPMECs）barrier dysfunction model,to reveal the effect of COPD mediated by pulmonary microvascular barrier dysfunction on AS,combined with the intervention effect of Tongxinluo capsule,to further support the scientific value of Vessel-collateral Theory in guiding the clinical treatment of cardiopulmonary diseases.PartⅠThe study of COPD mediated by pulmonary microvascular barrier dysfunction in aggravating AS and the intervention of dredging collateralsObjective:Based on AS model induced by high-fat diet,COPD combined with AS model was established by combining smoking to clarify the role of COPD in aggravating AS;The role of pulmonary microvascular barrier dysfunction in COPD with AS was revealed by detecting the changes of pulmonary microvascular barrier function;to observe the intervention effect of“Yiqi,Huoxue,Tongluo”representative drugs.Methods:C57BL/6N mice were used as normal control group,Apo E-/-mice were used as the research object,and the model of COPD combined with AS was established by smoking and high-fat diet in Apo E-/-mice.According to the completely randomized method,Apo E-/-mice were divided into five groups（n=10）:AS group,COPD+AS group,COPD+AS+Tongxinluo（TXL）group（TXL group）,COPD+AS+Atorvastatin（Ato group）,COPD+AS+TXL+Ato group（TXL+Ato group）.Before the experiment,0.5%sodium carboxymethyl cellulose（CMC-Na）solution was used to prepare the drug to the required concentration.The dosage of TXL was 1.5 g/kg/day,and Ato was 10 mg/kg/day.The volume of intragastric administration was 0.10ml/10g.After intragastric administration for 20 weeks,the lung function of mice was evaluated by pulmonary function analyzer;the pathological changes of lung tissue were observed by HE staining;the pulmonary inflammatory factors and systemic inflammatory factors were detected by ELISA and RT-q PCR;the pulmonary microvascular permeability was measured by Evans blue staining,and the ultrastructure of pulmonary microvascular was observed by transmission electron microscope;Western blot and RT-q PCR were used methods the m RNA and protein levels of tight junction proteins VE-cadherin,β-catenin and claudin5 were detected;the serum lipid level was detected by biochemical analyzer;the formation of aortic atherosclerotic plaque was observed by he and oil red O staining;the biomarkers of endothelial injury were detected by RT-q PCR.To reveal the role of pulmonary microvascular barrier dysfunction in COPD aggravated AS,and the intervention effect of Tongxinluo through pulmonary microvascular barrier protection on COPD aggravated AS.Results:1 Smoking can aggravate the decline of lung function induced by high fat diet in miceCompared with control group,FRC in as group and COPD+AS group increased significantly,cdyn and FEV50/FVC decreased significantly;RI in COPD+AS group increased significantly compared with control group,while RI in AS group had no significant difference compared with control group.Compared with AS group,FRC and RI were significantly increased,cdyn and FEV50/FVC were significantly decreased in COPD+AS group.2 Smoking can aggravate the increase of pulmonary microvascular permeability induced by high-fat diet in miceEvans blue leakage in lung tissue reflects pulmonary microvascular permeability.Compared with control group,Evans blue content in lung tissue of as and COPD+AS groups increased significantly.Compared with AS group,the content of Evans blue in COPD+AS group was significantly higher.3 Smoking can aggravate the damage of pulmonary microvascular ultrastructure induced by high fat diet in miceElectron microscopy was used to observe the ultrastructure of pulmonary endothelium.In AS group,the basal membrane（BM）of Qi blood barrier was not continuous and the tight junction（TJ）was fuzzy.In COPD+AS group,the cytoplasm（EN）of capillary endothelial cells was moderately edem Atous,the cell membrane was incomplete,the tight junction（TJ）between cells was blurred and nearly disappeared,and the basal membrane（BM）was fuzzy and discontinuous.4 Smoking can reduce the expression of tight junction protein in the lung tissue of mice induced by high-fat dietTight junction protein is located in the area of tight junction between two cells,which is the main adhesion protein to control and maintain the integrity of endothelial barrier.Compared with the control group,the m RNA and protein levels of VE-cadherin andβ-catenin in AS group and COPD+AS group were significantly decreased.Compared with AS group,the m RNA and protein levels of VE-cadherin andβ-catenin in COPD+AS group were significantly lower.Compared with control group,the expression of VE-cadherin in AS group and COPD+AS group was decreased,and the expression of VE-cadherin in COPD+AS group was lower than that in AS group.5 Smoking can aggravate the pathomorphological changes of lung tissue induced by high-fat diet in miceHe staining was used to observe the pathomorphological changes of lung tissue in mice.In the control group,the structure of bronchi and alveoli was clear,the structure of bronchial epithelial cells was complete,the arrangement of mucosal epithelial cells was orderly,the cilia were complete,the structure of alveoli was complete,there was no hyperemia and edema,the alveolar cavity was not enlarged,and there was no obvious exudation;in the as group,the structure of alveoli was slightly damaged,and there was a small amount around the submucosa.In COPD+AS group,there were hyperemia and edema in lung interstitium,infiltration of a large number of inflammatory cells in alveolar wall,collapse and abscission of myxocilia,abscission of bronchial epithelium,increase of secretion,disappearance of some alveolar structures and fusion into pulmonary bullae.The mean linear intercept（MLI）of alveoli was analyzed by image pro plus.Compared with control group,the MLI of as group and COPD+AS group increased significantly;compared with as group,the MLI of COPD+AS group increased significantly.6 Smoking can increase the level of inflammatory factors in the lung tissue of mice induced by high-fat dietCompared with control group,IL-1βand TNF-αm RNA in COPD+AS group were significantly up-regulated;compared with AS group,IL-1βand TNF-αm RNA in COPD+AS group were significantly up-regulated.ELISA results showed that compared with control group,the levels of IL-1βand TNF-αin AS group and COPD+AS group were significantly up-regulated;compared with as group,the levels of IL-1βand TNF-αin COPD+AS group were significantly up-regulated.7 Smoking can increase the serum levels of systemic inflammatory factors in mice induced by high-fat dietCompared with control group,IL-1β,IFN-γand TNF-αwere significantly up-regulated in AS group and COPD+AS group;compared with AS group,IL-1β,IFN-γand TNF-αwere significantly up-regulated in COPD+AS group.8 Smoking can aggravate the pathomorphological changes of aorta induced by high-fat diet in miceThe results of oil red O showed that the inner wall of aorta in the control group was smooth and transparent,and there was no atherosclerotic plaque;compared with the control group,a large number of plaques were found in the inner wall of aorta,especially in the aortic root in the AS group and COPD+AS group,and multiple diffuse plaques were found in the thoracic aorta and below;compared with the AS group,the degree of plaques in COPD+AS group was more serious.He staining showed that the surface of aortic intima in control group was smooth,the cells arranged orderly,and the structure of each layer was normal.In AS group,the intimal and intimal cells of the aorta were arranged in disorder,a large number of cytoplasm vacuolated,the aortic intima protruded from the fibrous plaque layer,inflammatory cells infiltrated,and foam was serious.In COPD+AS group,endothelial cells were isolated,obvious plaque and intimal hyperplasia were found in the lumen.Intimal thickening,calcification,smooth muscle fiber disturbance,intimal smooth muscle cell proliferation and necrosis,atheromatous plaques,foam cells accumulation,suggesting that the model was successfully constructed.9 Smoking can increase the serum lipid level of mice induced by high-fat dietCompared with the control group,the levels of TC,TG,LDL-C in the AS group and COPD+AS group were significantly increased,while the level of HDL was significantly decreased;compared with the AS group,the levels of TC,TG,LDL-C,HDL-C in the COPD+AS group had no significant difference.10 Smoking can increase the expression of aortic endothelial injury markers induced by high-fat diet in miceCompared with the control group,the m RNA levels of VCAM-1 and ICAM-1 in AS and COPD+AS groups were significantly increased.Compared with AS group,VCAM-1 and ICAM-1 m RNA in COPD+AS group increased significantly.11 Tongxinluo can improve the lung function of mice induced by smoking and high fat dietCompared with COPD+AS group,FRC and RI of TXL group and TXL+Ato group were significantly decreased,FEV50/FVC and cdyn were significantly increased,FRC of Ato group was significantly decreased,FEV50/FVC and cdyn were significantly increased.However,compared with COPD+AS group,Ato group had no significant effect on RI.Compared with Ato group,FRC and RI of TXL group and TXL+Ato group were significantly decreased,FEV50/FVC and cdyn of TXL+Ato group were significantly increased;compared with Ato group,FEV50/FVC and cdyn of TXL group had no significant difference.Compared with TXL group,FRC and RI in TXL+Ato group were significantly decreased,FEV50/FVC was significantly increased,while cdyn in TXL group was not significantly different from that in TXL+Ato group.12 Tongxinluo can reduce the pulmonary microvascular permeability induced by smoking plus high-fat diet in miceCompared with COPD+AS group,TXL group and TXL+Ato group significantly reduced the content of Evans blue in lung tissue,while Ato group had no significant effect on reducing the content of Evans blue in lung tissue.Compared with Ato group,the content of Evans blue in TXL group and TXL+Ato group was significantly decreased.Compared with TXL group,TXL+Ato group significantly reduced the content of Evans blue.13 Tongxinluo can improve the ultrastructure of pulmonary microvasculature induced by high fat dietThe edema of endothelium（EN）and epithelium（I）in TXL group was slightly lighter than that in Ato group,and the tight junction（TJ）partially disappeared and the gap increased.In the Ato group,there were edema of blood and Qi barrier,blurred basement membrane（BM）and partial disappearance of tight junction（TJ）.The damage of Qi blood barrier in TXL+Ato group was slightly lighter than that in TXL group,and the TJ was also slightly better than that in TXL group.14 Tongxinluo can increase the expression of tight junction protein in the lung tissue of mice induced by smoking combined with high-fat dietCompared with COPD+AS group,the m RNA and protein expressions of VE-cadherin andβ-catenin in TXL group,Ato group and TXL+Ato group were significantly increased.Compared with the Ato group,the m RNA and protein levels of VE-cadherin andβ-catenin in TXL group and TXL+Ato group were significantly increased.Compared with TXL group,the expression levels of VE-cadherin andβ-catenin m RNA and protein in TXL+Ato group were significantly increased.The results of immunofluorescence staining showed that VE-cadherin levels were restored in TXL group,Ato group and TXL+Ato combination therapy.Compared with Ato group,VE-cadherin levels were increased in TXL group and TXL+Ato group.Compared with TXL group,the level of VE-cadherin in TXL+Ato group increased significantly.15 Tongxinluo can improve the pathomorphological changes of lung tissue induced by smoking plus high-fat diet in miceCompared with COPD+AS group,TXL group had less hyperemia,edema and inflammatory infiltration in lung tissue;Ato group had less alveolar destruction and a small amount of inflammatory cells infiltration in submucosa;COPD+AS group had less hyperemia and edema in lung tissue,no obvious red blood cell exudation,cilium lodging and airway wall injury and abscission,alveolar structure was basically complete,and inflammatory cells infiltration process was stable compared with the model group,the degree of apoptosis was significantly decreased.Image Pro Plus analysis showed that the MLI of TXL group,Ato group and TXL+Ato group was significantly lower than that of COPD+AS group;compared with Ato group,the MLI of TXL group and TXL+Ato group was significantly lower;compared with TXL group,the MLI of TXL+Ato group was significantly lower.16 Tongxinluo can reduce the level of inflammatory factors in the lung tissue of mice induced by smoking combined with high-fat dietCompared with COPD+AS group,the m RNA levels of IL-1βand TNF-αin TXL group,Ato group and TXL+Ato group were significantly down regulated;compared with Ato group,the m RNA levels of IL-1βand TNF-αin TXL group and TXL+Ato group were significantly down regulated;compared with TXL group,the m RNA levels of IL-1βand TNF-αin TXL+Ato group were significantly down regulated.ELISA results showed that the levels of IL-1βand TNF-αin TXL group,Ato group and TXL+Ato group were significantly lower than those in COPD+AS group;compared with Ato group,the levels of IL-1βand TNF-αin TXL group and TXL+Ato group were significantly lower;compared with TXL group,the levels of IL-1βand TNF-αin TXL+Ato group were significantly lower.17 Tongxinluo can reduce the serum levels of systemic inflammatory factors in mice induced by smoking combined with high-fat dietThe levels of IL-1β,IFN-γand TNF-αin TXL group,Ato group and TXL+Ato group were significantly lower than those in COPD+AS group;compared with Ato group,the levels of IL-1β,IFN-γand TNF-αin TXL group and TXL+Ato group were significantly lower;compared with TXL group,the levels of IL-1β,IFN-γand TNF-αin TXL+Ato group were significantly lower.18 Tongxinluo can alleviate the pathomorphological changes of aorta induced by smoking plus high-fat diet in miceThe results of oil red O showed the plaque surface of TXL group,Ato group and TXL+Ato group was more severe compared with the TXL group,the plaque area in the Ato group and the TXL+Ato group decreased significantly;compared with the Ato group,the plaque area in the TXL+Ato group decreased significantly.He staining showed that the atherosclerotic pathological changes of aorta in TXL group,Ato group and TXL+Ato group were significantly less than those in COPD+AS group.The inner wall of aorta in TXL+Ato group was smoother,and the plaque decreased most significantly.19 Tongxinluo can reduce the serum lipid level of mice induced by smoking combined with high-fat dietCompared with COPD+AS group,the levels of TC,TG,LDL-C and HDL-C in TXL group,Ato group and TXL+Ato group were significantly down regulated;compared with TXL group,the levels of TC,TG and LDL-C in Ato group and TXL+Ato group were down regulated,and there was no significant difference;compared with Ato group,the level of LDL-C in TXL+Ato group was significantly down regulated.Compared with Ato group,HDL-C levels in TXL group and TXL+Ato group were significantly higher;compared with Ato group,HDL-C levels in TXL+Ato group had no significant difference.20 Tongxinluo can reduce the expression of aortic endothelial injury markers induced by smoking plus high-fat diet in miceCompared with COPD+AS group,VCAM-1 and ICAM-1 m RNA in TXL group,Ato group and TXL Ato group were significantly decreased.Compared with TXL group,the m RNA levels of ICAM-1 and VCAM-1 in Ato group and TXL+Ato group were significantly lower;compared with Ato group,the m RNA levels of VCAM-1 and ICAM-1 in TXL+Ato group were significantly lower.PartⅡEffect of cigarette smoke extract on barrier dysfunction induced by HPMECs and intervention of dredging collateralsObjective:To observe the effect of CSE on the barrier dysfunction of HPMECs in vitro,and to reveal the protective effect of Tongxinluo on the barrier function of HPMECs.Methods:HPMECs were cultured at 37℃in 5%CO2incubator.HPMECs were pretreated with TXL for 6 h,and then stimulated with CSE（100μg/ml）to establish the model of pulmonary microvascular barrier dysfunction.They were divided into Control group,CSE group,CSE+TXL200μg/ml group,CSE+TXL 400μg/ml group and CSE+TXL 800μg/ml group.The effect of TXL on the viability of HPMECs was detected by MTS colorimetry;the monolayer permeability of HPMECs was detected by the trans monolayer cell resistance（TER）and FITC dextran permeability;the expression of tight junction protein was detected by Western blot,RT-q PCR and immunofluorescence;ELISA and Western blot were used Inflammatory factors and biomarkers of endothelial injury were detected by RT-q PCR.Results:1 CSE decreased the viability of HPMECs and Tongxinluo increased the viability of HPMECsMTS results showed that the viability of HPMECs showed a significant dose-response relationship after 24 h of CSE induction.According to the IC50calculation formula,the concentration of 50%effective dose was 100μg/m L.Therefore,the modeling conditions were selected as the concentration of 100μg/ml and the time of 24 h.TXL has a protective effect on HPMECs,and TXL can promote the proliferation of HPMECs at 200μg/m L,400μg/m L and 800μg/m L,respectively.Therefore,200μg/m L,400μg/m L and 800μg/m L were selected as the intervention dose in the follow-up study.2 CSE increased the permeability of HPMECs,and Tongxinluo decreased itThe leakage of TER and FITC-dextran is one of the indicators reflecting the integrity of tight junction protein,which is often used to evaluate cell permeability.The permeability of microvascular endothelial cells was inversely proportional to the change of TER and positively proportional to the concentration of FITC-dextran.Compared with the control group,the concentration of FITC-dextran was significantly increased and the TER value was significantly decreased in the CSE group,which indicated that the pulmonary microvascular barrier function was damaged;compared with the CSE group,the concentration of fluorescence leakage was significantly decreased in the TXL 200μg/m L,400μg/m L and 800μg/m L,which significantly increased the TER value and protected the cell barrier function.3 CSE decreased the expression of tight junction protein in HPMECs,and Tongxinluo increased the expression of tight junction protein in HPMECsWestern blot was used to detect the expression of tight junction proteins VE-cadherin andβ-catenin.The results showed that compared with control group,CSE group significantly decreased the protein levels of VE-cadherin andβ-catenin;compared with CSE group,TXL 200μg/m L,400μg/m L and800μg/m L significantly increased the protein levels ofβ-catenin;TXL 800μg/m L significantly increased the protein levels of VE-cadherin,while TXL200μg/m L significantly increased the protein levels of VE-cadherin.The expression of VE-cadherin was not significantly affected by the concentration of 400μg/m L or 200μg/m L.RT-q PCR results showed that compared with control group,the expression of VE-cadherin,β-catenin and claudin5 m RNA in CSE group was significantly down regulated;compared with CSE group,TXL 200μg/m L,400μg/m L and 800μg/m L could increase the expression of VE-cadherin m RNA;compared with CSE group,TXL 200μg/m L and 800μg/m L significantly increased the expression ofβ-catenin m RNA,while TXL 400μg/m L had no effect on the expression ofβ-catenin.Compared with CSE group,TXL 200μg/m L and 400μg/m L significantly up-regulated claudin5m RNA expression,while TXL 800μg/m L had no significant effect on claudin5 m RNA expression.Immunofluorescence staining showed that compared with control group,the expression of VE-cadherin andβ-catenin in CSE group was down regulated,and the cell morphology was seriously damaged;compared with CSE group,the expression of TXL 400μg/m L VE-cadherin andβ-catenin was increased,and the cell morphology was improved.4 CSE increased the inflammatory response of HPMECs,while Tongxinluo decreased itELISA results showed that the levels of IL-6,IL-1βand hs CRP in the supernatant of CSE group were significantly higher than those in control group.Compared with CSE group,TXL 200,400 and 800μg/m L decreased the levels of IL-6,IL-1βand hs CRP.The expression of NF-κB was detected by Western blot.Compared with control group,the expression of NF-κB in CSE group was increased.Compared with CSE group,TXL 200μg/m L and TXL 800μg/m L significantly decreased the level of NF-κB.However,TXL 400μg/m L NF-κB had no significant difference compared with CSE group.In general,TXL can inhibit CSE mediated inflammation,which is consistent with the results of cigarette smoke combined with high-fat diet induced TXL inhibiting systemic inflammatory response in Apo E-/-mice in vivo.5 CSE increased the expression of endothelial injury markers in HPMECs,while Tongxinluo decreased the expression of endothelial injury markers in HPMECsRT-q PCR results showed that compared with control group,CSE significantly up-regulated VCAM1 and ICAM1 m RNA expression;compared with CSE group,TXL 200μg/m L,400μg/m L and 800μg/m L could significantly down regulate VCAM1 and ICAM1 m RNA expression.6 CSE increased HPMECs apoptosis and Tongxinluo inhibited HPMECs apoptosisWestern blot was used to detect the apoptotic protein.Compared with control group,CSE up-regulated the expression of Caspase3 and Caspase8;compared with CSE group,TXL 200μg/m L,400μg/m L and 800μg/m L down regulated the expression of Caspase3 and caspase8.The apoptosis rate was detected by FITC annexin V method.Compared with control group,CSE significantly increased the apoptosis rate;compared with CSE group,TXL 200μg/m L,400μg/m L,800μg/m L significantly decreased the apoptosis rate.PartⅢRac1/Cdc42 signaling pathway mediates pulmonary microvascular barrier dysfunction and intervention of dredging collateralsObjective:Objective to explore whether Rac1/Cdc42 signaling pathway regulates pulmonary microvascular barrier function,and to further explore the role of Rac1/Cdc42 pathway in barrier dysfunction induced by CSE in vitro,and to evaluate the intervention effect and mechanism of Tongxinluo on pulmonary microvascular barrier function.Methods:In HPMECs were cultured in 37℃,5%CO2incubator and stimulated with CSE（100μg/m L）for 24 hours to establish a barrier dysfunction model.The cells were divided into Control group,CSE group,CSE+TXL 200μg/m L group,CSE+TXL 400μg/m L group and CSE+TXL 800μg/m L group.The expression of Rac1 protein in HPMECs was detected by Western blot.The results showed that Rac1 protein expression increased in CSE induced HPMECs,while TXL decreased Rac1 protein expression,indicating that the increase of Rac1 protein represents the destruction of HPMECs barrier function.RT-q PCR was used to determine the optimal conditions for Rac1 si RNA silencing in HPMCs.HPMECs were divided into Control group and si RNA Rac1 group.The monolayer permeability of HPMECs was detected by TER and FITC-dextran,and the effects of si RNA Rac1 expression on the expression of VE-cadherin,β-catenin and Rac1/Cdc42 signaling pathway key factors were detected by Western blot.HPMECs were divided into control group,CSE group,CSE+si RNA Rac1 group,CSE+TXL group and CSE+TXL+si RNA Rac1 group.The expression of VE-cadherin andβ-catenin in HPMECs was detected by Western blot;the expression of VE-cadherin andβ-catenin in HPMECs was detected by immunofluorescence staining;the expression of VE-cadherin andβ-catenin in HPMECs cells was detected by Western blot.The protein expressions of Rac1,Cdc42 and p-Rac1+Cdc42 were detected by Western blot.Results:1 CSE induced up regulation of Rac1 protein level in HPMECs,while Tongxinluo could down regulate Rac1 protein levelRac1 protein can play a role in signal transduction in the change of intercellular space and the increase of monolayer permeability of endothelial cells.Western blot was used to detect the effect of CSE on Rac1 protein expression in HPMECs.Compared with the control group,CSE significantly increased Rac1 protein expression,while TXL 200μg/m L,400μg/m L and800μg/m L could down regulate Rac1 protein expression.2 Silencing of Rac1 gene inhibits microvascular dysfunctionRT-q PCR was used to detect the silencing effect of si RNA Rac1 on Rac1m RNA in HPMECs.The results showed that all three si RNAs could silence Rac1 protein in HPMECs,especially si RNA-001.Compared with the control group,si RNA Rac1 group down regulated the concentration of FITC-dextran and up-regulated the TER value of HPMECs,indicating that si RNA Rac1 can inhibit the damage of pulmonary microvascular barrier function.Western blot was used to detect the expression of VE-cadherin andβ-catenin.The results showed that compared with control group,VE-cadherin andβ-catenin were significantly up-regulated in si RNA Rac1 group.It was confirmed that silencing Rac1 gene expression with si RNA Rac1 in vitro could promote the expression of VE-cadherin andβ-catenin in HPMECs.3 Silencing Rac1 gene inhibits Rac1/Cdc42 pathwayWestern blot was used to detect the silencing effect of si RNA Rac1 on Rac1 gene expression in HPMECs.The results showed that compared with control group,the protein levels of Rac1,Cdc42 and p-Rac1+Cdc42 were significantly decreased after pretreatment with si RNA Rac1,which indicated that si RNA Rac1 inhibited the activation of Rac1/Cdc42 pathway.4 Tongxinluo protects HPMECs barrier function by inhibiting Rac1/Cdc42pathway4.1 Tongxinluo inhibits Rac1/Cdc42 signaling pathway in HPMECsWestern blot results showed that the protein levels of Rac1,Cdc42 and p-Rac1+Cdc42 in CSE group were significantly up-regulated compared with control group;compared with CSE group,the protein levels of Rac1,Cdc42and p-Rac1+Cdc42 in CSE+TXL group and CSE+si RNA Rac1 group were significantly down regulated,suggesting that Tongxinluo and si RNA Rac1 can inhibit Rac1/Cdc42 signaling pathway in HPMECs.Compared with Rac1group,the expression of Rac1,Cdc42 and p-Rac1+Cdc42 in CSE+si RNA Rac1+TXL group was significantly down regulated;compared with CSE+TXL group,the expression of Rac1,Cdc42 and p-Rac1+Cdc42 in CSE+si RNA Rac1+TXL group was down regulated.These results indicate that si RNA Rac1 can partially synergize the inhibition of TXL on Rac1/Cdc42signaling pathway.4.2 Tongxinluo up regulates the expression of tight junction protein by inhibiting Rac1/Cdc42 pathwayWestern blot was used to detect the protein expression of VE-cadherin andβ-catenin.The results showed that compared with control group,the protein expression of VE-cadherin andβ-catenin in CSE group was down regulated;compared with CSE group,the protein expression of VE-cadherin andβ-catenin in CSE+TXL group and CSE+si RNA Rac1 group was up-regulated;compared with CSE+TXL group,the protein expression of VE-cadherin andβ-catenin CSE+si RNA Rac1 group was up-regulated compared with CSE+si RNA Rac1 group,the expression of VE-cadherin andβ-catenin in CSE+si RNA Rac1+TXL group was up-regulated,suggesting that Rac1/Cdc42signaling pathway is involved in the protective effect of TXL on HPMECs.Immunofluorescence staining showed that the expression of VE-cadherin andβ-catenin decreased in CSE group;treatment with CSE+TXL could increase the expression of VE-cadherin andβ-catenin;while treatment with TXL+si RNA Rac1 could significantly increase the expression of VE-cadherin andβ-catenin.Conclusions:1 Based on the dysfunction of pulmonary microvascular barrier,we constructed COPD plus AS mouse model and CSE induced HPMECs induced barrier dysfunction model to reveal that pulmonary microvascular barrier dysfunction can aggravate AS by mediating COPD at the whole animal level and in vitro cell level.2“Yiqi,Huoxue,Tongluo”therapy and representative medicine Tongxinluo capsule can intervene the pathological process of COPD aggravating AS by protecting pulmonary microvascular barrier function,thus revealing the important clinical value of“Yiqi,Huoxue,Tongluo”therapy in the intervention of COPD and other respiratory diseases,and providing experimental data support for clinical treatment of heart and lung. |
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