| Research background and general ideaSmoking and passive smoking is the major risk factor for chronic obstructive pulmonary disease(COPD)and pulmonary hypertension(PH)secondary to COPD,studies have shown that smoking can start pulmonary artery smooth muscle cells'(PASMCs')derivative marker gene transcription and protein expression by up-regulation transforming growth factor beta1/ Smad(TGF-beta1/Smad)signal transduction pathway,resulting in PASMCs phenotypic transformation.Vascular smooth muscle cells(VSMCs)that have undergone phenotypic transformation are called synthetic phenotype VSMCs,which have the ability of abnormal proliferation,synthesis and secretion of various cytokines and extracellular matrix(extracellular matrix,ECM)and the ability to migrate to the intima,and then lead to pulmonary vascular remodeling(PVSR)which are characterized by intimal thickening and lumen stenosis of the pulmonary arteries,finally pulmonary vascular compliance decreased,pulmonary vascular resistance(PVR)is elevated and PH is formed.So it is deduced that blocking the PASMCs phenotypic transformation process and its downstream event of PASMCs' abnormal proliferation,migration to the intima,and synthesis and secretion of ECM by down-regulation TGF-beta 1 / Smad signal transduction pathways can improve the pulmonary vascular remodeling and pulmonary vascular resistance increases,thus improve the PH.Therefore,it is of great significance to study the regulation mechanism of PASMCs' phenotype transformation for the prevention and treatment of PVSR and PH secondary to COPD and an in-depth understanding of the mechanism of PVSR and PH secondary to COPD.Diosgenin is a steroidal saponins extracted from plant roots,which have pharmacological effects of inhibition of cell proliferation,promotion of cell apoptosis,anti-inflammatory,anti-tumor,anti-oxidant effects and protection of endothelial cells,is widely used in the treatment of cardiovascular diseases and tumor.PVSR is the main pathophysiological characteristics of PH,and the abnormal proliferation of PASMCs is the main pathogenesis of PVSR and PH secondary to COPD.Does Diosgenin have a therapeutic effect on PVSR and PH secondary to COPD caused by abnormal proliferation of such PASMCs? And what is its mechanism of action? Existing experiments have shown that Diosgenin can prevent and treat rats' pulmonary vascular remodeling and pulmonary hypertension induced by monocrotaline(MCT)by down-regulating the activity of TGF-?1 / Smad signal transduction pathway,inhibiting the abnormal proliferation of PASMCs and promoting its apoptosis,anti-inflammatory and anti-oxidation.But does the Diosgenin have a therapeutic effect on pulmonary vascular remodeling and PH induced by cigarette smoking by down-regulation of the TGF-beta 1 / Smad signal transduction pathway activity? And the next treatment target after down-regulation TGF-?1/Smad signal transduction pathway by inhibiting gene transcription to prevent PASMCs' phenotypic transformation and inhibition of PASMCs abnormal proliferation,migration to the intima,synthesis and secrete ECM to relieve pulmonary vascular remodeling and pulmonary hypertension is not clear.We have observed up-regulation of TGF-?1/Smad signal transduction pathway activity and PASMCs phenotypic transformation and PVSR in our preliminary experiment of pulmonary vascular reconstruction model of rats exposed to cigarette smoke,the up-regulation of TGFbeta 1/Smad signal transduction pathway activity and PASMCs phenotypic transformation are inhibited,and the PVSR was significantly improved in Diosgenin treatment group.So we deduce that Diosgenin alleviate the pulmonary artery remodeling and PH exposed to cigarette smoke by down-regulation TGF-?1/Smad signal transduction pathway and inhibiting the most upstream event of pulmonary artery remodeling namely PASMCs phenotypic transformation.Diosgenin was intervened in the rat model of pulmonary hypertension secondary to COPD established by smoke exposure in this study.The right ventricular systolic pressure,mean pulmonary artery pressure and right heart index were observed;The protein expression level of TGF-?1 / Smad2,p-Smad2,Smad3,P-Smad3,smooth muscle ?-actin(SM-?-actin)and osteopontin(OPN)in PASMCs of each group rats were measured by the western blotting method and by the immunohistochemistry method respectively;Pathomorphological changes of the lung' mall airways,alveolar structure and the morphometric characteristics of intra-acinar pulmonary muscule arteries were analyzed by optical microscope,the correlation between these results were analyzed;The ultrastructure changes of PASMCs were studied by transmission electron microscope.To clarify the hypothesis that Diosgenin inhibits pulmonary artery remodeling and pulmonary hypertension by inhibiting the the most upstream event of pulmonary artery remodeling namely PASMCs phenotypic transformation,blocking the downstream event of proliferation of PASMCs,migration to intima,and synthesis and secretion ECM by down-regulating the activity of TGF-?1 / Smad signal transduction pathway,which provides new ideas and targets for early intervention of pulmonary hypertension secondary to COPD.In order to confirm this hypothesis,the study protocol is as follows:First,the rat model of pulmonary hypertension secondary to COPD was established by cigarette smoke exposure and Diosgenin was intervened.The airway resistance of each group was measured through the pressure sensor of computer small animal ventilator,arterial blood gas analysis of each group was measured by the fast blood gas analyzer,right ventricular systolic pressure and mean pulmonary artery pressure of each group were measured and calculated by the BL-420 F biological function experiment system,and right heart index of each group was measured by the electronic analysis balance weighing,pathomorphological changes of the lung's small airway and alveolar structure were observed by optical microscope,the morphometric characteristics of intra-acinar pulmonary muscule arteries were observed by optical microscope and analyzed and calculated by DMR+Q550 pathological image analyzer and Leica Qwin analysis software.The protein expression level of TGF-?1 / Smad2,p-Smad,Smad3 and p-Smad3 in the lung tissue of rats were detected by western blott.To investigate the method of cigarette smoke exposure to establish the rat model of PH secondary to COPD preliminary;To investigate the effects of up-regulation of TGF-?1 / Smad signal transduction pathway on pulmonary vascular remodeling and PH in rats,the effects of Diosgenin on the regulation of TGF-?1 / Smad signal transduction pathway and its effects and its mechanism on pulmonary vascular remodeling and PH in rats.Second,the ultrastructural changes of intra-acinar pulmonary arteries' smooth muscle cell were observed by transmission electron microscope.The protein expression level of SM-?-actin and OPN in PASMCs were detected by immunohistochemistry.To compare the relationship of the protein expression level of TGF-?1 / Smad signa transduction pathway and the protein expression level of SM-?-actin and OPN in PASMCs,and the correlation between the expression level of phenotype-related proteins SM-?-actin and OPN in PASMCs and PVSR and PH.To investigate the role of TGF-?1 / Smad signal transduction pathway in phenotypic transformation of pulmonary artery smooth muscle cells,the relationship between phenotypic transformation of PASMCs and PVSR and PH,and to confirm the hypothesis of Diosgenin attenuates PVSR and PH by the way of inhibiting intra-acinar pulmonary arteries' smooth muscle cell phenotypic transformation in rats exposed to cigarette smoke by down-regulation TGF?1/Smad signal transduction pathway.Section I: Study on the relationship between TGF-?1 / Smad signal transduction pathway and PVSR and PH in rats exposed to cigarettesmoke Objectives:To investigate the expression level of TGF-?1 / Smad signal transduction pathway related protein in lung tissue of rats exposed to cigarette smoke and its correlation with PVSR and PH,To clarify the signal regulation mechanism of PVSR and PH secondary to COPD in rats exposed to cigarette smoke.Methods:36 health male SD rats aged for 6 weeks were divided into 3 guoups by the digital random method,12 rats per group,normal control group(group A,n = 12),rats were standard raised for 12 weeks,every day were put in the 0.22 mm3 organic glass capsule for 2 hours,but were not exposed to cigarette smoke,at the same time were lavaged with 3% sodium carboxymethyl cellulose solvent 0.5 ml.Cigarette smoke exposure group(group B,n = 12),rats were standard raised for 12 weeks,every day were put in the 0.22 mm3 organic glass capsule and exposed to cigarette smoke for 2 hours(5 cigarettes at a time,a total of 20 cigarettes),at the same time were lavaged with 3% sodium carboxymethyl cellulose solvent 0.5 ml.Cigarette smoke exposure + Diosgenin treatment group(group C,n = 12),rats were standard raised for 12 weeks,every day were purt in the 0.22 mm3 organic glass capsule and were exposed to cigarette smoke for 2 hours(5 cigarettes at a time,a total of 20 cigarettes),at the same time were lavaged with 100 mg/kg Diosgenin soluble in 3% sodium carboxymethyl cellulose solvent 0.5 ml.The modeling time is 12 weeks,three groups of rats in the organic glass capsule are free to eat and drink.At the end of 12 weeks,two rats in group B died,the other two groups had no death rats.10 rats were taken from group A and group C respectively according to random number method to perform the follow-up experiment and data analysis.The airway resistance of each group was measured through the pressure sensor of computer small animal ventilator,arterial blood gas analysis of each group was measured by the fast blood gas analyzer,right ventricular systolic pressure and mean pulmonary artery pressure of each group were measured and calculated by the BL-420 F biological function experiment system,and right heart index of each group was measured by the electronic analysis balance weighing,pathomorphological changes of the lung's small airway and alveolar structure were observed by optical microscope,the morphometric characteristics of intra-acinar pulmonary muscule arteries were observed by optical microscope and analyzed and calculated by DMR+Q550 pathological image analyzer and Leica Qwin analysis software;The TGF-beta 1 / Smad2,p-smad,Smad3,p-smad3 signal transduction pathway related proteins of lung tissue were detected by the western blott method.Compare the relationship of the expression level of TGF-?1 / Smad signal transduction pathway related proteins and RVSP,m PAP,RV / LV + S,intra-acinar pulmonary arteries' intima thickness,lumen area,internal elastic layer of mscule vascular circumference,the percentage of muscular artery,partial musclar artery and non-muscular artery.Results:Compared with group A,arterial oxygen pressure and oxygen saturation of group B mildly decreased,airway resistance increased,RVSP and m PAP increased,RV / LV + S increased,the ratio of muscular artery and partial musclar artery increased,the ratio of non-muscular artery decreased,the intra-acinar pulmonary arteries' intima thickening,lumen area deceased,internal elastic layer of vascular circumference decreased,the protein expression levels of TGF-?1 / Smad2,p-Smad2,Smad3 and p-Smad3 in rats' lung homogenate were increased,the difference was significant(P <0.01).The above indexes in group C were mildly changed,compared with group A,the difference was not significant(P> 0.05),compared with group B,the difference was significant(P <0.05).The correlation analysis showed that the protein levels of TGF-?1 / p-Smad2 and p-Smad3 were significantly positive correlation with those of RVSP,m PAP,RV / LV + S,intra-acinar pulmonary arteries' intima thickness,the percentage of muscular artery and partial musclar artery(r values were: 0.700,0.704,0.706,0.621,0.679,0.752,0.763,0.767,0.750,0.653,0.700,0.755,0.737,0.622,0.652,0.701,0.796,0.737 respectively,p <0.01 or 0.05),the protein levels of TGF-?1 / p-Smad2 and p-Smad3 were significantly negative correlation with lumen area,internal elastic layer of mscule vascular circumference and the percentage of non-muscular artery(r values were:-0.692,0.631,-0.734,-0.671,-0.763,-0.817,-0.661,-0.701,-0.0777,respectively,p <0.01 or 0.05).Lung tissue Pathology: Compared with group A,the small airway wall of group B had a large number of lymphocyte infiltration,local lymphoid follicle formation,smooth muscle layer intermittent fracture,airway epithelial cell cilia shedding.The alveolar cavity was enlarged and emphysema was formed.Partial alveolar septal fracture led to pulmonary bullae,the other alveolar septal was congestion,edema and had a large number of inflammatory cell infiltration;The intra-acinar pulmonary arteries' internal elastic layer disappeared,the layer of smooth muscle and intimal thicked,luminal narrowed,the pathomorphological changes of the lung's small airway and alveolar structure and the morphometric characteristics of intra-acinar pulmonary muscule arteries mentioned above combined with airway resistance,RVSP,m PAP and RV / LV + S increased,which is consistent with the clinical characteristics of PH secondary to COPD.Compared with group B,the infiltration of inflammatory cells in the small airway wall of group C was significantly lower,the small airway wall structure was clearer,airway epithelial cell cilia shedding was lighter,the structure of smooth muscle layer was more complete,and no obvious mucous goblet cell hyperplasia;Alveolar cavity slightly expanded and only mild emphysema was formed,but no lung bullae formation,alveolar septal edema and inflammatory cell infiltration was lower;The intra-acinar pulmonary arteries' internal and external elastic layer was more complete,the layer of initmal was no thickening,the layer of smooth muscle was slightly thicked and lumen diameter slightly reduced.conclusion:1.The up-regulation of TGF-?1 / Smad signal transduction pathway in lung tissue is one of the possible mechanisms of PVSR and PH in rats exposed to cigarette smoke.2.Diosgenin improves the PVSR and PH in rats exposed to cigarette smoke by down-regulating the activity of TGF-?1 / Smad signal transduction pathway.Section 2: Effects and its mechanism study of diosgenin onintra-acinar pulmonary arteries' smooth muscle cell phenotypictransformation in rats exposed to cigarette smoke Objective:To investigate the effect and its mechanism of Dio on intra-acinar pulmonary arteries' smooth muscle cell phenotypic transformation in rats exposed to cigarette smoke,the relationship of PASMCs phenotypic transformation and PVSR and PH,To explore the possible target for Diosgenin treatment of PVSR and PH in rats exposed to cigarette smoke.Methods:The rat model of PH secondary to COPD was established by cigarette smoke exposure and Diosgenin was intervened as the method of section one.The ultrastructural changes of intra-acinar pulmonary muscule arteries' PASMCs was observed by transmission electron microscope.The relationship between the expression level of TGF-?1 / Smad signal transduction pathway related protein and the expression level of SM-?-actin and OPN phenotypic related proteins in PASMCs,and the relationship between the expression level of SM-?-actin and OPN phenotypic related proteins in PASMCs of intra-acinar pulmonary muscle arteries and the PVSR related indicators namely intra-acinar pulmonary arteries' intima thickness,lumen area,internal elastic layer of mscule vascular circumference,the percentage of muscular artery,partial musclar artery and non-muscular artery and PH related indicators namely RVSP,m PAP and RV / LV+S were observed by correlation analysis.Results:Compared with group A,the protein expression level of SM-?-actin in group B was significantly decreased(P <0.01),and the protein expression level of OPN was significantly increased(P <0.01).The protein expression level of SM-?-actin in group C was increased(P <0.01),and the protein expression level of OPN was decreased(P <0.01).And there was significant difference compared with group B(P <0.01).There was no significant difference compared with group A(P> 0.05).The correlation analysis showed that the protein expression level of TGF-?1 / p-Smad2 and p-Smad3 were significantly positively correlated with the protein expression level of OPN protein(r values were:0.829,0.854,0.852 respectively,p all<0.01)and were negatively correlated with the protein expression level of SM-?-actin(r values were:-0.800,-0.856,-0.779 respectively,p all<0.01).The protein expression level of OPN were significantly positive correlation with those of RVSP,m PAP,RV / LV + S,intra-acinar pulmonary arteries' intima thickness,he percentage of muscular artery and partial musclar artery(r values were: 0.877,0.729,0.800,0.710,0.848,0.755,respectively,p all<0.01),and the protein expression level of SM-?-actin were significantly negatively correlation with those of RVSP,m PAP,RV / LV + S,intra-acinar pulmonary arteries' intima thickness,he percentage of muscular artery and partial musclar artery(r values were:-0.911,-0.686,-0.740,-0.623,-0.758,-0.741 respectively,p<0.01 or 0.05).The protein expression level of OPN were significantly negatively correlation with the lumen area,internal elastic layer of mscule vascular circumference and the percentage of non-muscular artery(r values were:-0.650,-0.671,-0892 p<0.01 or 0.05),and the protein expression level of SM-?-actin were significantly positively correlation with the the lumen area,internal elastic layer of mscule vascular circumference and the percentage of non-muscular artery(r values were: 0.712,0.658,0893 p<0.01 or 0.05).The observation of ultrastructural changes of intra-acinar pulmonary arteries' PASMCs by transmission electron microscope: Compared with group A,the PASMCs' volume of group B was increased and their shape was irregular;Most of the smooth muscle cells' long axis were perpendicular to the internal elastic membrane and are synthetic phenotype smooth muscle cells with lesser microfilaments and dense bodies and dense patches but more other organelles namely free ribosomes and rough endoplasmic reticulum,and rich in extracellular matrix,showing a large number of collagen fibers.Most of the intra-acinar pulmonary arteries' PASMCs of group C is contraction phenotype abundant in cytoplasmic microfilaments,dense bodies and dense patches,less in extracellular matrix and organelles,which was spindle-shaped and arranged slightly irregular,but the long axis is parallel to the internal elastic membrane.conclusion:1.TGF-?1 / Smad signal transduction pathway plays an important role in the phenotypic transformation of intra-acinar pulmonary arteries' PASMCs.2.The phenotypic tansformation of intra-acinar pulmonary arteries' PASMCs promote PVSR and PH in rats exposed to cigarette smoke.3.Inhibiting intra-acinar pulmonary arteries' PASMCs phenotypic transformation in rats exposed to cigarette smoke by down-regulation TGF?1/Smad signal transduction pathway is a possible target for Diosgenin treatment of PVSR and PH. |
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