| Objective:1.To explore the feasibility of establishing the rabbit model of SSc-PAH induced by MCT combined with BLM.2.To explore the expression and mechanism of HIF-1α in SSc-PAH model,and the relationship between HIF-1α and MIF.Methods:Part 1: 40 male rabbits were randomly divided into four groups: control group（n=10）,BLM induced model group（n=10）,Low-dose MCT induced model group（n=10）,Low-dose MCT combined with BLM induced model group（n=10）.The modeling method in BLM-induced model group: 100 u L BLM solution was injected intraperitoneally into every rabbit every day,and consecutive interventions were given at the same time until the 21 st day.The modeling method in Low-dose MCT induced model group: A one-time intraperitoneal injection of 40 mg/kg MCT solution was given on the first day.MCT combined with BLM induced model group:Combination of the above two modeling methods.Rabbits in the solvent control group were injected daily with the same volume of the above solvent.After 3 weeks of modeling,the pulmonary artery pressure and the right ventricular hypertrophy index（RVHI）were measured,anatomy was taken.After the dissection,the skin,pulmonary trunk and the right upper lobe were taken as pathological sections by paraffin embedding,section,and hematoxylin-eosin staining.To observed the pathological differences among the four groups.Part 2: 30 male rabbits were randomly divided into three groups: normal control group（n=10）,model group（n=10）,HIF-1α inhibitors intervention group（n=10）.The rabbits in the model group were modeled by MCT combined with BLM.In addition to being modeled as the model group,the models in the HIF-1α inhibitors intervention group was daily given intraperitoneally with HIF-1α inhibitor,2-methoxyestradiol（2-ME2）.The control group was injected intraperitoneally with the same volume of the above-mentioned solvent daily.After 3 weeks,the pulmonary artery pressure and RVHI were measured,pathology were observed.Western blotting and immunohist-ochemistry were used to detect the quantify and localization of HIF-1α and MIF protein in pulmonary tissues.Results:Part 1: After 3 weeks of modeling,there were 1,2,2 and 2 models died in normal control group,BLM-induced model group,Low-dose MCT induced model group and Low-dose MCT combined with BLM-induced model group,respectively.The skin of the model in BLM-induced model group and Low-dose MCT combined with BLM induced model group is thickened and hardened,gradually losing its elasticity.Pulmonary arterial pressure of the model in the MCT combined with BLM induced model group all reached 30 mm Hg or more,and the pulmonary artery pressure and RVHI were significantly higher than those in the other three groups,and all had statistical significance（P<0.05）.Pulmonary arterial pressure and RVHI of the rabbits in the BLM induced model group were slightly higher than those in the solvent control group,but neither had statistical significance（P>0.05）.In Low-dose MCT induced model group,the pulmonary artery pressure in the 62.5% of the models exceeded 30 mm Hg.The dermatological pathology of BLM-induced model and combination group was thickened,the hyperkeratosis of the hair follicle,the thickening of the vascular wall,the reduction or even disappearance of the skin appendages,and accompanied by a large number of collagen and fibroblasts proliferating in the dermis.The pulmonary arteries in the MCT combined with BLM induced model group showed obvious thickening of the vascular wall,stenosis or occlusion of the lumen,and papillary changes of the intima.In the pathology of the right upper lobe,the walls of the pulmonary arterioles in the MCT combined with BLM induced model group were significantly thickened,the lumen was narrowed or even obstructed,the vascular remodeling of the pulmonary microvessels and arterioles were observed,and a large number of inflammatory cells infiltrated,red blood cells were found in the alveolar cavities.Some models in the induced model group showed thickening of the wall of the pulmonary arteries.The pulmonary arterioles in the lungs were thickened,the lumens were narrowed,and a part of them had vascular remodeling.While the pathological structure of the pulmonary artery trunk and the right upper lobe of the solvent control group and the BLM induced model group nearly maintained a normal structure.Part 2: After 3 weeks of intervention,the pulmonary arterial pressure and RVHI were different among the three groups.These values in the model group were significantly higher than those in the other two groups,and all had statistical significance（P<0.05）.These values of the HIF-1α inhibitors intervention group were higher than those of the solvent control group,and were statistically significant（P<0.05）.Pathological results showed that the dermis of the model group was significantly thickened with collagen proliferating,the keratinization of hair follicles was observed,and the skin appendages were reduced or even disappeared.The HIF-1α inhibitors intervention group showed only slightly thickening of the dermis without reduction of the appendage or keratoses of the hair follicle;In model group,the vascular wall of pulmonary arterial trunk and pulmonary arterioles were significantly thickened with lumen stenosis.The pulmonary arterioles showed vascular remodeling,and inflammatory cells were observed infiltrated in the alveolar septum.In the HIF-1α inhibitors intervention group,pulmonary arterial trunk and pulmonary arterioles were slightly thickened without lumen stenosis or vascular remodeling.The skin,the pulmonary trunk,and the right upper lobe of the model in the solvent control group showed normal structures.Western blotting indicated that the expression of HIF-1α in pulmonary tissues in model group were higher than nomal control group（P < 0.05）.The result of western blotting showed that the expression of HIF-1α protein in the model group was significantly higher than it in the control group（P<0.001）.When HIF-1α protein was inhibited,the quantify of MIF protein was significantly down-regulated,and there was a statistically significant difference between the model group and HIF-1α inhibitors intervention group（P<0.001）.The results of immunohistochemistry showed: the expression of HIF-1αon the pulmonary artery wall in model of control group was weakly positive,and HIF-1α was strongly expressed in the endothelial cells of the pulmonary artery wall in the SSc-PAH model;Compared with the strong positive expression of MIF on endothelial cells of pulmonary arterial wall in the model group,when HIF-1α was inhibited,the expression of MIF protein was significantly weakened.Conclusion:1.The pulmonary arterial pressures of the model established by MCT combined with BLM all reached 30 mm Hg or more.The histology of the model conforms to the pathological features of SSc-PAH or may be one of the choices of the SSc-PAH model.2.The expression of HIF-1α protein in the SSc-PAH model was elevated,and it’s mainly expressed on the endothelial cells of the pulmonary artery wall.After the administration of 2-ME2 to inhibit the expression of HIF-1α protein,the pulmonary artery pressure decreased and the pathology improved,which suggesting that HIF-1αplays an key role in the pathogenesis of SSc-PAH. |
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