| BackgroundChronic mountain sickness(CMS)is defined as occurring when living at high altitude(>2500 m)residents with a clinical syndrome characterized by polycythemia and severe hypoxemia.Usually,the disease is associated with moderate or severe pulmonary hypertension,which may develop into high altitude cor pulmonale and lead to congestive heart failure.Hypoxia releases angiotensin ?(Ang?)and other vasoconstrictor substances,and increases vascular wall inflammation and oxidative stress levels in vivo by inducing active oxygen species and other cytokines,leading to multi-tissue damage.In the clinical treatment of CMS,targeted drugs for vascular dilation or calcium channel blocking drugs are mostly used,but these drugs cannot reverse the pulmonary vascular remodeling process of CMS,and there are many adverse reactions.As an Ang? receptor blocker,Irbesartan has been widely used in essential hypertension and diabetic nephropathy.Therefore,from the perspective of "old drugs for new use",irbesartan was taken as a potential candidate drug to preliminarily explore the possibility of CMS prevention and treatment.MethodsBased on the successful establishment of CMS rat model,irbesartan intervention was conducted to explore the mechanism of the occurrence and development of CMS and the protective effect of Irbesartan on the heart,lung and other tissues and organs of CMS rats through a variety of modern biotechnology which applied pathology,pharmacology and cell biology research methods.Further,from the perspectives of proteome,microbiome and serum metabolome,the changes of the body and the effects of drugs in the low-pressure and low-oxygen environment on the plateau were further discussed,so as to provide theoretical basis for finding effective prevention and control measures against CMS.1)To establish a simple and effective CMS animal model simulating the altitude of 5000 meters plateau environment,and lay an experimental foundation for CMS control by Irbesartan.2)The pathogenesis of myocardial injury in CMS rats was determined by irbesartan intervention,and the ameliorative effect of irbesartan was evaluated.3)Establish hypoxia injury model of myocardial cell in vitro and evaluate the effectiveness of irbesartan by cellular and molecular biological methods.4)Based on pathology,the protective effect of Irbesartan on lung of CMS rat model was studierd,and the basic mechanism of irbesartan against CMS was discussed.5)Based on proteomic methods,to study the occurence and development of CMS and the protective mechanism of Irbesartan against CMS,and to explore the potential protein markers of CMS.6)To study the effects of irbesartan on serum metabolomics of CMS rats,and to explore the differential expression profile and potential biomarkers of CMS,as well as the metabolic pathways involved in the occurrence and development of CMS.7)From the perspective of CMS,the composition and structure of intestinal microbial community were studied to find the microenvironment and different flora that lead to injury of CMS and explain the pathogenesis of CMS.ResultsCompared with plain control group(CG),body weight in chronic mountain sickness group(MG)decreased but the difference was not significant;PaO2,SaO2 and myocardial MDA were significantly decreased.PAP,Hb,Hct,EPO,myocardial SOD,GSH-Px and right ventricle hypertrophy index(RVHI)were increased.After irbesartan intervention,the above indicators were improved in all dose groups,especially in the high dose group(IB.H).Cardiac ultrasound results showed that left atrial diameter and right atrial diameter(transverse diameter)in MG group were significantly increased,the inner diameter of pulmonary artery,left ventricular systole,right ventricular inner diameter and diastolic upper and lower diameter were significantly reduced,the anterior wall of right ventricle was significantly thickened,cardiac ejection fraction decreased.After irbesartan intervention,IB.L group had a slight increase in diastolic diameter,pulmonary artery diameter,left ventricular systolic diameter and right ventricular anterior wall thickness.IB.M and IB.H groups increased the inner diameter of pulmonary artery,left ventricular systolic and diastolic,and ejection fraction increased.The results of HE staining and transmission electron microscopy showed that the myocardial interstitial vessels and subepicardial vessels in MG group were mildly hyperemic,the myocardium was blurred,the striations disappeared,and the myofibrils were disordered.Increased lipid droplets;Mitochondrial hyperplasia,shuttle type,crista fracture,mitochondrial local crista dissolved into floccule.In IB.L and IB.M groups,vascular hyperemia near epicardium was observed,eosinophilic lesions were occasionally observed,there were some inflammatory cells in the interstitium,granuloid lesions were rarely observed,myocardial myofibrils were orderly arranged,and z-line was slightly irregular.Endoplasmic reticulum slightly dilated;The mitochondrial cristae was orderly and regular,and there was slight edema around some mitochondria.In IB.H group,normal myocardial structure was observed,with occasional slight hyperemia,no eosinophilic cells or inflammatory cells were observed.Irbesartan inhibits hypoxia-induced cell damage.MTT results show that irbesartan has a significant proliferation effect on H9C2 cells treated with hypoxia for 48h,and has a certain protective effect on H9C2 cells within the range of 0-100 ?M.The IC50 of irbesartan to H9C2 cells after 48h hypoxia injury is 50.14 ?M.The results of cell apoptosis showed that H9C2 cells were significantly induced by hypoxic injury for 48h,5?M,25 ?M and 50 ?M of IB have protective effects on apoptosis of H9C2 induced by hypoxic,of which 50 ?M was the most effective.The results of flow cytometry and fluorescence microscopy showed that the content of Reactive oxygen species(ROS)in the hypoxia group was significantly increased compared with the normal group,ROS in the IB 5 ?M was not significantly different from that in the hypoxia group,however,ROS decreased in IB 25 ?M and IB 50 ?M group.The results of HE staining and transmission electron microscopy showed that the pulmonary artery wall elastic fibers were thickened,pulmonary artery wall plexus lesions,hyperplasia of middle layer,hyperplasia of intima were serious,and fibroblast layer of outer membrane was myosylated in CMS rats.In IB.L group,the thickness of the pulmonary artery wall was consistent,the intima hyperplasia was slight,the continuity of the fibrous cells of the pulmonary artery wall was good,the middle membrane of the wall was mild hyperplasia,and the outer membrane was mild fibrosis.In IB.M group,mild hyperplasia of pulmonary artery,smooth intima,mild hyperplasia of smooth muscle cells in the middle membrane,mild hyperplasia of fibroblasts in the outer membrane,and cluster lesions were observed.In IB.H group,the pulmonary artery intima was smooth and slightly thickened,the number of pulmonary artery wall cells was increased,the elastic fiber layer was clumped,and the outer membrane fibroblasts were myosylated.The results of HE staining and transmission electron microscopy showed that the lung tissues of MG group were observed under the microscope,which showed obvious alveolar septal hyperplasia,collapse and compensatory dilation,telangiectasia and hyperemia in the alveolar wall,alveolar edema and a large number of inflammatory cells infiltration.In IB.L group,alveolar septa were thickened,collapsed and compensatory dilated,and interstitial capillaries were dilated and hyperemic.In IB.M group,alveolar cavity was obvious,alveolar wall was thin,capillaries were slightly bleeding,but no inflammatory cell infiltration was observed.In IB.H group,alveolar septum thickened significantly,partial alveolar compensatory dilation or collapse,pulmonary interstitial capillaries dilated and congested,and the lesion degree was slightly lighter than that in medium dose group.A total of 550 proteins were identified by proteomics,of which 94 proteins were changed in the MG group compared with the CG group and 154 proteins were changed in the Irbesartan(IB)group compared with the MG group.40 differentially expressed proteins were found in the two comparison groups,and 27 proteins were significantly changed after cluster analysis.GO and KEGG pathway analysis revealed that cholesterol metabolism pathway is crucial in the occurrence of chronic altitude disease.ELISA and immunohistochemistry verified the differential proteins(Apo-A1,Apo-C1,Apo-E,IGF-1,Profilin1 and Colla1),and found that the expression levels of these six proteins were increased in the MG group.The expression level was decreased in the IB group.NMR metabolomics results showed that,compared with CG group,there were 20 different metabolites in serum of MG group(lipids,isoleucine,glutamine,leucine,citric acid,valine,creatine,3-hydroxybutyric acid,lactic acid,acetic acid,alanine,choline,glycoprotein,acetoacetic acid,pyruvate,taurine,proline,glycine,citrulline,?-glucose).And these 20 metabolites were reversed after irbesartan intervention,the difference was statistically significant.Ten metabolic pathways were found after enrichment analysis,including ketone body synthesis and decomposition,pyruvate metabolism,taurine metabolism and so on.The OTU analysis of intestinal flora showed that the sample size of each group in this study was sufficient.There were 981 OTU in the three groups(CG,MG and IB).Analysis of phylum composition in intestinal contents of rats showed that the proportion of Firmicutes in MG group was significantly higher than that in CG group and IB group,while the proportion of Bacteroidetes was lower than that in other two groups.Irbesartan intervention restored firmicutes and Bacteroides to levels similar to CG.The F/B ratio in the MG group was three times higher than that in the CG group,and was restored to normal by irbesartan.At the family level,the abundance of Lactobacillaceae,Peptostreptococcaceae,Erysipelotrichaceae and Bacteroidales?S24-7 decreased in CMS,but increased to the normal level after irbesartan was administered.The abundance of Lachnospiraceae and Prevotellaceae increased in CMS,and the abundance of these bacteria approached the normal level after irbesartan intervention.Shannon index increased and Simpson index decreased in CMS,and shannon and Simpson index were close to the normal level after irbesartan intervention.The results showed that high altitude hypoxic exposure could increase the diversity of intestinal flora,and irbesartan could restore the diversity of intestinal flora to normal state.The first two principal components of PCA analysis explained 32.52%and 20.91%of the total variation,indicating that there were significant differences between chronic mountain sickness rats and normal rats,and the bacterial community structure tended to be in the normal group after irbesartan administration.PLS-DA results showed that there were significant differences between MG and CG,and there were also significant differences between irbesartan and normal rats.The results of functional prediction analysis showed that the main biological functions of sequenced bacteria were concentrated:amino acid transport and metabolism;Transcription;Copy;Recombination and repair;Carbohydrate transport and metabolism;Translation;Ribosome structure and so on.ConclusionThe preliminary conclusions of this study are as follows:1)Irbesartan significantly improved CMS related indicators,corrected right heart hypertrophy and diastolic function of right heart in CMS rats,and protected myocardium by improving oxidative stress level and tissue structure.At the cellular level,irbesartan was found to reduce the apoptosis rate of hypoxic-injured cardiomyocytes by reducing intracellular ROS content.2)Irbesartan alleviated the injury of vascular endothelial cells and vascular remodeling by inhibiting Ang? receptor activity and thus IGF-1 after chronic hypoxia,and inhibits the injury of vascular endothelial cells by cholesterol metabolism by changing the concentration of cholesterol metabolization-related proteins(APO-A1,APO-C1 and APO-E).Finally,it can relieve pulmonary hypertension,inflammation and heart injury caused by chronic altitude hypoxia.3)Irbesartan can correct energy metabolism,amino acid metabolism and other pathways in rats with chronic altitude disease,significantly affect inflammatory level and nitric oxide(NO)metabolism in vivo,improve pulmonary artery contraction and remodeling process,providing a reliable theoretical basis for the intervention of irbesartan in chronic altitude disease.4)The diversity level of microflora in CMS rats was significantly increased,and there were significant differences between CMS rats and normal rats in the composition of microflora,showing obvious "pathogenic characteristics",and these abnormalities in composition and abundance were ameliorated by irbesartan.The study on the intestinal microbial diversity of CMS rats has enriched our understanding of the changes of intestinal microbial community in the altitude hypoxia environment,and has important implications for further studies on the pathogenesis and therapeutic drugs of CMS. |
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