Effects Of Series Of Alginate Oligosaccharide On Pulmonary Arterial Hypertension By Regulating The SGC Expression In Rats

Objective:Pulmonary hypertension is a pathophysiology state of abnormal increased pulmonary vascular resistance caused by a variety of reasons,and it is a hemodynamic disorder in various clinical diseases.The pathophysiology characteristics are abnormal productions of vasoactive substances and cytokines induced by hypoxemia,inflammation and abnormal blood flow shear force,which act on the target cells such as vascular endothelial cells and vascular smooth muscle cells.It can lead to abnormal constriction,growth and remodeling of pulmonary vessels and the accumulation of extracellular matrix.It is difficult to detect,diagnose and treat in the early stages because the clinical manifestations of pulmonary hypertension are lack of specificity.Clinically,the feature of the disease progression is the gradually increasing pulmonary vascular resistance,which leads to right heart failure and eventually causes systemic venous congestion and organ perfusion injury.Patients with pulmonary hypertension have poor prognosis,high mortality and disability rate.Pulmonary hypertension seriously affects the quality of life and threatens the health of patients.which is still a chronic and incurable disease at present.Cor pulmonale caused by pulmonary hypertension is a common cause of human death.So far,a variety of drugs of different mechanisms against pulmonary hypertension were developed,although some research achievements have been done,there is still a lack of effective treatment against pulmonary hypertension,especially drug treatment.Listed drugs with few species are expensive and poor curative effect,which have many side effects.Unlisted drugs are in the stage of experiment research and clinical trials,which have not yet been used in clinical practice.Therefore,the development of safe,effective,high-quality and affordable drugs to lower pulmonary hypertension is of great significance to the vast number of patients suffering from the disease and economic burden of pulmonary hypertension.Marine polysaccharides are abundant in sources.Marine animals,marine plants,and marine microorganisms can synthesize polysaccharides with large charges that are different from terrestrial plants.Ocean polysaccharides have biological activities such as anticoagulant,antithrombotic,hypoglycemic,antioxidant,anti-tumor,anti-inflammatory and so on.Yet,Ocean polysaccharide has the characteristics of strong gelling,large viscosity,water-insolubility and difficulty in absorption,which limit the application of polysaccharides in biomedical fields.In recent years,with the development of marine scientific research,oligosaccharide,a degradation product of polysaccharide with many biological activities,has gradually entered people’s vision.Alginate O1igosaccharides（AOS）is an oligomer formed by the degradation of sodium alginate and a linear block compound formed by theβ-D-mannose andα-L-guluronic acids connected by a 1-4glycoside bond.It has biological activities such as promoting plant growth,inducing antiretroactivity,anticoagulant,antibacterial,anti-inflammatory,anti-oxidant,anti-tumor,antiviral,neuroprotective,production of cytokines,and immunoregulation.The effects of oligosaccharide composition,molecular weight（degree of polymerization）and end sugar ring structure on the antioxidant activity of oligosaccharide are different.Soluble guanylase cyclase（sGC）with heme of protoporphyrin IX as cobasis,is a heterodimer consisting of two subunits,αandβ,with two isomers for each subunit that are namedα1,α2,β1andβ2,respectively.In lung tissue,sGC mainly exists in the form ofα1 andβ1 subunits.Nitric oxide（NO）plays an important role in the regulation of vascular tension,cell proliferation,migration and apoptosis as an endothelium-derived diastolic factor.sGC is the only receptor in the cell of NO.sGC catalyzes GTP to produce intracellular second messenger molecular cyclic guanosine monophosphate,participating in vasorelaxating,inhibiting platelet aggregation,regulating cell proliferation and apoptosis,and synapsing signal transmission.In addition to the classic endothelium-dependent NO-sGC pathway,which plays an important role in maintaining low resistance of the pulmonary arteries,an endothelium-independent sGC activation pathway related to reactive oxygen metabolites.,which named H₂O₂-sGC pathway,also participates in the regulation of vascular tension and organ blood flow.Based on the above reasons,this study initially explores whether it is possible to apply alginate o1igosaccharides’biological activities such as antioxidant,scavenging oxygen radicals and so on,to activate the H₂O₂-sGC pathway by regulating the sGC target,therefore to relieve rat pulmonary arterial hypertension.Then through a comparative study of the structural and dose effects of a series of alginate o1igosaccharides,we can screening the best drugs applied in the prevention and treatment of pulmonary hypertension,which will provide safer and more effective methods for clinical treatment of pulmonary hypertension.Methods:1.The rat pulmonary arterial hypertension model was prepared by a single injection of MCT 60 mg/kg.After 5 weeks,the rats pulmonary arterial acceleration time（PAT）,ejection time（ET）,pulmonary artery diameter（PAD）,right ventricular end-diastolic internal diameter（RVIDd）and right ventricular end-systolic internal diameter（RVIDs）were measured by the Vevo2100 ultra-high resolution animal ultrasound real-time molecular imaging system.Then we calculated the mean value of PAT/ET.The hemodynamics of rats were measured by means of the right heart catheter that was used to evaluate the models.2.Firstly,the effects of different structure alginate oligosaccharides on pulmonary arterial hypertension in rats were examined.Random groups of rats:PAH group（equivalent to PAH+M-3K-H group）,PAH+PGE1 group(PGE1 5ug·kg^-1·d^-1),PAH+M-3K-H group(M-3K 20mg·kg^-1·d^-1),PAH+M-6K-H group(M-6K 20mg·kg^-1·d^-1),PAH+G-3K-H group(G-3K 20mg·kg^-1·d^-1),PAH+G-6K-H group(G-6K 20mg·kg^-1·d^-1)and Control group(physiological saline 1ml·d^-1).Secondly,the effects of different doses of alginate oligosaccharides on pulmonary arterial hypertension in rats were monitored.Random groups of rats:PAH group（equivalent to PAH+M-3K-H group）,PAH+PGE1group(PGE1 5ug·kg^-1·d^-1),PAH+M-3K-L group(M-3K 5mg/kg^-1·d^-1),PAH+M-3K-M group(M-3K 10mg·kg^-1·d^-1),PAH+M-3K-H group(M-3K 20mg·kg^-1·d^-1)and Control group(normal saline 1ml·d^-1).By detecting the right ventricular hypertrophy index,the echocardiographic index,the ultrastructure of lung tissues and pulmonary arteries observing by HE staining and transmission electron microscopy,and the reconstruction of lung tissues and pulmonary arteries by masson staining,van Gieson staining and elastic fiber staining,the statistical comparison of each group of indicators was conducted.Then we evaluated the effects of alginate oligosaccharides on pulmonary arterial hypertension in rats,and observed the effects of different structures and doses of alginate oligosaccharides on pulmonary arterial hypertension in rats.3.Using a series of experimental methods such as immunohistochemical staining,in situ hybridization,westernblot,ELISA and so on,we studied whether different structures and doses of alginate oligosaccharides could relieve pulmonary arterial hypertension in rats by regulating sGC targets and observed the difference in activation of sGC targets.Results:1.After 5 weeks of intraperitoneal injection of MCT,rats developed symptoms such as wheezing,cyanosis of the lips,thick breathing sound,and the decreased activity tolerance.The results of cardiac ultrasound showed that the PAT and PAT/ET in the MCT group were significantly reduced compared to the Control group,and the differences were considered statistically significant(^**P<0.01).In the MCT group,RVIDd,RVIDs and PAD increased significantly,and the differences were considered statistically significant(^**P<0.01,^*P<0.05).The results of the right-heart catheter experiment showed that the RVSP in the Control group was 14.64±1.096 mmHg,and the RVSP in the MCT group was 56.30±1.243 mmHg.Compared with the Control group,the RVSP in the MCT group was significantly higher and the difference was considered statistically significant（^*P<0.01）.2.The results of the right ventricular hypertrophy index test showed that the RVHI in the PAH group was significantly higher than the Control group,^**P<0.01.Compared with the PAH group,RVHI was reduced to different degrees in each drug group,^##P<0.01,of which the significantly decrease was in PAH+M-3K-H group.Echocardiographic indexes comparison results show that compared with the Control group,RVIDd,RVIDs and PAD in PAH group were obviously higher,^**P<0.01 or^*P<0.05,PAT,PAT/ET decreased obviously,^**P<0.01.Compared with PAH group,the indexes in each drug group improved with various degree,PAH+M-3k-H group improved obviously among them.Histologic observations revealed that Alginate oligosaccharides can alleviate the structural disorders of lung tissues and pulmonary arteries in rats with pulmonary hypertension,reduce the thickness of pulmonary arterial vessels,expand the vascular cavity,reduce thrombosis in situ,alleviate inflammatory cell infiltration and collagen proliferation,and decrease vascular remodeling.The effects were related to oligosaccharide component and molecular weight.The effect of M component was better than that of G component,and the effect of 3K molecular weight was better than that of6K molecular weight.3.The results of immunohistochemistry and in situ hybridization experiment showed that,compared with the Control group,the expression protein levels of sGCα1and sGCβ1 in the PAH group were significantly lower,^**P<0.01.Compared with the PAH group,the expression protein levels of sGCα1 and sGCβ1 in each drug group was significantly higher,^##P<0.01or^#P<0.05.The results of Westernblot and ELISA experiments showed that compared with the Control group,the level of SOD,CAT,cGMP and the protein expression of sGCα1 and sGCβ1 in PAH group were significantly decreased,^**P<0.01.Compared with PAH group,the level of SOD,CAT,cGMP and the protein expression of sGCα1 and sGCβ1 in each dose group were increased with various degree,^##P<0.01 or^#P<0.05,PAH+M-3k-H group increased obviously among them.Also,the effects were related to oligosaccharide component and molecular weight.The effect of M component was better than that of G component,and the effect of 3K molecular weight was better than that of 6K molecular weight.Conclusions:1.Alginate oligosaccharides can relieve the structural disorders of lung tissues and reconstruction of pulmonary arteries in rats with pulmonary hypertension,thereby reducing pulmonary arterial pressure,reducing the right heart load,and improving the reconstruction of the right ventricle.2.Alginate oligosaccharides can increase the content of SOD and CAT in lung tissues by exerting the biological activity of scavenging oxygen free radicals and anti-oxidation.By activating the sGC targets and the H₂O₂-sGC-cGMP pathway,the alveolar vascular smooth muscle is relaxed,the pulmonary artery pressure is reduced,the alveolar ventilation/blood flow ratio is improved,so as to relieve the symptoms of pulmonary hypertension.3.The effects were related to oligosaccharide component and molecular weight.The effect of M component was better than that of G component,and the effect of 3K molecular weight was better than that of 6K molecular weight.