Effect And Mechanism Of Tibetan Medicine Tsantan Sumtang On Right Ventricular Function In Hypoxia-induced Pulmonary Hypertension Rats

Backgroud and Objective:At present,with the development of the plateau tourism and exploration,some acute and chronic plateau diseases have gradually become the focus of many researchers.High altitude pulmonary hypertension（HAPH）is one of the common high altitude diseases.It has been confirmed that right ventricular function is an important prognostic factor in patients with pulmonary hypertension.With the increase of pulmonary artery pressure,right ventricular afterload continues to increase,which leads to pathological hypertrophy of right ventricle.Long-term chronic hypoxia can directly lead to right ventricular cardiomyocyte loss and interstitial fibrosis,irreversible right ventricular function injury,and eventually right ventricular failure and even premature death.As a result,reducing right ventricular afterload by reducing pulmonary artery pressure or directly protecting right ventricular function has become a new treatment of PAH.Echocardiography is one of the main noninvasive methods of cardiac function examination,which is very important for the quantification of right ventricular systolic and diastolic function.Echocardiographic indexes including right ventricular diameter（RVID-DIA）,pulmonary artery acceleration time（PA-AT）and tricuspid annular plane systolic excursion（TAPSE）have been mature indexes for clinical evaluation of RV function.Since RV mainly depends on the change of afterload,the evaluation of RV function should include both pressure and volume measurement,which is called right ventricle-pulmonary artery coupling（RV-PA coupling）.fractional area change（FAC（%））,FAC/mPAP and TAPSE/PA-AT ratio were also considered as important indicators to evaluate RV-PA coupling.In addition,NT-proBNP was also an important predictor of RV function in PAH patients.Rho/Rho kinase pathway,as a negligible factor in motility,contraction,apoptosis and proliferation of cells,its impact on the cardiovascular system cannot be ignored.ROCK 1 and ROCK 2 are the two subtypes of Rho kinases,and studies have shown that ROCK 1 and ROCK 2 are closely related to hypoxic pulmonary hypertension and right heart hypertrophy.ROCK1 and ROCK2 are also important therapeutic targets in the treatment of pulmonary hypertension.NFATc3 and STAT3 are also closely related to right ventricular hypertrophy,which are the downstream factors of ROCK signaling pathway.In addition,RV fibrosis,oxidative stress and cardiomyocyte apoptosis are also important factors that cause RV decompensation and maladjustment.Tsantan Sumtang,which consists of Choerospondias axillaris（Roxb.）B.L.Burtt&A.W.Hill,Myristica fragrans Houtt.and Santalum album L.（1:1:1）,is a common prescription for the treatment of heart fever.The study showed that it could attenuate myocardial ischemia and improve left ventricular systolic and diastolic function in rats.Our previous study found that,Tsantan Sumtang reduced mean pulmonary artery pressure and improve right ventricular remodeling in hypoxia-induced pulmonary hypertension（HPH）rats.For further study on the effect and mechanism of Tsantan Sumtang on right ventricular function in HPH rats,the purpose of this research is showed as following:1)To research the effects of Tsantan Sumtang on right ventricular function in HPH rats;2)To detect the effect of Tsantan Sumtang on RV-PA coupling in HPH rats;3)To explore the mechanism of Tsantan Sumtang protecting right ventricular function in HPH rats;4)To investigate the material basis of Tsantan Sumtang with molecular docking.Methods1.The components of volatile oil in Tsantan Sumtang is detected by GC-MS.2.Groups:SD rats are divided into control group,hypoxia group and hypoxia+Tsantan Sumtang group(1.0 g·kg^-1·d^-1,1.25 g·kg^-1·d^-1,or 1.5g·kg^-1·d^-1).Hypoxia group and hypoxia+Tsantan Sumtang group were treated in a hypobaric hypoxic chamber（simulated high altitude hypoxia environment at 4500 m altitude）for 4 weeks.3.The relevant indicators of right ventricular systolic function were detected by echocardiography:right ventricular diameter（RVID-DIA）,pulmonary artery acceleration time（PA-AT）,and tricuspid annular plane systolic offset（TAPSE）.Computation RV-PA coupling related indicators:FAC（%）,TAPSE/PA-AT,TAPSE/PASP and FAC/mPAP.Then,the RVSP,mPAP,hematological indexes and Nt-proBNP content were measured.4.Rats are weighed and calculated the organ indexes:including right ventricular hypertrophy index（RV/（LVS）,RV/BW）.HE staining and transmission electron microscope（TEM）were used to evaluate the changes of morphology and ultrastructure in RV tissues of HPH rats.The effect of Tsantan Sumtang on apoptosis of HPH rats was evaluated by TUNEL staining and immunohistochemical staining.5.The level of SOD、MDA、GSH and GSH-PX of right heart tissue in HPH rat were detected to research the antioxidant ability of Tsantan Sumtang on right ventricular tissue of HPH rats.Western blotting、q RT-PCR analysis were used to detected the protein and mRNA expression levels of Bcl-2,Bax,caspase-3,ROCK1,ROCK2,NFATC3,P-STAT3 in HPH rats.6.Auto Dock Tools-1.5.6 software was used to investigate the material basis of Tsantan Sumtang.Result1.35 chemical constituents are identified in volatile oil of Tsantan Sumtang.Compounds 23,28 and 31 are Methyleugenol,1,3-Benzodioxole,4-methoxy-6-（2-propenyl）-and Isoelemicin,which were the main components in Myristica fragrans Houtt..Compounds 25、32 and 33 wereα-Bergamotene,α-Bisabolol andβ-Santalol respectively,which were the main components in Santalum album L.2.Effect of Tsantan Sumtang on right ventricular function in HPH rats:After hypoxia treatment,NT-proBNP content in HPH is increased,and the content of which is decreased after Tsantan Sumtang intervention.The RVID-DIA level in hypoxic group was decreased（P<0.05）than that of the control group,which is significantly decreased in Tsantan Sumtang group（P<0.05）.PA-AT and TAPSE are reduced in model group,but increased after Tsantan Sumtang intervention（P<0.05）.RV-PA coupling related indicators[TAPSE/PASP,FAC（%）,FAC/mPAP and TAPSE/PA-AT]are decreased under hypoxic conditions,which are increased after Tsantan Sumtang intervention（P<0.05）.3.The main physiological indexes of rats in hypoxia group including mPAP,RVSP,RV/BW and RVHI were significantly higher than those in the control group（P<0.05）.The Hct,HGB and RBC levels in serum of HPH rats were increased in hypoxia group,the WBC and PLT were lower than the control group（P<0.05）.4.In the hypoxia group,the physiological indexes of HPH rats,including mPAP,RVSP,RV/BW and RVHI,were higher than control group（P<0.05）,the levels of Hct,HGB and RBC in the serum of HPH rats were increased,while the levels of WBC and PLT were decreased（P<0.05）.After intervention with Tsantan Sumtang,the physiological indexes（mPAP,RVSP,RV/BW and RVHI）of HPH rats were decreased compared with hypoxia group,the levels of Hct,HGB and RBC were lower than those of in model group,and the levels of WBC and PLT were increased（P<0.05）.5.Effect of Tsantan Sumtang on the right ventricular structure of HPH rats:the myocardial fibers in the right ventricular tissue of HPH rats were obviously hypertrophy and sparse distribution.After the intervention of Tsantan Sumtang,the injury of myocardial tissue caused by hypoxia was significantly alleviated.The ultrastructural changes of heart tissue were observed by TEM,the chromatin of cardiomyocytes in the control group was evenly distributed,the mitochondria of cytoplasm were abundant,the structure was clear,and the muscle fibers were arranged neatly.However,in the hypoxic group,mitochondrial swelling was observed and mitochondrial structure was not obvious.After the intervention of Tsantan Sumtang,the damage of mitochondrial ultrastructure was reduced.Masson staining and immunohistochemical research showed that the fibrosis level of HPH rats in hypoxia group were increased significantly,and the fibrosis level is significantly decreased after Tsantan Sumtang intervention（P<0.05）.TUNEL staining showed that the apoptosis level of right ventricle increased in HPH rats,while it is decreased after Tsantan Sumtang intervention（P<0.05）.6.The contents of MDA in RV tissues is increased significantly,GSH-PX activity,GSH content and SOD activity were decreased of HPH rats in hypoxia group（P<0.05）.After Tsantan Sumtang intervention,it showed a increase in GSH content,SOD and GSH-PX activity,a reduction in MDA content compared with hypoxia group（P<0.05）.In hypoxia group,Bax,cleaved-caspase-3 expression level in HPH rats and Bax/Bcl-2 ratio were increased,while the Bcl-2 level was decreased compared with control group（P<0.05）.Tsantan Sumtang intervention reduced Bax and cleaved-caspase-3 expression,increased the Bcl-2 level compared hypoxia group（P<0.05）.Bax/Bcl-2 ratio was decreased by Tsantan Sumtang intervention.Meanwhile,after Tsantan Sumtang intervention,the ROCK1 and ROCK2 expression levels in HPH rats were decreased（P<0.05）.The mRNA and protein expression levels of NFATc3 and P-STAT3 which were the downstream factors of ROCK pathway are significantly lower（P<0.05）than those of hypoxia group.7.Molecular docking results showed that isoquercetin,ursolic acid,ricarin B,dehydrodiisobutylol and 2-（hydroxypropan-2-yl）-9-（3-methylbut-2-enyl）-2,3-dihydrof uro[3,2-g]chromen-7-one five monomer compounds had better binding ability to 4wot target protein,possibly potential compounds inhibiting ROCK proteins.Conclusion1.This study demonstrates that Tsantan Sumtang could restore RV function,improve RV-PA coupling,recover hemodynamic and hematological indexes,and protect RV against the structural maladaptive remodeling in HPH rats.2.The protective mechanism of Tsantan Sumtang on RV function in HPH rats was probably mediated by suppressing the ROCK signaling pathway and its downstream signaling molecules.3.The suppression of the ROCK signaling pathway may be the result of antioxidant and anti-apoptosis effects of Tsantan Sumtang4.Molecular docking results showed that Isoquercitrin,Licarin B,Ursolic Acid,Dehydrodiisoeugenol and 2-（2-hydroxypropan-2-yl）-9-（3-methylbut-2-enyl）-2,3-dihydrofuro[3,2-g]chromen-7-one are potential compounds for inhibiting ROCK proteins.