Effect And Mechanism Of Rhodiola Tangutica On Pulmonary Vascular Endothelium In Hypoxia-induced Pulmonary Hypertension Rats

Objective and BackgroundPulmonary hypertension（PH）is a devastating disease with poor prognosis and high mortality.Hypoxia induced pulmonary hypertension（HPH）is a persistent threat to human health,especially to people who live on high altitude plateau.Pulmonary artery endothelial cells（PAECs）are the first layer to be exposed to changed oxygen levels and involved in numerous pathophysiological processes,including in vasoconstriction,oxidative stress,cell growth and differentiation.Hypoxia as a major component of plateau environment,could lead to PAECs dysfunction.Endothelial-derived nitric oxide（NO）is the most important bioactive molecule,which could regulate endothelial homeostasis.In the early stage of our research group,the hypobaric chamber was used to simulate the hypoxia environment of plateau,and it was found that Rhodiola tangutica（R.tangutica）had an intervention effect on the pulmonary hypertension established of rats in hypobaric hypoxia environment.The bioactive fraction of R.tangutica was screened by the vascular ring perfusion method in vitro and verified that its vasodilatory effect was mainly acting on the vascular endothelium and showed dose dependence.L-NAME,as an endothelial nitric oxide synthase（e NOS）inhibitor,could effectively inhibit the production of nitric oxide（NO）and block the role of vasodilatory effect of the bioactive fraction of R.tangutica,indicating that NO occupies an important position in the role of diastolic pulmonary arterioles.In this study,the active ingredients in the bioactive fraction of R tangutica which have a protective effect on pulmonary artery endothelial cells（PAECs）were screened,and whether the mechanism was associated with NO-related signaling pathway proteins.Methods1.CCK-8 was used to screen the bioactive fraction from R.tangutica（ACRT）that were resistant to hypoxia-induced damage to PAECs,and NO detection kits was continued to be used to screen the screened ACRT which could increase the content of NO in hypoxia-induced PAECs supernatant.Combined with the proportion of the main components in the ACRT and the results of molecular docking by Autodock Tools,screened out the final bioactive fraction,then acted on the animal level and the cell level respectively.2.Animal level,SD rats were randomly divided into 7 groups with 15 rats in each group.Control group:normoxia environment,0.5%sodium carboxymethyl cellulose solution（CMC-Na）,intra-gastric gavage（ig）.Model group:Rearing in hypobaric hypoxic chamber,0.5%CMC-Na,ig.Rats with different drug interventions were housed in a hypobaric hypoxic chamber and administrated by intra-gastric gavage.Luteolin group:two doses of luteolin 50 mg/kg/d and 100 mg/kg/day.Sildenafil group:sildenafil 30 mg/kg/d.L-NAME group:L-NAME 40 mg/kg/d.L-NAME+Luteolin group:L-NAME（40 mg/kg/d）+Luteolin（100 mg/kg/d）.After 28 days,hemodynamic indexes,histopathological changes,pulmonary artery endothelial function,NO content and arginase activity in lung tissue,NO related pathway proteins（HIF-2α、HIF-1β、Arg2、AKT1、p-AKT1-T308、p-AKT1-S473、e NOS、p-e NOS-S1177）expression were measured to evaluate the effect of luteolin on HPH.3.Cellular level,PAECs were given in three different treatments:1)Normoxia,Hypoxia,Hypoxia+Luteolin（1μM）group,Hypoxia+Luteolin（1μM）group.The activity of PAECs and the expression of NO related pathway proteins（HIF-2α,HIF-1β,Arg2,AKT1,P-AKT1-T308,P-AKT1-S473,e NOS,P-ENOS-S1177）were determined.2)Normoxia,Hypoxia,Hypoxia+Luteolin（10μM）,Hypoxia+LY294002（10μM）,Hypoxia+Luteolin（10μM）+LY294002（10μM）.The expressions of AKT1 and its phosphorylated protein,e NOS and its phosphorylated protein were measured.3)Normoxia,Hypoxia,Hypoxia+Luteolin（10μM）,Hypoxia+L-NAME（100μM）,Hypoxia+Luteolin（10μM）+L-NAME（100μM）.NO content in cells supernatant was tested.Normoxia condition:21%O₂-5%CO₂,24 h.Hypoxia condition:1%O₂–5%CO₂–94%N₂,24 h.To reveal the protective mechanism of luteolin at the cellular level.Results1.CCK-8 and nitric oxide detection kit were used to screen the bioactive fraction from R.tangutica with pulmonary vascular endothelial protective effect,and finally screening out Luteolin,as a research object.Luteolin not only protects hypoxia-induced damage to pulmonary artery endothelial cells,but also increases the production of vasodilation substance NO.2.Animal level,the effects of luteolin on physiological indexes of HPH rats:compared with the control group,the mean pulmonary arterial pressure（m PAP）and[RV/LV+S]ratio in the model group were increased,and they were decreased after luteolin treatment（P<0.05）.Morphological analysis of pulmonary arteries stained with H&E showed that significant remodeling of pulmonary arteries in model group,and which was alleviated after luteolin treatment.The fibrosis analysis of pulmonary vessels using Masson staining showed that there was mild blue-stained fibrous tissue in the outer membrane of the artery in rats in control group,and there was almost no blue-stained fibrous tissue in middle and inner membrane of the artery.Scattered blue fibrous tissue appeared in the smooth muscle tissues of pulmonary artery in model group,additionally,the inner membrane thickened and fibrosis,the outer membrane fibrosis was obvious.Fibrosis degree was decreased after luteolin intervention Immunofluorescence double staining showed thatα-SMA protein was significantly decreased and v WF protein expression was increased after luteolin treatment（compared with model group,all P<0.05）.The results of ex vivo lung perfusion showed that after luteolin（100 mg/kg）intervention,the value ofΔm PAP was significantly increased(compared with model group（P<0.05）.The arginase activity in lung tissue was increased under hypoxia（P<0.05）,and decreased after luteolin intervention（P<0.05）.The results of NO measurement in lung tissue suggested that NO production was reduced after luteolin intervention（P<0.05）.Western Blotting results showed that the expression levels of HIF-2α-Arg axis and PI3K-AKT-e NOS-NO pathway related proteins was down-regulated after luteolin treatment（P<0.05）.3.Cellular level,NO content in cells supernatant was decreased after 1%O2intervention,and was increased after luteolin（10μm）treatment（P<0.05）.After treatment with e NOS inhibitor L-NAME（100μM）,the NO content was further decreased compared with 1%O2 intervention.After treatment with luteolin and L-NAME,the NO content was decreased compared with luteolin alone,but increased compared with L-NAME（P<0.05）.Western Blotting showed that there were no significant effect on the total protein levels of AKT1 and e NOS（P>0.05）.However,the phosphorylated protein levels of AKT1（p-AKT1-T308,p-AKT1-S473）and e NOS（p-e NOS-S1177）were significantly up-regulated（P<0.05）.Furthermore,PI3K inhibitor LY294002 was used to clarificate the mechanism of luteolin on PI3K-AKk T-e NOS-NO pathway.The results showed that there were no effect on the total protein expression of AKT1 and e NOS（P>0.05）.The phosphorylation of AKT1-S473 was inhibited after LY294002 treatment（P<0.05）,but had no significant effect on the phosphorylation of AKT1-T308（P>0.05）.The phosphorylation levels of AKT1-S473and AKT1-T308 were significantly increased after luteolin intervention（P<0.05）.Compared with 1%O₂ alone,the phosphorylation level of e NOS-S1177 protein was not further inhibited after LY294002 treatment（P>0.05）.The phosphorylation level of e NOS-S1177 protein was significantly increased after luteolin intervention（P<0.05）.After combined administration of luteolin and LY294002,the phosphorylation level of e NOS-S1177 protein was significantly increased compared with LY294002alone（P<0.05）.Compared with control group,HIF-2αexpression in PAECs in 1%O₂group was increased（P<0.05）.After luteolin 10μmol/L intervention,the expression of HIF-2αdecreased compared with1%O₂ group（P<0.05）.There was no significant difference in HIF-1βexpression in PAECs between control group and1%O₂ group（P>0.05）.Compared with1%O₂ group,the expression of HIF-1βwas decreased after luteolin 10μmol/L intervention（P<0.05）.Compared with control group,Arg2expression in 1%O₂ group was increased（P<0.05）.Compared with 1%O₂ group,the expression of Arg2 was decreased after luteolin（1μmol/L,10μmol/L）intervention（P<0.05）.Conclusion1.In the early stage,UHPLC-Q-TOF-MS/MS was used to identify the main chemical components in the bioactive fraction from R.tangutica.Luteolin,which has good protective effect on pulmonary artery endothelial cells,was screened by CCK8and NO kit.Luteolin can alleviate hypoxia-induced damage to pulmonary artery endothelial cells and can increase the production of the vasodilation substance NO.It is suggested that Luteolin is involved in the vasodilation effect of the bioactive fraction of R.tangutica.2.Through animal experiments,it was found that Luteolin can decrease the pressure of pulmonary hypertension,alleviate right ventricular hypertrophy and pulmonary vascular remodeling,protect pulmonary vascular endothelial function in HPH rats.3.The protective mechanism of Luteolin on pulmonary vascular endothelial function in HPH rats was mediated by inhibiting HIF-2α-Arg axis and regulating PI3K-AKT-e NOS-NO signaling pathway related proteins.