Study On Effect And Mechanism Of Magnesium Lithospermate B On Ameliorating Hypoxic Pulmonary Hypertension

ObjectiveHypoxic pulmonary hypertension(HPH)is the basis pathogenesis of plateau sickness and various related diseases,it involves multiple organ lesions and gut microbes disorders.Despite current advances has been made in the study of vascular signaling pathways as targets,effective preventive/therapeutic drugs are still lacking.As the main active component of Chinese traditional medicine Radix Salvia Miltiorrhiza,Magnesium Lithospermate B(MLB)has extensive pharmacological effects on cardiovascular and tissue hypoxia protection.Therefore,this thesis mainly studies the preventive effect of MLB on HPH and its mechanism,and preliminarily explores the potential mechanism of MLB to improve the gut flora imbalance caused by HPH.It is expected to provide a theoretical basis for MLB to prevent/treat HPH related diseases.MethodsThree parts of experimental contents and methods were adopted in the current study:(1)The preventive effect of MLB on HPH and its mechanismStochastic model was used to divide rats into five groups,normoxia group,hypoxia group,low and high dose MLB group,and positive drug control group.After 30 days of administration,the mPAP and RVHI of each group were measured,and the samples of heart,liver,spleen,lung and kidney were collected.HE staining was applied to determine the histopathological changes.Immunohistochemistry and Immunofluorescence were applied to quantify the expression of?-SMA and EndMT in pulmonary blood vessels of HPH rats,respectively.TUNEL staining applicated for measuring tissues cell apoptosis,and qRT-PCR were used to determine the expression of related factors and pathway target proteins in lung tissue.(2)MLB inhibits pulmonary vascular remodeling in HPH through ROCK2 signaling pathwayHPASMCs exposed normoxic(21%O₂)and hypoxic(5%O₂)treatments to simulate the oxygen status of human PASMCs.PCNA staining was used to detect HPASMCs proliferation.Scratch healing experiment was conducted to investigate the migration of HPASMCs.Flow cytometry was applied to determine the apoptosis of HPASMCs.Western blotting and qRT-PCR were applied to detect the expression of related factors and signal path target proteins in HPASMCs.(3)Preliminary study on the mechanism of MLB in ameliorating HPH through gut floraStochastic model was adopted to divide rats into normoxia group,hypoxia group,MLB and positive drug control group,the experiment lasted for 4 weeks,at 8:00 AM every Thursday.Feces were collected by stress defecation method and placed in sterile enzyme-free test tubes.They were saved in a refrigerator at-80?after liquid nitrogen quick freezing.16S r RNA was adopted to determine the abundance of rat fecal flora and analyze the structure and diversity of the flora.ResultsCompared with the normoxia rats,in the hypoxia rats,the mPAP and RVHI were significantly increased,the lung,liver,spleen,kidney and heart tissue had obvious pathological changes,which means the experimental model was successfully established.The apoptosis and the expressions of?-SMA,vWF,EndMT,HIF-1?,NF-?B,MCP-1,PCNA,CyclinD1,CDK4,RhoA,ROCK1 and ROCK2 were significantly increased in lung tissue;mPAP and RVHI significantly decreased by MLB intervention,which also improved the histopathological changes to show normal tissue morphology and structure similar to the normoxia rats.MLB intervention inhibited the apoptosis of lung tissue,and down-regulated the mRNA expression of?-SMA,vWF,EndMT,HIF-1?,NF-?B,MCP-1,PCNA,CyclinD1,CDK4,RhoA,ROCK1 and ROCK2 in a dose-dependent manner.The intervention of High-MLB showed a similar preventive effect to that of Sildenafil,and the related indexes of the two drugs intervention were numerically close to those of normal rats.Hypoxia caused the proliferation and migration of HPASMCs,and restrained the apoptosis.Compared with the normoxia,hypoxia up-regulated the expression of HIF-1?,NOX4,?-SMA,CyclinD1,CDK4,OPN,PCNA,RhoA and ROCK,and down-regulated the expression of Calponin1.Compared with the hypoxia group,MLB intervention inhibited the proliferation and migration of HPASMCs,and promoted the apoptosis.Meanwhile,it down-regulated the expression of HIF-1?,NOX4,?-SMA,CyclinD1,CDK4,OPN and PCNA and up-regulated the expression of Calponin1 by inhibiting the activation of RhoA/ROCK signaling pathway in a dose-dependent manner.There was no significant difference in the regulation of the remodeling core protein between the High-MLB and the normoxia group.Overexpression of ROCK2 accelerated the proliferation and migration of HPASMCs,and it significantly inhibited the apoptosis.It also up-regulated the expression of ROCK2 downstream genes HIF-1?,?-SMA,CyclinD1,CDK4,OPN and PCNA;while ROCK2 silence showed the opposite effects.Compared with the normoxia rats,high altitude hypoxia significantly increased the gut flora F/B ratio in hypoxia rats.The?diversity index,acetate and butyrate metabolizing bacteria decreased,and a total of 47 species information with significant differences were found at the level of Kingdom,Phylum,Class,Order,Family and Genus.The?diversity analysis showed significant differences in bacterial community structure.Compared with the hypoxia rats,?diversity index showed an upward trend in the MLB and Sildenafil administration rats.The richness and diversity of the gut flora were restored,and the level was close to that of the normoxia rats.But there was no significant difference between the two drug groups.In addition,the proportion of butyrate metabolizing bacteria increased,while the proportion of lactate metabolizing bacteria decreased.The relative abundance of Odoribacterium and Clostridium increased significantly in rats with MLB administration.ConclusionMLB can ameliorate HPH by significantly reducing mPAP,RVHI and pulmonary arteriole remodeling of HPH rats,while inhibiting the activation of related factors and proteins and RhoA/ROCK signaling pathway in the tissue of HPH rats.MLB inhibits pulmonary vascular remodeling in HPH by regulating the proliferation,migration,apoptosis and remodeling core protein expression of HPASMCs through ROCK2signaling pathway.The phenomenon that MLB ameliorated the gut flora imbalance caused by HPH may be related to the protection of flora,the increase of beneficial metabolites,and the anti-inflammatory and anti-hypertensive effects of specific flora.These findings may offer new ideas for the research of MLB-ameliorating-HPH mechanism,and provide experimental basis for the clinical diagnosis and prevention of HPH.