Study On The Mechanism Of YAP-targeted Compound Xueshuantong Regulating High Glucose-induced Endothelial Cell Functio

ObjectiveDiabetic retinopathy(DR)is one of the most serious complications of diabetes.DR is a disease that occurs in diabetic patients whose structure and function of blood vessels changes in the fundus.The compound Xueshuantong,which is composed of Panax notoginseng,Radix Salviae Miltiorrhizae,Radix Astragali and Radix Scrophulariae,has the ability of improving local microcirculation,dilating blood vessels,increasing blood flow,inhibiting angiogenesis,and preventing and treating DR.Previous research group found that the compound Xueshuantong could affect the expression of YAP in the retinal tissue of diabetic rats.YAP is a major effector molecule downstream of the Hippo signaling pathway and participates in the proliferation of endothelial cells and regulates angiogenesis.To further reveal the pharmacological mechanism of the compound Xueshuantong,the research group prepares different fractions of the compound Xueshuantong,uses gene silencing technology to inhibit the expression of YAP,and observes the effect of the compound Xueshuantong on the function of retinal vascular endothelial cells.Finallythe molecular mechanism and effective components of compound Xueshuantong which target YAP will be clarified,and the foundation for the secondary development of compound Xueshuantong will be layedMethods1.the effects of the differentingredients of the compound Xueshuantong Rhesus monkey retinal vascular endothelial cells were selected and divided into control group(5.5 mM glucose),high glucose group(25 mM glucose),and compound Xueshuantong groups(25 mM glucose+different ingredients with various doses).The CCK-8 assay was used to determine the cell viability,and the toxicity and effective doses of compound Xueshuantong were analyzed.Then the cell migration,the tube formation and the wound healing test were adopted to observe the effects of compound Xueshuantong on the high-glucose stimulated endothelial cells and to compare the effects of different ingredients of compound Xueshuantong.2.The effect of compound Xueshuantong on YAP and its target protein:The expression of YAP mRNA in cells was detected by Real time-PCR,and the expression levels of YAP,VEGF and VEGFR were detected by Western blotting.3.A sh-YAP plasmid was established and transfected into the endothelial cells.Verteporfin was used as a YAP inhibitor.Real time-PCR method or Western blot method was adopted to verify the reduced expression of YAP in endothelial cells.4.After YAP gene in the endothelial cell was inhibited,the effect of compound Xueshuantong on the cell migration and the tube formation was observed.Meanwhile,the effect of the compound Xueshuantong on the expression of VEGF and VEGFR was detected by Western blot.Results1.The effect of compound Xueshuantong on the cell viability was observed.Compared with the control group,the cell viability of the high glucose group increased.Compared with the high glucose group,the water extract,the component B and the component C of the compound Xueshuantong all can effectively inhibited cell viability.The component A and the component D,which were ineffective,increased cell viability.2.The effects of compound Xueshuantong on the cell migration was examined.Compared with the control group,the number of migrated cells in the high glucose group increased;compared with the high glucose group,the water extract,the component B and the component C decreased the number of migrated cells,and the water extract and the component C had better efficacy than the component B.3 The effects of compound Xueshuantong on the tube formation capacity was examined.Compared with the control group,the total tube length in the high glucose group increased;compared with the high glucose group,the water extract,the component C all reduced the total tube length,and the water extract had the best efficacy.4.The cell wound healing experiment was used to examine the effects of compound Xueshuantong on the cell migration.Compared with the control group,the high glucose increased the cell migration distance;compared to high glucose group,the water extract,the component B and the component C all reduced cell migration distance.5.Compared with the control group,the expression of YAP mRNA in the high glucose group increased.Compared with the high glucose group,the expression of YAP mRNA in the water extract decreased.Western Blot results showed that compared with the control group,the protein expression of YAP,VEGF and VEGFR increased in the high glucose group.Compared with the high glucose group,the protein expression of YAP,VEGF and VEGFR decreased in the group of water extract.6.A sh-YAP plasmid was successfully constructed and transfected into the cells,the PCR and WB results showed that the YAP expression was inhibited.Meanwhile,the YAP levels were reduced in VP stimulated cells.7.After sh-YAP plasmid was transfected,compared with the control group,the number of migrated cells and the total tube length of sh-YAP group decreased;compared with the compound Xueshuantong group,the number of migrated cells and the total tube length of sh-YAP+xst group and VP+xst group increased,and the efficacy of the water extract of compound Xueshuantong decreased.ConclusionThe water extract of compound Xueshuantong,who governs cell viability,migration and tube formation,exhibited the optimal efficacy.When YAP is inhibited,the ability of the compound Xueshuantong to modulate cell migration and the tube formation is weakened.And the effect of compound Xueshuantongon on the regulation of VEGF,the target gene of YAP,is also weakened.The efficacy of compound Xueshuantong is highly dependent on YAP.MeaningThis study not only clarifies that the compound Xueshuantong is a YAP-targeting traditional Chinese medicine compound that inhibits angiogenesis,examines the effective components of the compound Xueshuantong,but also provides a new target for prevention and treatment of DR.