Clinical Effect And Mechanism Of Tengfujiangya Tablet On Protecting Against Endothelial Progenitor Cells Injury In Patients With Hypertension Based On LncRNA-p21/p53 Pathway

Objective: The purpose is to evaluate the clinical efficacy and safety of Tengfujiangya Tablet.We also measure the endothelial progenitor cells(EPCs)in the peripheral circulation of patients with hypertension.Based on the result of clinical trial,we carry out the experiment in vitro to reveal the intrinsic mechanism and target of Tengfujiangya Tablet in protecting against EPCs injury.Methods: Clinical study1.A randomized,single-blind,placebo-controlled clinical study was conducted.90 hypertensive patients(grade 1)with Liver-Yang hyperactivity syndrome were selected according to inclusion and exclusion criteria.They were randomly divided into experimental drug group and placebo group(45 cases each).Tengfujiangya Tablet and placebo were given for 4 weeks respectively.Before and after the study,TCM syndrome scores,blood pressure and the level of NO and ET-1 in serum were recorded to evaluate the efficacy;blood biochemical tests,electrocardiogram tests and adverse reactions were recorded to evaluate the safety.2.Peripheral blood samples were collected before and after the clinical study,and the expressions of CD133,CD34 and KDR were measured by flow cytometry to detect EPCs in order to explore the intervention effect of "Tengfujiangya Tablet" on EPCs in patients with hypertension.We also recruited 30 healthy volunteers and collected peripheral blood for EPCs detection to conduct EPCs data analysis as a control group.Experimental study in vitro1.First of all,we carried out primary culture and identification of EPCs from rat bone marrow.AngiotensinⅡ(AngⅡ)which is the main injury factors in hypertensive patients was used to explore effect on the apoptosis,adhesion,migration and tubular differentiation of EPCs.By adding autophagy activator or inhibitor to the up-regulate or down-regulate autophagy level,we explored the role of autophagy in AngⅡ-induced EPCs injury.2.Based on previous high-throughput sequencing results,we detected the expression of lnc RNA-p21 in EPCs treatment with AngⅡ.After overexpressing or knocking down lnc RNA-p21 through viral transfection,we detected the changes of cellular function and autophagy level of EPCs to preliminarily verify whether lnc RNA-p21 can regulate cell autophagy,and also explore the mechanism of EPCs injury induced by AngⅡ and the regulation mechanism of autophagy from the perspective of lnc RNA.3.Based on the clinical study and the above experimental study,we detected the effect of "Gou-Lai component" which is the main effective component of "Tengfujiangya Tablet" on lnc RNA-p21 expression.After overexpression or knockdown of lnc RNA-p21,we detected the changes of EPCs cell function,autophagy level and the expression of p53,AMPK,TSC2,m TOR and ATg13 after the intervention of "Gou-Lai component".We want to explore the target of "Gou-Lai component" on regulating lnc RNA-p21 expression and autophagy level,which could reveal the mechanism of "Gou-Lai component" on protecting against EPCs injury.Results: Clinical study1.Compared with placebo group,"Tengfujiangya Tablet" not only significantly reduced the blood pressure and improved the clinical symptoms of hypertensive patients,but also increased NO content in serum and decreased ET-1 concentration in serum,indicating that it shows a good effect on antihypertensiion and vascular endothelial protection;Adverse events such as adverse reactions occurred less,and clinical safety of "Tengfujiangya Tablet" was better.2.Compared with normal people,the number of EPCs in peripheral blood of hypertensive patients with Liver-Yang hyperactivity syndrome was significantly decreased,and the proportion of late EPCs was higher."Tengfujiangya Tablet" increased the number of EPCs in peripheral blood of patients with hypertension,and increased the proportion of EPCs in early stage,which is conducive to improving the repair of vascular endothelial injury.Experimental study in vitro1.We successfully primarily cultured EPCs from rat bone marrow in vitro.After 14 days of culture in vitro,double staining of Dil-ac LDL/FITC-UEA-1 and flow cytometry analysis of cell-labeled proteins showed that the proportion of EPCs was about 80%.Our study also showed that AngⅡ reduced the level of autophagy of EPCs,leading to apoptosis,decreased of adhesion,migration and tube formation.Rapamycin,an autophagy activator,reduced the apoptosis of EPCs and improve the cellular function of EPCs by up-regulating autophagy.3-MA,an autophagy inhibitor,was showed to inhibit autophagy and aggravate the damage effect of AngⅡ on EPCs.2.Our studies have confirmed that AngⅡ significantly reduced lncRNA-p21 expression in EPCs.Overexpression of lnc RNA-p21 can significantly improve the level of autophagy and protect against EPCs injury induced by AngⅡ.Knocking down lnc RNA-p21 can reduce the level of autophagy,which aggravated the damage effect of AngⅡ and reduced adhesion,migration and tubular differentiation of EPCs.3.The results showed that "Gou-Lai components" repaired the cell dysfunction of EPCs induced by AngⅡ,such as adhesion,migration and tubular differentiation.Further mechanism study revealed that "Gou-Lai component" promoted p53 transcription by upregulating lnc RNA-p21,and then increased the phosphorylation of AMPK,up-regulated TSC2 expression,inhibited the phosphorylation and activation of m TOR,and up-regulated Atg13 expression and the level of autophagy to antagonize the functional damage of EPCs.Conclusions: "Tengfujiangya Tablet" has good clinical efficacy and safety,and can protect against EPCs damage in patients with hypertension.Studies in vitro show that "GouLai component" promotes p53 transcription by up-regulating lnc RNA-p21,and then upregulates the level of autophagy to antagonize the functional impairment of EPCs induced by AngⅡ.