A Study On Absorption Of The Triterpenoid Saponins In Ilex Pubescens By Vascular Endothelial Cells And The Pharmacodynamic Mechanism Based On Autophagy

ObjectiveAtherosclerosis(AS)is the cause of most cardiovascular diseases.In the course of atherosclerosis,endothelial dysfunction caused by inflammatory factors infiltrating vascular endothelial cells is an important factor.Improving inflammatory damage and apoptosis of endothelial cells has become an effective measure for preventing and treating atherosclerosis.Triterpenoid saponins of Ilex puhescens(IPTS)is the main active ingredient of Ilex pubescehs,which applied to treat cardiovascular disease(CVD)and vascular endothelial cell injury.However,it is difficult to explain a paradox that IPTS were low bioavailability while exerting noticeable cardiovascular protective effects in vivo.Therefore,exploring the specific absorption and transport mechanism of IPTS in vascular endothelial cells is an effective idea to explain this paradox.At the same time,the mechanism of action of IPTS has not been fully elucidated.The pharmacological effect of PI3K/Akt-autophagy on endothelial cell injury is significant,and it is important for vascular endothelial cell injury in early atherosclerosis.Therefore,this study is intended to be from PI3K/Akt-From the perspective of autophagy mechanism,the efficacy and mechanism of IPTS on tumor necrosis factor(TNF-α)-induced inflammatory injury and apoptosis of human umbilical vein endothelial cells(HUVEC)were explored,which provided a new perspective for clinical use.Methods1.Absorption and transport mechanism of IPTS in HUVECUltra-High-Performance Liquid Chromatography Coupled with Quadrupole Time-of-Flight Mass Spectrometry(UPLC-QTOF-MS)was used to qualitatively analyze the influx components of IPTS,and High performance liquid chromatography-tandem mass spectrometry(HPLC-MS/MS)method was established to quantitatively analye the absorption characteristics and transmembrane transport mechanisms of IPTS components in HUVEC cells,such as ilexgenin A(C1)and ilexsaponin B1(C3).2.Effects of IPTS on inflammatory injury and apoptosis of vascular endothelial cellsTNF-α was induced to induce inflammatory injury in HUVEC cells,and model cells were treated with IPTS.Via some indicators were tested to evaluate the pharmacodynamic effects of IPTS on inflammatory model cells,such as cell viability,apoptosis rate.mitochondrial membrane potential(JC-1)and expression of related apoptotic proteins(Caspase-3,Caspase-9),the expression of related inflammatory proteins(ICAM-1,MMP-9)and so on.3.The role of P13K/AKT-autophagy in inhibiting endothelial cell inflammatory injury and apoptosis by IPTSBy using autophagy activator resveratrol(RSV)and inhibitor 3-MA,PI3K/AKT signaling pathway activator IGF]and inhibitor LY294002 acted together on TNF-α-induced HUVEC cells under IPTS intervention,respectively.Expression of inflammatory proteins(ICAM-1,MMP-9.MCP-1),apoptotic proteins(Caspase-3,Caspase-9),apoptotic ratesh and mitochondrial membrane potential(JC-1),etc.PI3K/AKT and autophagy play a key role in IPTS-induced TNF-α-induced inflammatory injury and apoptosis in HUVEC cellsResult1.Among the 13 components of IPTS,9 components can be absorbed by HUVEC cells,including 7 triterpenoid saponins;The absorption of the components of IPTS,such as chlorophyll A(C1)and hollyin B1(C3),is time,temperature and concentration dependent.The absorption of both components has a bimodal phenomenon,and equilibrium can be achieved after 6 hours.;The uptake and transport of C1/C3 components are passively diffused and actively transported.The carrier protein SGLT1 may mediate the transmembrane transport of C1/C3,and verapamil can significantly increase C1/C3.Accumulation in cells:2.Compared with the TNF-α-induced model group,IPTS can effectively improve the cell morphology damage of HUVEC.increase cell activity,reduce the expression of inflammation-related proteins ICAM-1 and MMP-9,and achieve anti-inflammatory effects,and significantly inhibited Apoptosis can also reduce the decrease of mitochondrial membrane potential and nuclear staining,which can effectively prevent and improve HUVEC inflammatory injury and apoptosis.3.IPTS can significantly activate autophagy in model cells,and autophagy plays an important role in inhibiting TNF-α-induced inflammation and apoptosis of HUVEC cells;IPTS can significantly inhibit the PI3K/AKT/mTOR signaling pathway,while the PI3K/AKT/mTOR signaling pathway is involved in the regulation of autophagy to inhibit TNF-α-induced HUVEC cell inflammation and apoptosis,but regulates IPTS through autophagy.There is insufficient evidence to improve the mechanism of model cell inflammation and apoptosis.Therefore.whether there are other signaling pathways involved in the regulation of autophagy and the role of IPTS in inhibiting TNF-α-induced inflammation and apoptosis of HUVEC cells needs further investigation.Conclusion1.This study analyzed the absorption components.component absorption and transmembrane transport mechanism of IPTS in HUVEC cells by qualitative and quantitative analysis,and proved that IPTS can be specifically absorbed by vascular tissues and cells,and can be permanently distributed in vascular cells..2.Using TNF-α-induced inflammatory injury and apoptosis model of HUVEC cells as the research object,the effects of IPTS on inflammatory injury and apoptosis of model cells were investigated,and the cellular inflammation and apoptosis of IPTS inhibition model were explained from the perspective of autophagy.The mechanism of action of death.3.The mechanism of PI3K/AKT signaling pathway regulating IPTS to improve inflammatory injury and apoptosis in model cells through autophagy is insufficiently evidenced,so whether there are other signaling pathways involved in the regulation of autophagy and inhibition of TNF-α induced HUVEC by IPTS Further research is needed to play a role in cellular inflammatory damage and apoptosis.