Regulation On Endothelial Autophagy On Atherosclerosis With Propofol Through The PI3K/Akt/mTOR Pathway

Background Atherosclerosis（AS）is an age-associated chronic inflammatory disease induced by multiple factors.The exact cause of AS is still unclear.Currently,endothelial damage is considered the initial link of AS.Studies have shown that oxidized low-density lipoprotein（ox-LDL）can promotes vascular endothelial dysfunction and structural damage,which is one of the main factors causing endothelial damage,and is associated with cardiovascular diseases,especially AS.Propofol is a commonly used intravenous anesthetics that has been shown to relieve endothelial cell dysfunction through multiple mechanisms.However,it is unclear whether it alleviates endothelial cell damage caused by ox-LDL.Autophagy plays a critical physiological role in eukaryotic cells.In the case of energy or nutrient deficiency,it can prevent cell damage,thereby adapting to the ever-changing environment.Propofol has been shown to enhance autophagy to increase endothelial cell migration and angiogenesis,but its role in ox-LDL-induced endothelial cell damage and its mechanism are currently unclear.PI3K is a phosphatidylinositol kinase widely distributed in the cytoplasm.It is an important signal transduction molecule in the cells.It can be activated by a variety of extracellular signals such as hormones,growth factors and cytokines.Akt is finally activated by transferring from near the cell membrane to the cytoplasm.m TOR,as a key downstream signal of PI3K/Akt,can be activated by Akt phosphorylation to play a negative role in regulating autophagy.Studies have shown that the PI3K/Akt/m TOR signaling pathway is involved in the regulation of autophagy in AS.On the other hand,studies have also shown that propofol can inhibit the myocardial PI3K/Akt signaling pathway,but whether this inhibitory effect can regulate autophagy in endothelial cells after ox-LDL treatment remains to be clarified.In summary,this study speculates that propofol can upregulate autophagy of endothelial cells by inhibiting the PI3K/Akt/m TOR signaling pathway,therby reduing ox-LDL-induced endothelial damage.Objective In this study,HUVECs were incubated with ox-LDL and ApoE^-/-gene knockout mice were fed with high fatty diet were used as models to study the effect of propofol on damaged endothelial cells and the molecular mechanism of anti Atherosclerosis in vitro and in vivo.Methods1.HUVECs were treated with 0,10,20,50,100,and 150μg/m L ox-LDL for 24 h,or 100μg/m L ox-LDL for 6 h,12 h,24 h,48 h,72 h,or co-treated with 5,25 and 100μM propofol.Cell proliferation was detected by CCK-8 kit,apoptosis was detected by Annexin V/PI double staining and flow cytometry,cell damage was detected by LDH assay,and apoptosis-related proteins Bax,Bcl-2,Caspase3 and Cleaved-Caspase3 were detected by western blotting.The Caspase3 activity was detected by Caspase3 kit.2.HUVECs were treated with 100μg/m L ox-LDL for 6 h,12 h,24 h,and co-treated with 5,25 and 100μM propofol.Western blotting was used to detect autophagy-related proteins LC3-I,LC3-II,p62 and Beclin-1.Transmission electron microscopy was used to observe the intracellular autophagosomes.HUVECs were treated with 100μg/ml ox-LDL,and 10 n M Rpamycin（autophagy activator）and 100 n M Bafilomycin A1（autophage inhibitor）were used to interfere autophagy.Cell proliferation was detected by CCK-8 test;cell apoptosis was monited by Annexin V/PI double staining and flow cytometer assay;cell damage was analyzed by LDH test;the expression of autophagey-related protein LC3-I,LC3-II and Beclin-1 were detected by Western Blot assay.HUVECs were co-treated 100μg/m L ox-LDL with 100μM propofol,with presence or absence of autophagy inhibitor,100 n M Bafilomycin A1 or Beclin-1 si RNA to block cell autophagy.Transmission electron microscopy was used to observe the intracellular autophagosomes.Cell proliferation was detected by CCK-8 assay.Apoptosis was detected by Annexin V/PI double staining and flow cytometry,and the expressions of apoptosis-related proteins Bax,Bcl-2,Caspase3 and Cleaved Caspase3 were detected by western blotting.3.HUVECs were co-treated with 100μM propofol combined with 100μg/m L ox-LDL,with presence or absence of the PI3K activator 20μM 740Y-P.Western blotting was used to detect PI3K,p-PI3K,Akt,p-Akt,LC3-I,LC3-II,p62 and Beclin-1 expression.4.AS animal model was established in ApoE^-/-gene knockout male mice,fed with high-fat diet.Animals were administrated 75 mg/kg propofol with presence or absence of 22.4 mg/kg740Y-P（PI3K/Akt/m TOR signal activator）.Samples of orbital vein blood and aortic tissue samples were collected.The blood lipid content（TG,TC,HDL-C,LDL-C）was determined by the blood biochemical analyzer of each group;cryosectioning tissue of arcus aortae were stained with H&E and oil red O,and the pathological changes of the aorta and the formation of aortic plaque were observed under electron microscopy and microscope;Western blotting was used to detect LC3-I,LC3-II,p62 and Beclin-1 expression in aorta.Results1.Propofol improves the proliferation,and Decreases the apoptosis,cell damage and apoptosis pathway of ox-LDL-damaged HUVECs.HUVECs were treated with different concentrations of ox-LDL for 24 h.With the increase of ox-LDL concentration,the apoptosis level and cell damage of HUVECs gradually increased,and the cell survival rate gradually Decreased.HUVECs were treated with 100μg/m L ox-LDL for different duration.With the prolongation of duration,the apoptosis level and cell damage of HUVECs gradually increased,and the cell survival rate gradually Decreased.Propofol below 100μM had no obvious toxicity to HUVECs,but significantly improved the cell viability of HUVECs treated with 100μg/m L ox-LDL,Decreased cell apoptosis and cell damage,and gradually enhanced the expression level of anti-apoptotic protein Bcl-2,reduced the expression levels of pro-apoptotic proteins Bax and Cleaved-Caspase3 in a dose dependent effect manner,and decreased the activity of Caspase3 in HUVECs.2.Propofol upregulates ox-LDL-induced autophagy in HUVECsox-LDL treatment can induce autophagy in HUVECs at 24h.Autophagy activator Rapmycin further promoted the ox-LDL-induced autophagy,significantly upregulated protein expression of Beclin-1 and the ratio of LC3-II/I,which increased cell survival rate and reduced apoptosis and cell damages.In contrast,after the autophagy inhibitor Bafilomycin A1 blocked the occurrence of autophagy,it significantly downregulated Beclin-1 protein expression and the ratio of LC3-II/I,reduced the cell survival rate of HUVECs,and increased apoptosis and damage.Rapmycin or Bafilomycin A1 cannot significantly affect autophagy,proliferation,apoptosis,and damage in normal HUVECs,only in the presence of ox-LDL.Propofol further enhanced the autophagy level in ox-LDL-induced HUVECs,including up-regulated Beclin-1 protein expression and LC3-II/I ratio,and down-regulated p62 protein expression,the number of autophagosomes were increased and showed a dose dependent effect manner.However,the concentration of propofol below 100μM could not stimulate the autophagy of normal HUVECs,and only in the presence of ox-LDL could up-regulate Beclin-1 protein expression and LC3-II/I ratio.Propofol improved the proliferation,apoptosis,cell damage,and apoptotic pathway of ox-LDL-injured HUVECs.However,autophagy pathway inhibitors Bafilomycin A1 and Beclin-1 si RNA blocked autophagy and reduced the HUVECs protective effect of propofol,indicating that the protective effect of propofol on HUVECs damaged by ox-LDL was mediated by autophagy upregulation.3.Propofol ameliorates ox-LDL-induced HUVECs damage through enhancing autophagy via PI3K/Akt/m TOR pathway upregulates ox-LDL-induced autophagyHUVECs were pretreated with propofol and PI3K activator 740Y-P,and then ox-LDL was added to stimulate HUVECs.It was found that ox-LDL up-regulated the protein expression levels of p-PI3K and p-Akt,and activated the activity of PI3K/Akt signaling pathway.Propofol pretreatment significantly inhibited ox-LDL-activated PI3K/Akt signaling pathway activity,down-regulated p-PI3K and p-Akt protein expression levels,down-regulated p-m TOR/m TOR ratio and p62 protein expression levels,up-regulated LC3-II/I ratio and Beclin-1 protein expression level,while 740Y-P-activated-PI3K blocked propofol inhibiting PI3K/Akt/m TOR signaling pathway-mediated autophagy in HUVECs,up-regulated p-PI3K and p-Akt protein expression levels,up-regulated p-m TOR/m TOR ratio and p62 protein expression level,down-regulated LC3-II/I ratio and Beclin-1 protein expression level.4.Propofol improved autophagy level of aorta in ApoE^-/-mice fed with high-fat dietIt was verified that propofol could not only improved the pathological morphology of aorta and reduce the area of plaque formation in ApoE^-/-mice fed with high fat,but also up-regulated aortic Beclin-1 protein level,LC3-II/I ratio,and reduced p62 protein level.In the PI3K activator740Y-P co-treatment group,due to the activation of PI3K in ApoE-/-mice,it blocked the aortic atherosclerotic pathological improvement and autophagy enhancement effect of propofol,and lowered the level of aortic Beclin-1 protein and LC3-II/I ratio,and increased the level of p62protein.Conclusion This study shows that propofol can enhances autophagy by inhibiting the PI3K/Akt/m TOR signaling pathway,thereby reducing ox-LDL-induced endothelial cell damage and apoptosis.