Plantamajoside Improves Atherosclerosis Through The PTEN/AKT/GSK3? Signaling Pathway:Mechanism And Preliminary Clinical Studies

Objective:.To explore the main anti-inflammatory component of plantain through the clinical observation of the effects of plantain on the inflammatory response in atherosclerosis(AS)and to investigate the mechanism of plantamajoside(PMS)in improving the ox-LDL-induced inflammatory response and functional disorder of vascular endothelial cells through the PTEN/AKT/GSK3? signalling pathway,Method:1.Clinical study:Eleven patients with AS who met the eligibility criteria and five patients without AS(controls)were recruited.The patients with AS received treatment with plantain therapy added to the standard of care for 2 weeks;the pre-treatment and post-treatment interleukin(IL)-1?,monocyte chemoattractant protein(MCP)-1 and tumour necrosis factor(TNF)-a levels were observed in patients with AS and without AS.2.Experimental study:The PMS concentrations that did not have a significant effect on the growth of human umbilical vein endothelial cells(HUVECs)were identified by detecting the viability of HUVECs treated with different doses of PMS using CCK-8 assay.A cell model of AS with ox-LDL-induced vascular endothelial injury was prepared to examine the effects of PMS on the viability of and inflammatory response in HUVECs following ox-LDL induction and to explore the possible mechanism of PMS for improving the ox-LDL-induced inflammatory response and oxidative stress as well as apoptosis of HUVECsResults:1.Clinical study:The results of ELISA showed that after treatment with plantain therapy for 2 weeks,the IL-1?,MCP-1 and TNF-? levels in the plasma of patients with AS decreased from pre-treatment levels(particularly MCP-1,which had a statistically significant decrease,P=0.0322).UPLC-QTOF-MS assay showed that PMS was detectable in the plasma of patients with AS before using plantain therapy,indicating that plantain inhibits the inflammatory response in these patients through the action of PMS.2.Experimental study:(1)The PMS biological activity test showed that PMS could inhibit cell viability in a concentration-dependent manner.Higher concentrations(100 and 200 ?M)could significantly inhibit cell viability.Statistically significant differences from the 0 ?M(control)group were observed,indicating that high PMS concentrations are toxic to HUVECs.Therefore,further investigations with PMS concentrations<100 ?M(20 ?M,40 ?M and 80 ?M)were conducted.(2)A study of the effects of PMS on the viability of and the inflammatory response in HUVECs following ox-LDL induction showed that compared with the control group,the ox-LDL group demonstrated significantly inhibited HUVEC viability;increased cell apoptosis;up-regulated IL-1?,MCP-1 and TNF-? expression;increased ROS release;reduced NO release;increased MPO;reduced SOD,IKB-a(Ser32)phosphorylation level and NF-KBp65 expression in the nucleus;significantly increased LOX-1,ET-1,Bax,cleaved caspase-3 and PTEN expression;significantly reduced eNOS(Ser113),AKT(Ser473)and GSK3?(Ser9)phosphorylation levels and significantly down-regulated NF-icBp65 expression in the cytoplasm and I?B-? and Bcl-2 expression in total cellular protein.These results indicated that the ox-LDL-induced cell model of AS with vascular endothelial injury was successfully prepared.Further intervention with PMS 20?80 ?M significantly enhanced HUVEC viability;inhibited cell apoptosis;down-regulated IL-1?,MCP-1 and TNF-? expression;reduced ROS release;increased NO release:reduced MPO,enhanced SOD,I?B-?(Ser32)phosphorylation level and NF-?Bp65 expression in the nucleus;significantly reduced LOX-1,ET-1,Bax,cleaved caspase-3 and PTEN expression:significantly increased eNOS(Serl 13),AKT(Ser473)and GSK3?(Ser9)phosphorylation levels and significantly up-regulated the NF-?Bp65 expression in the cytoplasm and I?B-? and Bcl-2 expression in total cellular protein in a concentration-dependent manner.These results indicated that PMS acted to improve ox-LDL-induced AS with vascular endothelial injury,with the most prominent effect at 80 ?M.(3)PMS is involved in the ox-LDL-induced inflammatory response and oxidative stress and apoptosis of HUVECs through the PTEN/AKT/GSK3? signalling pathway.The study results showed that compared with the ox-LDL group,the presence of the AKT inhibitor(LY294002)resulted in a significantly reduced AKT(Ser473)phosphorylation level(from 2.09 ± 0.03 to 1.17± 0.03).Similarly,presence of PTEN inhibitor(SF1670)resulted in a significantly reduced PTEN expression(from 0.58 ± 0.03 to 0.43 ± 0.02)and a significant decrease in the corresponding GSK3?(Ser9)(from 2.27± 0.04 to 2.09± 0.07)and 1.12± 0.09,respectively,suggesting that the use of selective drugs achieved targeted inhibition of the PTEN/AKT/GSK3?signalling pathway.Furthermore,the addition of the AKT inhibitor(LY294002)reversed PMS's effects of enhancing HUVEC viability;down-regulating IL-1?,MCP-1 and TNF-?;down-regulating ROS and up-regulating SOD.In contrast,the addition of the PTEN inhibitor(SF1670)promoted PMS's effects of down-regulating IL-1?,MCP-1 and TNF-?;down-regulating ROS and down-regulating MPO.These results further confirm that PMS improved the ox-LDL-induced HUVEC injury and inflammatory response through the PTEN/AKT/GSK3? signalling pathway,and the effects were concentration-dependent.Conclusion:1.Plantain could inhibit the inflammatory response in patients with AS;PMS might be the main active component of plantain.2.PMS improves the ox-LDL-induced inflammatory response and functional disorder of vascular endothelial cells through the PTEN/AKT/GSK3? signalling pathway.