Effect Of Zedolongdiol On The Proliferation Of Vascular Smooth Muscle Cells And Endothelial Cell Injury

Vascular smooth muscle cells(VSMCs)proliferation and endothelial cells injury play a key role in atherosclerosis(AS)and in-stent restenosis(ISR).Platelet-derived growth factor-BB(PDGF-BB)is relased after vascular injury,which contributes significanlty to VSMCs proliferation.Oxidized low density lipoprotein(ox-LDL)is a major cause for endothelial cells injury.In response to ox-LDL,endothelial cells initiate oxidative stress and inflammatory response,which accelerate the progression of atherogenesis.Zedoarondiol,a sesquiterpene lactone compound extratcted from Zedoariae rhizoma(Curcuma zedaria Roscoe,e'zhu in Chinese),has been reported to inhibit inflammation in lipopolysaccharide-stimulated murine macrophages and attenuate D-galactosamine-induced liver injury in mice.Our previous study has demonstrated that stents coated with Zedoariae rhizoma essential components,mainly including zedoarondiol,suppressed neointimal formation and showed well-developed endothelium in a porcine coronary arterial balloon-injury model.However,the underlying mechanisms remain unclear.In the present study,we hypothesized that zedoarondiol inhibited PDGF-BB-induced VSMCs proliferation via adenosine monophosphate-activated protein kinase(AMPK)pathway and attenuated ox-LDL-induced endothelial cells injury via NF-E2-related factor 2(Nrf2)pathway.To test this hypothesis,we conducted studies from two aspects:(1)we investigated the effects of zedoarondiol on PDGF-BB-induced VSMCs proliferation and explored its mechanisms focused on adenosine monophosphate-activated protein kinase(AMPK)pathway;(2)we investigated the effects of zedoarondiol on ox-LDL-induced human umbilical vein endothelial cells(HUVECs)injury and explored its mechanisms focused on NF-E2-related factor 2(Nrf2)pathway.Part 1 Effects of zedoarondiol on PDGF-BB-induced VSMCs proliferationObjective: To demonstrate that zedoarondiol inhibited PDGF-BB-induced VSMCs proliferation through AMPK pathway.Methods: PDGF-BB(20ng/m L)was used to induce VSMCs proliferation.The inhibitory effects of zedoarondiol on PDGF-BB-induced VSMCs proliferation were evaluated by cell counting and the Cell Counting Kit-8(CCK-8)assay.DNA synthesis was examined by bromodeoxyuridine(Brd U)incorporation assay.Cell cycle was assessed by propidium iodide staining.Western blotting was performed to determine the expression of cyclin-dependent kinase 2(CDK2),cyclin E,p53,p21,total and phosphorylated adenosine monophosphate-activated protein kinase(AMPK),acetyl Co A carboxylase(ACC),mammalian target of rapamycin(m TOR),and p70 ribosomal protein S6 kinase(p70S6K).Results: PDGF-BB significantly increased VSMCs number,VSMCs viability,and DNA synthesis(P<0.05).Zedoarondiol suppressed PDGF-BB-induced VSMCs proliferation and DNA synthesis(P<0.05).In addition,zedoarondiol increased the proportion of cells in G0/G1 phase(P<0.05)and decreased the proportion of cells in S phase(P<0.05).Zedoarondiol did not induce apoptosis in VSMCs exposed to PDGF-BB(P>0.05)and had no significant effect on the viability of normal cultured VSMCs(P>0.05).Besides,zedoarondiol activated AMPK and ACC,inhibited the phosphorylation of m TOR and p70S6 K,increased the expression of p53 and p21,and decreased the expression of CDK2 and cyclin E(P<0.05).Pretreatment with zedoarondiol and compound C(an AMPK inhibitor)abrogated zedoarondiol-mediated inhibition on cell number,cell viability,and DNA synthesis in PDGF-BB-treated VSMCs(P<0.05).However,5-aminoimidazole-4-carboxamide 1-?-ribofuranoside(AICAR,an AMPK activator)enhanced zedoarondiol-mediated inhibition on cell number,cell viability,and DNA synthesis in PDGF-BB-treated VSMCs(P<0.05).Conclusion: Zedoarondiol inhibits PDGF-BB-induced VSMCs proliferation via AMPK-mediated down-regulation of the m TOR/p70S6 K pathway and up-regulation of the p53/p21 pathway.Part 2 Effects of zedoarondiol on ox-LDL-induced HUVECs injuryObjective: To demonstrate that zedoarondiol attenuated ox-LDL-induced endothelial cells injury through Nrf2 pathway.Methods: The protective effects of zedoarondiol on ox-LDL-induced HUVECs injury were evaluated by CCK-8 assay and released lactic dehydrogenase(LDH)activity assay.Oxidative stress was determined by methane dicarboxylic aldehyde(MDA)content and superoxide dismutase(SOD)activity.The level of reactive oxygen species(ROS)was measured by dichlorodihydrofluorescin diacetate(DCFH-DA)staining.The culture supernatant was collected for enzyme linked immune-sorbent assays(ELISA)of interleukine-1?(IL-1?),tumor necrosis factor-?(TNF-?),and monocyte chemoattractant protein-1(MCP-1).Immunofluorescence staining was used to observe NF-E2-related factor 2(Nrf2)translocation.Western blotting was performed to determine the expression of IL-1?,TNF-?,MCP-1,heme oxygenase-1(HO-1),and Nrf2.Results: Ox-LDL significantly decreased cells viability and SOD activity(P<0.05),and increased released LDH activity,MDA content,ROS generation,levels of IL-1?,TNF-?,and MCP-1 in the cell culture supernatant,and protein expression of IL-1?,TNF-?,and MCP-1(P<0.05).Zedoarondiol(20?g/m L)pretreatment obviously increased cells viability and SOD activity(P<0.05),reduced LDH activity,MDA content,and ROS generation(P<0.05)in ox-LDL-treated HUVECs.In addition,zedoarondiol(20?g/m L)pretreatment inhibited secretion and protein expression of IL-1?,TNF-?,and MCP-1(P<0.05).Zedoarondiol had no significant impact on cells viability,oxidative stree,and imflammatory response in mormal cultured HUVECs(P>0.05).Zedoarondiol induced Nrf2 translocation into the nucleus,increased expression of HO-1 and Nrf2 in the nucleus(P<0.05).In addition,pretreatment with zedoarondiol and all-trans-retinoic acid(ATRA),an inhibitor of Nrf2 abolished zedoarondiol-mediated protective effects on injured HUVECs(P<0.05).Conclusion: Zedoarondiol attenuates endothelial cells injury induced by ox-LDL by inhibiting oxidative stress and inflammation via Nrf2/HO-1 pathway.