Hydrogen-rich Saline Attenuates Vascular Smooth Muscle Cell Proliferation And Neointima Hyperplasia By Inhibiting ROS Production And Inactivating Ras-ERK1/2-MEK1/2and Akt Pathways

Percutanerous coronary intervention(PCI) is very important therapy forcoronary heart disease.Modern application of drug-eluting stents has improvedoutcome in these patients;however,5-10%of patients are still affected byrestenosis after coronary stent placement.VSMCs proliferation and migration in the arterial wall is crucial in thedevelopment of post-angioplasty restenosis and atherosclerosis.Reactive oxygen species (ROS) and oxidative stress are involved in thepathogenesis of intimal thickening in atherosclerosis and restenosis.Hydrogen is a potent antioxidant, able to selectively scavenging andneutralizing hydroxyl radicals (such as OH and ONOO-). Hydrogen act as ascavenging agent, selectively neutralizing ROS and exerting potent cellularprotective effects. The exact mechanisms by which hydrogen exerts its effectson the vascular wall remain unknown. Besides its antioxidant effects, there aresome indications that it may directly interact with some specific pathways.The present study aimed to examine the anti-proliferative effects of HRSSon abnormal vascular smooth muscle cells (VSMC) proliferation and toinvestigate the mechanisms responsible for these effects.We detected theeffects of HRSS on VSMC proliferation, migration,cell cycle,cellapoptosis,We further investigated the pathways involved in proliferation andmigration of VSMC.Inadition,In vitro rat balloon injury model was made inorder to mini procedure of PTCA and further investigated the effects of HRSSon neointima hyperplasia and possible mechanism.1Hydrogen-rich medium attenuates vascular smooth muscle cell proliferationby inhibiting ROS production and inactivating Ras-ERK1/2-MEK1/2and Akt pathways.We detected the effects of HRM on VSMC proliferation, migration,cellcycle,cell apoptosis,and then we further investigated the pathways involved inproliferation and migration of VSMC. The results were as follows:1.1HRM inhibit FBS-induced VSMCs proliferation and migrationMTT assay showed that VSMC viability in the HRM group wassignificantly lower than in the control group (32.4±5.2%vs.73.3±5.1%, P <0.01). Two VSMC cultures were treated with HRM for48h; Hydrogen wasthen ceased for one culture, while continued in the other. After the removal ofhydrogen, cells resumed viability. Cells without hydrogen treatment exhibiteda BrdU index of64.1±11.1%, compared to26.7±4.9%in the hydrogen group.VSMCs cell count was smaller in HRM-treated cells after48h and72h(40%and51%reductions, respectively, P<0.01). VSMCs treated with FBSfor24h showed greater mobility (FBS:58.3±13.9vs. hydrogen:18.6±5.8cells, P<0.01).1.2HRM prevent FBS-induced S-phase entry in VSMCsTreatment with HRM inhibited FBS-induced G1-S progression, asdemonstrated by the increase in G0/G1cells (68.8±2.2%) accompanied byconcurrent decrease in S-phase cells (16.7±2.0%). These results suggest thathydrogen may prevent FBS-induced S-phase entry in VSMCs via a G0-G1blocking mechanism.1.3Ras-MEK1/2-ERK1/2pathway are involved in the effect of HRM onFBS-stimulated VSMCsStimulation of cells with10%FBS induced Ras activation. Cells treatedwith HRM exhibited a slight inhibition of FBS-induced Rasactivation.Treatment of FBS-stimulated VSMCs with HRM dramaticallydecreased phosphorylated MEK1/2levels. Data showed that FBS also induceda profound increase in ERK1/2activation. Treatment with HRM significantlyinhibited FBS-stimulated phosphorylation of ERK1/2. In contrast, totalERK1/2protein levels were not altered by treatment with hydrogen.1.4The effect of HRM on Akt phosphorylation FBS induced a profound increase in Akt activation. The level of Aktphosphorylation after FBS stimulation was also significantly inhibited bytreatment with HRM.1.5The effect of HRM on PCNA expressionFBS-induced PCNA expression was significantly inhibited by HRM by50%(P<0.01).1.6ROS is involved in the effect of HRM on FBS-stimulated VSMCsFBS treatment significantly induced intracellular peroxide production.ROS generation in cells treated with HRM was395.6±16.1, compared with483.0±15.1for controls (P <0.01).8-OHdG levels in cells treated with HRM were1.3±0.0ng/ml, compared to1.6±0.1ng/mL in controls (P <0.01).Result shows that treatment with HRM significantly inhibited MDAgeneration (FBS3.7±0.2vs. hydrogen2.9±0.1, P <0.01).2Hydrogen-rich saline attenuates neointima hyperplasia by inhibitingROS production and inactivating Ras-ERK1/2-MEK1/2Rat balloon injury model was made in order to mini procedure of PTCAand further investigated the effects of HRSS on neointima hyperplasia andpossible mechanism. The results were as follows:2.1The effect of HRSS on neointima hyperplasiaBalloon-induced intimal hyperplasia was evident in treated rats comparedwith rats of the control group. Results showed that3doses of HRSS wereeffective for prevention of neointimal formation in a dose-dependent manner.These findings showed intima/media ratio reductions of36.9%,52.2%and71.8%(P <0.01) in HRSS treated groups using2.5ml,5ml, and10ml/kgdoses, respectively, compared with the balloon-injured control group.2.2Effects of hydrogen-rich saline on proliferated cell nuclear antigenimmunostainingPCNA-positive cells were abundant in the balloon injury group. However,PCNA immunostaining was much less apparent in cells treated with HRSS.Staining intensities were lower in HRSS-treated rats (P <0.01). 2.3Hydrogen-rich saline inhibited neointimal hyperplasia induced by ballooninjury via suppression of Ras-MEK1/2-ERK1/2pathwayTo determined whether the Ras-MEK1/2-ERK1/2signal pathway isinvoled in suppression of neointimal hyperplasia by hydrogen-rich saline invivo,we also evaluated the effect of hydrogen-rich saline on the ERK signalingcascade activated by balloon injury.Western blot analysis showed that theactivation of ERK1/2,MEK1/2,and Ras was strongly suppressed with a dose-dependent maner in hydrogen-rich saline treated samples on day14afterballoon injury,compared with control samples(P<0.01).These results furthersupport the concept that hydrogen plays an important role in the inhibition ofthe FBS or injured-activated ERK1/2signaling cascade and VSMCproliferation in vitro and in vivo.2.4Inhibitory effect of hydrogen-rich saline on ROS generation in hyperplasianeointimaTo further confirm that Hydrogen-rich saline induced inhibition ofneointimal hyperplasia was related to the anti-oxidative property ofhydrogen-rich saline,two oxidative stress markers were measured.Resultsshowed hydrogen-rich saline significantly inhibited the levels of carotid arteryMDA and8-OHdG in a dose-dependent manner.3HRM attenuated VSMC proliferation and migration through cell apoptoticpathway3.1HRM increase the proportion of apoptotic cells in VSMCsProportion of early apoptotic cells increased from1.36%(FBS treated) to2.1%(FBS+hydrogen treated)(P <0.05). These results also showed that theproportion of late apoptotic cells increased from6.96%to13.47%(P <0.01).3.2The effect of HRM on Bax/Bcl2ratioAfter HRM treatment, a significant increase in Bax/Bcl2ratio (P <0.01)indicated that VSMCs were, in fact, progressing towards apoptosis3.3The effect of HRM on8-OHdG level8-OHdG levels in cells treated with HRM were1.3±0.0ng/ml, comparedto1.6±0.1ng/mL in controls (P <0.01). 3.4The effect of HRM on MDA levelResult shows that treatment with HRM significantly inhibited MDAgeneration (FBS3.7±0.2vs. hydrogen2.9±0.1, P <0.01).Conclusion:1HRM inhibited proliferation and migration of VSMCs.the possiblemechnisam include:1.1HRM prevent FBS-induced S-phase entry in VSMCs by multiplepathways.1.2Ras-MEK1/2-ERK1/2pathway are involved in the effect of HRM onFBS-stimulated VSMCs proliferation and migration1.3P13k-Akt pathway are involved in the effect of HRM on FBS-stimulatedVSMCs proliferation and migration1.4Inhibiting the expression of PCNA at the nuclear level is one of theimportant mechnisam1.5Inhibitory effect of hydrogen-rich saline on ROS generation in hyperplasianeointima indicated HRM taken action by inhibiting oxidative stress.2HRSS inhibited neointimal hyperplasia.the possible mechnisam include:2.1Inhibitory effect of hydrogen-rich saline on ROS generation in hyperplasianeointima indicated HRSS taken action by inhibiting oxidative stress.2.2Inhibiting the expression of PCNA at the nuclear level is one of theimportant mechnisam.2.3Hydrogen-rich saline attenuates neointima hyperplasia by inhibitingROS production and inactivating Ras-ERK1/2-MEK1/2.3HRM attenuated VSMC proliferation and migration through cell apoptoticpathway3.1Early apoptosis and late apoptosis were significantly increased after HRMintervention.3.2Bax/BCL2ratio increased significantly indicated HRM could promote cellapoptosis.