Effect Of Mct And Glyceryltrioleate On The Proliferation Of Rat Aortic Smooth Muscle Cells

Background and objectiveTriglycerides, also known as fat, is a major source of energy in the bodyand formed by three molecules of fatty acids and one molecule of glycerol.According to their carbon chain length, triglycerides can be divided intothree types:1) Short-chain triglycerides (SCT), the carbon atoms number ofthe carbon chain of triglyceride is less than6;2) Medium ChainTriglycerides (MCT), the carbon atoms number of the carbon chain oftriglyceride is from6to12;3) Long Chain Triglycerides (LCT), the carbonatoms number of the carbon chain of triglyceride is more than12.Hypertriglyceridemia refers to plasma triglycerides≥150mg/dl(≥1.7mmol/L). Epidemiological studies showed that hypertriglyceridemiacould increase cardiovascular disease risk. Atherosclerosis is one of the mostcommon types of vascular lesions. The proliferation of vascular smoothmuscle cells is the distinctive clinical and pathological feature ofatherosclerosis. LCT is the main ingredient of body fat, studies have revealedthat oxidation of low density lipoprotein (ox-LDL) was associated with theproliferation of vascular smooth muscle cells. MCT can reduce the LDL-Clevel and inhibit the production of pro-inflammatory cytokines in the processof atherosclerosis. However, the effects of medium-chain triglycerides andglyceryl trioleate on proliferation of vascular smooth muscle cells have notbeen reported. Therefore，the aim of this work is to study the effects of medium-chain triglycerides and triolein on the proliferation of rat aorticvascular smooth muscle cells (RASMC), to detect their time-effect and thedose-effect relationships, and to provide experimental evidence for rationalcontrol hypertriglyceridemia.Method1. Six dose groups were set up for medium-chain triglyceride andglycerol trioleate respectively, such as62.5mg/dl group,125mg/dl group,250mg/dl group,500mg/dl group,1000mg/dl group, and2000mg/dl. At thesame time, a control group was set up. Six time points of observation were setup for each group, such as24h,48h,72h,96h,120h, and144h.2. Establishment of culture system and identification of rat aortic smoothmuscle cell.3. Oil red o staining was used to observe lipid infiltration and cell shapechanges.4. MTT assay was used to detect effect of MCT and glyceryl trioleate onthe proliferation of RASMC.5. Flow cytometry was used to detect effect of MCT and glyceryltrioleate on the cell cycle of RASMC.6．Flow cytometry was used to detect effect of MCT and glyceryltrioleate on the apoptosis of RASMC.Results1. Immunofluorescence results of vascular smooth muscle cellsUnder the inverted biological microscope, the cell shape is long spindle,nuclei are round or oval. Growth of cells can be duplication,"peak-Valley-like" growth. Immunofluorescence results showed thatanti-myosin antibody staining positive, indicating the cells are SMC.2. Oil red o stain resultsFor medium-chain triglycerides, lipid infiltration started in125mg/dl, the number of infiltrating cells was increased along with the increase ofconcentration. High concentrations of2000mg/dl, changes in cell morphologyare more obvious. For example, somata get bigger and circles, the number ofcellular protrusions and irregular shape were increased.For glyceryl trioleate, compared with the control group, lipid infiltrationwas obvious and associated with the concentration of glyceryl trioleate. Asignificant change began at125mg/dl, changes in cell morphology wereobvious, and somata got bigger and lose their long fusiform appearance. Thereis no normal cell when exposed with2000mg/dl of glyceryl trioleate.3. MTT assay resultsFor medium-chain triglycerides, on24h and48h, the proliferation of rataortic smooth muscle cell was associated with medium-chain triglycerideconcentration. Inhibition effect was observed at≥250mg/dl, and the lowestproliferation rates were detected at1000mg/dl on72h,96h,120h, and144h.At low concentration (<250mg/dl), the proliferation rate was not differentbetween the test and control groups.For glyceryl trioleate, on24h, the proliferation rate was declined withconcentration increasing at low concentration (≤125mg/dl), and promotingproliferation effect was observed at≥250mg/dl, proliferation rate up to themaximum of115.9%at the1000mg/dl (t=–3.7377, p=0.0022). On48h and72h, at <500mg/dl, proliferation rate was increased with concentrationincreasing; at>500mg/dl, the inhibition of proliferation was appeared and thelowest proliferation rate was found at2000mg/dl. From96h to144h, onlyinhibiting proliferation effects were observed.4. Flow cytometry analysis on cell cycleFor medium-chain triglycerides, on24h, G0/G1-phase percentage dropfirstly and then slowly rise with the concentration increasing compared withthe control group; Percentage of s-phase was weak increasing and at500mg/dl up to the maximum; Percentage of G2/M-phase was increasing and at250mg/dl up to the maximum. On48h, at≤250mg/dl, percentage of theG0/G1period has negative correlation with concentrations, the minimumvalue occurs at250mg/dl; Percentage of s-phase first increased and thendeclined (the turning point at250mg/dl); Percentage of G2/M-phase hadalmost no change with the concentration increasing. On144h, G0/G1-phasewas negative relationship and S-phase was positive correlation, with theconcentration, respectively; Percentage of G2/M-phase was almost zoro at thehigher concentration (>250mg/dl).For glyceryl trioleate, the cell cycle has the same trend on24h,48h and72h. Percentage of G0/G1-phase at62.5mg/dl was lower compared with thecontrol group, and no change was observed from62.5mg/dl to1000mg/dl. At2000mg/dl, the percentage increased and returned to control level; Percentageof s-phase was higher than the control group at62.5mg/dl, after that nochange was found, and finally the percentage of s-phase was lower than thecontrol group at2000mg/dl; Percentages of the G2/M-phase firstly increasedand then decreased, the above trend was very weak on24h and was relativelyobvious on48h; Percentage of G0/G1-phase were the same on96h,120h and144h, there was a weak negative correlation with the concentration; On96h,120h and144h, there was a posivtive relationship between1000mg/dl andpercentage of S-phase (15.5%, t=-5.2561, p=0.0001;13.8%, t=-11.8158,p<0.0001;24.9%, t=-20.8513, p<0.0001; respectively); Percentage ofG2/M-phase showed a weak decreasing with the concentration increasing on96h, showed a weak increasing with the concentration increasing and themaximum value was8.6%(t=-18.5389, p<0.0001) at2000mg/dl and120h.G2/M percentage was almost0and independent of concentration on144h.5. Flow cytometry analysis on cell apoptosisFor medium-chain triglycerides, on24h and≤500mg/dl, the effect of promoting apoptosis and a negative relationship with concentration wereobserved. On48h, percentage of apoptosis was increased with theconcentration increasing (except62.5mg/dl and500mg/dl, slightly less thanthe control group). The trends of inhibiting apoptosis were the same andindependent of concentration on72h,96h and120h. There was a lowerapoptosis percentage on144h compared with control group on72h,96h and120h; A mild promoting apoptosis was observed at2000mg/dl(6.4%,t=-2.5669, p=0.0224) compared with control group.For glyceryl trioleate, the percentage of apoptosis was higher than that ofcontrol group on24h.<500mg/dl, apoptosis was negative associated withconcentration;>500mg/dl, apoptosis increased again. On48h, the apoptosisrate was not different between the test and the control grpups at≤1000mg/dl,but an obvious increase was found at2000mg/dl (13%, t=-9.4442, p<0.0001).The trends of apoptosis at≤1000mg/dl were the same on72h,96h and120h,the inhibition effects were not association with the concentration. Theapoptosis percentage increased obviously at2000mg/dl. The highest apoptosispercentage was deceted on144h, and the effect of promoting apoptosis waspositive relationship with concentration from125mg/dl to1000mg/dl.ConclusionMedium-chain triglyceride and glycerol trioleate were components ofbody triglycerides and source of energy, and may play role on the morphologyand function of vascular smooth muscle. Our results indicated that theireffects on the proliferation of RASMC were a time-dependent andconcentration-dependent.Medium-chain triglyceride could promote proliferation at lowerconcentration (≤250mg/dl) and short time (24h,48h). The cell cycle resultsshowed a decreasing percentage of G0/G1-phase and increasing of S-phase.The effect of promoting apoptosis was observed. The inhibition of proliferation was deceted at500mg/dl from72h to144h, at the same time, thepercentage of S-phase was reduced and the inhibition of apoptosis wasobserved.On24h, glycerol trioleate could inhibite the proliferation of RASMC at125mg/dl; On24h and≥250mg/dl,48h-72h and≤500mg/dl, the effects ofpromoting proliferation were detected; On48h-72h and>500mg/dl,≥96h, theinhibition of proliferation was observed in each test group. The results of flowcytometry revealed that the percentage of S-phase was higher than that ofcontrol group from24h to72h and independent of concentration. From96h to144h, the percentage of S-phase was more higher and positive associationwith concentrations on the test groups. The effect of promoting apoptosis anda negative relation with concentration was found at24h; the promotation-orinhibition-effect was not detected at48h; the effect of inhibiting apoptosis wasfound from72h to120h; an effect of promoting apoptosis was detected at2000mg/dl and144h.Whether medium-chain triglycerides or triolein, theirs impact on the rataortic smooth muscle cell proliferation is complex. The promoting orinhibiting proliferation effect is closely associated with their concentrationand exposure-time.The cell proliferation is closely related with cell cycle. The effect ofpromoting proliferation could be characterized by reduced G0/G1-phase andincreased S-phase, and the effect of inhibiting proliferation could becharacterized characterized by increased G0/G1and reducted S-phase.