Rapamycin On The Lipid Homeostasis Of Mesangial Cells And Mechanisms Discussed

BackgroundIt has been confirmed that immunosuppressant rapamycin has atheroprotective effect though it may cause hyperlipidemia.Prevention of foam cells formation by reducing intracellular cholesterol accumulation comprises one mechanism of this effect.Glomerulosclerosis shares similar pathological mechanisms with atherosclerosis,and foam cell-like changes are frequently present in glomerular sclerotic lesions.It has been demonstrated that human mesangial cells(HMCs) express low-density lipoprotein receptor(LDLR) which mediates the binding and internalization of low-density lipoprotein(LDL).HMCs also express ATP-binding cassette transporter A1(ABCA1) which mediates cholesterol efflux.The expression of ABCA1 is regulated by peroxisome proliferators activated receptor-γ(PPARγ)and liver X receptorα(LXRα).In pathological conditions,increased lipoprotein uptake and/or reduced cholesterol efflux lead to foam cell formation in mesangial cells.To investigate the effects of rapamycin on cholesterol homeostasis of glomerular mesangial cells,we assessed changes of intracellular cholesterol content of human mesangial cells(HMCs) after rapamycin treatment and probed into the underlying mechanisms,aimed to provide some laboratory basis for the clinical application of rapamycin.ObjectiveTo investigate the effects of rapamycin on cholesterol homeostasis of glomerular mesangial cells under basic and inflammatory state and probe into the underlying mechanisms.MethodsIntracellular cholesterol accumulation was measured by oil red O staining and high performance liquid chromatography(HPLC).The effect of rapamycin on mRNA and protein changes of low-density lipoprotein receptor(LDLR) and ATP-binding cassette transporter A1(ABCA1) in the absence or presence of IL-1βwere assayed by quantitative real-time PCR and Western-blot.The effects of rapamycin on PPARγand LXRαmRNA expression were assayed by quantitative real-time PCR.The effects of rapamycin on ABCA1 expression was further assessed after blockade of PPARγand LXRαwith specific antagonists.Transient expression of three types of mammalian target of rapamycin(mTOR)- mTOR-WT(wild type),mTOR-RR(rapamycin resistant,with kinase activity),mTOR-RR-KD(rapamycin resistant and without kinase activity) was got by plasmid transfection.Results1.Oil red O staining showed no obvious difference between rapamycin-treatment IL-1βnon-treatment HMCL and IL-1βnon-treatment control cells in rapamycin concentrations of 10,50,100,200ng/ml.The 5 ng/mL IL-1βremarkably increased lipid droplet accumulation in HMCL and rapamycin in the concentration ranged from 10-100ng/ml reduced the increased lipid droplet accumulation caused by IL-1β.2.Quantitative measurement of cholesterol with HPLC showed that the concentrations of TC and CE in Rapamycin treatment groups had no significant difference with that of control group.The concentrations of TC and CE in IL-1β-treated cells were 1.43±0.14 and 2.49±0.93 times that of the control cells respectively(P＜0.05).TC and CE concentrations were decreased significantly with rapamycin 10,50,and 100 ng/mL in the IL-1βtreated HMCL compared with those treated with IL-1βalone(P＜0.05).No differences were found in the different concentration of rapamycin-treated cells.FC concentrations between groups had no significant difference.3,Rapamycin time-dependently regulated the expression of LDLR mRNA.2 hours after the treatment of 100ng/ml Rapamycin,LDLRmRNA was up-regulated, which was 2.06±0.03 times that of 0 hour(P＜0.01).Then LDLR mRNA expression was down-regulated rapidly and reached the lowest amount at the 8^th hour,which was 0.45±0.03 times that of the 0 hour(P＜0.01).There was no significant difference between LDLR mRNA expression at the 24^th and the 8^th hour.At each time point, LDLR protein expression was lower than that of the 0 hour,but the difference didn't reach statistic significance.4,At the 24 hour time point,Rapamycin showed dose-dependent down-regulation of the expression of LDLR mRNA.At concentration of 10ng/ml,50ng/ml and 100ng/ml,LDLRmRNA expression was 0.47±0.06,0.34±0.04 and 0.38±0.06 times that of the control,which were all significantly lower than that of the control group(P＜0.01).Althogh 50ng/ml Rapamycin had the most obvious effect, no significant difference were found between 100ng/ml and 50ng/ml.LDLR protein expression was lower than that of the control group in all the Rapamycin treatment groups,but didn't reach statistic significance.5,IL-1βsignificantly up-regulated HMCL LDLR mRNA expression,which was 1.48 times that of the control group(P＜0.01).Rapamycin at concentrations of 10,50, and 100 ng/mL dose-dependently prevented the increased LDLR mRNA and protein expression induced by IL-1β,which were all significantly decreased as compared with that of the cells treated with IL-1βalone(P＜0.01).6,100ng/ml Rapamycin time-dependenly regulated ABCA1 mRNA and protein expression in HMCL cells.2 hours after Rapamycin treatment,ABCA1 mRNA and protein expression were significantly down-regulated than that at the 0 hour (P＜0.01).Then ABCA1 mRNA and protein expression was up-regulated and reached peak at the 8^th hour,which were significantly higher than that at the 0 hour (P＜0.01).At the 24 hour time point,ABCA1 mRNA and protein were similar with that at the 0 hour。7,At the 8^th hour after treatment,Rapamycin dose-dependenly up-regulated the expression of ABCA1 both in mRNA and protein level(P＜0.01).At mRNA level,100ng/ml had the most obvious effect,at protein level,50ng/ml had the most obvious effect.8,IL-1βsignificantly down-regulated ABCA1 mRNA and protein expression in HMCL(P＜0.01).Rapamycin at concentrations of 10,50,100 ng/mL dose-dependently showed increase of ABCA1 mRNA and protein expression suppressed by IL-1β(P＜0.01).And those increases of ABCA1 mRNA was significant different at the concentrations of rapamycin 10,50,100 ng/mL(P＜0.01),which was most obvious at 100ng/ml.At the protein level,50,100 ng/mL rapamycin significantly up-regulated ABCA1 expression suppressed by IL-1β(P＜0.01),10ng/ml had no significant effect.9,100ng/ml Rapamycin time-dependently regulate PPARγand LXRαmRNA expression in HMCL.2 hours after Rapamycin treatment,both PPARγand LXRαmRNA were significantly down-regulated compared with that at the 0 hour (P＜0.01),with 0.63±0.04 and 0.49±0.04 times that of the 0 hour respectively.Then the expression of them was upregulated and reached peak at the 8^th hour,which were 2.42±0.05,1.99±0.06 times that at the 0 hour respectively,both were significantly increased(P＜0.01).At the 24 hour time point,PPARγmRNA decreased to approach the level at the 0 hour.LXRαmRNA was 1.68±0.04 times that at the 0 hour,which was still significantly higher than the later(P＜0.01)。10,At the 8^th hour after treatment,,Rapamycin dose-dependently up-regulated the expression of both PPARγand LXRαmRNA.10ng/ml,50ng/ml,100ng/ml could all significantly increasd PPARγand LXRαmRNA expression(P＜0.01).100ng/ml had the most obvious effect on PPARγmRNA expression,which was 1.92±0.06 times that of the control and 50ng/ml had the most obvious effect on LXRαmRNA expression,which was 1.73±0.07 times that of the control.11,IL-1βsignificantly down-regulated both PPARγand LXRαmRNA expression in HMCL,which were 0.41±0.05 and 0.25±0.04 times that of the control respectively(P＜0.01 ).Rapamycin at concentrations of 10,50,100ng/ml dose-dependently increased PPARγand LXRαmRNA expression suppressed by IL-1β.Among them,100ng/ml had the most obvious effect.At this concentration, PPARγmRNA expression were 1.04±0.04 times that of the control and LXRαmRNA expression was 0.78±0.04 times that of the control,which were all significantly increased compared with that of the cells treated with IL-1βalone(P＜0.01). 12,When the activity of PPARγwas blocked,100ng/ml Rapamycin could still significantly up-regulate the expression of ABCA1 mRNA(P＜0.01),which was 1.98±0.04 times that of the control and had no significant difference compared with that of cells treated with Rapamycin without PPARγblochage.While when the activity of LXRαwas blocked,ABCA1 mRNA expression was only 0.81±0.03 times that of the control,which was significantly decreased compared with both control group and that of cells treatd with Rapamycin without LXRαblochage(P＜0.01)。13,Transient expression of mTOR-WT,mTOR-RR,mTOR-RR-KD all reduced ABCA1mRNA expression significantly(P＜0.01),which all could be overroded by rapamycin(p＜0.01)。Conclusions1.Rapamycin had no obvious influence on intracellular cholesterol concentration of HMCL under normal condition but could significantly ameliorate intracellular cholesterol homeostasis under inflammatory state.2.Rapamycin contribute to maintanace of HMCL cells intracellular cholesterol homeostasis by regulate the expression of both influx pathway of LDLR and efflux pathway of PPARγ-LXRα-ABCA1.3.Rapamycin regulate the expression of ABCA1 through LXRα,but PPARγwas not the only way by which Rapamycin regulate the expression of ABCA1.4.mTOR could inhibite the expression of ABCA1and its not the only way that Rapamycin regulate the expression of ABCA1.