| AIMOver proliferation of vascular smooth muscle cells(VSMCs) is a common patholigical change of vascular hyperplasia diseases,such as diabetes,hypertension and atherosclerosis. Insulin resistance(IR) is one of the important factors in the proliferation of VSMCs.Therefore, it is essential to inhibt proliferation of VSMCs for treatment of vascular hyperplasia diseases.Mammalian target of rapamycin(mTOR) is an important regulator of the growth of mammalians in response to the availability of nutrients and cellular energy supplies,which is effects on translational control,apoptosis modulation,cell cycle regulation,metabolic modulation and neuronal function.Recent studies in yeast and mammals indicate that mTOR exists in two different complexes which have been termed mTORC1 and mTORC2,mTOR interacts with regulatory associated protein of mTOR(Raptor) to form mTORC1 or with rapamycin insensitive companion of mTOR(Rictor) to form mTORC2.Cell proliferation is induced by the phosphorylation and activation of 70 kDa ribosomal protein s6 kinase(p70 S6K) and cell cycle transport factor via mTOR.The application of RAP was limited because of adverse effects,such as immune suppression especially.There was fewer investigation of active component from Chinese medicinal materials on anti-proliferation by mTOR.Cardamonin(Car)(2',4'-dihydroxy-6'-methoxychalcone) is naturally occuring chalcone. Some studies demonstrate that Car has various biological activities which include anti-inflammaory,anti-allergy,anti-oncosis,antioxidant and anti-mutagenic effect.In another study,Car caused vasorelaxation via NO-mediated processes and by protein kinase C-mediated pathway,To date,very few anti-proliferation effects of VSMCs in the mimic IR state have been ascribed to this compound.In the present study,we investigate the mechanism of Car on anti-proliferation of human umbilical artery smooth muscle cells(HUASMCs) which is cultured in the mimic IR state by detecting the activity of immunodepression and by determing mRNA expression of mTOR and its binding protein.METHODS 1 Cell cultureHUASMCs were cultured with 10%calf serum in RPMI 1640 medium(normal group) and with the mimic IR medium(including 10%calf serum in RPMI 1640 with 100U·L-1 insulin and 2.5×102 mol·L-1 glucose,IR group) at 37℃in humidified atmosphere containing 5%CO2.Human lymphocytes were isolated by density centrifugation.Cells were cultured with 10%calf serum in RPM1 1640 medium at 37℃in humidified atmosphere containing 5% CO2.2 Experimented protocolHUASMCs were cultured in the normal medium and in the mimic IR medium.Then six groups were founded.They are control,PD98059(3×10-5 mol·L-1),PD98059 and LY294002 (10-5 mol·L-1),PD98059 and LY294002 plus PA(10-4 mol·L-1),RAP(10-7 mol·L-1) and different doses of Car(10-8 mol·L-1,10-7 mol·L-1,10-6 mol·L-1,10-5 mol·L-1,10-4 mol·L-1), respectively.Human lymphocytes were cultured in normal medium and in the phytohemagglutinin (PHA)(5 mg·L-1) medium.Then four groups were set.They are control,PHA,RAP and CsA(10-7 mol·L-1,10-5 mol·L-1) positive drug and different doses of Car10-7 mol·L-1,3×10-7 mol·L-1,10-6 mol·L-1,3×10-6 mol·L-1,10-5 mol·L-1,3×10-5 mol·L-1,10-4 mol·L-1),respectively.3 MeasurementHUASMCs proliferation was assayed by MTT method(λ= 490 nm) after drugs administrated for 3 days.The total RNA of HUASMCs was extracted by Trizol reagent following the manufacturer's instructions.The expression of mRNA encoding mTOR,Raptor and Rictor was assayed by Real-time PCR.The expression content was calculated by Livak method using internal control ofβ-actin.RESULTS1 The proliferation of HUASMCs cultured in the mimic IR medium was significantly increased(0.545±0.028 vs 0.422±0.047,P＜0.01).In normal and mimic IR culture medium,cells proliferation was inhibited by higher dose of Car(10-5 mol·L-1,10-4 mol·L-1). The proliferation of HUASMCs was decreased by PD98059,and enhanced by RAP and Car (10-6 mol·L-1,10-4 mol·L-1) respectively(P＜0.01).The HUASMCs proliferation induced by PA was obviouly inhibited by RAP and Car(10-6 mol·L-1,10-4 mol·L-1) after treated with PD98059 and LY294002 in normal medium(P＜0.05,P＜0.01 )and in the mimic IR medium (P＜0.05,P＜0.01),and the effect of Car in higher dosage had more powerful(P＜0.01).2 Pretreated with PD98059,the expression of mTOR was decreased by RAP and Car of both dosage(P＜0.01,P＜0.05,respectively) in the mimic IR medium.The mRNA expression of Raptor was significant decreased by RAP and Car(10-6 mol·L-1) in the normal medium,whereas was increased by RAP and Car(10-5 mol·L-1) in the mimic IR state.Rictor mRNA expression was reduced by RAP,but Car(10-6 mol·L-1,10-5 mol·L-1) has the oppsited effects(P＜0.01).The mRNA expression of mTOR was obviously promoted by PA(P＜0.01),and inhibited by RAP(P＜0.05).Car(10-6 mol·L-1) increased the expression of mTOR mRNA(P＜0.05). The mRNA expression of Raptor was significantly increased by the lower dosage of Car(10(-6) mol·L-1),whereas decreased by the higher dosage of Car(10-5 mol·L-1).And Car of both dosage can reduce the mRNA expression of Rictor.3 The IL-2 level of human lymphocytes was increased by PHA(10.400±0.582 vs 15.575±0.794,P＜0.01).It is showed that the effect of Car on IL-2 secretion has a concentration-dependent manner both in normal medium and in the PHA medium.Car(10-5 mol·L-1,3×10-5 mol·L-1) had no effects on the IL-2 secretion.CONCLUSIONSIn conclusion,Car had nhibition effects at higher dose on HUASMCs in normal clutrure and mimic IR medium.In the mimic IR state,HUASMCs proliferation was inhibited by Car in the present of PD98059 or PA,whereas the RNA expression of mTOR,Raptor and Rictor was deceased.It is impliacted that there are intimated relationship of the anti-proliferation mechanism by mTOR.Meanwhile,Car had no effects on the IL-2 secretion of human lymphocytes.
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