| IntroductionOver proliferation of vascular smooth muscle cells (VSMCs) is a commonpathological change of vascular hyperplasia diseases such as hypertension and type 2diabetes. There are many factors involved in the proliferation of VSMCs, and one ofthe most important is insulin resistance (IR). Therefore, it is essential to improve andreverse the structure changes induced by over proliferation of VSMCs for treatment ofcardiovascular diseases.Binding with the receptor, two important signal transduction pathways involvedin the cell proliferation induced by insulin, including mitogen activated protein kinase(MAPK) pathway and phosphatidylinositol-3 kinase (PI3K)-protein kinase B (PKB)-mammalian target of rapamycin (mTOR) pathway. PD98059 is a specific inhibitorof MAPK. Nitric oxide (NO) is a downstream signal of PI3K pthway, which plays animportant role in cell proliferation. The activity of nitric oxide synthase (NOS) andthe production of NO are decreased in the IR status.The mammalian target of rapamycin (roTOR) is a serine/threonine kinase thatplays a key role in the regulation of cell growth and proliferation. It belongs tophosphatidylinositol kinase-related kinase (PIKK) family of protein kinases. 70-kDaribosomal protein s6 kinase (p70 S6K) is the downstream targets of roTOR, which canpromote differentiation of VSMC by phosphorylation at Thr389and Thr229.Baicalin (Ba) is one of the effectory compositions extracted from the Scutellariabaicalensis georgi. A lot of data show that baicalin has the effects on cleaning oxygen free radicals, decreasing the ischemia-reperfusion injury, improving microcirculation,relaxing the blood vessel, protecting the myocardial cells and inhibiting arrhythmias.It is confirmed that baicalin can inhibit proliferation of VSMCs in normal culturedcondition induced by blood-serum. It is reported that baicalin promote VSMCsapoptosis in lower dose and has the opposite effect in higher dose, which mechanismis unknown. In present study, we investigate the mechanism of Ba onanti-proliferation of HASMCs which cultured in the mimic IR medium by detectingthe NOS activity, NO production and by determing mRNA expression of mTOR andp70 S6K using RT-PCR method.Methods1 Cell cultureHuman aortic smooth muscle cells(HASMCs) were cultured with 10% calfserum 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 2.5×10-2 mol·L-1glucose, IR group).2 Groups and drugs interventionIn experiment 1, HASMCs were cultured in normal medium and in the mimic IRmedium, respectively. Then four groups were set, they are control, Ba (the finalconcentration was 10-7 mol·L-1,10-6 mol·L-1,10-5 mol·L-1 and 10-4 mol·L-1), Ba plusL-NAME (10-4 mol·L-1) and L-NAME, respectively.In experiment 2, the control, PD98059(3×10-5 mol·L-1), PD98059 and PA(10-4mol·L-1) groups were set when HASMCs cultured in the normal medium and in themimic IR medium. RAP(10-7 mol·L-1), Ba(10-6 mol·L-1, 10-4 mol·L-1) or Pu(10-6mol·L-1) were adminstrated after treated with PD98059, PD98059 combined with PA,respectively.3 MeasurementHASMCs proliferation was assayed by MTT method (λ=490nm) after drugsadministrated for 3 days. NO production in culture solution and NOS activities ofcultured HASMCs were determined by assay kits, respectively. The total RNA from HASMCs was isolated by Trizol reagent following the manufacturer's instructions.The expression of mRNA encoding mTOR and p70 S6K was assayed by RT-PCR.The PCR conditions were set at 94℃for 4 min followed by 35 cycles of 94℃for 40 s,56.5℃for 45 s, and 72℃for 45 s. The final extension was run in 72℃for 10 min.Each PCR product was separated on 1.8% agarose gel. Scanning the density of bandsand photography were performed after electrophoresis.ResultsThe proliferation of cultured HASMCs in the mimic IR medium wassignificantly increased(0.3437±0.0165 vs 0.7380±0.0474, P＜0.01). HASMCsproliferation were promoted by lower dose of Ba (10-5 mol·L-1) and significantlyinhibited by higher dose of Ba (10-4 mol·L-1) in normal culture medium(0.3437±0.0165 vs 0.1658±0.0229, P＜0.01). And in mimic IR culture medium, HASMCsproliferation were inhibited by Ba in both groups(P＜0.01). NO production and NOSactivities of HASMCs cultured with two kinds of culture medium were remarkablyincreased by Ba, and these effects of Ba were partially blockaded by L-NAME andwere enhanced by the mimic IR medium stimulation.The proliferation of cultured HASMCs in normal culture medium wassignificantly decreased by PD98059, which was enhanced by rapamycin and Ba (10-4mol·L-1) (P＜0.01), respectively. In the present of PD98059, HASMCs proliferationwas inhibited by Ba (10-6 mol·L-1,10-4 mol·L-1) and Pu (10-6 mol·L-1) in mimic IRculture medium (P＜0.05, respectively). The mRNA expression of roTOR and p70S6K of VSMCs was reduced by Ba of both doage and Pu (10.6 mol·L-1) after treatedwith PD98059 in normal culture medium and in mimic IR culture medium, and theeffect of Ba in higher dosage had more powerful (P＜0.01). HASMCs proliferationinduced by PA were obviously inhibited by rapamycin after treated with PD98059 innormal culture medium and in mimic IR culture medium (P＜0.01,)respectively. Theexpression of mTOR and p70 S6K was decreased by Ba of both dosage and Pu (10-6mol·L-1) in the same conditions, the effect of Ba in higher dosage also was morepowerful than that of Ba in lower dosage. ConclusionsIn conclusion, Ba had stimulation effects at lower dose and had inhibition effectson HASMCs in normal culture medium. It may partially involve in NOS activities andNO production. In the present of PD98059, HASMCs proliferation was inhibited byBa, whereas the mTOR and p70 S6K expression of HASMCs was decreased by Ba.Pretreated with PD98059 and PA, Ba had the same inhibition effects on proliferationand the expression ofmTOR and p70 S6K in HASMCs. |
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