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目的:研究艳山姜挥发油(EOFAZ)对氧化低密度脂蛋白(ox-LDL)诱导的人主动脉内皮细胞(HAECs)损伤的保护作用。方法:体外传代培养HAECs,EOFAZ保护作用研究分为7组,分别为空白组(无血清ECM),模型组(200 mg·L~(-1)oxLDL),EOFAZ高质量浓度组(200 mg·L~(-1)ox-LDL+100μg·L~(-1)EOFAZ),EOFAZ低质量浓度组(200 mg·L~(-1)ox-LDL+10μg·L~(-1)EOFAZ),阿司匹林组(Asp,200 mg·L~(-1)ox-LDL+2.5×10~(-4)mol·L~(-1)Asp),卡维地洛组(Car,200 mg·L~(-1)ox-LDL+1×10-6mol·L~(-1)Car),阿托伐他汀钙组(Atorv,200 mg·L~(-1)ox-LDL+1×10-6mol·L~(-1)Atorv);一氧化氮合酶(NOS)信号研究分为5组,分别为空白组(无血清ECM),模型组(200 mg·L~(-1)ox-LDL),EOFAZ组(200 mg·L~(-1)ox-LDL+100μg·L~(-1)EOFAZ),NOS抑制剂组(200 mg·L~(-1)ox-LDL+100μmol·L~(-1)L-NAME或300μmol·L~(-1)L-NMMA),EOFAZ加NOS抑制剂组(200 mg·L~(-1)ox-LDL+100μg·L~(-1)EOFAZ+100μmol·L~(-1)L-NAME或300μmol·L~(-1)L-NMMA)。噻唑蓝(MTT)法分析细胞存活率,酶标仪法及Griess试剂法分别检测培养上清液中乳酸脱氢酶(LDH)活性和一氧化氮(NO)含量。化学法检测内皮型一氧化氮合酶(eNOS)和诱导型一氧化氮合酶(iNOS)活性。实时荧光定量聚合酶链式反应(Real-time PCR)检测iNOS及eNOS mRNA表达水平。结果:与模型组比较,EOFAZ明显提高ox-LDL诱导损伤的HAECs的细胞存活率,抑制LDH外漏及iNOS活力(P<0.05,P<0.01),促进NO及eNOS的产生(P<0.05,P<0.01)。Real-time PCR分析表明,EOFAZ以及相关抑制剂显著下调iNOS mRNA表达水平(P<0.05,P<0.01),上调eNOS mRNA表达水平(P<0.05)。结论:EOFAZ对ox-LDL诱导损伤的HAECs具有保护作用,其作用机制与调控eNOS和iNOS的表达水平有关。
Objective: To study the protective effect of Eolazurenil on the injury of human aortic endothelial cells (HAECs) induced by ox-LDL. Methods: HAECs were cultured in vitro and the protective effect of EOFAZ was divided into seven groups: blank group (serum free ECM), model group (200 mg · L -1 oxLDL) and EOFAZ high concentration group (200 mg · L -1) (-1) ox-LDL + 100μg · L -1 EOFAZ), EOFAZ low concentration group (200 mg · L -1 ox-LDL + 10μg · L -1 EOFAZ) , Asp (200 mg · L -1 ox-LDL + 2.5 × 10 -4 mol·L -1 Asp), Carvedilol group (200 mg · L -1) (-1) ox-LDL + 1 × 10-6mol·L -1 Car), atorv, 200 mg · L -1 ox-LDL + 1 × 10- 6 mol·L -1 Atorv). The signal of nitric oxide synthase (NOS) was divided into five groups: blank group (serum-free ECM), model group (200 mg · L -1 ox- LDL and 200 mg · L -1 ox-LDL + 100 μg · L -1 EOFAZ in EOFAZ group and 200 μmol·L -1 ox-LDL + (-1) L-NAME or 300μmol·L -1 L-NMMA), EOFAZ plus NOS inhibitor group (200 mg · L -1 ox-LDL + 100μg · L -1 ) EOFAZ + 100μmol·L -1 L-NAME or 300μmol·L -1 L-NMMA). Cell viability was assayed by MTT assay. Lactate dehydrogenase (LDH) activity and nitric oxide (NO) contents in culture supernatants were detected by microplate reader and Griess reagent. The activity of endothelial nitric oxide synthase (eNOS) and inducible nitric oxide synthase (iNOS) was detected by chemical method. Real-time quantitative polymerase chain reaction (Real-time PCR) was used to detect the mRNA expression of iNOS and eNOS. Results: Compared with the model group, EOFAZ significantly increased the cell viability, inhibited the leakage of LDH and the iNOS activity (P <0.05, P <0.01), and promoted the production of NO and eNOS in HAECs induced by ox-LDL P <0.01). Real-time PCR analysis showed that EOFAZ and related inhibitors significantly down-regulated iNOS mRNA expression (P <0.05, P <0.01) and up-regulated eNOS mRNA expression (P <0.05). CONCLUSION: EOFAZ has a protective effect on HAECs induced by ox-LDL, and its mechanism is related to the regulation of the expression of eNOS and iNOS.