氧化苦参碱对高脂饮食诱导胰岛素抵抗载脂蛋白E基因敲除小鼠肝脏胆固醇代谢调控基因的影响

Effect of Oxymatrine on Hepatic Cholesterol Metabolism Regulated Genes in Insulin Resistance ApoE^(-/-) Mice Induced by High Fat Diet

目的探讨氧化苦参碱对高脂饮食诱导载脂蛋白E(ApoE)基因敲除(ApoE^(-/-))小鼠模型肝脏胆固醇代谢调控基因的影响。方法 2014年4月—2015年1月,将17只SPF级C57BL/6J小鼠作为正常组(NC组),给予基础饲料饲养;68只SPF级ApoE^(-/-)小鼠给予高脂饲料饲养;均喂养16周后,再将高脂饲料喂养的ApoE^(-/-)小鼠按照随机数字表法分为胰岛素抵抗模型组(IR组)、氧化苦参碱低剂量组(OXYL组)、氧化苦参碱中剂量组(OXYM组)、氧化苦参碱高剂量组(OXYH组),每组17只,OXYL、OXYM、OXYH组分别给予氧化苦参碱25 mg·kg^(-1)·d^(-1)、50mg·kg^(-1)·d^(-1)、100 mg·kg^(-1)·d^(-1)连续灌胃8周,各组实验过程中均给予等体积的纯水。分别检测各组小鼠葡萄糖输注率(GIR),空腹血糖(FBG)水平,血清总胆固醇(TC)、三酰甘油(TG)、低密度脂蛋白胆固醇(LDL-C)、高密度脂蛋白胆固醇(HDL-C)水平,空腹胰岛素(FINS)水平,并计算胰岛素抵抗指数(HOMA-IR),并检测肝脏组织TC水平。采用RT-q PCR和Western blotting法检测肝脏组织固醇调节元件结合蛋白-2(SREBP-2)、固醇调节元件结合蛋白裂解激活蛋白(SCAP)、胰岛素诱导基因-2(INSIG2)、羟甲基戊二酸单酚辅酶A还原酶(HMGCR)、低密度脂蛋白受体(LDLr)mRNA及其蛋白水平。结果 IR组FBG水平,血清TC、TG、LDL-C、HDL-C水平均高于NC组(P<0.05);OXYL组血清TC、TG、LDL-C、HDL-C水平均低于IR组,OXYM、OXYH组FBG水平,血清TC、TG、LDL-C、HDL-C水平均低于IR组(P<0.05)。IR组GIR低于NC组,OXYL组、OXYM组、OXYH组GIR均高于IR组(P<0.05)。IR组FINS水平、HOMA-IR均高于NC组(P<0.05);OXYL组HOMA-IR低于IR组(P<0.05);OXYM、OXYH组FINS水平、HOMA-IR均低于IR组(P<0.05)。IR组肝脏组织SREBP-2、HMGCR、LDLr mRNA及其蛋白水平低于NC组,SCAP、INSIG2 mRNA及其蛋白水平高于NC组(P<0.05);OXYL组肝脏组织LDLr mRNA及其蛋白水平高于IR组(P<0.05);OXYM、OXYH组SREBP-2、HMGCR、LDLr mRNA及其蛋白水平高于IR组,SCAP、INSIG2 mRNA及其蛋白水平低于IR组(P<0.05)。结论氧化苦参碱能通过调控肝脏胆固醇相关基因的表达改善高脂饮食诱导ApoE^(-/-)的血糖、血脂和胰岛素抵抗情况。

Objective To investigate the effect of oxymatrine on genes regulated hepatic cholesterol metabolism in ApoE -/- mice induced by high fat diet. Methods From April 2014 to January 2015, seventeen SPF grade C57BL/6J mice were selected as normal control （NC） group and fed on the basal diet. Sixty - eight SPF grade ApoE-/- mice fed on high fat diet. After 16 weeks of feeding, according to the random number table method, ApoE-/- mice were averagely divided into insulin resistance model group （ 1R group）, low oxymatrine dose group （ OXYL group）, middle oxymatrine dose group （ OXYM group） and high oxymatrine dose group （OXYH group） . There were seventeen mice in each group. In OXYL group, OXYM group and OXYH group, 25 mg·kg-1· d-1, 50 mg·kg-1· d-1and 100mg·kg-1· d-1 oxymatrine were given by continuous oral garage for 8 weeks, and all groups were given equal volume of pure water. Glucose infusion rate （GIR）, blood glucose （FBG） level, serum leves of total cholesterol （ TC）, three glycerol （ TG）, low density lipoprotein cholesterol （ LDL-C）, high density lipoprotein cholesterol （HDL-C）, fasting insulin （FINS） level in each group of mice were measured, and the insulin resistance index （HOMA- IR） was calculated, and the TC level of liver tissue was detected. The expression of SREBP-2, SCAP, INSIG2, HMGCR and LDLr mRNA and its protein were detected using RT- qPCR and western -blotting methods in mice liver tissue. Results The FBG level, serum levels of TC, TG, LDL-C and HDL-C in IR group was significantly higher than NC group （P 〈 0.05） . The serum levels of TC, TG, LDL-C and HDL-C in OXYL group was significantly lower than IR group （ P 〈 0.05） . The FBG level, serum levels of TC, TG, LDL-C and HDL-C of in OXYM and OXYH group were significantly lower than IR group （ P 〈 0. 05） , CIR. in iR group was lower than NC group （ P 〈l）. 05 ） ; FiR in OXYL group, OXYI-I group and OXYM group was higher than IR group （ P 〈 0.05 ） ; The FINS level and HOMA - IR in IR group were higher than NC group （P 〈 0. 05 ） . The HOMA - IR in OXYL group was lower than IR group （P 〈 0. 05 ） . The FINS level, HOMA - IR in OXYH group and OXYM group were lower than IR group （ P 〈 0. 05 ） . The TC level in mice liver tissue of IR group was higher than NC group （ P 〈 0. 05 ） . The TC level in mice liver tissue of OXYL group, OXYM group and OXYH group was lower than IR group （ P 〈 0. 05） . The expressions of SREBP-2, HMGCR, LDLr mRNA and their protein in mice liver tissue of IR group were lower than NC group, and the expressions of SCAP, INSIG2 mRNA and their protein in mice liver tissue of IR group were higher than NC group （P 〈 0. 05） . The expression of LDLr mRNA and its protein in mice liver tissue of OXYL group were higher than IR group （P 〈 0. 05） . The expressions of SREBP-2, HMGCR, LDLr mRNA and their protein in mice liver tissue of OXYH group and OXYM group were higher than IR group, and the expressions of SCAP, INSIG2 mRNA and their protein in mice liver tissue of OXYH group and OXYM group were lower than IR group （P 〈 0. 05 ） . Conclusion Oxymatrine could regulate the expression of genes related hepatic cholesterol and improve the blood glucose, lipid and insulin resistance in ApoE -/- mice induced by high fat diet.