桑叶有效部位对高胰岛素诱导人肝癌HepG2细胞胰岛素抵抗的影响

Effective Ingredients from Mori Folium on Improving High Insulin-induced Insulin Resistance in Human HepG2 Cells

目的:观察桑叶有效部位(总多糖、总黄酮、总生物碱)影响核因子-κB(NF-κB)通路对高胰岛素诱导人肝癌Hep G2细胞胰岛素抵抗的改善作用。方法:以人肝癌Hep G2细胞为研究对象,在含10 mg·L-1胰岛素的培养基中培养48 h,建立IR-HepG2模型;采用桑叶有效部位观察对Hep G2细胞胰岛素抵抗的影响,从而确定最佳给药浓度。实验分为空白组,10mg·L-1胰岛素处理组(模型组),总多糖组,总黄酮组,总生物碱组,小白菊内酯组。采用葡萄糖氧化酶-过氧化物酶法(GODPOD)法检测培养液中残存葡萄糖含量,RT-q PCR法检测NF-κB,I-κβ激酶-β(IκKβ)及核因子κB抑制蛋白α(IκBα)mRNA的表达,Western blot检测蛋白NF-κB,IκKβ及IκBα的相对表达量。结果:经桑叶有效部位改善Hep G2细胞胰岛素抵抗的筛选得出各给药组最佳给药剂量分别为总多糖组、总黄酮组、总生物碱组、小白菊内酯组最佳给药质量浓度分别为200,100,10mg·L-1,20μmol·L-1。与空白组比较,高胰岛素刺激后,NF-κB,IκKβ含量明显升高(P<0.01),NF-κB结合蛋白IκBα明显降低(P<0.01),表明高胰岛素刺激后NF-κB通路已被部分激活;而桑叶有效部位干预后,与模型组相比,只有黄酮组NF-κB,IκKβ的含量明显降低(P<0.05),抑制IκBα的降解(P<0.05);多糖组和生物碱组影响作用不明显。结论:高胰岛素诱导人肝癌Hep G2细胞发生胰岛素抵抗后,NF-κB通路在一定程度上被激活,推测胰岛素抵抗与NF-κB炎症通路间存在一定的关系;而桑叶黄酮可影响NF-κB通路从而改善Hep G2细胞胰岛素抵抗状态。

Objective： To investigate the effect of Mori Folium effective parts （total polysaccharides, flavonoids and total alkaloids） on high insulin-induced insulin resistance of human hepG2 cells by nuclear faetor-κB （NF-KB） pathway. Method： The insulin resistance cell model was induced by insulin stimulated human HepG2 cells for 48 h. The experimental groups were the control group, the model group （ 10 μg·mL^-1 insulin） , total polysaccharide （MFP） group, total flavonoid （MFF） group, the total alkaloid group （MFA） , parthenolide （PTL） group. Residual glucose content of liquid culture was detected using glucose oxidase-peroxidase method （GOD- POD）. Expressions of NF-κB, I-κB kinaseβ （IκKβ） and inhibitor κB protein α （IκBα） mRNA were detected using RT-qPCR. The relative expressions of protein NF-κB, NF-κβ and IKBawere detected using Western blot. Result： The optimal doses against insulin resistance for MFP, MFF, MFA and PTL were 200, 100, 10, 20μmol·L^-1, respectively. After insulin stimulation, NF-κB, IκKβ activity were enhanced significantly （P 〈0. 01 ） , NF- KB binding protein IκBα decreased significantly （P 〈 0.01 ）, which indicated that NF-KBpathway had beenactivated. After intervention of the mulberry effective parts, only flavonoids could lower NF-κB, IκKβ activity significantly （P 〈0. 05 ）, inhibite the degradation of IκBα （P 〈0. 05）. The results was not significant in polysaccharide group and alkaloid group. Conclusion： After high insulin-induced insulin resistance in HepG2 cells, NF-KB pathway was activated to some extent, suggesting that there is a certain relationship between insulin resistance and NF-KB inflammatory pathways. Meanwhile, mulberry flavonoids could improve the insulin resistance by NF-KB pathway.