盐酸小檗碱对胰岛素抵抗HepG2细胞α7烟碱型乙酰胆碱受体及相关因子影响的研究

Effect of berberine hydrochloride on α7nAchR and related factors in insulin resistant HepG2 cells

目的探讨盐酸小檗碱作用α7烟碱型乙酰胆碱受体(nAchR)与改善氓的分子机制。方法建立IR HepG2细胞模型,用盐酸小檗碱(BBR)、烟碱(NIC)干预,并设正常对照(NC)组和模型组。用现代分子生物学检测细胞培养液葡萄糖消耗量、乙酰胆碱酯酶(AchE)、乙酰胆碱(Ach)、白介素-6(Ⅱ-6)、核因子-κB(NF-κB p65)、IκB激酶βSer181(ⅡKKβSer181)、α7nAchR、磷酸肌醇3激酶(PI3K)、葡萄糖转运因子4(GluT4)等相关指标,用荧光标记方法检测细胞摄取葡萄糖的荧光强度。结果葡糖糖消耗量NC组、HepG2细胞模型组、BBR组和NIC组依次为(25.33±0.77)mmol/L、(11.95±1.94)mmol/L、(16.85±0.33)mmol/L和(16.27±1.09)mmol/L。细胞内2-NBDG(荧光标记葡萄糖)荧光强度增强,NC组、HepG2细胞模型组、BBR组和NIC组依次为(118.17±14.30)、(92.33±9.35)、(120.50±26.26)和(127.67±16.07)。BBR能抑制细胞AchE活性,抑制率为53%,NIC对AchE活性无影响。BBR和NIC能激活α7nAchR,抑制IKKβSer181和NF-κBp65的表达,降低炎性细胞因子IL-6,上调PI3K、GluT4,与HepG2细胞模型组比较,差异有统计学意义(P<0.05或P<0.01)。结论 BBR可能通过抑制AchE活性以激活α7nAchR,从而抑制IKKβSer181、NFκB p65和炎症因子IL-6的表达,促进PI3K、GluT4上调,发挥其改善IR、降低葡萄糖的作用。NIC虽不能抑制AchE活性,但它是特异性激动剂,可直接激活α7nAchR。

Objective To investigate the effect of berberine hydrochloride on alpha 7 nicotinic acetylcholine receptor （nAchR） and the molecular mechanism of insulin resistance （IR） improvement. Methods The IR HepG2 ceil model was established. Berberine hydrochloride（BBR） and nicotine（NIC） were used for intervention. The cells were divided into normal control （NC） group and model group. Glucose consumption in cell culture fluid, acetylcholinesterase （AchE）, acetylcholine （Ach）, interleukin -6 （IL-6）,nuclear factor kappa B （NFκB p65） Ser181 kappa B kinase beta （IKKβSer181）, alpha 7nAchR, phosphatidylinositol 3 kinase （PI3K）,glucose transporter 4 （GLUT4） and other related indicators were measured by modern molecular biological methods. The cellular uptake of glucose was tested by the fluorescence of fluorescent markers. Results Glucose consumption of HepG2 cells were （25.33±0.77） mmol/L, （11.95± 1.94） mmol/L, （16.85± 0.33） mmol/L and （16.27±1.09） mmol/L in NC group,HepG2 cell model group, BBR group and NIC group. The 2-NBDG （fluorescent labeled glucose） fluorescence intensity was enhanced in NC group, HepG2 cell model group, BBR group and NIC group [-（118. 17±14. 30）, （92.33±9.35）, （120.50±26.26） and （127.67±16.07）] respectively. BBR could inhibit the activity of AchE cells, with an inhibition rate of 53 %. NIC had no effect on the activity of AchE cells. BBR and NIC could activate alpha 7nAchR, inhibit the expression of IKK beta Ser181 and NF-κB p65, reduce the inflammatory cytokines IL-6, up-regulate PI3K and GLUT4. The difference was statistically significant（P〈0.05 or P〈0.01）. Conclusion BBR may activate alpha 7nAchR by inhibiting the activity of AchE, and inhibits the expression of IKK beta Serl81, NFκB p65, and IL-6 inflammatory cytokines, promotes PI3K and GLUT4 upregulation, improve IR, and thereby reduce glucose. NIC is a specific agonist to directly activate alpha 7nAchR, although it cannot inhibit the activity of AchE.