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目的:观察黄芩苷对人结肠癌上皮细胞株HT~(-2)9的炎症模型中磷脂酰肌醇3激酶(PI3K)/丝氨酸苏氨酸蛋白激酶(Akt)/核转录因子-κB(NF-κB)信号通路的影响,并探讨其机制。方法:以肿瘤坏死因子(TNF)-α及脂多糖(LPS)共同诱导HT~(-2)9细胞,建立细胞炎症模型。实验分为6组,空白组加入完全培养基孵育,实验过程中不予任何药物干预,模型组给予h TNF(20μg·L~(~(-1)))黄芩苷孵育12 h后,再给LPS(1 mg·L~(~(-1)))孵育15 h;柳氮磺胺吡啶组在模型组的基础上,加入柳氮磺胺吡啶(SASP,500μmol·L~(~(-1)));黄芩苷组在模型组基础上给予不同剂量(1,10,100μg·L~(~(-1)))黄芩苷孵育24 h。噻唑蓝(MTT)法检测黄芩苷(1,10,100μg·L~(~(-1)))对细胞生长的影响;免疫印迹(Western blot)法检测黄芩苷(1,10,100μg·L~(~(-1)))对PI3K,Akt,NF-κB等蛋白表达,酶联免疫吸附(ELISA)法检测黄芩苷(1,10,100μg·L~(~(-1)))对细胞上清中TNF-α,白细胞介素(IL)-6等含量的影响。结果:与模型组比较,黄芩苷组、柳氮磺胺吡啶组的细胞上清液中TNF-α,IL-6,IL-8,IL~(~(-1))分泌量明显减少(P<0.05),且两药联合应用对TNF-α,IL-6,IL-8,IL~(~(-1))分泌量的减弱效应更强(P<0.01)。与模型组比较,黄芩苷组(1,10,100μg·L~(~(-1))),柳氮磺胺吡啶组(500μmol·L~(~(-1)))中的PI3K蛋白磷酸化水平,Akt磷酸化水平,NF-κB活化入核水平,环氧化酶~(-2)(Cox~(-2))蛋白,β-连环蛋白(β-catenin)蛋白,天冬氨酸蛋白水解酶-9(Caspase-9)蛋白,人凋亡相关因子配体(Fas L)蛋白表达均明显减少(P<0.05)。结论:黄芩苷能减轻HT~(-2)9细胞炎症反应,抑制PI3K磷酸化,下调Akt的活化,抑制NF-κB的活化入核,从而抑制TNF-α,IL-6等炎症因子的分泌,发挥其抗炎效应,提示黄芩苷可能通过以抑制Akt的活化,抑制NF-κB的核转位,发挥其抗炎作用。
OBJECTIVE: To observe the effects of baicalin on the phosphorylation of PI3K / Akt / NF (NF-κB) in the inflammatory model of human colon cancer cell line HT-2 (-2) -κB) signaling pathway and to explore its mechanism. Methods: HT - (-2) 9 cells were induced by tumor necrosis factor (TNF) - α and lipopolysaccharide (LPS) to establish a model of cellular inflammation. The experiment was divided into 6 groups, the blank group was incubated with complete medium, without any intervention during the experiment. The model group was given baicalin of h TNF (20μg · L ~ (-1)) for 12 hours, LPS (1 mg · L ~ (-1)) for 15 h. SASP (500 μmol·L ~ (-1)) was added to the sulfasalazine group on the basis of model group. ); Baicalin group was given different doses (1, 10, 100μg · L ~ (-1)) of baicalin for 24h on the basis of model group. The effect of baicalin (1, 10, 100μg · L ~ (-1))) on the cell growth was detected by MTT assay. Baicalin (1,10,100μg · L ~ (-1) (-1,10,100μg · L ~ (-1))) on the expression of PI3K, Akt, NF-κB and other proteins by enzyme-linked immunosorbent assay (ELISA) In the TNF-α, interleukin (IL) -6 content. Results: Compared with the model group, the secretions of TNF-α, IL-6, IL-8 and IL-1 in the baicalin and sulfasalazine groups were significantly decreased (P < 0.05), and the combined effect of the two drugs had a weaker effect on the secretion of TNF-α, IL-6, IL-8 and IL-1 (P <0.01). Compared with the model group, PI3K phosphorylation level in the baicalin group (1, 10, 100μg · L ~ (-1)) and sulfasalazine group (500μmol·L ~ (-1) , Akt phosphorylation, activation of NF-κB into the nucleus, Cox-2, β-catenin and aspartate proteolysis Caspase-9 protein and Fas L protein expression were significantly decreased (P <0.05). CONCLUSION: Baicalin can alleviate the inflammatory response of HT-2-9 cells, inhibit the phosphorylation of PI3K, down-regulate the activation of Akt, inhibit the activation of NF-κB and inhibit the secretion of inflammatory cytokines such as TNF-α and IL- , Exert its anti-inflammatory effect, suggesting that baicalin may inhibit the activation of Akt, inhibit the nuclear translocation of NF-κB, exert its anti-inflammatory effect.