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目的运用脂多糖(lipopolysaccharide,LPS)刺激分化的结肠癌Caco-2细胞作为一种人体外肠上皮细胞的炎症模型,探讨清化肠饮的抗炎作用及其分子机制。方法制备清化肠饮,培养人结肠上皮细胞Caco-2,应用E LISA方法测定肿瘤坏死因子-α(TNF-α)和白细胞介素-8(lL-8),Western blot分析法检测抑制性卡巴蛋白(inhibitory Kaba protein,IκB-α)、磷酸化抑制性卡巴蛋白(phosphory lated inhibitory Kaba protein,p-IκB-α)、核转录因子p50(nuclear transcriotion factor p50,p50)、核转录因子RelA(nuclear transcription factor ReIA,RelA)蛋白。结果与未给予LPS刺激比较,LPS刺激能诱导Caco-2细胞TNF-α和IL-8的释放(P<0.05)。清化肠饮处理可降低LPS诱导的TNF-α和IL-8的分泌,并呈剂量依赖性(P<0.05)。0、5、10、50μg/mL不同浓度清化肠饮对Caco-2细胞存活率无明显影响,各浓度间比较,差异无统计学意义(P>0.05)。清化肠饮可抑制LPS刺激后的IκB-α的磷酸化,p-IκB-α的表达情况随着清化肠饮浓度的增加而下降(P<0.05),IκB-α无明显变化(P>0.05)。p50、RelA表达水平随着清化肠饮浓度的增加而下降(P<0.05),均呈剂量依赖性。结论清化肠饮抑制LPS介导的NF-κB活化可能是其治疗IBD的机制之一。
Objective To investigate the anti-inflammatory effect and its molecular mechanism of Qinghuashangyin on Caco-2 cells stimulated by lipopolysaccharide (LPS) as an inflammatory model of human intestinal epithelial cells. Methods Qingnao decoction was prepared and Caco-2 was cultured in human colon epithelial cells. Tumor necrosis factor-α (TNF-α) and interleukin-8 (IL-8) Inhibitory Kaba protein (IκB-α), phosphorylated inhibitory kaba protein (p-IκB-α), nuclear transcriotion factor p50 (p50), nuclear transcription factor RelA ( nuclear transcription factor ReIA, RelA) protein. Results Compared with no LPS stimulation, LPS stimulation induced the release of TNF-α and IL-8 in Caco-2 cells (P <0.05). Qinghua Decoction treatment can reduce LPS-induced secretion of TNF-α and IL-8 in a dose-dependent manner (P <0.05). There was no significant difference in the survival rate of Caco-2 cells when different concentrations of Qinghuaichangyin were treated with 0, 5, 10, 50μg / mL. There was no significant difference between each concentration (P> 0.05). Qinghuachangyin can inhibit the phosphorylation of IκB-α after LPS stimulation. The expression of p-IκB-α decreased with the increase of concentration of Qinghuwei intestines (P <0.05), while there was no significant change of IκB-α > 0.05). The expression of p50 and RelA decreased with the increase of Qingnao intestines concentration (P <0.05), all in a dose-dependent manner. Conclusion Qinghuaiyin inhibits the activation of NF-κB induced by LPS may be one of its mechanisms for the treatment of IBD.