参苓白术散含药血清抗脂多糖(LPS)诱导的肠隐窝上皮细胞 (IEC-6)通透性变化机制的研究

The study of mechanism the serum of SLBZS improve perpeability change on IEC-6 induced by LPS

目的:探讨参苓白术散抗脂多糖(lipopolysaccharide,LPS)诱导肠上皮隐窝细胞(intestinal crypt cel ls,IEC-6)通透性变化的机制研究。方法:将IEC-6细胞分为空白组、LPS模型组、LPS+参苓白术散含药血清低、中、高剂量组、10%FBS。对以下指标进行检测:IEC-6细胞凋亡、细胞内钙离子浓度、IEC-6电阻值变化、检测各组中磷酸化Rho激酶(ROCKⅡ)、肌球蛋白轻链激酶(MLCK)以及凋亡因子Caspase-3表达。结果:LPS显著引起IEC-6细胞凋亡而参岺白术散的含药血清明显抑制细胞凋亡。与空白组比较,模型组IEC-6细胞跨膜电阻的TEER值显著下降(P<0.01);与LPS模型组比较,参苓白术散含药血清低、中、高剂量组IEC-6细胞跨膜电阻的TEER值显著升高,实验结果具有统计学意义(P<0.01)。LPS模型组的钙离子浓度明显高于空白组(P<0.01);不同浓度的参苓白术散含药血清组的钙离子浓度明显低于LPS模型组(P<0.01)。与空白组比较,模型组中磷酸化ROCKⅡ、MLCK蛋白表达明显升高(P<0.05);与模型组比较,不同剂量参苓白术散含药血清组中磷酸化ROCKⅡ、MLCK蛋白表达明显降低(P<0.05)。结论:参苓白术散含药血清对LPS诱导的IEC-6细胞损伤具有明显地抑制作用,与抑制细胞凋亡及IEC-6细胞通透性的改善有关。

Objective： To investigate the serum of SLBZS treat permeability change induced by lipopolysaccharide （LPS） on intestinal crypt cells （IEC-6） study of the mechanism. Methods： IEC-6 cells were divided into control group, model group, Shenling Baizhu powder of low （4%）, medium （8%） and high （16%） dose group with LPS, 10%FBS. Building inflammation model of IEC-6 cells with 200 μg/mL lipopolysaccharide. The Annexin V-FITC/PI Hoechst staining were used to detect the LPS on IEC-6 cell apoptosis. The Flou 3-AM method of time- resolved was used to detect the intraceUular calcium ion concentration in each group. Test the IEC-6 cell TEER, Western blot detected the Phosphorylated ROCKII, MLCK, and the expression of apoptosis factor Caspase-3. Results： LPS significantly induced IEC-6 apoptosis and was reduced by SLBZS serum. The calcium ion concentration of the model group was significantly higher than the control group （P〈0.01）, Shenling Baizhu powder containing serum group and different concentration of calcium ion concentration was significantly lower than that of model group （P〈0.01）. The TEER value of IEC-6 cells, compared with blank group, model group transepithelial electrical resistance of IEC-6 decreased significantly （P〈0.01）; compared with model group, and SLBZS serum levels of low, medium and high dose group of IEC-6 across cell membrane resistance TEER a significant rise in value （P〈0.01）. The model group compared with the control group, the expression of Pho-ROCK II, MLCK protein increased significantly （P〈0.05）; while different dose medicated serum group could decrease the expression of Pho-ROCKII, MLCK protein decreased （P〈0.05）. Conclusion： SLBZS drug- containing serum can significant inhibit IEC-6 cell damage induced by LPS, it may be related to inhibit cell apoptosis and the improvement of the IEC-6 cell permeabili.