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目的探讨参苓白术散抗脂多糖(LPS)诱导的炎症性肠病(IBD)损伤的作用和机制。方法采用LPS(200μg·m L~(-1))造模,将细胞分为空白组(10%空白血清),模型组(10%空白血清+LPS),参苓白术散低、中、高剂量组(4%含药血清+LPS、8%含药血清+LPS、16%含药血清+LPS),FBS对照组(10%FBS+LPS)、Rho激酶(ROCK)阻断剂Y27632组(10μmol·L~(-1) Y27632+16%含药血清+LPS)、肌球蛋白亲链激酶(MLCK)阻断剂ML-7组(10μmol·L~(-1) ML-7+16%含药血清+LPS)。建立肠隐窝上皮细胞(IEC-6)与小鼠T淋巴细胞瘤细胞(Cyc-Tag)共培养体系。将共培养细胞接种于Transwell 12孔板中,采用Millipore-ERS电阻仪检测共培养体系下IEC-6细胞跨膜电阻值(TEER);采用Western Blotting法分别检测共培养体系中和Cyc-Tag单独培养下ROCK、MLCK、磷酸化ROCK(Pho-ROCK)及磷酸化MLC(Pho-MLC)蛋白的表达情况。结果共培养体系中,与空白组比较,模型组的IEC-6细胞TEER值显著下降(P<0.01),ROCK、Pho-ROCK、MLCK及Pho-MLC蛋白表达量显著增加(P<0.01);与模型组比较,参苓白术散低、中、高剂量组及Y27632组、ML-7组的IEC-6细胞TEER值均显著升高(P<0.01),ROCK、Pho-ROCK、MLCK及Pho-MLC蛋白表达量均有明显下调(P<0.05,P<0.01)。Cyc-Tag细胞单独培养下,与空白组比较,模型组的ROCK、Pho-ROCK、MLCK及Pho-MLC蛋白表达量显著增加(P<0.01);与模型组比较,参苓白术散低、中、高剂量组及Y27632组、ML-7组的ROCK、Pho-ROCK、MLCK及Pho-MLC蛋白表达量均有明显的下调(P<0.05,P<0.01)。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,可能与调节ROCK/MLCK信号通路及改善IEC-6细胞通透性有关。
Objective To investigate the effect and mechanism of Shenling Baizhu San on lipopolysaccharide (LPS) -induced inflammatory bowel disease (IBD) injury. Methods LPS (200μg · m L -1) model was used to divide the cells into blank group (10% blank serum), model group (10% blank serum + LPS), low, middle and high Dose group (4% serum containing LPS, 8% serum containing LPS, 16% serum containing LPS), FBS control group (10% FBS + LPS), Rho kinase inhibitor Y27632 group (10μmol·L -1 Y27632 + 16% drug-containing serum + LPS), MLLC group (10μmol·L -1 ML-7 + 16% Drug-containing serum + LPS). Establishment of intestinal crypt epithelial cells (IEC-6) and mouse T lymphocyte co-culture system (Cyc-Tag) system. The co-cultured cells were seeded into Transwell 12-well plates, and the transmembrane resistance (TEER) of IEC-6 cells in the co-culture system was measured by Millipore-ERS resistance assay. Western Blotting was used to detect the expression of Cyc-Tag alone The expression of ROCK, MLCK, phospho-ROCK (Pho-ROCK) and phospho-MLC (Pho-MLC) proteins were cultured in vitro. Results Compared with the blank group, the TEER value of IEC-6 cells in model group decreased significantly (P <0.01) and the expression of ROCK, Pho-ROCK, MLCK and Pho-MLC protein increased significantly Compared with the model group, the TEER values of IEC-6 cells in low, medium and high dose Shenling Baishan Powder group, Y27632 group and ML-7 group were significantly increased (P <0.01), while those of ROCK, Pho-ROCK, MLCK and Pho MLC protein were significantly down-regulated (P <0.05, P <0.01). Compared with the blank group, the expression of ROCK, Pho-ROCK, MLCK and Pho-MLC in Cyc-Tag cells increased significantly (P <0.01) compared with the blank group; Compared with the model group, ROCK, Pho-ROCK, MLCK and Pho-MLC in ML-7 group were significantly decreased (P <0.05, P <0.01). Conclusion Shenling Baizhu San containing serum can significantly inhibit LPS-induced IEC-6 cell injury, which may be related to the regulation of ROCK / MLCK signaling pathway and the improvement of IEC-6 cell permeability.