低聚半乳糖和聚葡萄糖联用预防Caco-2肠道细胞屏障损伤

Protective effects of galacto-oligosaccharide and polydextrose on Caco-2 intestinal cell barrier injury model

目的探究不同配比的低聚半乳糖(galacto-oligosaccharide,GOS)和聚葡萄糖(polydextrose,PDX)联用对Caco-2肠道细胞屏障损伤模型的保护作用。方法培养同一批Caco-2细胞至形成肠道细胞屏障模型,随机分为无钙损伤模型组、含钙空白对照组(1.8 mmol/L Ca^(2+))、低比-低剂量组(1.8 mmol/L Ca^(2+)+2 mg/mL GOS+2 mg/mL PDX)、低比-中剂量组(1.8 mmol/L Ca^(2+)+4 mg/mL GOS+4 mg/mL PDX)、低比-高剂量组(1.8 mmol/L Ca^(2+)+8 mg/mL GOS+8 mg/mL PDX)和高比-低剂量组(1.8 mmol/L Ca^(2+)+0.8 mg/mL GOS+3.2 mg/mL PDX)、高比-中剂量组(1.8 mmol/L Ca^(2+)+1.6 mg/mL GOS+6.4 mg/mL PDX)、高比-高剂量组(1.8 mmol/L Ca^(2+)+3.2 mg/mL GOS+12.8 mg/mL PDX),共8组,每组做3个平行。培养4 d后测定各组细胞的跨膜电阻值(trans epithelial electrical resistance,TEER)和表观渗透系数(apparent permeability coefficient,Papp),通过免疫荧光法观察ZO-1、Occludin、Claudin-1紧密连接蛋白的形态,并运用蛋白芯片法测定各组细胞炎性相关因子的表达水平。结果与损伤模型组相比,含钙空白对照组的跨膜电阻值显著增高(P<0.05),而表观渗透系数显著降低(P<0.05);与含钙空白对照组相比,低比-中剂量组和高比-高剂量组的跨膜电阻值显著增高(P<0.05),而表观渗透系数显著降低(P<0.05),且部分炎性因子显著下调(P<0.05)。除了模型组的紧密连接蛋白结构受损以外,其他各组细胞屏障均完好。结论与单独Ca^(2+)作用相比,两种益生元联用可增强Caco-2细胞屏障的致密性和降低其细胞旁路的通透性,并能显著降低部分炎性细胞因子的表达水平,有效保护肠道细胞屏障。

OBJECTIVE To explore the protective effect of different ratios of galactose oligosaccharide(GOS)and polydextrose(PDX)on intestinal cell barrier damage model of Caco-2.METHODS The same batch of Caco-2 cells were cultured to form a cell barrier model and randomly divided into damaged model group without calcium,calcium-containing blank control group(1.8 mmol/L Ca^(2+)),low-ratio/low-dose group(1.8 mmol/L Ca^(2+)2 mg/mL COS+2 mg/mL PDX)and low-ratio/medium-dose group(1.8 mmol/L Ca^(2+)4 mg/mL GOS+4 mg/mL PDX),low-ratio/high-dose group(1.8 mmol/L Ca^(2+)8 mg/mL COS+8 mg/mL PDX)and high-ratio/low-dose group(1.8 mmol/L Ca2^(2+)0.8 mg/mL GOS+3.2 mg/mL PDX),high-ratio/medium-dose group(1.8 mmol/L Ca^(2+)+1.6 mg/mL GOS+6.4 mg/mL PDX),high-ratio/high-dose group(1.8 mmol/L Ca2^(2+)3.2 mg/mL COS+12.8 mg/mL PDX),a total of 8 groups,three parallel groups were performed in each group.The Trans Epithelial Electrical Resistance value and apparent permeability coefficient value of each group were determined after 4 d culture,and the morphology of tight junction proteins ZO-1,Occludin and Claudin-1 were observed by immunofluorescence method,and the expression levels of inflammatory related factors in each group were determined by protein microarray method.RESULTSSCompared with damaged model group,TEER ratio in calcium-containing blank control groupwas significantly increased(P<0.05),while Papp value was significantly decreased(P<O.05);Compared with calcium-containing blank control group,TEER ratio in low-ratio/medium-dose group and high-ratio/high-dose group was significantly increased(P<0.05)while Papp value was significantly decreased(P<0.05),and they could significantly down-regulate some inflammatory response related cytokines.The cell barrier was intact in all groups except for the compact junction protein structure in the model group.CONCLUSION Compared with Ca*alone,the combination of two prebiotics can enhance the density of Caco-2 cell barrier and reduced the permeability of cell bypass.And it can significantly reduce the expression level of some inflammatory cytokines and effectively protect the intestinal cell barrier.