溶血磷脂酸对结肠炎小鼠肠道离子转运体SLC26A3表达的影响及机制分析
发布时间：2018-08-19 10:37
【摘要】：目的：腹泻是溃疡性结肠炎常见的临床症状,其产生主要与水盐(包括Cl-、 HCO3-和Na+)分泌和吸收不平衡相关。SLC26A3是位于肠上皮细胞顶端膜的离子转运蛋白,参与调控肠道Cl-、HCO3-和水的吸收与分泌。已有体外研究证实溶血磷脂酸(LPA)可提高SLC26A3的基因表达和离子转运功能,并可能成为治疗炎症性腹泻的候选药物,但是关于LPA在体内是否能提高SLC26A3表达并改善炎症性腹泻症状尚无研究报道,本课题首先对此进行观察。既往研究提示,LPA可以通过提高SLC26A3的启动子活性而上调SLC26A3的细胞膜表达和离子转运功能,但对LPA是否有助于提高SLC26A3细胞膜表达的稳定性和持续性却未见研究报道。鉴于SLC26A3的糖基化在肠道环境中对维持SLC26A3细胞膜表达的稳定性具有重要意义和PDZ结构蛋白NHERF4对SLC26A3黏膜表达持续性的影响,课题利用Caco2细胞观察LPA对SLC26A3糖基化水平的影响和NHERF4对LPA促SLC26A3蛋白细胞膜表达的影响,以期能进一步了解LPA调控SLC26A3细胞膜表达的相关机制。 方法：利用4%葡聚糖硫酸钠(DSS)构建C57BL/6J小鼠急性结肠炎动物模型,设立正常对照组,观察小鼠的体质量、肠道出血情况、粪便含水量、结肠大体组织炎症损害和镜下病理组织学表现,参考Butzner JD标准进行结肠损伤组织学大体评分,参考Sutherland LR标准进行结肠炎疾病活动指数(DAI)评分,运用实时定量荧光聚合酶链反应和蛋白免疫印迹实验分析炎症结肠的SLC26A3mRNA水平和蛋白表达,评估该动物模型应用于观察分析LPA对炎症性腹泻和离子转运体SLC26A3黏膜表达的影响的可行性。然后,对DSS诱导的急性结肠炎小鼠给予LPA灌肠治疗,设立正常小鼠组、模型组、磷酸盐缓冲溶液(PBS)灌肠处理组做为对照,观察LPA对结肠炎小鼠炎症性腹泻相关指标的影响,如体质量的下降、DAI评分、稀便的程度和黏膜炎症损伤程度,并观察分析LPA对中远段炎症结肠的SLC26A3mRNA水平和蛋白表达的影响,以评估LPA做为治疗炎症性腹泻候选药物的可能性。利用Caco2细胞,将LPA与Caco2细胞共孵育,以共孵育时间0小时、6小时、12小时、18小时为观察时间点,观察LPA对SLC26A3的基因表达、总蛋白表达、糖基化蛋白表达的影响。利用胰蛋白酶对SLC26A3蛋白的降解作用,设立LPA处理Caco2细胞组和LPA空白Caco2细胞组,以细胞和胰酶共孵育时间Omin、5min、10min、30min为观察时间点,观察分析LPA对SLC26A3细胞膜表面表达稳定性的影响。构建NHERF4质粒,转染Caco2细胞,设立转染NHERF4质粒的Caco2组(pIRES2-ZsGreenl-NHERF4-Caco2组,即NHERF4-Caco2组)、转染空质粒的Caco2组(pIRES2-ZsGreen1-NP-Caco2组,即NP-Caco2组)、LPA处理的转染NHERF4质粒的Caco2组(LPA+pIRES2-ZsGreenl-NHERF4-Caco2组,即LPA+NHERF4-Caco2组)、LPA处理的转染空质粒的Caco2组(LPA+pIRES2-ZsGreen1-NP-Caco2组,即LPA+NP-Caco2组)、LPA处理的Caco2组(即LPA+Caco2组)和LPA空白Caco2组(即Caco2组),以LPA与Caco2细胞共孵育12小时为观察时间点,分析Caco2细胞转染NHERF4质粒后,LPA对SLC26A3的细胞表达的影响是否发生改变,并观察分析LPA对NHERF4蛋白表达的影响,初步评价LPA、SLC26A3、NHERF4之间的相互作用关系。 结果：1.在构建DSS诱导的C57BL/6J小鼠急性结肠炎动物模型实验中,模型组小鼠表现出粪便含水量增加、血便、炎症活动指数DAI值迅速上升、体质量明显下降。实验结束时,与对照组相比,模型组的结肠大体组织学炎症评分升高(p0.05),结肠明显缩短(正常组vs.模型组：7.53±0.3cm vs.4.8±0.8cm,p0.05),炎症结肠黏膜中SLC26A3mRNA表达明显下降(正常组vs.模型组：14.03±1.4vs.1.0,p=0.00),蛋白表达明显下降。2.在观察LPA对结肠炎小鼠炎症性腹泻和对炎症结肠段SLC26A3表达影响的实验中,所有饮用DSS的小鼠均表现出体质量下降,PBS处理组(实验前vs.实验后：17.02±0.19gvs.14.46±0.97g,p=0.001)和模型对照组(实验前vs.实验后：16.90±0.49g vs.13.5±0.90g,p=0.001)体质量明显下降,LPA处理组的体质量下降趋势明显减缓(实验前vs.实验后：17.36±0.67g vs.16.05±0.92g,p=0.06)。LPA处理组的粪便含水量上升幅度(18.89±8.67%)较PBS处理组(29.48±6.71%)和模型对照组(28.97±6.95%)明显减缓(p=0.049,p=0.041)。LPA处理组SLC26A3mRNA(2.27±0.4)较模型对照组(1.0)明显上升(p=0.03),模型对照组和PBS处理组(1.41±0.45)SLC26A3mRNA差异无显著性(p=0.09)。LPA处理组、模型对照组、PBS处理组的SLC26A3蛋白表达水平较正常对照组均降低,但LPA处理组SLC26A3蛋白表达较模型对照组和PBS处理组升高。3.在观察LPA对SLC26A3糖基化水平影响的实验中,LPA孵育12小时的Caco2细胞与LPA空白Caco2细胞相比,SLC26A3基因表达量增加1.67±0.03倍,蛋白表达量亦增加(相对β-actin的表达量,0.92±0.10vs.0.46±0.05,p0.05),SLC26A3的细胞膜表达与细胞浆表达比增加(膜表达/浆表达：2.17±0.17vs.1.72±0.12,p=0.023),提示LPA在提高Caco2细胞SLC26A3总蛋白表达的同时,也有助于SLC26A3的细胞膜定位和表达。在观察LPA对SLC26A3蛋白抵抗胰酶降解的影响的实验中,LPA空白Caco2细胞的细胞膜SLC26A3蛋白在与胰酶共孵育10min后明显减少,而LPA孵育12小时的Caco2细胞的细胞膜SLC26A3蛋白在与胰酶共孵育后含量未出现明显减少,提示LPA可提高细胞膜SLC26A3蛋白抵抗胰酶降解的能力。4.在观察LPA与NHERF4在SLC26A3表达中的相互作用的实验中,与LPA+NP-Caco2组相比,LPA+NHERF4-Caco2组SLC26A3蛋白表达量明显减少(相对β-actin的表达量,0.27±0.042vs.0.56±0.022,p=0.003),提示NHERF4可弱化LPA的促SLC26A3蛋白表达作用。与NHERF4-Caco2组相比LPA+NHERF4-Caco2组NHERF4的蛋白表达量无明显改变,提示LPA对NHERF4的蛋白表达无明显影响。 结论：LPA可提高炎症结肠黏膜的SLC26A3表达,并减轻DSS诱导的结肠炎性腹泻的严重程度,提示其可做为治疗结肠炎相关性腹泻的候选药物。LPA在Caco2细胞可提高SLC26A3的蛋白表达和糖基化水平并增强SLC26A3抗胰蛋白酶降解的能力。NHERF4可弱化LPA促进Caco2细胞SLC26A3蛋白表达的作用,但LPA对NHERF4的表达影响不显著。提示LPA可提高SLC26A3细胞膜表达的稳定性,但对SLC26A3细胞膜表达持续性的影响尚需进一步实验探讨。
[Abstract]:OBJECTIVE: Diarrhea is a common clinical symptom of ulcerative colitis. Its production is mainly related to the imbalance of secretion and absorption of water and salt (including Cl-, HCO3 - and Na +). SLC26A3 is an ion transporter located at the apical membrane of intestinal epithelial cells. It is involved in regulating the absorption and secretion of Cl-, HCO3 - and water in the intestine. Increasing gene expression and ion transport of SLC26A3 may be a candidate drug for the treatment of inflammatory diarrhea. However, there is no report on whether LPA can increase the expression of SLC26A3 and improve the symptoms of inflammatory diarrhea in vivo. This study first observed this effect. Previous studies suggest that LPA can increase the promoter activity of SLC26A3. Sexually up-regulated cell membrane expression and ion transport of SLC26A3, but no studies have been reported on whether LPA contributes to the stability and persistence of cell membrane expression of SLC26A3. In view of the important significance of glycosylation of SLC26A3 in intestinal environment for maintaining the stability of cell membrane expression of SLC26A3 and the role of PDZ structural protein NHERF4 in the adhesion of SLC26A3 The aim of this study was to investigate the effect of LPA on the glycosylation level of SLC26A3 and the effect of NHERF4 on the membrane expression of SLC26A3 protein promoted by LPA in Caco2 cells.
Methods: Acute colitis model of C57BL/6J mice was established by using 4% dextran sodium sulfate (DSS). Normal control group was set up to observe the body mass, intestinal bleeding, fecal water content, inflammation damage of colon gross tissues and microscopic histopathological manifestations. Butzner JD standard was used for histological gross score of colon injury. Sutherland LR was used to assess the activity index (DAI) of colitis. Real-time quantitative fluorescence polymerase chain reaction and Western blot were used to analyze the expression of SLC26A3 mRNA and protein in inflammatory colon. The animal model was used to observe and analyze the effect of LPA on inflammatory diarrhea and the expression of SLC26A3, an ion transporter. Feasibility. Then, the mice with acute colitis induced by DSS were given LPA enema treatment. Normal mice, model group, phosphate buffer solution (PBS) enema treatment group were set up as control group. The effects of LPA on inflammatory diarrhea-related indexes such as body mass loss, DAI score, degree of stool dilution and degree of mucosal inflammation injury were observed. The effects of LPA on the expression of SLC26A3 mRNA and protein in the middle and distal inflammatory colon were observed and analyzed to evaluate the possibility of LPA as a candidate drug for the treatment of inflammatory diarrhea. The effects of trypsin on the degradation of SLC26A3 protein were studied. LPA-treated Caco2 cells and LPA-treated blank Caco2 cells were established. Cell and trypsin co-incubation time of Omin, 5 min, 10 min and 30 min were used to observe the effect of LPA on the stability of membrane expression of SLC26A3 cells. NHERF4 plasmid was constructed, Caco2 cells were transfected with NHERF4 plasmid, Caco2 cells were transfected with NHERF4 plasmid, Caco2 group (pIRES2-ZsGreenl-NHERF4-Caco2 group, namely NHERF4-Caco2 group), Caco2 group transfected emptemptplasmid (pIRES2-ZsGreen1-NP-NP-Caco2 group, NP-Caco2 group, NP-Caco2 group, LPA-LPA-treated with LPA transfected NHERF4 plasmid, Caco2 group (A+pES2-ZES2-ZsGreenl-ZsGreenl-NHERF4-Caco4-Caco2 group), LPA+LPA+LPA+pES2-Z4-Caco 2 group, L Cell expression of SLC26A3 was analyzed by incubation of LPA with Caco 2 (LPA+pIRES2-ZsGreen 1-NP-Caco 2 group), LPA with Caco 2 (LPA+Caco 2 group) and LPA with Caco 2 (Caco 2 group) for 12 hours. The effect of LPA on the expression of NHERF4 protein was observed and analyzed. The interaction among LPA, SLC26A3 and NHERF4 was preliminarily evaluated.
Results: 1. In the animal model of acute colitis induced by DSS in C57BL/6J mice, the water content of feces, blood stool, DAI value of inflammatory activity index increased rapidly, and body weight decreased significantly. At the end of the experiment, compared with the control group, the colon gross histological inflammation score of the model group increased (p0.05), colon bright. The expression of SLC26A3 mRNA in inflammatory colonic mucosa was significantly decreased (vs. normal vs. model group: 14.03 + 1.4 vs. 1.0, P = 0.00), and the expression of SLC26A3 protein was significantly decreased (vs. model group: 7.53 + 0.3 cm vs. 4.8 cm, P 0.05). The mice who drank DSS showed a significant decrease in body mass. The body mass of the PBS treatment group (before and after the experiment vs. 17.02 + 0.19 g vs. 14.46 + 0.97 g, P = 0.001) and the model control group (before and after the experiment vs. 16.90 + 0.49 g vs. 13.5 + 0.90 g, P = 0.001) decreased significantly, and the body mass of the LPA treatment group decreased significantly (before and after the experiment vs. 17.36). The increase of fecal water content in LPA treatment group (18.89+8.67%) was significantly slower than that in PBS treatment group (29.48+6.71%) and model control group (28.97+6.95%) (p=0.049, p=0.041). SLC26A3 mRNA (2.27+0.4) in LPA treatment group was significantly higher than that in model control group (1.0), model control group and PBS treatment group (1.41+0.45). There was no significant difference in SLC26A3 mRNA (p = 0.09). The expression of SLC26A3 protein in LPA treatment group, model control group and PBS treatment group was lower than that in normal control group, but the expression of SLC26A3 protein in LPA treatment group was higher than that in model control group and PBS treatment group. Compared with LPA blank Caco2 cells, SLC26A3 gene expression increased by 1.67+0.03 times and protein expression increased (relative expression of beta-actin, 0.92+0.10 vs.0.46+0.05, p0.05). The ratio of membrane expression to cytoplasmic expression of SLC26A3 increased (membrane expression/plasma expression: 2.17+0.17 vs.1.72+0.12, p=0.023), suggesting that LPA increased the expression of SLC26A3 in Caco2 cells. The expression of SLC26A3 protein was also helpful for the localization and expression of SLC26A3. In the experiment of observing the effect of LPA on the resistance of SLC26A3 protein to trypsin degradation, the expression of SLC26A3 protein in the cell membrane of LPA blank Caco2 cells decreased significantly after 10 minutes incubation with trypsin, while that in the cell membrane of Caco2 cells incubated with LPA for 12 hours decreased significantly after incubation with trypsin. There was no significant decrease in the content of SLC26A3 protein after co-incubation, suggesting that LPA could enhance the ability of SLC26A3 protein to resist trypsin degradation. 4. In the experiment of observing the interaction between LPA and NHERF4 in the expression of SLC26A3, the expression of SLC26A3 protein in LPA+NHERF4-Caco2 group was significantly lower than that in LPA+NP-Caco2 group (relative expression of beta-actin, 0.27 +0.042 vs. 0). The expression of NHERF4 protein in LPA+NHERF4-Caco2 group had no significant change compared with NHERF4-Caco2 group, suggesting that LPA had no significant effect on the expression of NHERF4 protein.
CONCLUSION: LPA can increase the expression of SLC26A3 in inflammatory colonic mucosa and reduce the severity of DSS-induced inflammatory diarrhea, suggesting that LPA can be used as a candidate drug for the treatment of colitis-associated diarrhea. LPA in Caco2 cells can increase the protein expression and glycosylation level of SLC26A3 and enhance the anti-trypsin degradation ability of SLC26A3. It is suggested that LPA can improve the stability of SLC26A3 cell membrane expression, but the effect of LPA on the persistence of SLC26A3 cell membrane expression needs further study.
【学位授予单位】：华中科技大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R965
本文编号：2191413
[Abstract]:OBJECTIVE: Diarrhea is a common clinical symptom of ulcerative colitis. Its production is mainly related to the imbalance of secretion and absorption of water and salt (including Cl-, HCO3 - and Na +). SLC26A3 is an ion transporter located at the apical membrane of intestinal epithelial cells. It is involved in regulating the absorption and secretion of Cl-, HCO3 - and water in the intestine. Increasing gene expression and ion transport of SLC26A3 may be a candidate drug for the treatment of inflammatory diarrhea. However, there is no report on whether LPA can increase the expression of SLC26A3 and improve the symptoms of inflammatory diarrhea in vivo. This study first observed this effect. Previous studies suggest that LPA can increase the promoter activity of SLC26A3. Sexually up-regulated cell membrane expression and ion transport of SLC26A3, but no studies have been reported on whether LPA contributes to the stability and persistence of cell membrane expression of SLC26A3. In view of the important significance of glycosylation of SLC26A3 in intestinal environment for maintaining the stability of cell membrane expression of SLC26A3 and the role of PDZ structural protein NHERF4 in the adhesion of SLC26A3 The aim of this study was to investigate the effect of LPA on the glycosylation level of SLC26A3 and the effect of NHERF4 on the membrane expression of SLC26A3 protein promoted by LPA in Caco2 cells.
Methods: Acute colitis model of C57BL/6J mice was established by using 4% dextran sodium sulfate (DSS). Normal control group was set up to observe the body mass, intestinal bleeding, fecal water content, inflammation damage of colon gross tissues and microscopic histopathological manifestations. Butzner JD standard was used for histological gross score of colon injury. Sutherland LR was used to assess the activity index (DAI) of colitis. Real-time quantitative fluorescence polymerase chain reaction and Western blot were used to analyze the expression of SLC26A3 mRNA and protein in inflammatory colon. The animal model was used to observe and analyze the effect of LPA on inflammatory diarrhea and the expression of SLC26A3, an ion transporter. Feasibility. Then, the mice with acute colitis induced by DSS were given LPA enema treatment. Normal mice, model group, phosphate buffer solution (PBS) enema treatment group were set up as control group. The effects of LPA on inflammatory diarrhea-related indexes such as body mass loss, DAI score, degree of stool dilution and degree of mucosal inflammation injury were observed. The effects of LPA on the expression of SLC26A3 mRNA and protein in the middle and distal inflammatory colon were observed and analyzed to evaluate the possibility of LPA as a candidate drug for the treatment of inflammatory diarrhea. The effects of trypsin on the degradation of SLC26A3 protein were studied. LPA-treated Caco2 cells and LPA-treated blank Caco2 cells were established. Cell and trypsin co-incubation time of Omin, 5 min, 10 min and 30 min were used to observe the effect of LPA on the stability of membrane expression of SLC26A3 cells. NHERF4 plasmid was constructed, Caco2 cells were transfected with NHERF4 plasmid, Caco2 cells were transfected with NHERF4 plasmid, Caco2 group (pIRES2-ZsGreenl-NHERF4-Caco2 group, namely NHERF4-Caco2 group), Caco2 group transfected emptemptplasmid (pIRES2-ZsGreen1-NP-NP-Caco2 group, NP-Caco2 group, NP-Caco2 group, LPA-LPA-treated with LPA transfected NHERF4 plasmid, Caco2 group (A+pES2-ZES2-ZsGreenl-ZsGreenl-NHERF4-Caco4-Caco2 group), LPA+LPA+LPA+pES2-Z4-Caco 2 group, L Cell expression of SLC26A3 was analyzed by incubation of LPA with Caco 2 (LPA+pIRES2-ZsGreen 1-NP-Caco 2 group), LPA with Caco 2 (LPA+Caco 2 group) and LPA with Caco 2 (Caco 2 group) for 12 hours. The effect of LPA on the expression of NHERF4 protein was observed and analyzed. The interaction among LPA, SLC26A3 and NHERF4 was preliminarily evaluated.
Results: 1. In the animal model of acute colitis induced by DSS in C57BL/6J mice, the water content of feces, blood stool, DAI value of inflammatory activity index increased rapidly, and body weight decreased significantly. At the end of the experiment, compared with the control group, the colon gross histological inflammation score of the model group increased (p0.05), colon bright. The expression of SLC26A3 mRNA in inflammatory colonic mucosa was significantly decreased (vs. normal vs. model group: 14.03 + 1.4 vs. 1.0, P = 0.00), and the expression of SLC26A3 protein was significantly decreased (vs. model group: 7.53 + 0.3 cm vs. 4.8 cm, P 0.05). The mice who drank DSS showed a significant decrease in body mass. The body mass of the PBS treatment group (before and after the experiment vs. 17.02 + 0.19 g vs. 14.46 + 0.97 g, P = 0.001) and the model control group (before and after the experiment vs. 16.90 + 0.49 g vs. 13.5 + 0.90 g, P = 0.001) decreased significantly, and the body mass of the LPA treatment group decreased significantly (before and after the experiment vs. 17.36). The increase of fecal water content in LPA treatment group (18.89+8.67%) was significantly slower than that in PBS treatment group (29.48+6.71%) and model control group (28.97+6.95%) (p=0.049, p=0.041). SLC26A3 mRNA (2.27+0.4) in LPA treatment group was significantly higher than that in model control group (1.0), model control group and PBS treatment group (1.41+0.45). There was no significant difference in SLC26A3 mRNA (p = 0.09). The expression of SLC26A3 protein in LPA treatment group, model control group and PBS treatment group was lower than that in normal control group, but the expression of SLC26A3 protein in LPA treatment group was higher than that in model control group and PBS treatment group. Compared with LPA blank Caco2 cells, SLC26A3 gene expression increased by 1.67+0.03 times and protein expression increased (relative expression of beta-actin, 0.92+0.10 vs.0.46+0.05, p0.05). The ratio of membrane expression to cytoplasmic expression of SLC26A3 increased (membrane expression/plasma expression: 2.17+0.17 vs.1.72+0.12, p=0.023), suggesting that LPA increased the expression of SLC26A3 in Caco2 cells. The expression of SLC26A3 protein was also helpful for the localization and expression of SLC26A3. In the experiment of observing the effect of LPA on the resistance of SLC26A3 protein to trypsin degradation, the expression of SLC26A3 protein in the cell membrane of LPA blank Caco2 cells decreased significantly after 10 minutes incubation with trypsin, while that in the cell membrane of Caco2 cells incubated with LPA for 12 hours decreased significantly after incubation with trypsin. There was no significant decrease in the content of SLC26A3 protein after co-incubation, suggesting that LPA could enhance the ability of SLC26A3 protein to resist trypsin degradation. 4. In the experiment of observing the interaction between LPA and NHERF4 in the expression of SLC26A3, the expression of SLC26A3 protein in LPA+NHERF4-Caco2 group was significantly lower than that in LPA+NP-Caco2 group (relative expression of beta-actin, 0.27 +0.042 vs. 0). The expression of NHERF4 protein in LPA+NHERF4-Caco2 group had no significant change compared with NHERF4-Caco2 group, suggesting that LPA had no significant effect on the expression of NHERF4 protein.
CONCLUSION: LPA can increase the expression of SLC26A3 in inflammatory colonic mucosa and reduce the severity of DSS-induced inflammatory diarrhea, suggesting that LPA can be used as a candidate drug for the treatment of colitis-associated diarrhea. LPA in Caco2 cells can increase the protein expression and glycosylation level of SLC26A3 and enhance the anti-trypsin degradation ability of SLC26A3. It is suggested that LPA can improve the stability of SLC26A3 cell membrane expression, but the effect of LPA on the persistence of SLC26A3 cell membrane expression needs further study.
【学位授予单位】：华中科技大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R965
【参考文献】
相关期刊论文 前2条
1 吴瑛;汪芳裕;魏娟;朱东海;;甘露糖结合凝集素在小鼠葡聚糖硫酸钠结肠炎发病中的机制研究[J];医学研究生学报;2010年02期
2 冯仁田,潘宏志,何维,Hirotomo Ochi;Balb/C小鼠免疫系统结构与功能的增龄性变化[J];中华老年医学杂志;2000年03期
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