LGG通过上调SERT基因表达治疗感染后肠易激综合征的作用机制研究

Serotonin transporter (SERT) is a universally existing cross-membrane transport protein that plays a key role in 5-hydroxytryptamine (5-HT) reuptake. The human SERT (5-HTT) gene is located on chromosome 17q11, named SLC6A4, which is composed of 14–15 exons. The 5-HTT-linked polymorphic region (5-HTTLPR) is located upstream of the transcription start site, which plays a key role in the etiology of irritable bowel syndrome (IBS) by modulating the transcriptional activity of the 5-HTT. 5-HT is an important gastrointestinal hormone that modulates intestinal fluid secretion, gut motility, and gastrointestinal sensation. Serum 5-HT levels decrease in patients with IBS with constipation (C-IBS) and increase in patients with IBS with diarrhea (D-IBS), But is inactivated after reuptake by SERT in intestinal or nerve cells. Downregulation of SERT is implicated in the pathophysiology of various functional gut disorders. Faure et al. found that SERT mRNA was lower in children with IBS than in the control. In our previous study, we found that the S/S genotype results in lower SERT expression and causes 5-HT accumulation at the affected site. Ordinarily, 5-HT can activate the intestinal functions of motion and secretion, which are often related to D-IBS. Chen et al. found that transgenic mice with targeted deletion of SERT frequently exhibit diarrhea interspersed with transient constipation..Lactobacillus rhamnosus GG (LGG) is one of the best-studied Lactobacillus strains in clinical trials. Oksanen et al. found that LGG can effectively reduce the incidence of diarrhea without side effects. LGG also has been shown to induce remission and prevent recurrence of IBD in humans and in non-human colitis models. Yan et al. purified two soluble proteins from LGG supernatant (LGG-s) and demonstrated that LGG-s and soluble proteins produced by LGG-s activated Akt, inhibited cytokine-induced epithelial cell apoptosis, and promoted cell growth in human and mouse colon epithelial cells and cultured mouse colon explants. The role of probiotics on IBS has been proposed over the past decade and the suggestion that probiotics can relieve IBS symptoms has been confirmed by many studies..In recent study, we examined serotonin transporter (SERT) mRNA and serotonin transporter protein (SERT-P) levels in HT-29 cells and mice intestinal epithelial cell after treated with Lactobacillus rhamnosus GG supernatant(LGG-s). We observed that the SERT mRNA and SERT-P in HT-29 cells were higher than control when treated with LGG-s for 24h. This indicates that LGG-s can up-regulate SERT mRNA and SERT-P levels in HT-29 cells and mice intestinal epithelial cells. LGG-s increasing SERT expression and consequently decrease 5-HT levels offer potential mechanism for probiotics therapy for IBS..The purpose of the present study was to investigate the regulative mechanisms of LGG on SERT gene expression and the pharmacological action in the treatment of Post-infectious Irritable Bowel Syndrom

文献及课题组已发表前期工作证实腹泻型肠易激综合征5-羟转运体（SERT）低表达，SERT是5-HT再摄取的主要转运蛋白，SERT低表达可使肠黏膜5-HT过多蓄积导致腹泻。益生菌用于治疗感染后肠易激综合征（PI-IBS）且疗效良好，我们的预实验表明，鼠李糖乳杆菌GG(LGG)上清液可上调HT29细胞及小鼠结肠黏膜的SERT表达，LGG是否通过上调SERT表达在PI-IBS治疗中发挥作用及其机制值得研究。本研究拟①用SDS-PAGE、MALDI-TOF等筛查LGG上清液差异蛋白；②用Co-IP及酵母双杂交进行蛋白质-蛋白质相互作用研究；③用GST pull-dwon assay、EMSA、CHIP对LGG上调SERT表达的机制及信号通路进行研究；④构建SERT差异表达PI-IBS小鼠及大鼠模型并进行LGG治疗PI-IBS的动物实验及临床研究。本课题旨在为临床益生菌治疗PI-IBS提供理论依据。

本研究应用鼠李糖乳杆菌（LGG）等多种益生菌上清在细胞实验和动物实验中对结肠上皮细胞的SERT 表达进行研究并同时对LGG基因分析。发现如下：1.益生菌（LGG、嗜酸乳杆菌、长双歧杆菌、枯草杆菌、粪肠球菌、屎肠球菌）上清均可上调肠道上皮细胞SERT的表达，且具有时间和浓度依赖性；2.通过对感染后肠易激综合征（PI-IBS）动物模型研究发现，其结肠的SERT 表达下降，枯草杆菌、粪肠球菌等益生菌均可上调SERT表达并改善PI-IBS动物模型症状；3.LGG上清液>10kd蛋白对SERT的表达上调起到了重要作用，且发现其上调表达SERT过程需要EGFR信号通路的参与，这可能是LGG上清治疗肠易激综合征（IBS）的潜在机制，为临床应用LGG治疗IBS提供理论依据。4.本课题同时在两株不同来源LGG中发现了基因变异，这可能是导致分泌蛋白Msp1/p75表达差异的原因，而这一原因有可能与SERT上调表达差异有关。目前已证实在枯草杆菌、长双歧杆菌中同样发现了分子量与Msp1/p75相似的上清液分泌蛋白，然而其功能需要进一步的研究。以上结果表明：1.LGG以及其他益生菌（嗜酸乳杆菌、长双歧杆菌、枯草杆菌、粪肠球菌、屎肠球菌）均能够在肠上皮细胞中上调SERT表达，且这种上调作用与浓度相关； 2.LGG以及其他益生菌（枯草杆菌、屎肠球菌）可用于改善PI-IBS动物模型症状且疗效良好； 3.LGG上清液中大分子蛋白对SERT上调起重要作用，并通过EGFR通路上调SERT表达； 4.两株不同来源LGG中发现基因变异，其导致分泌蛋白Msp1/p75表达差异，其在LGG上调表达SERT缓解IBS患者症状中可能起着重要的作用。