孕期脂多糖诱导的孤独症样大鼠肠道5-羟色胺相关代谢通路的变化

Changes of colonic 5-hydroxytryptamine-related metabolic pathways in autism-like rats induced by lipopolysaccharide during pregnancy

目的:探讨孕期脂多糖(LPS)诱导的孤独症样子代大鼠肠道5-羟色胺(5-HT)相关代谢通路的变化。方法:将20只SD大鼠随机平分为LPS组和磷酸盐缓冲液(PBS)组(n=10),分别在怀孕9.5 d时腹腔注射LPS(100µg/kg)和等体积PBS,所产子代大鼠即为LPS组(n=60)和PBS组(n=60)。采用三箱社交实验和旷场实验检测子代大鼠社会交往能力和重复刻板行为。检测粪便含水量和胃肠转运时间以评估子代大鼠肠道蠕动功能。ELISA检测子代大鼠血清二胺氧化酶(DAO)、IgA和5-HT含量,以及粪便IgA含量。转录组学及色氨酸靶向代谢组学分析子代大鼠结肠组织、16S多样性检测子代大鼠粪便中肠道微生物菌群变化。Western blot技术测定子代大鼠结肠组织色氨酸羟化酶1(TPH1)和TPH2的表达水平。结果:孕期LPS处理可诱导子代大鼠出现孤独症样行为,并导致粪便含水量增加(P<0.01),胃肠转运时间和结肠转运时间缩短(P<0.01)。同时观察到LPS组子代大鼠血清5-HT、DAO和IgA含量及粪便IgA含量均有升高趋势。转录组学富集到色氨酸代谢通路以及犬尿酸相关通路;色氨酸靶向代谢组学显示LPS子代大鼠结肠中犬尿酸途径存在异常变化;16S多样性检测结果显示LPS组棒状杆菌(Coryne⁃bacterium)比例增加;且LPS组结肠组织TPH1和TPH2蛋白表达水平显著升高(P<0.01)。结论:妊娠期LPS暴露不仅导致子代大鼠孤独症样社交障碍,而且还会合并胃肠蠕动加快、肠上皮通透性增加等不良症状,究其原因可能是由于LPS处理激活了结肠组织中5-HT限速酶过度表达,导致肠道中5-HT含量升高所致。

AIM:To investigate the changes of colonic 5-hydroxytryptamine(5-HT)-related metabolic pathways in the autism-like offspring rats induced by lipopolysaccharide(LPS)during pregnancy.METHODS:Twenty SD rats were randomly divided into LPS group and phosphate-buffered saline(PBS)group(n=10),which were intraperitoneally injected with LPS(100µg/kg)and equal volume of PBS on the 9.5th day of pregnancy,and the offspring rats were the LPS group(n=60)and PBS group(n=60).Three-box test and open-field test were used to detect the social interaction ability and repetitive stereotyped behavior of offspring rats.Fecal water content and gastrointestinal transit time were used to evaluate the intestinal peristalsis function of offspring rats.The serum diamine oxidase(DAO),IgA and 5-HT levels,and the fecal IgA level in the offspring rats were determined by ELISA.Transcriptomics and tryptophan-targeted metabolomics were conducted in the colon tissue of offspring rats,and 16S DNA diversity was used to detect the changes of intestinal microbial flora in the feces of offspring rats.The expression levels of tryptophan hydroxylase 1(TPH1)and TPH2 proteins in the colon tissues of offspring rats were determined by Western blot.RESULTS:Exposure to LPS during pregnancy induced autism-like behavior in offspring rats,and also resulted in the increase in fecal water content and the decreases in gastrointestinal transit time and colonic transit time(P<0.01).Meanwhile,the serum levels of 5-HT,DAO and IgA and the fecal level of IgA showed an increasing tendency in the offspring rats of LPS group.Tryptophan metabolic pathways and kynuric acid-related pathways were enriched after transcriptomics analysis,and the abnormal changes of kynuric acid pathway were shown in the colon of offspring rats in LPS group via tryptophan-targeted metabolomics.The 16S diversity detection results displayed the increased ratio of Corynebacterium in LPS group.Moreover,the expression levels of TPH1 and TPH2 proteins were significantly elevated in the colon of offspring rats in LPS group(P<0.01).CONCLUSION:Exposure to LPS during pregnancy not only leads to autism-like behavior of offspring rats,but also possibly causes accelerated gastrointestinal peristalsis and increased intestinal epithelial permeability.The reason may be that LPS treatment activated the over-expression of TPH in colon tissue,resulting in increased intestinal 5-HT level in the offspring rats.