辣蓼中性多糖单糖组成分析、抗小鼠大肠杆菌性腹泻作用及其分子机制

Monosaccharide composition analysis of neutral polysaccharide from Polygonum hydropiper and its effect and mechanism against Escherichia coli-induced diarrhea in mice

目的分析辣蓼中性多糖(NPP)的单糖组成,评价其抗大肠杆菌性腹泻作用,并基于高通量转录组测序技术探讨其作用机制。方法水提醇沉法制备辣蓼粗多糖,通过反复柱色谱分离纯化得到中性多糖组分NPP,采用1-苯基-3-甲基-5-吡唑啉酮(PMP)柱前衍生化-高效液相色谱(HPLC)法分析NPP单糖组成。雄性BALB/c小鼠随机分为正常对照组、模型组、模型+恩氟沙星5 mg·kg^(-1)组、模型+NPP 40和80 mg·kg^(-1)组。除正常对照组外,其余各组小鼠ip给予大肠杆菌菌液(1.0×10^(12)CFU·L^(-1),10 mL·kg^(-1)),每日1次,连续8 d造摸;正常对照组小鼠注射同体积生理盐水。给药组于造模前2 d ig给药,每日2次,连续给药10 d;正常对照组和模型组小鼠ig给予等量生理盐水。HE染色观察小鼠十二指肠组织病理变化;高通量转录组测序技术分析正常对照组、模型组和模型+NPP组小鼠肠组织的差异表达基因(DEG),并对DEG进行GO功能富集和KEGG信号通路富集分析;采用实时荧光定量PCR(RT-qPCR)检测DEG mRNA表达量。结果NPP主要由葡萄糖、半乳糖、阿拉伯糖及半乳糖醛酸组成,单糖在各自浓度范围内线性关系良好(R^(2)≥0.9995),平均加样回收率为99.03%~102.41%,各单糖摩尔比为13.66∶10.33∶2.34∶1.00。HE染色结果表明,模型组小鼠十二指肠细胞水肿变性,少量细胞坏死或凋亡;模型+NPP组十二指肠组织结构清晰,仅见少量炎症细胞浸润。转录组测序结果显示,与正常对照组相比,模型组共筛选出1020个DEG;与模型组相比,模型+NPP组共筛选出960个DEG。GO功能分析显示,DEG主要富集于细胞过程、单有机体过程、代谢过程、细胞等生物学过程;KEGG通路分析显示,DEG主要富集于细胞因子-细胞因子受体相互作用通路、Toll样受体信号通路和趋化因子信号通路等炎症相关信号通路。RT-qPCR验证结果表明,选取的8个DEG的差异表达倍数趋势与转录组测序结果基本一致。结论建立的PMP-HPLC方法简单可行、重复性好,可用于NPP的分析检测及质量控制。NPP对大肠杆菌性腹泻小鼠的受损十二指肠具有明显的修复作用,其作用机制可能与调控机体的趋化因子信号通路,Toll样受体信号通路等有关。

OBJECTIVE To analyze the monosaccharide composition of neutral polysaccharide from Polygonum hydropiper(NPP)and investigate its mechanism against Escherichia coli(E.coli)induced diarrhea via high-throughput transcriptome sequencing technology.METHODS Crude polysaccharide of Polygonum hydropiper was prepared via water extraction and alcohol precipitation before being separated and purified by repeated column chromatography to obtain NPP.The monosaccharide composition of NPP was analyzed using the 1-pheny-3-methyl-5-pyrazolone(PMP)precolumn derivatization-high perfor⁃mance liquid chromatography(HPLC)method.Male BALB/c mice were randomly divided into the normal control group,model group,model+enrofloxacin(5 mg·kg^(-1))group and model+NPP(40 and 80 mg·kg^(-1))groups.Except the normal control group,mice in the other groups were ip given Escherichia coli solution(1.0×10^(12)CFU·L^(-1),10 mL·kg^(-1))once a day for 8 consecutive days.Mice in the normal control group were ip given the same volume of saline.The administration groups were ig administered 2 days before mod⁃eling,twice a day,for 10 consecutive days.The normal control group and model group were ig given the same amount of saline.Histopathological analysis of duodenal samples was performed by HE staining.Transcriptome sequencing technology was used to detect the gene expression profiles of duodenal tissues in the normal group,model group and NPP 80 mg·kg^(-1)group.Differentially expressed genes(DEGs)were screened out,the GO enrichment and KEGG pathway analysis of DEGs were conducted,and the expression levels of DEGs were verified by real-time quantitative PCR(RT-qPCR).RESULTS NPP was mainly composed of glucose,galactose,arabinose,galacturonic acid with the molar ratios of 13.66∶10.33∶2.34∶1.00.The linearity of each monosaccharide in its concentration range was good(R2≥0.9995)and the average recoveries ranged from 99.03%to 102.41%.HE staining showed that a large number of intestinal cells in the model group had obvious edema and degeneration,and a small number of cells had necrosis or apoptosis.The intestinal structure in the model+NPP group was clear,and only a few inflammatory cells were observed.RNA-seq results showed that compared with the normal group,1020 DEGs were screened in the model group.Compared with the model group,960 DEGs were screened in model+NPP 80 mg·kg^(-1)group.GO functional analysis showed that DEGs were mainly enriched in the cell process,single organism process,metabolism process and cells.KEGG pathway analysis displayed that DEGs were mainly enriched in inflammatory-related signaling pathways such as cytokine-cytokine receptor interaction pathway,Toll-like receptor signaling pathway,chemokine signal⁃ing pathway.RT-qPCR results showed that the fold change of the selected eight DEGs was basically consistent with that of transcriptome sequencing.CONCLUSION The established PMP-HPLC analytical method is simple,feasible and reproducible,which could be used for the determination and quality control of NPP.The mechanism of NPP in the treatment of enterophathogenic E.coli-induced diarrhea may be related to the regulation of chemokine signaling pathway and Toll-like receptor signaling pathway.