一株猪肠道Lactobacillus animalis LGM对结肠炎小鼠Th细胞分化转录因子基因表达的影响

Effects of Lactobacillus animalis LGM on transcription factors gene expressions in DSS-induced colitis mice

摘要:

[目的]从体外和体内研究Lactobacillus animalis LGM对Th细胞分化转录因子T-bet、GATA-3、ROR-γt和Foxp3的调节作用，以及探究L. animalis LGM对小鼠结肠炎的影响。[方法]本试验采用改良型的Hungate滚管技术从猪结肠内容物中分离一株L. animalis LGM，根据其16S rRNA序列进行鉴定。收集L. animalis LGM培养液上清，与细菌脂多糖（LPS，2μg/mL）同时孵育Caco-2细胞24 h，体外研究L. animalis LGM对Caco-2细胞内Th细胞分化转录因子（T-bet，GATA3，ROR-γt和Foxp3） mRNA表达的影响；配制L. animalis LGM细菌悬液，研究L. animalis LGM灌胃对DSS诱导结肠炎小鼠症状及结肠Th细胞分化转录因子和细胞因子（IFN-γ，IL-4，IL-17和IL-10） mRNA表达的影响，表达结果采用荧光定量PCR法检测。[结果]与对照组相比，L. animalis LGM培养液上清显著上调Caco-2细胞内ROR-γt与Foxp3 mRNA表达（P<0.05），显著下调GATA3、IL-4、IL-17和TGF-β mRNA表达（P<0.05）。L. animalis LGM灌胃显著上调小鼠结肠内ROR-γt和Foxp3的表达（P<0.05），显著降低了促炎因子IL-4和IL-17的表达（P<0.05），阻止了小鼠结肠长度缩短（P<0.05）。[结论]猪肠道分离L. animalis LGM表现出对Th细胞分化转录因子的选择性调节，显著上调Caco-2细胞及结肠炎小鼠ROR-γt与Foxp3 mRNA表达。降低DSS诱导结肠炎小鼠炎症水平，对DSS诱导结肠炎起保护作用，有助于维护肠道环境稳态。

Abstract:

[Objective] Investigating the relationship between Lactobacillus animalis LGM and transcription factors (T-bet, GATA3, ROR-γt and Foxp3) gene expressions in vivo or in vitro and observing the effects of L. animalis LGM gavage on DSS-induced colitis.[Methods] L. animalis LGM was isolated from pig colon contents using Hungate rolling tube technique. Bacterial supernatants were extracted and incubated Caco-2 cells with LPS (2 μg/mL) simultaneously. Bacterial suspension was adjusted to 1×10⁹ CFU/0.2 mL and gavaged to DSS-induced mice. Gene expressions of transcription factors (T-bet, GATA-3, ROR-γt and Foxp3) and intracellular cytokines (IL-4, IL-17, INF-γ and IL-10) were analyzed by real-time PCR. [Results] In Caco-2 cells culture experiment, compared to control and LPS groups, L. animalis LGM supernatants treatment significantly downregulated the GATA-3, IL-4, IL-17 and TGF-β gene expressions and upregulated Foxp3 and ROR-γt gene expressions (P<0.05). In DSS-induced colitis mice, compared to the DSS group, L. animalis LGM administration restored the colonic length and increased ROR-γt and Foxp3 gene expressions (P<0.05). [Conclusion] L. animalis LGM isolated from pig colon contents in the present study have distinctive effects on transcription factors. ROR-γt and Foxp3 expressions were significantly upregulated both in Caco-2 cells and DSS-induced colitis mice. L. animalis LGM showed protection on DSS-induced colitis and gut inflammation.