粪菌移植对 LPS 诱导小鼠重症肺炎的改善作用及分子机制

Ameliorative effect and mechanism of fecal microbiota transplantation on LPS-induced severe pneumonia in mice

摘要

目的 探讨粪菌移植（FMT）对脂多糖（LPS）诱导重症肺炎小鼠TLR4/NF-κ B 信号通路的影响及机制。方法 30 只 6∼8 周龄雄性 SPF 级 BALB/c 小鼠随机均分为正常对照组（control）、重症肺炎模型组（LPS）、FMT组。LPS组和 FMT 组鼻腔滴人 LPS造模，control组滴人无菌生理盐水；造模后 6 h，FMT组灌胃粪菌移植液，其余两组灌胃等体积无菌生理盐水，持续干预 5 d 。第 6 天检测血清及支气管肺泡灌洗液（BALF）中炎症细胞水平、IL-1 β 、IL-6、TNF-α 含量，观察肺组织病理改变并评分，通过免疫组化及Western blot检测肺组织TLR4、p-NF-кB p65、NF-кB p65蛋白表达，收集粪便进行 16 S rRNA测序。结果 与 control组相比，LPS 组血清及 BALF 中中性粒细胞百分比显著升高（P<0.001），血清 IL1β 、IL-6、TNF-α 水平及 BALF 中 IL1β 、TNF-α 水平显著升高（P<0.05），肺组织炎症浸润明显、病理评分升高（P<0.01），TLR4、p-NF-кB p65表达上调（P<0.01），NF-кB p65无显著变化，肠道菌群失衡。与 LPS 组相比，FMT 组上述炎症指标均显著降低（P<0.05），肺组织病理损伤改善、评分降低（P<0.01）， TLR4、 p -NF-κB p65表达下调（P<0.05），肠道菌群结构恢复。结论 FMT 可通过抑制 TLR4/NF-κB 通路减轻肺部免疫炎症反应，改善 LPS 诱导的小鼠重症肺炎。

Abstract

Objective To investigate the effect of fecal microbiota transplantation（FMT）on the TLR4/NF-κ B signaling pathway in mice with LPS-induced severe pneumonia. Methods Thirty 6-8-week-old male SPF BALB/c mice were randomly divided into normal control group，severe pneumonia model group（LPS），and FMT group.The mice in LPS group and FMT group were intranasally instilled with LPS into the nasal cavity to establish the model，while the mice in control group were instilled with sterile normal saline. From 6 h after the model establishment，the mice in FMT group were intragastrically gavaged with fecal microbiota suspension for 5 d， while the mice in the other two groups were intragastrically gavaged with the same volume of sterile normal saline.At day 6，the levels of inflammatory cells，and the contents of IL-1 β，IL-6，and TNF-α in serum and bronchoalveolar lavage fluid（BALF）were detected. The pathological changes of lung tissue and the scores were observed.The protein expressions of TLR4，p-NF-κB p65，and NF-κB p65 in lung tissue were detected by immunohistochemistry and Western blot.The feces were collected for 16S rRNA sequencing. Results Compared with control group，the percentage of neutrophils in serum and BALF significantly increased in LPS group（P<0.001）；the levels of IL-1 β，IL-6，and TNF-α in serum were all significantly elevated（P<0.05），while only IL-1 β and TNF-α levels in BALF significantly increased（P<0.05）；the inflammatory infiltration in lung tissue was obvious，and the pathological score increased（P<0.01）； the expressions of TLR4 and p-NF-κB p65 were up-regulated（P<0.01），while NF-κB p65 showed no significant change；the gut microbiota imbalance was observed.Compared with LPS group，the above inflammatory indicators were significantly reduced in FMT group（P<0.05）；the pathological damage of lung tissue was improved，and the score was decreased（P<0.01）；the expressions of TLR4 and p-NF-κ B p65 were down-regulated（P<0.05），and the gut microbiota structure was restored. Conclusion FMT can improve LPS-induced severe pneumonia in mice by alleviating the pulmonary immune inflammatory response via the inhibition of TLR4/NF-кB pathway.

关键词

粪菌移植 / 脂多糖 / 重症肺炎 / TLR4/NF-кB信号通路 / 肠一肺轴 / 肠道菌群 / 炎症反应 / 小鼠

Key words

fecal microbiota transplantation / lipopolysaccharide（LPS） / severe pneumonia / TLR4/NF-кB signaling pathway / gut-lung axis / gut microbiota / inflammatory response / mice

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基金资助

陕西省重点研发计划项目(S2024-YF-YBSF-0840)