目的:探讨粪菌移植技术对脓毒症小鼠肠粘膜屏障的影响。方法:按随机数字表法将75只雄性ICR小鼠分为3组:假手术组、脓毒症组和脓毒症 + 粪菌移植组,每组25只。采用盲肠结扎穿孔方法制作脓毒症小鼠模型。各组取10只小鼠观察7 d存活率;剩余小鼠分别于制模后12、24、48 h心脏取血后各处死5只,测定血清IL-6、IL-10、TNF-α和高迁移率族蛋白B1(HMGB1)水平;并在光镜下观察脓毒症小鼠小肠组织病理学改变并进行评分。结果:粪菌移植治疗组小鼠7 d存活率较脓毒症组小鼠组改善(P < 0.05);与假手术组比较,脓毒症组造模后12、24及48 h血清IL-6、IL-10、TNF-α、HMGB1均有差异(P < 0.05);与脓毒症组相比,在造模后12、24及48 h粪菌治疗干预后,粪菌移植组小鼠的血清TNF-α、IL-6、IL-10及HMGB1均明显下降(P < 0.05),但仍高于假手术组(P < 0.05);光镜下小肠组织病理学改变,与假手术组相比,在造模后12、24及48 h,脓毒症组小鼠肠组织损伤评分明显升高(P < 0.05);与脓毒症组相比,造模后12 h粪菌移植组小鼠肠组织损伤评分无显著差异(P > 0.05),而24、48 h的时间点粪菌移植组小鼠肠组织损伤评分偏低(P < 0.05)。结论:粪菌移植治疗能够降低严重脓毒症小鼠炎症反应并减轻由其引起的肠组织损伤,从而改善脓毒症进程,提高存活率。 Objective: To investigate the effects of fecal microbiota transplantation on serum inflammatory fac-tors and intestinal injury in mice with sepsis. Methods: A total of 75 male C57BL/6 mice were ran-domly and equally divided into three groups: sham operation, sepsis, and sepsis + fecal microbiota transplantation. Severe sepsis model was induced by cecal ligation and puncture in mice. Ten mice in each group were monitored for the 7-d survival rate, and the remaining were killed 12, 24, and 48 h after the model was established. The levels of IL-6, IL-10, and high mobility group protein B1 (HMGB1) were measured. Results: The 7-day survival rate of severe sepsis mice was 0, and the 7-day survival rate of fecal bacteria transplantation group was increased to 50%; the difference was statistically significant compared to the model group (P < 0.05). Compared to the sham operation group, the sepsis group had higher serum creatinine levels at 12, 24, and 48 h after modeling, and the levels of TNF-α, IL-6, IL-10, and HMGB1 were significantly increased (P < 0.05). Compared to the sepsis group, the levels of serum TNF-a, IL-6, IL-10, and HMGB1 in the fecal bacteria transplantation group were significantly decreased (P < 0.05) after 12, 24, and 48 h of fecal bacteria treatment (P < 0.05), but still higher than those in the sham operation group (P < 0.05). Compared to the sepsis group, the intestinal tissue injury score of the fecal bacteria transplantation group did not show a significant difference at 12 h after modeling (P > 0.05), but the intestinal tissue injury score of the fecal bacteria transplantation group was lower at 24 and 48 h after modeling (P < 0.05). Conclusion: Fecal microbiota transplantation decreases the inflammatory response and intestinal tissue dam-age in severe sepsis mice and improves the survival rate.
目的:探讨粪菌移植技术对脓毒症小鼠肠粘膜屏障的影响。方法:按随机数字表法将75只雄性ICR小鼠分为3组:假手术组、脓毒症组和脓毒症 + 粪菌移植组,每组25只。采用盲肠结扎穿孔方法制作脓毒症小鼠模型。各组取10只小鼠观察7 d存活率;剩余小鼠分别于制模后12、24、48 h心脏取血后各处死5只,测定血清IL-6、IL-10、TNF-α和高迁移率族蛋白B1(HMGB1)水平;并在光镜下观察脓毒症小鼠小肠组织病理学改变并进行评分。结果:粪菌移植治疗组小鼠7 d存活率较脓毒症组小鼠组改善(P < 0.05);与假手术组比较,脓毒症组造模后12、24及48 h血清IL-6、IL-10、TNF-α、HMGB1均有差异(P < 0.05);与脓毒症组相比,在造模后12、24及48 h粪菌治疗干预后,粪菌移植组小鼠的血清TNF-α、IL-6、IL-10及HMGB1均明显下降(P < 0.05),但仍高于假手术组(P < 0.05);光镜下小肠组织病理学改变,与假手术组相比,在造模后12、24及48 h,脓毒症组小鼠肠组织损伤评分明显升高(P < 0.05);与脓毒症组相比,造模后12 h粪菌移植组小鼠肠组织损伤评分无显著差异(P > 0.05),而24、48 h的时间点粪菌移植组小鼠肠组织损伤评分偏低(P < 0.05)。结论:粪菌移植治疗能够降低严重脓毒症小鼠炎症反应并减轻由其引起的肠组织损伤,从而改善脓毒症进程,提高存活率。
脓毒症,粪菌移植,肠道菌群
Peng Zhao, Xinli Yang, Qing Mi, Baohai Shi*
Department of Pediatrics, Tai’an Central Hospital, Tai’an Shandong
Received: May 8th, 2022; accepted: May 25th, 2022; published: Jun. 10th, 2022
Objective: To investigate the effects of fecal microbiota transplantation on serum inflammatory factors and intestinal injury in mice with sepsis. Methods: A total of 75 male C57BL/6 mice were randomly and equally divided into three groups: sham operation, sepsis, and sepsis + fecal microbiota transplantation. Severe sepsis model was induced by cecal ligation and puncture in mice. Ten mice in each group were monitored for the 7-d survival rate, and the remaining were killed 12, 24, and 48 h after the model was established. The levels of IL-6, IL-10, and high mobility group protein B1 (HMGB1) were measured. Results: The 7-day survival rate of severe sepsis mice was 0, and the 7-day survival rate of fecal bacteria transplantation group was increased to 50%; the difference was statistically significant compared to the model group (P < 0.05). Compared to the sham operation group, the sepsis group had higher serum creatinine levels at 12, 24, and 48 h after modeling, and the levels of TNF-α, IL-6, IL-10, and HMGB1 were significantly increased (P < 0.05). Compared to the sepsis group, the levels of serum TNF-a, IL-6, IL-10, and HMGB1 in the fecal bacteria transplantation group were significantly decreased (P < 0.05) after 12, 24, and 48 h of fecal bacteria treatment (P < 0.05), but still higher than those in the sham operation group (P < 0.05). Compared to the sepsis group, the intestinal tissue injury score of the fecal bacteria transplantation group did not show a significant difference at 12 h after modeling (P > 0.05), but the intestinal tissue injury score of the fecal bacteria transplantation group was lower at 24 and 48 h after modeling (P < 0.05). Conclusion: Fecal microbiota transplantation decreases the inflammatory response and intestinal tissue damage in severe sepsis mice and improves the survival rate.
Keywords:Sepsis, Fecal Microbiota Transplantation, Gut Microbiota
Copyright © 2022 by author(s) and Hans Publishers Inc.
This work is licensed under the Creative Commons Attribution International License (CC BY 4.0).
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脓毒症(Sepsis)是由感染引起的急危重症,重度脓毒症可继发严重的多脏器功能衰竭和脓毒性休克,其病死率可达50%以上 [
成年雄性C57BL/6小鼠6~8周龄,体重20~25 g,由山东大学医学科学院实验动物中心(济南)饲养并提供,控制动物房室温于22℃~24℃,相对湿度约50%,自然昼夜光线照明,动物可自由进食水,常规饲养1周使其适应饲养环境后用于实验研究。按照随机数字表法将实验动物分为三组(n = 75):假手术组、脓毒症组和脓毒症 + 粪菌移植组。本实验中以及后续实验中所涉及实验步骤及过程均经泰安市中心医院动物实验管理委员会批准。
采用盲肠结扎穿孔术(cecal ligation and puncture, CLP) [
按摩刺激对照组小鼠腹部排便,收集新鲜粪便,置于消毒的离心管中,将收集的粪便称重,加入37℃无菌生理盐水混匀,双层无菌纱布过滤掉残渣,6000 rpm,4℃离心5分钟,沉淀颗粒物,取上清液制成400 mg/ml浓度的FMT混悬液,每次FMT后留存500 uL菌液−80℃存放用于菌群分析。参考人体粪菌移植标准 [
制模后每组各取10只于标准条件下饲养,连续观察7 d并记录存活率,观察期间及时移除死亡动物尸体,以免对饲养环境造成污染。余各组15只小鼠于造模后12 h、24 h及48 h各处死5只备检。
于造模后12 h、24 h及48 h麻醉小鼠,沿原切口切开腹壁并充分暴露,在距回盲瓣2 cm处取回肠组织并制备组织匀浆。采取酶联免疫吸附试验(ELISA)测定肠道组织中促炎性细胞因子肿瘤坏死因子-α (Tumour necrosis factor-α, TNF-α)、白细胞介素-6 (Interleukin-6, IL-6)、和高迁移率族蛋白1 (High mobility group box-1, HMGB1)以及抗炎性细胞因子白细胞介素-10 (Interleukin-10, IL-10)的水平评价肠道组织的炎症反应情况。
于造模后12 h、24 h及48 h麻醉小鼠,沿原切口切开腹壁并充分暴露,在距回盲瓣2 cm处取回肠组织备用。组织用10%的福尔马林固定后,依次经梯度乙醇脱水、石蜡包埋、组织切片、二甲苯脱蜡后HE染色。光镜下观察结肠组织的病理变化,并进行肠组织损伤评分 [
统计学分析:采用SPSS18.0统计软件进行数据分析。采用Fisher确切概率法进行组间比较。计量资料以均数 ± 标准差(x ± s)表示,组间比较采用单因素方差分析,两两比较采用LSD检验,采用Kaplan-Meier生存分析方法。P < 0.05表示差异有统计学意义。
1) 粪菌移植治疗后小鼠体重变化趋势,可缓解小鼠体重下降(图1);粪菌移植治疗对脓毒症小鼠存活率的影响(图2):粪菌移植治疗显著提高了脓毒症小鼠的存活率(P < 0.05)。
图1. 各组小鼠体重变化水平
图2. 各组小鼠7天生存分析
2) 粪菌移植治疗对严重脓毒症小鼠血清炎性因子的影响(表1):与假手术组相比,在造模后12、24及48 h,脓毒症组小鼠的血清TNF-α、IL-6、IL-10及HMGB1均明显升高(P < 0.05);与脓毒症组相比,在造模后12、24及48 h粪菌治疗干预后,粪菌移植组小鼠的血清TNF-α、IL-6、IL-10及HMGB1均明显下降(P < 0.05),但仍高于假手术组(P < 0.05)。光镜下小肠组织病理学改变(图3),与假手术组相比,在造模后12、24及48 h,脓毒症组小鼠肠组织损伤评分明显升高(P < 0.05);与脓毒症组相比,造模后12 h粪菌移植组小鼠肠组织损伤评分无显著差异(P > 0.05),而24、48 h的时间点粪菌移植组小鼠肠组织损伤评分偏低(P < 0.05)。
术后时间 | 分组 | TNF-α (pg/ml) | IL-6 (pg/ml) | IL-10 (pg/ml) | HMGB1 (pg/mg) | 肠组织病理学 评分 |
---|---|---|---|---|---|---|
12 h | 假手术组 | 32.97 ± 1.45 | 21.87 ± 1.28 | 17.06 ± 2.20 | 11.06 ± 1.71 | 0.2 ± 0.45 |
脓毒症组 | 434.95 ± 5.64* | 175.79 ± 3.11* | 77.26 ± 6.33* | 23.78 ± 2.38* | 1.6 ± 0.55* | |
脓毒症 + FMT组 | 143.28 ± 5.16*# | 99.86 ± 5.31*# | 101.06 ± 3.86*# | 19.70 ± 4.32* | 1.6 ± 0.55* | |
24 h | 假手术组 | 33.23 ± 0.84 | 21.93 ± 1.99 | 17.38 ± 1.65 | 11.68 ± 1.28 | 0.6 ± 0.55 |
脓毒症组 | 435.61 ± 4.26* | 169.01 ± 5.65* | 70.00 ± 6.39* | 170.18 ± 7.04* | 3.6 ± 0.55* | |
脓毒症 + FMT组 | 126.99 ± 3.76*# | 91.38 ± 1.35*# | 101.65 ± 6.74*# | 91.42 ± 6.40*# | 1.8 ± 0.84*# | |
48 h | 假手术组 | 34.89 ± 1.06 | 22.39 ± 1.87 | 17.20 ± 1.53 | 10.96 ± 1.15 | 0.6 ± 0.55 |
脓毒症组 | 437.82 ± 4.36* | 160.76 ± 6.14* | 72.62 ± 4.99* | 118.89 ± 6.11* | 4.4 ± 0.55* | |
脓毒症 + FMT组 | 125.48 ± 2.95*# | 96.90 ± 5.90*# | 101.56 ± 5.37*# | 65.39 ± 2.23*# | 2.2 ± 0.45*# |
表1. 粪菌移植治疗对严重脓毒症小鼠不同时间点血清炎性因子及肠组织病理学评分( x ¯ ± s )
*:P < 0.05,与假手术组比较;#:P < 0.05,与脓毒症组比较。
图3. 光镜下观察术后12 h、24 h和48 h各组小鼠小肠组织病理学改变
本研究主要是通过经典的盲肠结扎穿孔诱导重度脓毒症 [
粪菌移植(FMT) [
临床研究发现脓毒症患者中肠道菌群存在高度特异性,其主要包括细菌菌数的多样性显著下降,微生物的生理功能丢失及单一菌群的过度生长 [
脓毒症是有感染诱发的,免疫系统对入侵的感染性微生物的不受控制的过度反应,其特征是促炎介质如IL-6、TNF-α和HMGB1的大量产生可能损害器官功能,导致不同器官的异常凋亡,导致多器官功能障碍综合征和死亡 [
综上所述,本研究证实粪菌移植能够明显提高严重脓毒症小鼠的存活率,改善肠组织病理特征,减轻炎性因子的释放。为其应用于临床脓毒症患者的治疗提供了理论依据。但在我们的研究中,并未能解释该结果的具体机制,我们考虑可能与肠道菌群的重新建立有关,需要进一步进行实验阐明。
泰安市科学技术发展计划(项目编号:2018NS0212)。
赵 鹏,杨新利,米 青,史宝海. 粪菌移植对脓毒症小鼠血清炎性因子和肠损伤的影响Effects of Fecal Microbiota Transplantation on Serum Inflammatory Factors and Intestinal Injury in Sepsis Mice[J]. 临床医学进展, 2022, 12(06): 5114-5120. https://doi.org/10.12677/ACM.2022.126741