隔日禁食对帕金森病模型小鼠肠道屏障的保护作用及机制

Protective effect and mechanisms of alternate day fasting on intestinal barrier function in Parkinson’s disease model mice

摘要:

【背景】帕金森病是一种神经退行性疾病，常伴有胃肠功能障碍等非运动症状。肠道菌群紊乱与肠上皮通透性增强是引起肠道屏障功能障碍的主要因素。饮食限制可改善肠道菌群的构成、维持肠上皮稳态。本文假设隔日禁食对帕金森病模型小鼠肠道屏障有保护作用，其机制可能与纠正肠道菌群的失调以及促进肠紧密连接蛋白的表达有关。【目的】探究隔日禁食对帕金森病模型小鼠肠道屏障的保护作用及其机制。【方法】32只C57BL/6小鼠随机分成生理盐水+自由饮食组(NS+AL组，n=8)、生理盐水+隔日禁食组(NS+ADF组，n=8)、MPTP+自由饮食组(MPTP+AL组，n=8)、MPTP+隔日禁食组(MPTP+ADF组，n=8)共4组。隔日禁食方案以48 h为一个实验周期，前24 h采取禁食，后24 h采取自由摄食，在第12?14个周期内连续5 d给予小鼠腹腔注射1-甲基-4苯基-1,2,3,6-四氢吡啶(1-methyl-4-phenyl-1,2,3,6-tetrathydropyridine，MPTP)建立帕金森病模型。在隔日禁食17个周期结束后收集小鼠粪便，通过16S rRNA基因高通量测序检测小鼠肠道菌群的变化；小鼠行为学测试后收集其空肠组织，通过HE染色观察肠道病理组织学变化，通过RT-qPCR方法检测AMPK、Occludin、ZO-1的mRNA表达水平(Prkaa1、Ocln、Tjp1)，通过Western blotting方法检测ZO-1的蛋白表达水平。【结果】行为学测试结果显示，与NS+AL组相比，MPTP+AL组小鼠运动能力显著下降(P<0.01)，而MPTP+ADF组小鼠运动障碍有所改善(P<0.01)。HE染色可见NS+AL组小鼠空肠绒毛结构完整、排列紧密，MPTP+AL组空肠绒毛破碎甚至脱落，而MPTP+ADF组则显示出空肠绒毛相对完整、排列紧密。肠道菌群测序结果显示，MPTP+AL组相较于NS+AL组，肠道菌群的丰度和多样性显著升高(P<0.001)，而相较于MPTP+ADF组并无显著变化；各组间小鼠的肠道菌群构成具有显著差异，相对物种丰度在科水平的检测结果显示，与NS+AL组相比，MPTP+AL组艾克曼菌科(Akkermansiaceae)丰度明显升高(P<0.05)，而MPTP+ADF组相较于MPTP+AL组该科菌的丰度显著下降(P<0.01)。RT-qPCR检测结果发现，相较于NS+AL组，MPTP+AL组小鼠空肠Prkaa1 (P<0.01)、Ocln (P<0.01)、Tjp1 (P<0.01)表达水平显著降低，其中，MPTP+ADF组与MPTP+AL组小鼠相比Prkaa1 (P<0.01)和Tjp1 (P<0.01)表达水平均有显著升高，MPTP+ADF组的Ocln表达水平也比MPTP+AL组高，但差异无显著性。Western blotting结果显示，ZO-1在MPTP+ADF组小鼠表达水平相较于MPTP+AL组显著上升(P<0.05)。【结论】隔日禁食对帕金森病模型小鼠空肠屏障具有保护作用，其机制可能与维持肠道菌群艾克曼菌科相对丰度及菌群稳态、提高空肠紧密连接表达有关。

Abstract:

[Background] Parkinson’s disease is a neurodegenerative disorder accompanied by gastrointestinal dysfunction and other non-motor symptoms. Intestinal microflora disorder and increased intestinal epithelial permeability are the main factors that affect the function of intestinal barrier. Dietary restriction can improve the composition of intestinal microflora and maintain intestinal epithelial homeostasis. We hypothesize that dietary restriction might have a protective effect on intestinal barrier function in Parkinson’s disease model mice and the mechanism might be related to the correction of intestinal microflora disorder and the promotion of intestinal tight junction protein expression. [Objective] To investigate the protective effect and its mechanism of alternate day fasting on intestinal barrier in Parkinson’s disease model mice. [Methods] Thirty-two C57BL/6 mice were randomly divided into normal saline+fed libitum group (NS+AL, n=8), normal saline+alternate day fasting group (NS+ADF group, n=8), MPTP+fed libitum group (MPTP+AL group, n=8) and MPTP+alternate day fasting group (MPTP+ADF group, n=8). We took 48 h as an experimental cycle of the alternate day fasting program, with fasting in the first 24 h and free feeding in the second 24 h. We injected 1-methyl-4-phenyl-1,2,3,6-tetrathydropyridine (MPTP) into mice intraperitoneally to build Parkinson’s disease model in the 12th?14th cycles (for five consecutive days) of ADF. After the end of the 17th cycle, we collected the feces of mice and detected the changes of intestinal microflora by high-throughput 16S rRNA gene sequencing, then we detected the behavioral test of mice. After collect jejunum tissues of mice, we observed the histopathological changes of intestine by HE staining. We detected mRNA expression levels of AMPK, Occludin and ZO-1 by RT-qPCR (Prkaa1, Ocln, Tjp1), and detected protein expression levels of ZO-1 by Western blotting in tissue. [Results] The results of behavioral test showed that compared with NS+AL group, the motor ability of mice in MPTP+AL group decreased significantly (P<0.01), while the motor ability of mice in MPTP+ADF group improved (P<0.01). HE staining showed that the jejunum villi in the NS+AL group were structurally completed and closely arranged, while the jejunum villi in the MPTP+AL group were broken or even shed. Compared with the MPTP+AL group, the MPTP+ADF group showed that the jejunum villi were intact and closely arranged. Sequencing results of intestinal microflora showed that the abundance and diversity of intestinal microflora in MPTP+AL group were significantly higher than that in NS+AL group (P<0.001), while there was no significant change between MPTP+AL group and MPTP+ADF group. There were significant differences in intestinal microflora structure between four groups. The relative species abundance at family level showed that compared with NS+AL group, Akkermansiaceae abundance in MPTP+AL group was significantly increased (P<0.05), while in MPTP+ADF group this was significantly decreased (P<0.01). The results of RT-qPCR showed the expression levels of Prkaa1 (P<0.01), Ocln (P<0.01), Tjp1 (P<0.01) were significantly decreased in the MPTP+AL group, while the expression levels of Prkaa1 (P<0.01) and Tjp1 (P<0.01) were significantly increased in the MPTP+ADF group. The expression level of Ocln in the MPTP+ADF group was also higher than the MPTP+AL group, but the difference was not significant. Western blot results showed ZO-1 expression in MPTP+ADF group was significantly higher than that in MPTP+AL group (P<0.05). [Conclusion] Alternate day fasting has protective effect on intestinal barrier in Parkinson’s disease model mice and the mechanism may be related to maintaining the relative abundance at family level of Akkermansiaceae and increasing the expression of intestinal tight junctions.