复合益生菌制剂对儿童功能性便秘肠道菌群及5-羟色胺影响

The effects of compound probiotics on intestinal flora and 5-hydroxytryptamine in children with functional constipation

目的 探讨添加复合益生菌制剂对儿童功能性便秘肠道菌群及5-羟色胺(5-HT)的动态影响,为临床治疗提供依据。方法 选取2022年7月至2023年7月在济南市儿童医院门诊收治的临床诊断为儿童功能性便秘的患儿作为研究对象,连续给予复合益生菌口服,按不同治疗时段分为治疗前组、治疗1月组、治疗2月组、治疗3月组,同期选取我院儿童保健科体检的同年龄段健康儿童为健康对照组,采用16S rDNA扩增子测序的方法检测各组肠道菌群的组成并分析其结果;同时,采用ELISA法测定各组血浆5-HT的水平并分析其结果。结果 共纳入便秘患儿10例,其中男6例、女4例,年龄7.16±2.2岁;健康儿童18例,其中男10例、女8例,年龄7.5±2.63岁。两组间性别及年龄的差异均无统计学意义(P>0.05)。各组肠道菌群Alpha多样性差异无统计学意义(P>0.05)。Beta多样性分析中,主坐标分析显示便秘各组肠道菌群结构与健康组肠道菌群结构差异较大;PERMA-NOVA分析显示,便秘治疗前组、治疗1月、治疗2月、治疗3月组菌群结构与健康对照组相比,存在显著差异(P<0.01),便秘各组治疗前后差异无统计学意义(P>0.05)。在门水平中各组主要菌群丰度排名前4位均依次为厚壁菌门、放线菌门、拟杆菌门、变形菌门。其中便秘治疗前组(0.402±0.11)、治疗1月组(0.408±0.108)厚壁菌门丰度较健康对照组(0.615±0.16)显著降低,差异有统计学意义(P<0.05);便秘治疗前组(0.206±0.101)拟杆菌门丰度较健康对照组(0.064±0.052)显著增高,差异有统计学意义(P<0.05)。在属水平中,双歧杆菌为各组首位优势菌种。与健康对照组(0.095±0.061,0.033±0.034)相比,便秘治疗前组(0.037±0.028,0.002±0.002)、治疗1月组(0.029±0.02,0.009±0.013)、治疗2月组(0.034±0.015,0.004±0.005)拟杆菌属及链球菌属丰度降低,差异有统计学意义(P<0.05)。便秘治疗前组(0.482±0.214)、治疗1月组(0.743±0.189)、治疗2月组(0.676±0.221)患儿血清5-HT水平低于健康对照组(1.071±0.149),差异有统计学意义(P<0.05);便秘治疗后1月组、治疗3月组(0.901±0.152)患儿血清5-HT水平较便秘治疗前组明显升高,差异有统计学意义(P<0.05)。结论 功能性便秘儿童存在肠道菌群结构改变的情况,便秘与肠道菌群之间可能存在相关性,添加多种益生菌可以改善便秘患儿肠道菌群结构,并提高体内5-HT水平。

Objective To investigate the dynamic effects of adding compound probiotics on intestinal flora and 5-hydroxytryptamine(5-HT) in children with functional constipation and provide evidence for clinical treatment. Methods From July 2022 to July 2023,Children diagnosed with functional constipation in the outpatient department of Jinan Children's Hospital were selected as the research objects. And compound probiotics were given orally continuously. According to different treatment periods, they were divided into pre-treatment group, treatment 1 month group, treatment 2 month group and treatment 3 month group, and normal children were assigned as the control group. The 16S rDNA amplicon sequencing method was used to detect the intestinal bacteria to analyze the composition and compare the results.Meanwhile, the plasma 5-HT levels in each group were determined by ELISA and the results were analyzed. Results A total of 10 children with functional constipation were included. 6 males and 4 females, aged(7.16±2.2) years;and 18 cases in the healthy control group, 10 males and 8 females, aged(7.5±2.63) years. The differences in gender and age among the two groups were no statistically signifificant(P>0.05). There was no significant difference in the Alpha diversity of intestinal flora between the each groups(P>0.05). In the Beta diversity, principal coordinates analysis showed that intestinal flora structure of the constipation group was significantly different from the healthy group.PERMANOVA analysis showed that the microflora structure of pre-treatment group, treatment 1 month group, treatment 2 month group and treatment 3 month group had statistical significance compared with healthycontrol group(P<0.01).There were no significant difference of the microflora structure in each functional constipation groups(P>0.05).At the phylum level, Firmicutes, Actinomycetes, Bacteroidetes and Proteobacteria were the most abundant flora in the intestinal microbial communities of each groups. Among them, the abundance of Firmicutes in pre-treatment group(0.402±0.11)and treatment 1 month group(0.408±0.108) were lower than healthy control group(0.615±0.16)(P<0.05). The abundance of Bacteroidetes in pre-treatment group(0.206±0.101)was higher than healthy control group(0.064±0.052)(P<0.05). At the genus level, Bifidobacterium was the dominant species in each groups. The abundance of Bacteroides and Streptococcus in pre-treatment group(0.037±0.028,0.002±0.002),treatment 1month group(0.029±0.02,0.009±0.013) and treatment 2 month group(0.034±0.015,0.004±0.005)were lower than healthy control group(0.095±0.061,0.033±0.034)(P<0.05). The serum 5-HT in pre-treatment group(0.482±0.214),treatment 1 month group(0.743±0.189) and treatment 2 month group(0.676±0.221)were lower than healthy control group(1.071±0.149)(P<0.05). The serum 5-HT of children in treatment 1 month group and treatment 3 month group(0.901±0.152)were significantly higher thanpre-treatment group(P<0.05). Conclusion There are changes in the structure of intestinal flora in children with functional constipation, There may be a correlation between constipation and intestinal flora. The addition of multiple probiotics can improve the intestinal flora structure and increase the level of 5-HT in children with constipation.