基于16S核糖体RNA高通量测序探讨加味四君子汤对严重烫伤兔肠道菌群多样性的影响

罗锦花; 詹剑华; 廖文强; 程兴; 黄凯

doi:10.3760/cma.j.cn501120-20200923-00421

基于16S核糖体RNA高通量测序探讨加味四君子汤对严重烫伤兔肠道菌群多样性的影响

doi: 10.3760/cma.j.cn501120-20200923-00421

南昌大学第一附属医院烧伤与创面修复中心，南昌　　330006

基金项目:

江西省科技计划 20171BBG70061, 2020BBG73020

详细信息

通讯作者:
詹剑华，Email：zjhseasky@163.com

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出版历程
- 收稿日期: 2020-09-23

Investigating the effects of Modified Sijunzi Decoction on the diversity of intestinal microflora of severe scald rabbits based on 16S ribosomal RNA high-throughput sequencing

Burn and Wound Repair Center, the First Affiliated Hospital of Nanchang University, Nanchang 330006, China

Funds:

Science and Technology Program of Jiangxi Province of China 20171BBG70061, 2020BBG73020

More Information

Corresponding author: Zhan Jianhua, Email: zjhseasky@163.com

摘要

摘要: 目的基于16S核糖体RNA（16S rRNA）高通量测序，探讨加味四君子汤对严重烫伤兔肠道菌群多样性的影响。方法采用实验研究方法。选取90只6~8个月龄雌雄不限日本大耳兔，按照随机数字表法分为正常对照组、单纯烫伤组、烫伤+低剂量组、烫伤+中剂量组、烫伤+高剂量组，每组18只。正常对照组兔自由饮食、饮水，单纯烫伤组、烫伤+低剂量组、烫伤+中剂量组、烫伤+高剂量组兔制作30%体表总面积Ⅲ度烫伤模型后分别给予生理盐水、0.2 g/mL加味四君子汤、1.0 g/mL加味四君子汤、5.0 g/mL加味四君子汤灌胃，持续给药7 d。于分组处理第1、3、7天，采用酶联免疫吸附测定法检测各组兔回肠黏膜组织中的肿瘤坏死因子α（TNF-α）、白细胞介素1β（IL-1β）、IL-10水平，各组每个时间点的样本数为6。根据前述实验结果，另外选取9只兔分为正常对照组、单纯烫伤组、烫伤+中剂量组，每组3只，各组兔分组及处理方法同前。于分组处理第7天，采用高通量测序技术对3组兔回肠黏膜菌群的16S rRNA V3、V4区进行测序；采用QIIME软件统计菌群优质序列数目；使用RDP Classifier软件分析菌群的门、纲、目、科、属水平；采用Illumina MiSeq测序技术对菌群行α多样性（Ace、Chao1、Simpson、Shannon指数）和β多样性分析。对数据行析因设计方差分析、SNK检验及Bonferroni校正。结果与正常对照组相比，单纯烫伤组、烫伤+低剂量组、烫伤+高剂量组分组处理第1、3、7天和烫伤+中剂量组分组处理第1、3天兔回肠黏膜组织中的TNF-α水平均明显升高（P<0.01），单纯烫伤组、烫伤+低剂量组、烫伤+中剂量组、烫伤+高剂量组分组处理第1、3、7天兔回肠黏膜组织中的IL-1β水平均明显升高（P<0.05或P<0.01），单纯烫伤组、烫伤+低剂量组、烫伤+中剂量组、烫伤+高剂量组分组处理第1、3、7天兔回肠黏膜组织中的IL-10水平均显著降低（P<0.01）。与单纯烫伤组相比，烫伤+低剂量组、烫伤+中剂量组、烫伤+高剂量组分组处理第3、7天及烫伤+中剂量组分组处理第1天兔回肠黏膜组织中的TNF-α水平均明显降低（P<0.01），烫伤+低剂量组、烫伤+中剂量组、烫伤+高剂量组分组处理第3、7天及烫伤+中剂量组分组处理第1天兔回肠黏膜组织中的IL-1β水平均明显降低（P<0.01），烫伤+低剂量组分组处理第7天和烫伤+中剂量组分组处理第1、3、7天以及烫伤+高剂量组分组处理第3、7天兔回肠黏膜组织中的IL-10水平均显著升高（P<0.05或P<0.01）。与烫伤+低剂量组相比，烫伤+中剂量组分组处理第1、3、7天和烫伤+高剂量组分组处理第3、7天兔回肠黏膜组织中的TNF-α水平均明显降低（P<0.01），烫伤+中剂量组分组处理第1、3、7天和烫伤+高剂量组分组处理第3、7天兔回肠黏膜组织中的IL-1β水平均显著降低（P<0.05或P<0.01），烫伤+中剂量组分组处理第1、3、7天和烫伤+高剂量组分组处理第7天兔回肠黏膜组织中的IL-10水平均显著升高（P<0.05或P<0.01）。与烫伤+中剂量组相比，烫伤+高剂量组分组处理第1、3、7天兔回肠黏膜组织中的TNF-α水平均明显升高（P<0.01），烫伤+高剂量组分组处理第1、3、7天兔回肠黏膜组织中的IL-10水平均明显降低（P<0.01），烫伤+高剂量组分组处理第7天兔回肠黏膜组织中的IL-1β水平均显著降低（P<0.01）。与第1天比较，单纯烫伤组分组处理第3、7天以及正常对照组分组处理第3天兔回肠黏膜组织中的TNF-α水平均明显升高（P<0.05或P<0.01），单纯烫伤组分组处理第3、7天兔回肠黏膜组织中的IL-1β水平均明显升高（P<0.01），烫伤+低剂量组、烫伤+高剂量组分组处理第7天和烫伤中剂量组分组处理第3、7天兔回肠黏膜组织中的IL-10水平均明显升高（P<0.05或P<0.01）；烫伤+高剂量组分组处理第3、7天及烫伤+中剂量组分组处理第7天的兔回肠黏膜组织中的TNF-α水平均明显降低（P<0.05或P<0.01），烫伤+中剂量组分组处理第7天兔回肠黏膜组织中的IL-1β水平明显降低（P<0.01），单纯烫伤组分组处理第7天兔回肠黏膜组织中的IL-10水平明显降低（P<0.01）。与第3天比较，单纯烫伤组分组处理第7天兔回肠黏膜组织中的TNF-α、IL-1β水平及正常对照组、烫伤+低剂量组、烫伤+中剂量组和烫伤+高剂量组分组处理第7天兔回肠黏膜组织中的IL-10水平均明显升高（P<0.05或P<0.01），烫伤+低剂量组、烫伤+中剂量组、烫伤+高剂量组分组处理第7天兔回肠黏膜组织中的TNF-α水平均明显降低（P<0.05），烫伤+中剂量组、烫伤+高剂量组分组处理第7天兔回肠黏膜组织中的IL-1β水平均明显降低（P<0.05或P<0.01），单纯烫伤组分组处理第7天兔回肠黏膜组织中的IL-10水平明显降低（P<0.01）。分组处理第7天，正常对照组、单纯烫伤组、烫伤+中剂量组兔回肠黏膜中的菌群分别获96 023、107 365、95 921条优质序列。3组兔回肠黏膜中的菌群在门、纲、目、科、属的分类水平上均分别以拟杆菌门和厚壁菌门、梭菌纲和拟杆菌纲、梭菌目和拟杆菌目、瘤胃菌科和梭菌科及拟杆菌科、梭菌属和拟杆菌属及瘤胃菌属菌群为主，但各组菌群组成不同。3组兔回肠黏膜中菌群的Ace、Chao1、Simpson、Shannon指数均没有显著差异（P>0.05），β多样性无明显差异。结论严重烫伤后兔回肠黏膜炎症反应明显，并呈时间依赖性增加。加味四君子汤可减轻炎症反应，其治疗效果最佳质量浓度为1.0 g/mL。高通量测序法能够精准地反映肠道菌群的结构组成，加味四君子汤对严重烫伤后肠道菌群具有一定的调节作用。
- 植物制剂 /
- 高通量核苷酸序列分析 /
- 加味四君子汤 /
- 严重烫伤 /
- 肠道屏障 /
- 肠道菌群 /
- 16S核糖体RNA
Abstract: Objective To investigate the effects of Modified Sijunzi Decoction on the diversity of intestinal microflora of in severe scald rabbits based on 16S ribosomal RNA (16S rRNA) high-throughput sequencing. Methods The experimental research method was adopted. Ninety Japanese big-ear rabbits regardless gender, aged 6 to 8 months, were randomly divided into normal control group, scald alone group, scald+low-dose group, scald+medium-dose group, and scald+high-dose group, with 18 rabbits in each group. The rabbits in normal control group were free to eat and drink, and the rabbits in scald alone group, scald+low-dose group, scald+medium-dose group, and scald+high-dose group were intragastrically administered normal saline, 0.2 g/mL Modified Sijunzi Decoction, 1.0 g/mL Modified Sijunzi Decoction, and 5.0 g/mL Modified Sijunzi Decoction, respectively for 7 days after sustaining full-thickness scalding of 30% total body surface area. On the 1^st, 3^rd, and 7^th day after grouping, the levels of tumor necrosis factor α (TNF-α), interleukin 1β (IL-1β), and IL-10 in ileal mucosa tissue of rabbits in each group were determined by enzyme-linked immunosorbent assay, and the number of samples in each group at each time point was 6. According to the above experimental results, another 9 rabbits were selected and divided into normal control group, scald alone group and scald+medium-dose group, with 3 rabbits in each group. The grouping and treatment methods of rabbits in each group were the same as before. On the 7^th day after grouping, the V3, V4 region of 16S rRNA of ileum mucosa of rabbits in three groups were sequenced by high-throughput sequencing technology. The number of quality bacteria was counted by QIME software. The classifications of phylum, class, order, family and genus of microflora were analyzed by RDP Classifier software. The α diversity (Ace, Chao1, Simpson, and Shannon indexes) and β diversity were analyzed by Illumina MiSeq sequencing technology, and the number of experiment samples in each group was 3. Data were statistically analyzed with analysis for variance of factorial design, SNK test, and Bonferroni correction. Results Compared with that in normal control group, the levels of TNF-α of ileal mucosa tissue of rabbits in scald alone group, scald+low-dose group, and scald+high-dose group on the 1^st, 3^rd, and 7^th day after grouping and scald+medium-dose group on the 1^st and 3^rd day after grouping were all significantly increased (P<0.01), the levels of IL-1β in ileal mucosa tissue of rabbits in scald alone group, scald+low-dose group, scald+medium-dose group and scald+high-dose group on the 1^st, 3^rd, and 7^th day after grouping were all significantly increased (P<0.05 or P<0.01), and the levels of IL-10 in ileal mucosa tissue of rabbits in scald alone group, scald+low-dose group, scald+medium-dose group, and scald+high-dose group on the 1^st, 3^rd, and 7^th day after grouping were all significantly decreased (P<0.01). Compared with that in scald alone group, the levels of TNF-α in ileal mucosa tissue of rabbits in scald+low-dose group, scald+medium-dose group, and scald+high-dose group on the 3^rd and 7^th day after grouping, and scald+medium-dose group on the 1^st day after grouping were all significantly decreased (P<0.01), and the levels of IL-1β in ileal mucosa tissue of rabbits in scald+low-dose group, scald+medium-dose group, and scald+high-dose group on the 3^rd and 7^th day after grouping and scald+medium-dose group on the 1^st day after grouping were all significantly decreased (P<0.01), and the levels of IL-10 in ileal mucosa tissue of rabbits in scald+low-dose group on the 7^th day after grouping and scald+medium-dose group on the 1^st, 3^rd, and 7^th day after grouping and scald+high-dose group on the 3^rd and 7^th day after grouping were all significantly increased (P<0.05 or P<0.01). Compared with that in scald+low-dose group, the levels of TNF-α in ileal mucosa tissue of rabbits in medium-dose scald alone group on the 1^st, 3^rd, and 7^th day after grouping and in high-dose scald alone group on the 3^rd and 7^th day after grouping were significantly decreased (P<0.01), and the levels of IL-1β in ileal mucosa tissue of rabbits in medium-dose scald alone group on the 1^st, 3^rd, and 7^th day after grouping and in high-dose scald alone group on the 3^rd and 7^th day after grouping were all significantly decreased (P<0.05 or P<0.01), and the levels of IL-10 in ileal mucosa tissue of rabbits in scald+medium-dose group on the 1^st, 3^rd, and 7^th day after grouping and in scald+high-dose group on the 7^th day after grouping were all significantly increased (P<0.05 or P<0.01). Compared with that in scald medium-dose group, the levels of TNF-α in ileal mucosa tissue of rabbits in scald+high-dose group on the 1^st, 3^rd, and 7^th day after grouping were all significantly increased (P<0.01), and the levels of IL-10 in ileal mucosa tissue of rabbits in scald+high-dose group on the 1^st, 3^rd, and 7^th day after grouping were all significantly decreased (P<0.01), and the levels of IL-1β in ileal mucosa tissue of rabbits in scald+high-dose group on the 7^th day after grouping was significantly decreased (P<0.01). Compared with that on the 1^st day after grouping, the levels of TNF-α in ileal mucosa tissue of rabbits in scald alone group on the 3^rd and 7^th day after grouping and in normal control group on the 3^rd day after grouping were all significantly increased (P<0.05 or P<0.01), and the levels of IL-1β in ileal mucosa tissue of rabbits in scald alone group both on the 3^rd and 7^th day after grouping were significantly increased (P<0.01), and the levels of IL-10 in ileal mucosa tissue of rabbits in both scald+low-dose group and scald+high-dose group on the 7^th day after grouping and scald+medium-dose group both on the 3^rd and 7^th day after grouping were significantly increased (P<0.05 or P<0.01), and the levels of TNF-α in ileal mucosa tissue of rabbits in scald+high-dose group on the 3^rd and 7^th day after grouping and in scald+medium-dose group on the 7^th day after grouping were all significantly decreased (P<0.05 or P<0.01), and the level of IL-1β in ileal mucosa tissue of rabbits in scald+medium-dose group on the 7^th day after grouping was significantly decreased (P<0.01), and the level of IL-10 in ileal mucosa tissue of rabbits in scald alone group on the 7^th day after grouping was significantly decreased (P<0.01). Compared with that on the 3^rd day after grouping, the levels of TNF-α and IL-1β in ileal mucosa tissue of rabbits in scald alone group and the levels of IL-10 in ileal mucosa tissue of rabbits in normal control group, scald+low-dose group, scald+medium-dose group, and scald+high-dose group on the 7^th day after grouping were all significantly increased (P<0.05 or P<0.01); and the levels of TNF-α in ileal mucosa tissue of rabbits in scald+low-dose group, scald+medium-dose group, and scald+high-dose group on the 7^th day after grouping were all significantly decreased (P<0.05), and the levels of IL-1β in ileal mucosa tissue of rabbits both in scald+medium-dose group and scald+high-dose group on the 7^th day after grouping were significantly decreased (P<0.05 or P<0.01), and the levels of IL-10 in ileal mucosa tissue of rabbits in scald alone group on the 7^th day after grouping was significantly decreased (P<0.01). On the 7^th day after grouping, the high-quality sequences obtained from the microflora in ileum mucosa of rabbits in normal control group, scald alone group, and scald+medium-dose group were 96 023, 107 365, and 95 921, respectively. At the classification level of phylum, class, order, family, and genus of the microflora in ileum mucosa of rabbits in three groups were all Bacteroidetes and Firmicutes, Clostridium and Bacteroidetes, Clostridium and Bacteroidetes, Rumenobacteriaceae and Clostridium and Bacteroideaceae, Clostridium and Bacteroidetes and rumen bacteria mainly, while the percentage of microflora in each group was different. There were no significant differences in Ace, Chao1, Simpson, Shannon indices (P>0.05), and no obvious difference in β diversity of microflora in ileal mucosa tissue of rabbits among three groups. Conclusions After severe scalding, the inflammatory response of rabbit ileal mucosa tissue is obvious and increased in a time-dependent manner. Modified Sijunzi Decoction can reduce inflammation with optimal therapeutic concentration of 1.0 g/mL. The technology of high-throughput sequencing can reflect the structural composition of the intestinal microflora accurately. The ileal microflora of the severe scald rabbit can be regulated by the administration of Modified Sijunzi Decoction.
- Plant preparations /
- High-throughput nucleotide sequencing /
- Modified Sijunzi Decoction /
- Severe scald /
- Intestinal barrier /
- Intestinal flora /
- 16S ribosomal ribonucleic acid
罗锦花：直接参与酝酿和设计实验、实施研究、分析数据、起草文章；詹剑华：酝酿和设计实验，行政、技术或材料支持，指导实验；廖文强：参与文章的数据采集、分析、解释及文章的撰写；程兴：参与文章的数据分析及解释；黄凯：支持性贡献

所有作者均声明不存在利益冲突

患者烧伤后，具有较高的死亡风险。烧伤后机体的免疫和炎症反应模式是否与患者预后相关，尚不清楚。该研究的目的是探讨重症烧伤患者的全身免疫和炎症反应与预后之间的关系。选取重度烧伤患者为研究对象，在入院即刻及第2、7、14和28天收集血液标本，通过流式细胞术进行细胞计数和细胞因子分析。结果显示，烧伤患者入院时外周血中恒定自然杀伤T细胞（iNKT）、γδT细胞和黏膜相关恒定T细胞的数量明显下降，这些细胞发挥着对抗抗原应激和维持内环境稳态的重要作用。同时，单核细胞表达人类白细胞抗原DR（HLA-DR）、自然杀伤（NK）细胞表达NKG2D受体和中性粒细胞表达CD16的水平均显著降低。入院时，外周血中CD8 T细胞、γδT细胞、B细胞和iNKT计数较低的患者更易发生细菌感染，而HLA-DR/单核细胞的延迟恢复和晚期细胞因子分泌失调则与脓毒症休克的发生有关。患者早期（第1周）的免疫反应模式与90 d内病死率没有显著相关性；然而延迟、持续的细胞因子谱改变和后期低HLA-DR/单核细胞水平在死亡患者中更加明显。

刘双庆，编译自《Front Immunol》,2021,12:586195；姚咏明，审校

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1 3组兔分组处理第7天的回肠黏膜菌群在属分类水平的构成差异

注：烫伤+中剂量组兔烫伤后，给予终质量浓度1.0 g/mL加味四君子汤灌胃；A1、A2、A3为正常对照组3只兔样本，B1、B2、B3为单纯烫伤组3只兔样本，C1、C2、C3为烫伤+中剂量组3只兔样本

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2 3组兔分组处理第7天的回肠黏膜中菌群等级丰度曲线

注：烫伤+中剂量组兔烫伤后，给予终质量浓度1.0 g/mL加味四君子汤灌胃；图中纵坐标为经过log₂处理的数据；A1、A2、A3为正常对照组3只兔样本，B1、B2、B3为单纯烫伤组3只兔样本，C1、C2、C3为烫伤+中剂量组3只兔样本

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3 3组兔分组处理第7天的回肠黏膜菌群β多样性分析

注：烫伤+中剂量组兔烫伤后，给予终质量浓度1.0 g/mL加味四君子汤灌胃；A1、A2、A3为正常对照组3只兔样本，B1、B2、B3为单纯烫伤组3只兔样本，C1、C2、C3为烫伤+中剂量组3只兔样本；主坐标成分表示菌群分布距离相对集中结果的解释度

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表1 5组兔分组处理第1、3、7天回肠黏膜组织的炎症水平（pg/mL，x¯±s）

组别	兔数（只）	第1天			第3天			第7天
组别	兔数（只）	TNF‐α	IL‐1β	IL‐10	TNF‐α	IL‐1β	IL‐10	TNF‐α	IL‐1β	IL‐10
正常对照组	18	108.2±13.2	13.24±0.10	923±4	128.8±3.6^f	11.37±0.72	909±17	114.2±1.9^j	12.10±1.51	937±16^k
单纯烫伤组	18	254.1±26.3^a	33.41±1.96^a	638±28^a	321.5±15.8^ag	42.91±2.74^ag	602±80^a	344.7±10.5^agk	53.25±2.45^agj	447±30^agj
烫伤+低剂量组	18	256.0±28.7^a	31.55±2.93^a	660±27^a	260.4±10.7^ab	30.64±3.99^ab	658±13^a	242.6±11.8^abk	34.72±2.29^ab	696±26^abgj
烫伤+中剂量组	18	187.4±17.9^abc	26.26±2.08^abc	774±38^abc	174.3±19.9^abc	20.53±6.78^abh	816±27^abci	130.8±40.0^bcfk	16.14±2.18^abcgk	843±28^abcgk
烫伤+高剂量组	18	240.7±18.0^ad	30.05±12.02^e	663±27^ad	209.8±11.6^abcdf	26.51±1.61^abh	692±38^adi	201.0±12.4^abcdgk	23.76±0.53^abcgj	749±33^abdghk
F值		152.84	36.38	341.86	555.94	171.24	153.22	387.98	1325.73	833.79
P值		<0.001	<0.001	<0.001	<0.001	<0.001	<0.001	<0.001	<0.001	<0.001
注：烫伤+低剂量组、烫伤+中剂量组、烫伤+高剂量组兔烫伤后，分别给予等体积终质量浓度0.2 g/mL、1.0 g/mL、5.0 g/mL加味四君子汤灌胃；TNF为肿瘤坏死因子，IL为白细胞介素；各组各时间点的样本数均为6；TNF-α处理因素主效应，F=716.67，P<0.001；时间因素主效应，F=17.42，P<0.001；两者交互作用，F=21.16，P<0.001；IL-1β处理因素主效应，F=349.68，P<0.001；时间因素主效应，F=8.19，P<0.001；两者交互作用，F=15.97，P<0.001；IL-10处理因素主效应，F=819.03，P<0.001；时间因素主效应F=0.58，P=0.560；两者交互作用，F=21.82，P<0.001；与正常对照组相比，^aP<0.01、^eP<0.05；与单纯烫伤组相比，^bP<0.01、ⁱP<0.05；与烫伤+低剂量组相比，^cP<0.01、^hP<0.05；与烫伤+中剂量组相比，^dP<0.01；与组内第1天相比，^fP<0.05、^gP<0.01；与组内第3天相比，^jP<0.01、^kP<0.05

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表2 3组兔分组处理第7天的回肠黏膜中菌群α多样性分析（x¯±s）

组别	兔数（只）	Ace指数		Chao1指数		Simpson指数	Shannon指数
正常对照组	3	2 536	±	2 396	±	0.991±	9.78±
正常对照组	3	661		518		0.011	0.22
单纯烫伤组	3	2 205	±	2 040	±	0.923±	8.26±
单纯烫伤组	3	532		519		0.140	2.38
烫伤+中剂量组	3	2 490	±	2 303	±	0.992±	9.69±
烫伤+中剂量组	3	169		142		0.012	0.23
F值		0.78		1.10		1.48	2.27
P值		0.478		0.359		0.258	0.138
注：烫伤+中剂量组兔烫伤后，给予终质量浓度1.0 g/mL加味四君子汤灌胃

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