Impact of Polyethylene Glycol (PEG) Bowel Preparation on Intestinal Microbiota Composition and Diversity in Patients Undergoing Appendectomy
Conditions: Appendicitis, Appendicitis Acute
Sponsor: Huaping Xie

Research Plan
A clinical trial study on the effects of
PEG laxatives on gut microbiota in
patients undergoing appendectomy
Version Number: V2.0
Version date: 2024.1.9
Principal Investigator: Xie Huaping
Research Institution: Tongji Hospital, Tongji Medical
College,
Huazhong
University
of
Science
and
Technology

I、Research Background
Colonoscopy is a must-have item in colorectal cancer check-ups, and for high-risk groups,
annual check-ups are the most effective way of early prevention; A large amount of polyethylene
glycol (PEG2000-4000) is used as a laxative and lubricant in colonoscopy at present; The extensive
use of PEG can cause long-term and irreversible damage to the gut microbiota, posing a great
threat[ 1] to health.
The appendix, located at the base of the cecum, is a slender tubular structure that protrudes
from the posterior wall inside the cecum and was once thought to be part of an evolutionary
redundancy. Appendicitis has long been the most common surgical acute abdominal disease, and
once acute appendicitis occurs, traditional treatments often opt for the removal of the appendix. In
recent years, researchers have revealed that appendectomy is associated[2] with mental disorders,
colorectal cancer, cardiovascular diseases, etc. Besides being an important organ involved in
immunity, the appendix also has the function of storing and protecting intestinal microbiota. When
there is a disruption of the gut microbiota in the human body, the microorganisms retained in the
appendix are released into the gut to reduce the impact[3] of the disruption. Previous researchers'
metagenomic sequencing of gut microbiota in patients who have undergone appendectomy shows
significant changes in their gut microbiota compared to the normal population, which in turn affects
central nervous system function through the microbiota-gut-brain axis and poses potential risks to
host health and behavior, such as Parkinson's disease[4-7]. Therefore, appendectomy may have
potential adverse effects on the human body, such as significantly altering the composition of the
gut microbiota, reducing the ability to restore intestinal homeostasis, and lowering[8, 9] the
production of short-chain fatty acids (SCFAs).
This study will observe the effects ofPEG on the gut microbiota, and by analyzing the changes
in the gut microecology before and after colonoscopy, design better intestinal cleansing drugs and
intestinal repair probiotics to further reduce the adverse effects of colonoscopy on patients. At the
same time, explore the interaction between the appendix and the gut microbiota and its significance.

2. Objectives of the study
The study intends to recruit a group of people who have undergone appendectomy for
colonoscopy, (1) to observe changes in gut microbiota before and after colonoscopy in the
appendectomy population; (2) To analyze whether the changes in gut microbiota before and after
colonoscopy in the appendectomy population were different from the recovery of the normal
population.
Iii. Study Design
This study uses a parallel controlled monitoring clinical trial approach to investigate the
short-term and long-term effects of PEG use on the gut microbiota of patients, including tracking
analysis of the recovery and impairment of the gut microbiota, and comparing the recovery and
impairment of the microbiota with those who did not undergo appendectomy to design
corresponding microecological restoration probiotics. This study used bioinformatics methods to
statistically analyze the data information of the samples.
4. Selection of subjects
1. Inclusion criteria
The subjects were mainly recruited who met the following conditions: 1) Had undergone
appendectomy or appendectomy for right-sided colon cancer; 2) were aged between 18 and 75 years
old. 3) Prepare for colonoscopy; 4) Be in good health and have no history ofmajor organ disease.
Control subjects were recruited to meet the following conditions: 1) age 18-75 years; 2) No
appendectomy or right colon cancer appendectomy; 3) Good health, no history of major organ
disease.
2. Exclusion criteria
Subjects excluded the following conditions: (1) contraindications for colonoscopy: such as
cardiopulmonary insufficiency, etc. (2) Intolerance to PEG laxatives; (3) Pregnant women; (4)
Mental disorders. Among them, the colonoscopy population without appendectomy was used as the
control; 4) Use of antibiotics within half a year.
Control subjects excluded the following conditions: (1) presence of organic disease; Such as
cardiopulmonary insufficiency, etc. (2) Pregnant women; (3) Mental disorders; 4) Use ofantibiotics

within half a year.
5. Estimation of sample size
About 10 people were included in this study.
Vi. Study Process
The technical route of this study is as follows:

Vii. Possible risks and Preventive measures
The possible discomforts ofthis study include: psychological and sensory discomforts
caused by fecal samples being mainly taken from the subjects' homes, including nausea and
vomiting. The fecal sampling box comes with a pair of gloves and two sheets oftoilet paper.
VIII、Data collection and statistical analysis
Fecal samples of patients were sent to the company for metagenomic sequencing to obtain
metagenomic data of microorganisms in the fecal samples.
The raw files obtained from the DNBSEQ-T7 platform were converted into short reads (raw
data) via base call, and these short reads were recorded in FASTQ format [30] containing sequence
information and corresponding sequencing quality information.
Sequence artifacts, including reads containing adapter contamination, low-quality nucleotides
and unidentifiable nucleotides (N), undoubtedly set obstacles for subsequent reliable bioinformatics
analysis. Therefore, quality control is a necessary step to ensure meaningful downstream analysis.
Fastp(version 0.23.1)[31] was used to perform basic statistics on the quality of the original
reads.
The data processing steps are as follows:
(1) Discard the paired reads if one of the reads contains adapter contamination;
(2) Abandon paired reads if the number ofuncertain bases exceeds 10% in each read;
(3) Discard pairs ofreads if the proportion of low-quality bases (Phred quality <5) in any read
exceeds 50%.
After passing the quality inspection, a total of 0.2g of DNA is required for each sample as the
input material for DNA library preparation. First, genomic DNA samples are ultrasonically crushed
by a Covaris ultrasonic disruptor to meet a size requirement of 350bp. After terminal repair, adding
A tail, adding sequencing headers, fragment screening, PCR amplification, and then using the
AMPure XP system (Beverly, USA) to purify the product, the library quality was evaluated and
quantified on the Agilent 5400 system (Agilent, USA), Quantification was performed using QPCR
(1.5 nM). The 5 'ends ofeach library were phosphorylated and cyclized. Then cyclic amplification is
carried out to produce DNA nanospheres. Finally, the DNA nanospheres were loaded into flow cells
for DNBSEQ-T7 sequencing at Novogene Bioinformatics Technology Co., LTD. (Beijing, China).
The entire library preparation process is completed, resulting in the final DNA library.

Additionally, calculate the Bray-Curtis distance using the vegan package for principal
coordinate analysis (PCoA). Linear discriminant analysis effect size (LEfSe) analysis was used for
the Kruskal-Wallis test and the Wilcoxon rank sum test, and a p <0.05 was considered statistically
significant. The LDA score was set to 2.0 to identify the characteristics with significant differences
between groups. This study retained taxa with an average relative abundance greater than
0.0001(0.01%). The bar charts were visualized using the "ggplot2" package in R software (version
4.0.5)(https://www.r-project.org/).
All data were expressed as mean ±standard error (SEM). The differences between groups were
analyzed by one-way ANOVA using GraphPad Prism 7.0 software, and the results of the analysis
were obtained by Tukey Test.
Ix. References
[1] Tropini, C., E. Lin Moss, et al. (2018). "Transient Osmotic Perturbation Causes Long-Term
Alteration to the Gut Microbiota." Cell 173: 1742-1754.e1717.
[2] SONG M Y, ULLAH S, YANG H Y, et al. Long-term effects of appendectomy in
humans: is it the optimal management of appendicitis? [J]. Expert Review of Gastroenterology &
Hepatology, 2021, 15(6): 657-64.
[3] GIRARD-MADOUX M J H, DE AGUERO M G, GANAL-VONARBURG S C, et al.
The immunological functions of the Appendix: An example of redundancy? [J]. Seminars in
Immunology, 2018, 36(C): 31-44.
[4] CAI S T, FAN Y Y, ZHANG B Z, et al. Appendectomy Is Associated With Alteration of
Human Gut Bacterial and Fungal Communities [J]. Frontiers in Microbiology, 2021, 12.
[5] GUINANE C M, TADROUS A, FOUHY F, et al. Microbial Composition of Human
Appendices from Patients following Appendectomy [J]. Mbio, 2013, 4(1).
[6] NAKAHARA K, NAKANE S, ISHII K, et al. Gut microbiota ofParkinson's disease in an
appendectomy cohort: a preliminary study [J]. Scientific reports, 2023, 13(1): 2210.
[7] VITETTA L, VITETTA G, HALL
S. The Brain-Intestinal Mucosa-Appendix-
Microbiome-Brain Loop [J]. Diseases (Basel, Switzerland), 2018, 6(2).
[8] BROWN K, ABBOTT D W, UWIERA R R E, et al. Removal of the cecum affects
intestinal fermentation, enteric bacterial community structure, and acute colitis in mice [J]. Gut
Microbes, 2018, 9(3): 218-35.
[9] GHOSH S, DAI C, BROWN K, et al. Colonic microbiota alters host susceptibility to

infectious colitis by modulating inflammation, redox status, and ion transporter gene expression [J].
American Journal of Physiology-Gastrointestinal and Liver Physiology, 2011, 301(1): G39-G49.