Clinical Experimental Study on the Impact of PEG Laxatives for Intestinal Preparation on Gut Microbiota in Patients Undergoing Cholecystectomy
Conditions: Cholecystectomy
Sponsor: Tongji Hospital

Research Protocol
Clinical Experimental Study on the
Impact of Bowel Preparation with
PEG Laxatives on Gut Microbiota in
Patients Undergoing
Cholecystectomy
Version:V1.0
Version Date:2025.3.26
Principal Investigator:XXX
Research Institution: Tongji Hospital Affiliated to
Tongji Medical College, Huazhong University of
Science and Technology

1.Research Background
Colonoscopy is a necessary item for colorectal cancer screening. For high-risk populations,
annual screening is the most effective method for early prevention. Currently, in colonoscopy, a
large amount of polyethylene glycol (PEG2000-4000) is used as a laxative and lubricant. The
extensive use of PEG can cause long-term and irreversible damage to gut microbiota, posing
significant risks to health[1].
The gallbladder is a sac-like organ located below the liver, whose main functions include
storing and concentrating bile, regulating biliary pressure, participating in the digestion and
absorption of fats, and protecting the intestinal mucosa[2]. Due to the prevalence of gallbladder
diseases such as gallstones and cholecystitis, cholecystectomy is a common surgical procedure
worldwide[3]. Laparoscopic cholecystectomy (LC) has become the preferred surgical method for
benign gallbladder diseases due to its advantages of minimal invasiveness and rapid recovery[4].
However, cholecystectomy may affect the digestive function of patients[5,6]. After surgery, bile
acids are continuously discharged into the duodenum, which may lead to symptoms such as
indigestion and steatorrhea[6,7], and even promote the development of colorectal cancer[8]. In
addition, cholecystectomy may affect the composition of gut microbiota, thereby impacting host
health[9].
This study will observe the impact of PEG on gut microbiota, analyze changes in the
intestinal microecology before and after colonoscopy, design better bowel cleansing agents and
intestinal repair probiotics, and further reduce the adverse effects of colonoscopy on patients who
have undergone cholecystectomy. Additionally, it will explore the interaction between the
gallbladder and gut microbiota and its significance.
2. Research Objectives
The study plans to recruit a group of individuals who have undergone cholecystectomy for
colonoscopy: (1) Observe changes in gut microbiota before and after colonoscopy in
cholecystectomy patients; (2) Analyze whether the recovery of gut microbiota changes before and
after colonoscopy in cholecystectomy patients differs from that in the normal population.
3. Study Design
This study adopts a parallel controlled observational clinical trial method to investigate the

short-term and long-term effects of PEG use on patients' gut microbiota, including tracking and
analyzing the recovery and damage of gut microbiota. It will also compare the recovery and
damage status of microbiota with those in individuals who have not undergone cholecystectomy,
and design corresponding microecological repair probiotics based on this. Bioinformatics methods
will be used for statistical analysis of sample data.
4. Selection of Subjects
1. Inclusion Criteria
Subjects will be mainly recruited who meet the following criteria: 1) Have undergone
cholecystectomy; 2) Aged 18-75 years; 3) Planning to undergo colonoscopy; 4) In good health
with no history of major organ diseases.
Control subjects will be recruited who meet the following criteria: 1) Aged 18-75 years; 2)
Have not undergone cholecystectomy; 3) In good health with no history of major organ diseases.
2. Exclusion Criteria
Subjects will be excluded if they meet the following conditions: (1) Have contraindications
for colonoscopy, such as cardiopulmonary insufficiency; (2) Unable to tolerate PEG laxatives; (3)
Pregnant women; (4) Mental disorders. The control group consists of individuals undergoing
colonoscopy who have not undergone cholecystectomy; (5) Have used antibiotics within half a
year.
Control subjects will be excluded if they meet the following conditions: (1) Have organic
diseases, such as cardiopulmonary insufficiency; (2) Pregnant women; (3) Mental disorders; (4)
Have used antibiotics within half a year.
5. Sample Size Estimation
The number of participants included in this study is approximately 20.
6. Study Process
The technical route of this study is as follows:

7. Potential Risks and Preventive Measures
Potential discomforts in this study include psychological and sensory discomfort caused by
fecal sample collection at home, such as nausea and vomiting. Each fecal sampling kit is equipped
with a pair of gloves and two pieces of toilet paper.

8.Data Collection and Statistical Analysis
Patients' fecal samples will be sent to the company for metagenomic sequencing to obtain
microbial metagenomic data from the fecal samples.
Raw files obtained from the DNBSEQ-T7 platform are converted into short reads (raw data)
through base calling, and these short reads are recorded in FASTQ format, which contains
sequence information and corresponding sequencing quality information.[30]
Sequence artifacts, including reads containing adapter contamination, low-quality nucleotides,
and
unidentifiable
nucleotides
(N),
undoubtedly
pose
obstacles
to
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 raw reads.
The data processing steps are as follows:
(1) If one of the reads contains adapter contamination, discard the paired reads;
(2) If the number of ambiguous bases exceeds 10% in any read, discard the paired reads;
(3) If the proportion of low-quality bases (Phred quality < 5) exceeds 50% in any read,
discard the paired reads.
After passing quality control, each sample requires a total of 0.2g DNA as input material for
DNA library preparation. First, genomic DNA samples are ultrasonically sheared using a Covaris
ultrasonic disruptor to meet the size requirement of 350bp. Then, end repair, A-tailing, addition of
sequencing adapters, fragment screening, PCR amplification are performed, followed by
purification of products using the AMPure XP system (Beverly, USA). Library quality is
evaluated and quantified on the Agilent 5400 system (Agilent, USA), and quantified by QPCR
(1.5 nM). The 5' end of each library is phosphorylated and circularized. Then, cyclic amplification
is performed to generate DNA nanoballs. Finally, these DNA nanoballs are loaded onto the flow
cell for DNBSEQ-T7 gene sequencing at Novogene Bioinformatics Technology Co., Ltd. (Beijing,
China). The entire library preparation process is completed to obtain the final DNA library.
In addition, Bray-Curtis distance was calculated using the vegan package for Principal
Coordinate Analysis (PCoA). Linear Discriminant Analysis Effect Size (LEfSe) analysis was used
for Kruskal-Wallis test and Wilcoxon rank-sum test, with p < 0.05 considered statistically

significant. The LDA score was set to 2.0 to identify features with significant differences between
groups. Taxa with an average relative abundance greater than 0.0001 (0.01%) were retained in this
study. Bar charts were visualized using the "ggplot2" package in R software (version 4.0.5)
(https://www.r-project.org/).
All data are expressed as mean ± standard error of the mean (SEM). Differences between
groups were analyzed using one-way analysis of variance (One Way ANOVA) with GraphPad
Prism 7.0 software, and inter-group difference results were obtained via Tukey's test.
9. 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]
Shaffer EA. Review article: control of gall-bladder motor function. Aliment Pharmacol
Ther. 2000;14 Suppl 2:2-8. doi:10.1046/j.1365-2036.2000.014s2002.x
[3]Editorial Board of Chinese Journal of Digestion, Hepatobiliary Disease Cooperative
Group, Chinese Society of Gastroenterology, Chinese Medical Association. Chinese Consensus on
Medical Diagnosis and Treatment of Chronic Cholecystitis and Gallstones (2018) [J]. Chinese
Journal of Digestion, 2019, 39(2): 73-79. DOI: 10.3760/cma.j.issn.0254-1432.2019.02.001.
[4]
Warchałowski Ł, Łuszczki E, Bartosiewicz A, et al. The Analysis of Risk Factors in the
Conversion from Laparoscopic to Open Cholecystectomy. Int J Environ Res Public Health.
2020;17(20):7571. Published 2020 Oct 18. doi:10.3390/ijerph17207571
[5]
Xu F, Chen R, Zhang C, et al. Cholecystectomy Significantly Alters Gut Microbiota
Homeostasis and Metabolic Profiles: A Cross-Sectional Study. Nutrients. 2023;15(20):4399.
Published 2023 Oct 17. doi:10.3390/nu15204399
[6]
Li YD, Liu BN, Zhao SH, Zhou YL, Bai L, Liu EQ. Changes in gut microbiota
composition and diversity associated with post-cholecystectomy diarrhea. World J Gastroenterol.
2021;27(5):391-403. doi:10.3748/wjg.v27.i5.391
[7]
Xu Y, Wang J, Wu X, et al. Gut microbiota alteration after cholecystectomy contributes
to post-cholecystectomy diarrhea via bile acids stimulating colonic serotonin. Gut Microbes.
2023;15(1):2168101. doi:10.1080/19490976.2023.2168101

[8]
Jiang X, Jiang Z, Cheng Q, Sun W, Jiang M, Sun Y. Cholecystectomy promotes the
development of colorectal cancer by the alternation of bile acid metabolism and the gut
microbiota.
Front
Med
(Lausanne).
2022;9:1000563.
Published
2022
Sep
23.
doi:10.3389/fmed.2022.1000563
[9]
Amaral Raposo M, Sousa Oliveira E, Dos Santos A, et al. Impact of cholecystectomy on
the gut-liver axis and metabolic disorders. Clin Res Hepatol Gastroenterol. 2024;48(7):102370.
doi:10.1016/j.clinre.2024.102370