Basic Study
Copyright ©The Author(s) 2025. Published by Baishideng Publishing Group Inc. All rights reserved.
World J Gastrointest Surg. Apr 27, 2025; 17(4): 103494
Published online Apr 27, 2025. doi: 10.4240/wjgs.v17.i4.103494
Single-nucleus RNA sequencing and spatial transcriptomics reveal the mechanism by which Xiaozhiling injection treats internal hemorrhoids
Min-Hui Ke, Shu-Yan Huang, Wei-Gan Lin, Zhen-Guo Xu, Xia-Xia Zheng, Xian-Bao Liu, You-Min Cheng, Zuan-Fang Li
Min-Hui Ke, Zhen-Guo Xu, Xia-Xia Zheng, Xian-Bao Liu, Department of Proctology, The Second People’s Hospital Affiliated with Fujian University of Traditional Chinese Medicine, Fuzhou 350003, Fujian Province, China
Shu-Yan Huang, Wei-Gan Lin, Zuan-Fang Li, Fujian University of Traditional Chinese Medicine, Fuzhou 350122, Fujian Province, China
You-Min Cheng, Fujian Academy of Chinese Medical Sciences, Fuzhou 350003, Fujian Province, China
Co-first authors: Min-Hui Ke and Shu-Yan Huang.
Author contributions: Ke MH contributed to research guidance, manuscript review, and financial support; Ke MH and Huang SY contributed to experiment design, article writing, and drawing, and they contributed equally to this article as co-first authors; Lin WG contributed to discussion of the article; Xu ZG contributed to the writing of the manuscript related to pathology; Zheng XX, Liu XB, Cheng YM, and Li ZF contributed to the performance of animal experiments; and all authors thoroughly reviewed and endorsed the final manuscript.
Supported by the National Natural Science Foundation of China, No. 81774118; the Foreign Cooperation Project of Science and Technology Department of Fujian Province, No. 2023I0021; and the Medical Innovation Project of Fujian Province, No. 2024CXB013.
Institutional animal care and use committee statement: All procedures involving animals were reviewed and approved by the Institutional Animal Care and Use Committee of Second People’s Hospital Affiliated with Fujian University of Traditional Chinese Medicine, approval No. FJATCM-IAEC2018015.
Conflict-of-interest statement: All the authors report no relevant conflicts of interest for this article.
ARRIVE guidelines statement: The authors have read the ARRIVE guidelines, and the manuscript was prepared and revised according to the ARRIVE guidelines.
Data sharing statement: Our data are robust and can be shared upon publication.
Open Access: This article is an open-access article that was selected by an in-house editor and fully peer-reviewed by external reviewers. It is distributed in accordance with the Creative Commons Attribution NonCommercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited and the use is non-commercial. See: https://creativecommons.org/Licenses/by-nc/4.0/
Corresponding author: Min-Hui Ke, Department of Proctology, The Second People’s Hospital Affiliated with Fujian University of Traditional Chinese Medicine, No. 282 Wusi Road, Gulou District, Fuzhou 350003, Fujian Province, China. 48330132@qq.com
Received: November 21, 2024
Revised: January 21, 2025
Accepted: February 11, 2025
Published online: April 27, 2025
Processing time: 128 Days and 16.6 Hours
Abstract
BACKGROUND

Hemorrhoids, a prevalent chronic condition globally, significantly impact patients' quality of life. While various surgical interventions, such as external stripping and internal ligation, procedure for prolapse and hemorrhoids, and tissue selecting technique, are employed for treatment, they are often associated with postoperative complications, including unsatisfactory defecation, bleeding, and anal stenosis. In contrast, Xiaozhiling injection, a traditional Chinese medicine-based therapy, has emerged as a minimally invasive and effective alternative for internal hemorrhoids. This treatment offers distinct advantages, such as reduced dietary restrictions, broad applicability, and minimal induction of systemic inflammatory responses. Additionally, Xiaozhiling injection effectively eliminates hemorrhoid nuclei, prevents local tissue necrosis, preserves anal cushion integrity, and mitigates postoperative complications, including bleeding and prolapse. Despite its clinical efficacy, the molecular mechanisms underlying its therapeutic effects remain poorly understood, warranting further investigation.

AIM

To investigate the molecular mechanism underlying the therapeutic effect of Xiaozhiling injection in the treatment of internal hemorrhoids.

METHODS

An internal hemorrhoid model was established in rats, and the rats were randomly divided into a modeling group [control group (CK group)] and a treatment group. One week after injection, Stereo-seq and electron microscopy were used to study the changes in gene expression and subcellular structures in fibroblasts.

RESULTS

Single-cell sequencing revealed differences in the expression and transcript levels of the genes collagen 3 alpha 1, decorin, and actin alpha 2 in fibroblasts between the CK group and the treatment group. Spatial transcriptome analysis revealed that genes of the sphingosine kinase 1 (Sphk1)/sphingosine-1-phosphate (S1P) pathway spatially overlapped with key genes of the transforming growth factor beta 1 pathway, namely, Sphk1, S1P receptor, and transforming growth factor beta 1, in the treatment group. The proportion of fibroblasts was lower in the treatment group than in the CK group, and Xiaozhiling treatment had a significant effect on the proportion of fibroblasts in hemorrhoidal tissue. Immunohistochemistry revealed a significant increase in the expression of a fibroblast marker. Electron microscopy showed that the endoplasmic reticulum of fibroblasts contained a large amount of glycogen, indicating cell activation. Fibroblast activation and the expression of key genes of the Sphk1-S1P pathway could be observed at the injection site, suggesting that after Xiaozhiling intervention, the Sphk1-S1P pathway could be activated to promote fibrosis.

CONCLUSION

Xiaozhiling injection exerts its therapeutic effects on internal hemorrhoids by promoting collagen synthesis and secretion in fibroblasts. After Xiaozhiling intervention, the Sphk1-S1P pathway can be activated to promote fibrosis.

Keywords: Internal hemorrhoids; Xiaozhiling injection; Fibroblast; Marker gene; Single-cell sequencing; Spatial transcriptome sequencing

Core Tip: Single-cell sequencing revealed differences in the expression and transcription levels of genes collagen 3 alpha 1, decorin, and actin alpha 2 in fibroblasts between the control groups and treatment groups. The activation of fibroblasts and the expression of key genes in the sphingosine kinase 1-sphingosine-1-phosphate pathway were observed at the injection site, suggesting that the sphingosine kinase 1-sphingosine-1-phosphate pathway could be activated to promote fibrosis after the intervention of Xiaozhling.