Basic Study
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World J Gastroenterol. Mar 7, 2025; 31(9): 102256
Published online Mar 7, 2025. doi: 10.3748/wjg.v31.i9.102256
Runchangningshen paste activates NLRP6 inflammasome-mediated autophagy to stimulate colonic mucin-2 secretion and modulates mucosal microbiota in functional constipation
Xue-Jiao Liu, Ye-li-ya Ye-er-tai, Yue-Bo Jia, Chen-Heng Wu, Xiang-Xiang Wang, Ke-Ming Yang, Xuan Yao, Jiang-Hong Ling
Xue-Jiao Liu, Ye-li-ya Ye-er-tai, Yue-Bo Jia, Chen-Heng Wu, Jiang-Hong Ling, Department of Gastroenterology, Shuguang Hospital Affiliated to Shanghai University of Traditional Chinese Medicine, Shanghai 200021, China
Xiang-Xiang Wang, School of Traditional Chinese Medicine, Shanghai University of Traditional Chinese Medicine, Shanghai 201203, China
Ke-Ming Yang, Department of Gastroenterology, Shenzhen Traditional Chinese Medicine Hospital, Shenzhen 518033, Guangdong Province, China
Xuan Yao, Guangdong Shaxi Pharmaceutical Co., Ltd, Zhongshan 528471, Guangdong Province, China
Co-first authors: Xue-Jiao Liu and Ye-li-ya Ye-er-tai.
Co-corresponding authors: Xuan Yao and Jiang-Hong Ling.
Author contributions: Liu XJ and Ye-er-tai YLY designed the experiments, analyzed the data, and wrote the manuscript; Liu XJ and Jia YB conducted the experimental research; Wu CH, Wang XX, and Yang KM provided the research software and experimental methods; Yao X and Ling JH contributed to the experimental design and financial support; All authors agree to be accountable for all aspects of work ensuring integrity and accuracy and approved the final manuscript. Liu XJ and Ye-er-tai YLY contributed equally to this work as co-first authors. Yao X and Ling JH contributed equally to this work as co-corresponding authors. The first corresponding author is my doctoral advisor, Ling JH, who has been instrumental in the conceptualization and design of our research project. Ling JH has provided invaluable guidance throughout the study, from the development of the hypothesis to the interpretation of the results. Her expertise in the field has been crucial in ensuring the scientific rigor and relevance of our work. The second corresponding author is Yao X, the manager at Guangdong Shaxi Pharmaceutical Co., Ltd, the company that supplied the medications used in our study. Yao X has been instrumental not only in facilitating our research by ensuring the availability of the necessary pharmaceuticals but also in supporting the dissemination of our findings by taking responsibility for the payment of the article’s publication fees. Both corresponding authors have made significant contributions to the research and its publication, and their involvement is critical for any further correspondence regarding the manuscript.
Supported by National Natural Science Foundation of China, No. 82174309; and Shanghai Shuguang Hospital Affiliated to Shanghai University of Traditional Chinese Medicine Siming Fund Special Project for Scientific Research, No. SGKJ-202304.
Institutional review board statement: No human experimentation was involved.
Institutional animal care and use committee statement: All procedures involving animals were reviewed and approved by the Shanghai University of TCM Animal Experimental Ethics Committee (Protocol No. PZSHUTCM2306150012).
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: No additional data are available.
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: Jiang-Hong Ling, PhD, Professor, Department of Gastroenterology, Shuguang Hospital Affiliated to Shanghai University of Traditional Chinese Medicine, No. 185 Pu'an Road, Huangpu District, Shanghai 200021, China. ljh18817424778@163.com
Received: October 13, 2024
Revised: December 6, 2024
Accepted: January 2, 2025
Published online: March 7, 2025
Processing time: 127 Days and 23.4 Hours
Abstract
BACKGROUND

Runchangningshen paste (RCNSP) is a paste made of four medicinal and edible homologous Chinese medicine mixed with honey. It is known for its ability to nourish yin and blood as well as to loosen the bowel to relieve constipation. The pathophysiology of functional constipation (FC) is associated with a reduction in mucin-2 (MUC2) secretion and microbial dysbiosis.

AIM

To investigate the underlying mechanism of RCNSP against FC through MUC2 and the gut mucosal microbiota.

METHODS

Ultra-performance liquid chromatography tandem mass spectrometry characterized RCNSP composition to elucidate the material basis of action. FC model was induced via loperamide gavage (16 mg/kg) twice daily for 7 days. Applying defecation function and gastrointestinal motility to assess constipation severity. Hematoxylin and eosin and Alcian blue-periodic acid-schiff staining analyzed colonic mucosal morphology. Transmission electron microscope was used to observe the ultrastructure of goblet cells (GCs). Immunofluorescence colocalization, quantitative PCR, and western blot assessed the impact of RCNSP on gene and protein expression within the NLRP6/autophagy pathway. 16S rDNA was employed to sequence the gut mucosal microbiota.

RESULTS

RCNSP contained 12 components with potential laxative effects. It enhanced defecation function, accelerated gastrointestinal motility, and maintained colonic mucosal integrity. RCNSP treatment significantly increased GC abundance and MUC2 production while preserving GC ultrastructure. At the molecular level, RCNSP enhanced the colocalized expression of key regulatory proteins and modulated mRNA and protein expressions in the NLRP6/autophagy pathway. Through 16S rDNA sequencing analysis, RCNSP significantly altered the mucosal microbiota composition. Specifically, it increased beneficial bacterial strains while reducing harmful ones. Simultaneously, RCNSP reduced butyrate-producing bacteria like Proteobacteria, Enterobacteriaceae, Blautia, and Eubacterium and decreased hydrogen sulfide-producing species, such as Prevotellaceae. It also reduced bile acid-inhibiting species, such as g_Eubacter_coprostanoligenes_group and Erysipelotrichaceae while increasing bile acid-producing species, such as Colidextribacter.

CONCLUSION

Our findings suggested that RCNSP ameliorated constipation through a dual mechanism: It stimulated colonic MUC2 secretion by activating NLRP6 inflammasome-mediated autophagy and modulated the composition of the mucosal microbiota.

Keywords: Traditional Chinese medicine; Functional constipation; NLRP6; Autophagy; Mucosal microbiota; Mucin; Goblet cells

Core Tip: Runchangningshen paste (RCNSP) is used to treat functional constipation. Based on the ultra-performance liquid chromatography tandem mass spectrometry, animal experiments, and the gut microbiota sequencing, we found that RCNSP could activate the inflammasome-mediated autophagy pathway to promote mucin-2 secretion and modulate the mucosal microbiota. In addition, mucosal microbiota might also promote mucin-2 secretion by activating the NLRP6-mediated autophagy pathway. These findings offer valuable information on the therapeutic efficacy of RCNSP and enhance the theoretical basis of traditional Chinese medicine regarding the nourishment of yin and blood for treating functional constipation.