Elsevier

Phytomedicine

Volume 68, March 2020, 153182
Phytomedicine

Gegen Qinlian decoction maintains colonic mucosal homeostasis in acute/chronic ulcerative colitis via bidirectionally modulating dysregulated Notch signaling

https://doi.org/10.1016/j.phymed.2020.153182Get rights and content

Abstract

Background

Gegen Qinlian decoction (GQ) is a well-known traditional Chinese medicine that has been clinically proven to be effective in treating ulcerative colitis (UC). However, its therapeutic mechanism has not been fully elucidated. Notch signaling plays an essential role in the regeneration of the intestinal epithelium.

Purpose

This study was designed to ascertain the mechanism by which GQ participates in the recovery of the colonic mucosa by regulating Notch signaling in acute and chronic UC models.

Methods

Acute and chronic UC mice (C57BL/6) were established with 3 and 2% dextran sulfate sodium (DSS), respectively, and treated with oral administration of GQ. The expression of the Notch target gene Hes1 and the Notch-related proteins RBP-J, MAML and Math1 was analyzed by western blotting. PTEN mRNA levels were detected by qRT-PCR. Mucin production that is characteristic of goblet cells was determined by Alcian blue/periodic acid-Schiff staining and verified by examining MUC2 mRNA levels by qRT-PCR. Cell proliferation was assayed by immunohistochemistry analysis of Ki67. HT-29 and FHC cells and Toll-like receptor 4 knockout (TLR4−/−) acute UC mice were also used in this study.

Results

GQ restored the injured colonic mucosa in both acute and chronic UC models. We found that Notch signaling was hyperactive in acute UC mice and hypoactive in chronic UC mice. GQ downregulated Hes1, RBP-J and MAML proteins and augmented goblet cells in the acute UC models, whereas GQ upregulated Hes1, RBP-J and MAML proteins in chronic UC mice, reducing goblet cell differentiation and promoting crypt base columnar (CBC) stem cell proliferation. Hes1 mRNA was suppressed in TLR4−/− UC mice, and GQ treatment reversed this effect. In vitro, GQ reduced Hes1 protein in Notch-activated HT29 and FHC cells but increased Hes1 protein in Notch-inhibited cells.

Conclusions

GQ restored the colonic epithelium by maintaining mucosal homeostasis via bidirectional regulation of Notch signaling in acute/chronic UC models.

Introduction

Studies have demonstrated that the secretion of trefoil peptides and mucine glycopeptides by goblet cells is essential in the initial phase (minutes to several days) of intestinal mucosal repair progress, termed “restitution” (Podolsky, 1997). The replacement of lost cells through proliferation and differentiation is crucial in the “second phase” of intestinal restoration (Nusrat et al., 1992). However, in some clinical conditions, such as ulcerative colitis (UC), the homeostasis and regeneration of intestinal epithelia is severely disturbed and present persistent mucosal ulceration on the inner lining of the large intestine and spontaneously replasing inflammation (Algieri et al., 2014; Hernandez-Chirlaque et al., 2016). In view of this, promoting intestinal regeneration and restoring intestinal mucosal homeostasis is an effective strategy for the treatment of UC. (Okamoto and Watanabe, 2004).

Crypt base columnar (CBC) stem cells are responsible for the regeneration of intestinal tissue (Barker et al., 2007). Replicating colonic CBC stem cells can self-renew or produce rapidly proliferating transit-amplifying cells that differentiate into two major differentiated cell types: absorptive colonocytes and mucus-secreting goblet cells (Leushacke and Barker, 2014). Notch signaling controls the fate of CBC stem cells and critically influences absorptive versus secretory cell fate decisions in the intestinal epithelium (Noah and Shroyer, 2013). The Notch pathway is a conserved mechanism of cell-cell interaction that regulates cell differentiation, proliferation, survival and development (Ortiz-Masia et al., 2016). In mammalian cells, ligation of Notch receptors to ligands leads to proteolytic cleavage of Notch by an intracellular γ-secretase complex and releases the Notch intracellular domain (NICD). Then, the NICD translocates to the nucleus and binds to DNA-binding protein recombinant-recognition-sequence-binding protein at the Jκ site (RBP-J) (Hu et al., 2008; Wang et al., 2017). This interaction recruits other co-factor proteins of the mastermind-like (MAML) family, and the RBP-J–NICD–MAML complex affects the activation of Notch target gene, hairy and enhancer of split (Hes) (Boriushkin et al., 2019; Wang et al., 2017). Hes is a crucial mediator of the effect of Notch on cell fate determination, and activation of Notch signaling increases Hes1 expression accompanied by augmented proliferation of CBC stem cells and repressed differentiation of secretory goblet cells in the extended proliferation zone, whereas disruption of Notch signaling decreases Hes1 expression and activates mouse atonal homoleg 1 (Math1) transcription coupled with a loss of CBC stem cell proliferation and secretory cell hyperplasia (Okamoto and Watanabe, 2004; Pellegrinet et al., 2011; VanDussen et al., 2012). Therefore, homeostasis and regeneration of the intestine is abrogated when Notch signaling is disrupted.

Research has shown that traditional Chinese medicine (TCM) is a source of pharmaceutical material that can be used in the treatment of various diseases with fewer associated adverse effects (Zhang et al., 2019). Gegen Qinlian decoction (GQ) composed of four quality-assured herbs, Scutellaria baicalensis Georgi., Coptis chinensis Franch., Pueraria lobata (Willd.) Ohwi. and Glycyrrihiza uralensis Fisch., has been used as an efficacious medicine against acute diarrhea for approximately 2000 years and has recently been clinically proven effective in the treatment of UC. In our previous study, we reported that GQ alleviated the hyperactive immune response by suppressing TLR4/NF-κB signaling and enhancing the antioxidant effect in acute colitis mice (Li et al., 2016a). To further explore the multiple targets and therapeutic mechanism of GQ, in this study, we focused on the role of GQ in the regeneration of the colonic mucosa through restoring dysregulated Notch signaling in DSS-induced acute and chronic UC mice (Algieri et al., 2014; Koboziev et al., 2011). We observed that Hes1 expression was upregulated in acute UC mice and was downregulated in chronic UC mice. Therefore, we hypothesized that GQ exerts its homeostatic effect on the colonic mucosa by modulating dysregulated Notch signaling in UC mice.

Section snippets

Reagents

DSS (36–50 kDa) was purchased from MP Biomedicals (California, USA). Antibodies against Hes1 (1:1000), MAML (1:1000) and Toll-like receptor 4 (TLR4) (1:1000) were purchased from Cell Signaling Technology (Colorado, USA), RBP-J (1:1000), Math1 (1:1000), Glyceraldehyde phosphate dehydrogenase (GAPDH) (1:5000) and β-actin (1:5000) antibodies were obtained from ABclonal Biotech Co., Ltd (Wuhan, China). Lipopolysaccaride (LPS) was purchased from Sigma-Aldrich (Missouri, USA). Tumor necrosis factor α

Quality control of GQ

The HPLC fingerprint of GQ at a wavelength of 270 nm is shown in Fig. 1. Six components in GQ were identified and quantified based on the retention times and peak areas with those of the reference standards.

GQ repaired the colonic mucosa through downregulation of Hes1 expression in acute UC mice

The acute UC mice were improved by GQ treatment, as shown in Figs. S3 and 2F. The weight loss of the mice in the L-GQ, H-GQ and BBR groups was ameliorated by administration of different doses of GQ and BBR (Fig. S3B). On the 12th day, the body weight of the mice in the L-GQ, H-GQ and BBR

Discussion

As an effective TCM prescription for treating diarrhea, GQ alleviates intestinal disorders by modulating the gut microbiota, reducing excessive inflammation and repairing the intestinal mucosa (Liu et al., 2019a,b; Lv et al., 2019; Wu and Tan, 2019; Wu et al., 2019; Xu et al., 2015). However, the molecular mechanism by which GQ repairs the damaged intestinal mucosa remains unclear. Notch signaling is crucial in regulating both the differentiation and proliferation of intestinal epithelial cells

Conclusion

In conclusion, our results revealed for the first time that GQ restored the regeneration and homeostasis of the colonic mucosa via bidirectionally modulating dysregulated Notch signaling in acute/chronic UC models. GQ enhanced hypoactive Notch signaling in chronic UC mice and promoted the proliferation of the epithelium and differentiation of absorption cell lines, while GQ suppressed hyperactive Notch signaling in acute UC mice to promote the differentiation of goblet cells and secretion of

CRediT authorship contribution statement

Yaxing Zhao: Conceptualization, Funding acquisition, Data curation, Formal analysis, Writing - review & editing. Haofan Luan: Funding acquisition, Data curation, Formal analysis, Writing - review & editing. Hui Gao: Funding acquisition, Writing - review & editing. Xiaojun Wu: Writing - review & editing. Yubin Zhang: Conceptualization, Writing - review & editing. Ruiyan Li: Conceptualization, Writing - original draft, Writing - review & editing, Data curation, Formal analysis.

Declaration of Competing Interest

The authors have no competing interests.

Acknowledgment

This work was supported by funding from the Opening Project of Shanghai Key Laboratory of Compound Chinese Medicines (Shanghai University of Traditional Chinese Medicine) (No. 17DZ2273300) and the Basic Research Project of China Pharmaceutical University (No. 2632018PY06) to Ruiyan Li; and the National Natural Science Foundation of China (No. 81573484) to Yubin Zhang.

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