Elsevier

Phytomedicine

Volume 68, March 2020, 153179
Phytomedicine

Original Article
Wogonoside alleviates colitis by improving intestinal epithelial barrier function via the MLCK/pMLC2 pathway

https://doi.org/10.1016/j.phymed.2020.153179Get rights and content

Abstract

Background

Intestinal epithelial barrier dysfunction, which involves myosin light chain kinase (MLCK) activation, contributes to the occurrence and progression of inflammation in inflammatory bowel disease (IBD). Wogonoside helps maintain intestinal homeostasis in mice with dextran sulfate sodium (DSS)-induced colitis, but it is unclear whether it modulates intestinal barrier function.

Purpose

Here, we demonstrate that wogonoside protects against intestinal barrier dysfunction in colitis via the MLCK/pMLC2 pathway both in vivo and in vitro.

Methods

Caco-2 cell monolayers treated with the proinflammatory cytokine TNF-α showed barrier dysfunction and were assessed in the absence and presence of wogonoside for various physiological, morphological, and biochemical parameters. Colitis was induced by 3% DSS in mice, which were used as an animal model to explore the pharmacodynamics of wogonoside. We detected MLCK/pMLC2 pathway proteins via western blot analysis, assessed the cytokines IL-13 and IFN-γ via ELISA, tested bacterial translocation via fluorescence in situ hybridization (FISH) and a proper sampling of secondary lymphoid organs for bacterial culture. In addition, the docking affinity of wogonoside and MLCK was observed with DS2.5 software.

Results

Wogonoside alleviated the disruption of transepithelial electrical resistance (TER) in TNF-α exposured Caco-2 cell; FITC-dextran hyperpermeability; loss of the tight junction (TJ) proteins occludin, ZO-1 and claudin-1 in Caco-2 cell monolayers; and bacterial translocation in colitic mice. Moreover, wogonoside reduced the levels of the proinflammatory cytokines IL-13 and IFN-γ to maintain intestinal immune homeostasis. Transmission electron microscopy (TEM) confirmed that wogonoside ameliorated the destruction of intestinal epithelial TJs. Wogonoside not only inhibited the cytoskeletal F-actin rearrangement induced by TNF-α, stabilized the cytoskeletal structure, suppressed MLCK protein expression, and reduced MLC2 phosphorylation. In addition, the results of molecular docking analysis showed that wogonoside had a high affinity for MLCK and formed hydrogen bonds with the amino acid residue LYS261 and π bonds with LYS229.

Conclusion

Collectively, our study indicates that wogonoside alleviates colitis by protecting against intestinal barrier dysfunction, and the potential mechanism may involve regulation of TJs via the MLCK/pMLC2 signaling pathway. Meanwhile, our study also explains the success of S. baicalensis in the treatment of ulcerative colitis (UC).

Introduction

Recently, many studies have demonstrated that inflammatory bowel disease (IBD) pathogenesis coincided with damage to the intestinal epithelial barrier, causing IBD to be included in a newly identified category named "barrier organ diseases" (Lopetuso et al., 2015). The epithelial barrier prevents the contents in intestinal lumen accessing into submucous or even lamina propria; these contents include food, gut microbiota and their products. Unsurprisingly, mild or severe disruption of intestinal barrier enhances inflammation or directly induces experimental IBD (Kinchen et al., 2018). Consequently, renovating the integrity of intestinal epithelial barrier is critical for the treatment of IBD.

The barrier function of the intestinal epithelium is maintained partially by tight junctions (TJs) between adjacent epithelial cells. TJs are dynamically complex with multiple functions and consist of TJ proteins, primarily occludins, claudins, junctional adhesion molecules (JAMs), other transmembrane proteins, and more than 50 intracellular proteins, such as zonula occludens (ZO) and filamentous actin (Zeisel et al., 2018). TJs maintain the stability of intestinal epithelial permeability under normal physiological conditions by sealing the gap between adjacent intestinal epithelial cells. Abnormal expression or distribution of some TJ proteins has been observed and is accompanied by damage to intestinal barrier function in IBD patients. In patients with Crohn's disease (CD) or ulcerative colitis (UC), the TJ chains were reduced and broken (Krug et al., 2018). In addition, abnormal expression of TJ proteins (claudin-1, ZO-1 and occludin) in intestinal tissue promoted intestinal permeability and pathogen infiltration, induced immunologic dysfunction and the development of IBD (Deng et al., 2018; Pope et al., 2014). Therefore, regulating TJs is an effective therapy for alleviating or treating IBD and may alleviate the prolonged recovery of IBD.

Tumor necrosis factor-α (TNF-α), an important promoter of intestinal epithelial barrier injury in colitis, binds to TNF receptor 2 on the surface of intestinal epithelial cells, which leads to myosin light chain kinase (MLCK) transcription and enzyme activation (Su et al., 2013). MLCK, a specific substrate enzyme of myosin light chain2 (MLC2), specifically phosphorylated the Ser18 and Thr19 residues of MLC2. Then, phosphorylated MLC2 (pMLC2) activated the ATPase of the myosin heavy chain head, producing energy that caused cytoskeletal actin filaments to slide (Laukoetter et al., 2006; Ye and Ma, 2008). The actin-globulin ring around the TJ shrank, and then, the cell membrane containing TJ proteins was endocytosed, occludin and other transmembrane proteins were degraded, and TJ relaxation occurred to form an intercellular space, resulting in increased intestinal epithelium permeability (Quiros and Nusrat, 2014; Soliman et al., 2018) and destruction of the intestinal epithelial barrier. Moreover, the bacterial infiltration caused by barrier destruction promotes the binding of pathogen-associated molecular patterns (PAMPs) to pattern recognition receptors (PRRs), such as TLR4, TLR5 and NLRP3; activates immunocytes to produce immune responses; and secretes cytokines, such as IL-13 and IFN-γ. A previous report (Wu et al., 2014) confirmed that excessive secretion of IFN-γ increased paracellular permeability, which is involved in the activation of the MLCK pathway. Activation of the MLCK signaling promoted the secretion of IL-13 and leads to the increase in the expression of the TJ protein claudin-2 and the flux of paracellular cations (Weber et al., 2010). Consequently, suppressing the activation of MLCK protects the intestinal epithelial barrier.

Wogonoside (WG; MW: 460.39, C22H20O11, Fig. 1A), the glucuronide metabolite of wogonin, is one of the major flavonoids derived from Scutellaria baicalensis Georgi (Lamiaceae) and has been shown to have a wide range of pharmacological activities (Shen and Fan, 2016; Lee et al., 2014). In recent years, increasing evidence has indicated that wogonoside has significant anti-inflammatory activity (Sun et al., 2015, 2016; Tang et al., 2017) and has an effect on IBD (Sun et al., 2015), but its treatment of the intestinal barrier in IBD has rarely been reported. Thus, we studied the effect of wogonoside on dextran sulfate sodium (DSS)-induced colitic mice and explored the mechanism of wogonoside protecting intestinal epithelial barrier function in vitro and in vivo; this effect may be closely associated with the inhibition of the MLCK/pMLC2 signaling pathway.

Section snippets

Drugs and reagents

Wogonoside (≥ 98% purity) was obtained from Chengdu Feipude Biological Technology Co. Ltd. (Chengdu, China). DSS was purchased from MP Biomedicals (Los Angeles, CA, USA). Sulfasalazine (SASP) tablets were obtained from Shanxi Tongda Pharmaceutical Co. Ltd. (Shanxi, China). Human TNF-α was purchased from PeproTech (Los Angeles, CA, USA). Rat anti-ZO-1 and mouse anti-Occludin were purchased from Santa Cruz Biotechnology (Los Angeles, CA, USA). Rabbit anti-Claudin1 was purchased from Abcam

Effect of wogonoside on colitic mice

The weight gain rate of mice in wogonoside (12.5 mg/kg 25 and 50 mg/kg) or SASP (500 mg/kg) groups was higher about 5% than that of mice treated with 3%DSS alone on the 9th and 10th day (p< 0.05, Fig. 2A). Compared with mice in the control group, colitic mice in the 3% DSS group had a significantly shortened colon (5.49 ± 0.19 cm vs. 7.12 ± 0.26 cm) due to inflammation, congestion and edema of the colon, and these symptoms were alleviated after administration of wogonoside (6.08 ± 0.14,

Discussion

The integrity of the intestinal epithelial barrier is believed to be a key determinant of the onset and acceleration of IBD (Mohanan et al., 2018; Shin and Kim, 2018). TJs play a vital role in the intestinal epithelial barrier. Once damaged, the barrier integrity is destroyed, and a series of inflammatory processes are triggered. Our studies demonstrated that wogonoside ameliorated the severity of colitis, such as ulcerative colitis (UC), by regulating intestinal epithelial TJs and protecting

Conclusion

Our study confirmed that wogonoside (12.5 mg/kg, 25 mg/kg and 50 mg/kg) alleviated intestinal inflammation in colitis and has a protective effect on intestinal epithelial barrier function both in vivo and in vitro. Simultaneously, it has been shown that wogonoside regulates intestinal epithelial TJs mainly by inhibiting the MLCK/pMLC2 signaling pathway. As a consequence, the findings in this current study provide novel insight that wogonoside ameliorates colitis by protecting the function of

Declaration of Competing Interest

There are no conflicts of interest to declare.

Acknowledgments

This work was supported by the National Natural Science Foundation of China (no. 81703785 & no. 81673668), the Characteristic Innovation Projects of Colleges and Universities in Guangdong Province, China (no. 2017KTSCX042), and the Natural Science Foundation of Guangdong Province (no. 2017A030313657).

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