Isoliquiritigenin protects against angiotensin II-induced fibrogenesis by inhibiting NF-κB/PPARγ inflammatory pathway in human Tenon's capsule fibroblasts

https://doi.org/10.1016/j.exer.2020.108146Get rights and content

Highlights

  • ISL reduced Angiotensin II-mediated inflammation in MPMs and HTFs.

  • ISL prevented inflammation by inhibiting NF-κB/PPARγ inflammatory pathway.

  • ISL attenuated Angiotensin II-induced fibrogenesis in HTFs.

  • NF-κB/PPARγ may be a key target in conjunctival fibrosis after trabeculectomy.

  • ISL may be the potential agent to cure conjunctival fibrosis after trabeculectomy.

Abstract

Purpose

To examine the protective effects of Isoliquiritigenin (ISL) in angiotensin II (ANG II)-induced inflammation and fibrosis on Human Tenon's capsule Fibroblasts (HTFs) and Mouse Peritoneal Macrophages (MPMs). This study also investigated the potential mechanism of action of ISL.

Method

Methyl-thiazolyl tetrazolium (MTT) assay was used to test ISL toxicity. An ELISA and an RT-qPCR assay detected the inflammatory cytokines (TNF-α, IL-6, COX-2, and ICAM-1). A Western blot investigated the expression levels of inflammation-related signals [nuclear factor-κB (NF-κB), peroxisome proliferator-activated receptor γ (PPARγ)], and fibrogenesis, including fibronectin and alpha-smooth muscle actin (α-SMA)]. Protein expressions of α-SMA were measured by immunofluorescence.

Results

Pre-treatment with ISL (10 or 20 μM) dose-dependently decreased the mRNA levels of TNF-α, IL-6, ICAM-1, and COX-2 induced by ANG II (1 μg/ml) in both MPMs and HTFs. ANG II remarkably increased the amount of P65 in the nuclei and decreased the amount of P65 in the cytoplasm. Additionally, ANG II reduced PPARγ expression levels in a time-dependent manner. Furthermore, these effects which were induced by ISL were remarkably neutralized by ISL pre-treatment. Finally, ANG II markedly elevated the expression of fibronectin and α-SMA.

Conclusion

ISL could alleviate ANG II-induced fibrogenesis by inhibiting the NF-κB/PPARγ inflammatory pathway. In addition, ISL may be a potential agent for the treatment of conjunctival fibrosis. Most importantly, the NF-κB/PPARγ signaling pathway could be an effective therapeutic target for the prevention and treatment of conjunctival fibrosis after glaucoma surgery.

Introduction

Globally, glaucoma is one of the leading causes of vision loss (Quigley and Broman, 2006). Since the last 40 years, the most widely performed surgery for its treatment has been a trabeculectomy. It is a filtering surgery to lower the elevated intraocular pressure (IOP) caused by a glaucoma. However, previous studies have shown that the success rate after a trabeculectomy ranges from 81.2% to 85%; which decreases to 65%–39.3% after long-time follow-up (Lindemann et al., 2017; Matlach et al., 2015; Panarelli et al., 2016; Wong et al., 2013). The reason for the failure after a trabeculectomy is either the vigorous wound healing or scarring of the conjunctiva (bleb).

A trabeculectomy involves creating filtering blebs to decrease the IOP. However, the blebs often fail due to cicatrization of the overlying conjunctiva and Tenon's capsule caused by the wound healing process after the filtration surgery. The mechanisms involved in the pathogenesis of conjunctival fibrosis after a trabeculectomy include over expression of the renin-angiotensin system (Shi et al., 2015), inflammation (Deva et al., 2012), proliferation (Yu-Wai-Man et al., 2017), and angiogenesis (Kim et al., 2015). Among these mechanisms, abnormal activation of the renin-angiotensin system (RAS) seems to be the most probable cause. Angiotensin II (ANG II), one of the most important components of the RAS and its receptors, are known to be stimulating factors for fibrogenesis in the human Tenon's capsule. In a study on rabbits, ANG II showed an increasing trend in the Tenon's capsule as well as its receptors after trabeculectomy (Shi et al., 2015).

Multiple signaling pathways and molecular mechanisms can be activated by ANG II, a powerful proinflammatory mediator, directly or indirectly, to trigger inflammatory responses and fibrosis (Bataller and Brenner, 2005; Guo et al., 2001; Huang et al., 2010). However, whether ANG II induces inflammation at an early stage after trabeculectomy remains unknown. We hypothesized that the profibrotic response to ANG II can be attenuated by blocking the inflammatory response. Although several small molecules with anti-fibrosis or anti-metabolic properties have shown protection against trabeculectomy, till date, novel therapeutic agents for treatment have not yet been found.

Isoliquiritigenin (ISL), a dietary flavonoid with a chalcone structure, is an important constituent of Glycyrrhizae Radix (Peng et al., 2015). ISL is associated with a variety of biological and pharmacological functions (Jung et al., 2014; Yerra et al., 2017). Our previous study demonstrated that ISL markedly inhibited lipopolysaccharide (LPS)-induced acute inflammatory responses including related lung and liver injuries (Chen et al., 2018). Additionally, ISL can also attenuate chronic inflammatory diseases such as osteoarthritis (Ji et al., 2018) and adipose tissue inflammation (Watanabe et al., 2016). These studies show that ISL, a natural compound, could be a potential novel candidate for anti-inflammatory treatment. Therefore, we hypothesize that ISL is a potential anti-inflammatory drug against inflammation caused by ANG II and conjunctival fibrosis. An increasing number of studies suggest that peroxisome proliferator-activated receptor-gamma (PPARγ) and nuclear factor-κB (NF-κB) signaling pathways play a part in regulating the gene expression of pro-inflammatory cytokines (Chen et al., 2018; Jin et al., 2016). However, the effect of ISL on conjunctival fibrosis after glaucoma surgery is not known yet. This study was designed to elucidate the potential impact of ISL on conjunctival fibrosis after trabeculectomy and explore the possible mechanisms.

Section snippets

Reagents and chemicals

For this study, ANG II (Sigma, Louis, MO, USA) and ISL (Aladdine, Shanghai, China) were used. The structure of ISL is shown in Fig. 1. Antibodies against fibronectin, alpha-smooth muscle actin (α-SMA), inhibitors of IκB, p65, PPARγ, and GAPDH were supplied by Cell Signaling Technology (Danvers, MA, USA); the Cox-2 antibody was supplied by Santa Cruz Biotechnology (Santa Cruz, CA), and Lamin B was purchased from Abcam (Cambridge, UK). DAPI (4′, 6-diamidino-2-phenylindole) was obtained from

Cytotoxicity

The MTT assay in human Tenon's capsule fibroblasts (HTFs) and mouse peritoneal macrophages (MPMs) were tested for different concentrations of ISL (0, 1.25, 2.5, 5, 10, 20 and 40 μM) for 24 h. As displayed in Fig. 1B–C, the ISL treatment exerted no significant toxicity on the MPMs (p > 0.05, Fig. 1B) even at a concentration of 40 μM, but little toxicity of HTFs by treatment with 40 μM (p < 0.01, Fig. 1C).

ISL inhibited ANG II-stimulated inflammatory responses in MPMs

We analyzed whether ISL could inhibit an in vitro ANG II-induced increase of TNF-α and IL-6

Discussion

Trabeculectomy is most commonly performed surgery to treat glaucoma. As a destructive negative result, the conjunctival fibrosis after a trabeculectomy was considered as the most essential impact on surgical failure. Although several potential agents targeting conjunctival fibrosis have been developed in recent years, their IOP-lowering effect has not yet been proven to be effective. This study indicates that ISL protects against ANG II-induced HTFs fibrosis. The beneficial actions of ISL are

Conclusions

This study indicates that ISL has a potent anti-inflammatory effect by the regulation of NF-κB inactivation and PPARγ activation in ANG II-induced HTFs fibrosis (Fig. 6). The results suggest that the NF-κB/PPARγ signaling system is a vital therapeutic target for the prevention and treatment of conjunctival fibrosis after glaucoma surgery. Although many studies have analyzed the anti-inflammatory and anti-fibrosis action of chalcones (like ISL), the exact molecular mechanism is yet to be

Authors contribution

H.Y.; Designed and performed experiments and analyzed data. Y.X.; Performed experiments and co-wrote the paper. X.C.; Co-wrote the paper. L.S.; Supervised the research. R.L.; Designed experiments, co-wrote and revised the paper.

Declaration of cometing interest

Authors declare no conflicts of interest.

Acknowledgement

This study was supported by Science and Technology Project of Wenzhou Science and Technology Bureau [2020Y0871 to R.L., Y20190125 to X.C.], the Natural Science Foundation of Zhejiang Province [LQ18H120010 to R.L.].

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