Indirubin-pregnane X receptor-JNK axis accelerates skin wound healing

Yuka Tanaka,Masutaka Furue,Hiroshi Uchi,Takamichi Ito

doi:10.1038/s41598-019-54754-2

Abstract

Indirubin is a potent anti-inflammatory phytochemical derived from indigo naturalis. It is also endogenously produced in the intestine and detected in the circulation in mammals. Indirubin exerts its biological functions via two xenobiotic receptor systems: aryl hydrocarbon receptor (AHR) and pregnane X receptor (PXR); however, its effects on wound healing remain elusive. To investigate whether indirubin promotes wound healing, we utilized an in vitro scratch injury assay and in vivo full-thickness mouse skin ulcer model and assessed wound closure. Indirubin significantly accelerated wound closure in both the scratch assay and the skin ulcer model. Using inhibitors of cell proliferation or migration, indirubin was found to upregulate the migratory but not the proliferative capacity of keratinocytes. Activation of AHR/PXR by indirubin was confirmed by their nuclear translocation and subsequent upregulation of CYP1A1 (AHR), or UGT1A1 mRNA (PXR) and also by luciferase reporter assay (PXR). Although both AHR and PXR were activated by indirubin, its pro-migratory capacity was canceled by PXR inhibition but not by AHR inhibition and was dependent on the JNK pathway. Moreover, activated PXR was detected in the nuclei of re-epithelialized keratinocytes in human skin ulcers. In conclusion, this study shows that the indirubin-PXR-JNK pathway promotes skin wound healing.

Highlights

Indigo naturalis (Sei Tai in Japanese or Qing Dai in Chinese), prepared from leaves of plants such as Baphicacanthus cusia, Polygonum tinctorium, Isatis indigotica, and Indigofera tinctoria, has long been used as a traditional herbal drug to treat inflammatory and leukemic disorders[1,2], and skin disorders such as eczema, aphthae, eruptions, furuncles, and psoriasis[3]
Indirubin inhibits the proliferation and accelerates the differentiation of keratinocytes[7] and induces Foxp3-expressing www.nature.com/scientificreports cells (B) were treated with dimethyl sulfoxide (DMSO) (0.1%) or indirubin (100 nM) for 6 h and the nuclear translocation of pregnane X receptor (PXR) was assessed by immunocytochemistry
Scale bar = 100 μm. (C,D) normal human epidermal keratinocytes (NHEKs) (C) or HaCaT cells (D) were treated with DMSO (0.1%) or indirubin (1, 100, or 10,000 nM) for 6 h and assessed for UGT1A1 expression (n = 6). (E) Cells were treated with DMSO (0.1%), indirubin (100 nM), or rifampicin

Summary

Introduction

Indigo naturalis (Sei Tai in Japanese or Qing Dai in Chinese), prepared from leaves of plants such as Baphicacanthus cusia, Polygonum tinctorium, Isatis indigotica, and Indigofera tinctoria, has long been used as a traditional herbal drug to treat inflammatory and leukemic disorders[1,2], and skin disorders such as eczema, aphthae, eruptions, furuncles, and psoriasis[3]. The binding of indirubin to AHR induces its cytoplasmic-to-nuclear translocation and upregulates the transcription of downstream genes such as cytochrome P450 1A1 (CYP1A1)[11]. The AHR activation by indirubin inhibits cell proliferation and induces terminal differentiation by upregulating involucrin, which may contribute to some of its efficacy on psoriasis[3,7]. Conflicting evidence has suggested that indirubin enhances intestinal epithelial wound healing through the activation of another xenobiotic receptor, pregnane X receptor (PXR, known as nuclear receptor subfamily 1 group I member 2, NR1I2)[17,18]. Indirubin activates PXR and upregulates the expression of its downstream responsive genes, such as CYP3A4 (a potent xenobiotic-catabolizing enzyme). Indirubin accelerates keratinocyte wound healing via PXR, but not AHR

Methods

Results

Conclusion