Nrf2-mediated mucoprotective and anti-inflammatory actions of Artemisia extracts led to attenuate stress related mucosal damages

Abstract

The aim of this study was to compare biological actions between isopropanol and ethanol extracts of Artemisia including antioxidant, anti-inflammatory, and cytoprotective actions. Antioxidant activities were evaluated using 2,2-diphenyl-1-picrylhydrazyl (DPPH) method and confocal microscopy on lipopolysaccharide-induced RGM1 cells, cytoprotection effects evaluated by detecting heme oxygenase-1 (HO-1), Nf-E2 related factor2 (Nrf2) and heat shock protein 70 (HSP70), and anti-inflammatory effects investigated by measuring inflammatory mediators. Water immersion restraint stress was imposed to provoke stress related mucosal damages (SRMD) in rats. Isopropanol extracts of Artemisia showed the higher DPPH radical scavenging activity and lesser LPS-induced reactive oxygen species productions and increased HO-1 expression through increased nuclear translocation of Nrf2 transcription factor compared to ethanol extracts. The increased expression of HSP70 and decreased expression of endothelin-1 were only increased with isopropanol extracts. A concentration-dependent inhibition of LPS-induced COX-2 and iNOS even at a rather lower concentration than ethanol extract was achieved with isopropanol extracts. Cytokine protein array revealed Artemisia extracts significantly attenuated the levels of CXCL-1, CXCL-16, and MCP-1. These orchestrated actions led to significant rescue from SRMD. Conclusively, Artemisia extracts imposed significant antioxidant and anti-inflammatory activity against SRMD and isopropanol extracts were superior to ethanol extracts in these beneficiary actions of Artemisia.

Keywords: HO-1; anti-inflammation; artemisia asiatica; isopropanol extracts; stress related mucosal damages.

Fig. 1

Comparison of antioxidative activities according to extract solvent. (A) Chromatography of ethanol extracts and isopropanol extracts of Artemisia. Peak of eupatilin and jaceosidin was significantly higher in isopropanol extracts compared to ethanol extracts of Artemisia. Molecular structure and chemical name was shown. (B) Higher DPPH radical reduction activity in isopropanol extracts than ethanol extracts of Artemisia. To compare the antioxidant capacity between the ethanol extracts and the isopropanol extracts of Artemisia, DPPH scavenging capability was measured. (C) Confocal imaging of DCF-DA according to extracts. The intracellular accumulation of ROS induced by LPS was measured by using DCF-DA as a probe capable of detecting peroxides. LPS caused increased intracellular accumulation of ROS in RGM1 cells, which was apparently abolished by pretreatment with isopropanol extracts. (D) The data were presented as mean ± SD. for three different experiments performed in triplicate.

Fig. 2

Comparison of HO-1 expression according to extraction solvent. Isopropanol extracts induces HO-1 protein (A) and mRNA expression (B). HO-1 protein and mRNA expression were increased following Artemisia extracts treatment in a dose-dependent manner irrespective of extraction solvent. However, 30 µg/ml isopropanol extract showed significantly increased expression of HO-1. HO-1 protein (A) and mRNA (B) expression were analyzed by Western blotting and RT-PCR, respectively. (C) Cell viability. Artemisia extracts were tested for their cytotoxic activity using the MTT colorimetric assay. The data were presented as mean ± SD. for three different experiments performed in triplicate.

Fig. 3

Comparison of Nrf2 activation according to extract solvent. (A) Isopropanol extracts of Artemisia activated nuclear translocation of Nrf2. Nuclear fraction analyzed to determine the nuclear Nrf2 levels by Western blotting. Nuclear translocation of Nrf2 with isopropanol extracts was significantly increased compared to ethanol extracts in RGM1 cells. (B) Confocal imaging of Nrf2. Nrf2 nuclear translocation was examined under confocal microscope after its immunofluorescence staining. Increased nuclear translocation of Nrf2 was seen in isopropanol extracted group compared to ethanol extracts treated group, as evidenced with yellow color. (C) Nrf2 dependence of HO-1 induction. After RGM1 cells were transfected with nonspecific or Nrf2 siRNA, HO-1 protein expression was analyzed by Western blotting. The isopropanol extracts-induced up-regulation of HO-1 was abolished by silencing of Nrf2 expression with specific siRNA.

Fig. 4

Comparison of inflammatory mediators including iNOS and COX-2 according to extract solvent. (A) Isopropanol extracts reduces COX-2 protein expression. COX-2 protein expression was analyzed by Western blotting. Isopropanol extracts in the presence of LPS inhibited COX-2 protein in RGM1 cell in a dose-dependent manner. (B) Isopropanol extracts of Artemisia reduced more COX-2, iNOS, and IL-6 mRNA expression than ethanol extracts. COX-2, iNOS, and IL-6 mRNA expression were analyzed by RT-PCR. Isopropanol extracts in the presence of LPS inhibited COX-2, iNOS, and IL-6 mRNA expression in RGM1 cell in a dose-dependent manner. (C) Protein array for inflammatory mediators. CXCL-1, CXCL-16, and MCP-1 were inhibited in density in the isopropanol extracts treated group than ethanol extracts treated group. The low panel shows the relative intensity of decreased each cytokine.

Fig. 5

Comparison of endothelin-1 (ET-1) and HSP70 expression according to extract solvent. (A) Isopropanol extracts of Artemisia significantly reduced endothelin-1 protein expression than ethanol extracts. ET-1 is known as strong vasoconstriction factor engaged in gastric mucosal damages as exemplified in stress-induced ulcer, Helicobacter pylori-associated ulcer, and duodenal ulcerogenesis. In protein array, isopropanol extracts significantly decreased ET-1 expression compared to ethanol extracts under LPS-stimulation, inferring cytoprotective characteristics of isopropanol extracts of Artemisia. (B) Isopropanol extracts induced HSP70 protein expression. The HSP70 as a molecular chaperone has been suggested to exert its gastroprotective action by protecting mitochondria and by interfering with the stress-induced apoptotic program. The treatment with the isopropanol extracts of Artemisia provoked significant up-regulation of HSP70 protein, while ethanol extracts did not increased the levels of these proteins.

Fig. 6

Water Immersion Restraint Stress (WIRS)-induced gastric damages and rescuing action of Artemisia extracts. (A) Gross morphology (B) RT-PCR for IL-8, IL-1β, TNF-α, CXCL-1, CXCL-16 (C) RT-PCR for COX-2 and HO-1 followed with western blot for COX-2 and HO-1 (D) The western blot for Nrf2 using cytoplasmic fraction (E ) RT-PCR for HSP70 and western blot for HSP27, HSP60, and HSP70. (F) The changes of gastric mucosal levels of lipoxinA4 (LAX4) according to group.

Fig. 7

Isopropranol extracts of Artemisia could rescue from stress related gastric damages, in which higher antioxidative, anti-inflammatory, and cytoprotective mechanisms were intervened. Rescuing action of Artemisia against stress-induced gastritis Improved biological actions of Artemisia with isopropanol extraction was validated in animal model. Isopropanol extracts of Artemisia offered three kinds of beneficial actions compared to ethanol extracts, higher antioxidative activities reflected with higher HO-1 and Nrf2 activation, higher anti-inflammatory actions accompanied with attenuated inflammatory cytokines, and higher chance of cytoprotection including HSP70 induction as well as decreased endothelin-1, all of these benefits from isopropanol extraction can orchestrate higher clinical efficacy of gastritis treatment.