Galli gigeriae endothelium corneum: its intestinal barrier protective activity in vitro and chemical composition

Abstract

Background: Galli gigeriae endothelium corneum (GGEC) has been effectively used for centuries for the treatment of functional dyspepsia (FD) in clinical practice in Asian countries. However, its potential mechanism and chemical composition remains undertermined.

Methods: In this study, the chemical profile of GGEC ethyl acetate extract (EAE) was evaluated by HPLC-Q-TOF-MS/MS. The effects of EAE on intestinal barrier function and inflammation were investigated in IEC-6 cells and RAW264.7 cells.

Results: The results showed that 33 compounds were tentatively identified, including 12 soy isoflavones, 7 bile acids for the first time in EAE. EAE significantly reinforced intestinal barrier function via increasing the tight junction protein levels of ZO-1 and Occludin, reducing the mRNA expression levels of interleukin (IL)-1β and IL-6 in tumor necrosis factor alpha (TNF-α)-challenged IEC-6 cells. The scratch wound assay showed that EAE accelerated wound healing of IEC-6 cells. EAE evidently reduced the level of NO in a dose-dependent manner with an IC₅₀ value of 18.12 μg/mL, and the mRNA expression of TNF-α, IL-1β, IL-6, iNOS and COX-2 in LPS-treated RAW264.7 cells.

Conclusion: This study revealed the intestinal barrier protective effects and chemical profile of GGEC, and the results indicated that GGEC strengthened the intestinal barrier by up-regulating protein expression of tight junctions and limiting inflammatory responses.

Keywords: Anti-inflammatory; Galli gigeriae endothelium corneum; Gastrointestinal barrier; HPLC-QTOF–MS/MS; Identification; Wound healing.

Fig. 1

Total ion chromatograms (TIC) in the positive-ion mode (a) and negative-ion mode (b) from HPLC-QTOF–MS/MS of EAE. EAE: Galli gigeriae endothelium corneum ethyl acetate extract

Fig. 2

EAE exerted protective effects on TNF-α-treated IEC-6 cells. When cells reached confluence in the transwell system, Cells were pretreated with EAE (0.1, 0.03, and 0.01 mg/mL) for 1 h, followed by TNF-α (50 ng/mL) stimulation for 24 h. a TEER values were shown as percentage relative to the control. b Permeability of FD-4 in TNF-α-treated IEC-6 cell. IEC-6 cells were pretreated with EAE (0.1, 0.03, and 0.01 mg/mL) for 1 h, followed by TNF-α (50 ng/mL) stimulation for 24 h. The relative ratios of ZO-1/GAPDH and Occludin/GAPDH were calculated based on the densities of bands on Western blots. The protein expression levels of ZO-1 (c) and occludin (d) were increased in TNF-α-treated IEC-6 cells while pretreated with EAE. EAE decreased the mRNA expression levels of IL-6 (e) and IL-1β (f) in TNF-α-stimulated IEC-6 cells. Mean ± SD (n = 3 independent experiments). *p < 0.05, **p < 0.01 vs. the control group. #p < 0.05, ##p < 0.01 vs. the TNF-α-stimulated group. EAE: galli gigeriae endothelium corneum ethyl acetate extract

Fig. 3

The anti-inflammatory effects of EAE on RAW264.7 cells. a The cytotoxic effect of EAE on macrophages viability in LPS-stimulated RAW264.7 cells. b The effect of EAE on nitric oxide (NO) production in LPS- stimulated RAW264.7 cells. c The IC₅₀ for inhibition of LPS-induced NO. The effect of EAE on the mRNA expression level of TNF-α (d), IL-6 (e), IL-1β (F), iNOS (g) and COX-2 (H) in LPS-stimulated RAW 264.7 cells. RAW264.7 cells were stimulated with or without 1 μg/mL of LPS for 24 h. Mean ± SD (n = 3 independent experiments). ***p < 0.001 versus control group; #p < 0.05, ##p < 0.01 and ###p < 0.001 versus LPS-only treatment group. EAE: galli gigeriae endothelium Corneum ethyl acetate extract

Fig. 4

Effects of EAE on wound healing of IEC-6 cells. a The cytotoxic effect of EAE on cell viability in IEC-6 cells. b Wound healing activity on cell migration of EAE. c The representative image of the wound healing assay. Mean ± SD (n = 4 independent experiments). *p < 0.05, **p < 0.01 vs. the control group. EAE: galli gigeriae endothelium corneum ethyl acetate extract