Augmented reduction in colonic inflammatory markers of dextran sulfate sodium-induced colitis with a combination of 5-aminosalicylic acid and AD-lico™ from Glycyrrhiza inflata

Abstract

The primary aim of this study was to determine whether the oral administration of AD-lico™, a functional extract from Glycyrrhiza inflata in combination with 5-aminosalicylic acid (5-ASA) could ameliorate the inflammatory symptoms in dextran sulfate sodium (DSS)-induced colitis in rodents. This DSS rodent model is used to study drug candidates for colitis, as part of the spectrum of diseases falling under the inflammatory bowel disease (IBD) category. Here, with oral AD-lico™ administration, there was a substantial disruption of the colonic architectural changes due to DSS and a significant reduction in colonic myeloperoxidase (MPO) activity, a marker of colitis. In the same samples, there were also reduced levels of colonic and serum IL-6 in the oral AD-lico™ treated rats. This study also addressed the possible mechanisms for AD-lico™ mediated changes on colonic inflammation markers. These included the observations that AD-lico™ dampened the IL-6 proinflammatory-signaling pathway in THP-1 human monocytic cells and reduced the TNFα-mediated upregulation of surface adhesion molecule ICAM-1 in human umbilical vein endothelial cells (HUVECs). Finally, it was shown that AD-lico™ could be combined with 5-ASA in reducing the inflammatory markers for colorectal sites affected by colitis, a first study of its kind for a combination therapy.

Keywords: Dextran sulfate sodium; colitis; inflammation; inflammatory bowel disease; interleukin-6; tumor necrosis factor.

Figure 1.

Inhibition of morphological changes in colon cross-sections due to DSS treatment. A 1-cm segment was removed from the distal end of the colon and fixed in 4% paraformaldehyde solution prior to embedding in paraffin. The degree of inflammation and morphological injury caused by 5% DSS exposure was assessed by hematoxylin and eosin staining according to the standard protocols (magnification, ×100). This is a representative of three experiments. G1, vehicle; G2, 5% DSS; G3, 5% DSS + AD-lico™ 25 mg/kg; G4, 5% DSS + 5-ASA 50 mg/kg; G5, 5% DSS + 5-ASA 50 mg/kg + AD-lico™ 25 mg/kg

Figure 2.

Inhibition of myeloperoxidase (MPO) activity. The rat colons were rinsed with cold PBS, blotted dry and frozen immediately in liquid nitrogen. They were then stored at −80°C until assayed for MPO activity using the o-dianisidine method. G1, normal control; G2, 5% DSS; G3, 5% DSS + AD-lico™ 25 mg/kg BW; G4, 5% DSS + AD-lico™ 100 mg/kg BW; G5, 5% DSS + 5-ASA 50 mg/kg BW; G6, 5% DSS + 5-ASA 50 mg/kg BW + AD-lico™ 25 mg/kg BW. *p < 0.05. This is a representative of three experiments.

Figure 3.

Inhibition of IL-6 levels in (A) colon and (B) serum samples from DSS treated rats. Levels of IL-6 in the serum and tissue were measured using an enzyme-linked immunosorbent assay (ELISA), as described in Materials and Methods. G1, normal control; G2, 5% DSS; G3, AD-lico™ 25 mg/kg BW; G4, 5-ASA 50 mg/kg BW; G5, 5-ASA 50 mg/kg BW + AD-lico™ 25 mg/kg BW. *p < 0.05. This is a representative of three experiments.

Figure 4.

AD-lico™ inhibiting IL-6 downstream signaling markers in IL-6 treated THP-1 cells. JAK2 and ERK activation and STAT-3 phosphorylation increases due to activation of the THP-1 cells by IL-6 were dose-dependently blocked by AD-lico™ pretreatment of the cells for 2 h. Genistein, a known kinase inhibitor, served as a positive control in the experiments. This is a representative of three experiments (A). Proposed pathway components of IL-6 signaling in THP-1 cells (B).

Figure 5.

AD-lico™ blocking upregulation of surface ICAM-1 in HUVECs treated with TNFα. HUVECs at 7,000 per well in 200 µl in complete F-12 K media were plated overnight in cell culture wells. Following 16 h, the cells were starved for 12 h in F-12 K media but only containing 2% FBS. The cells were then pretreated with serial dilutions of AD-lico™ stock for 3 h in culture. Subsequently, the cells were induced for an upregulation of endogenous ICAM-1 with 10 ng/mL of human TNFα for 8 h. Cell surface expression of ICAM-1 by HUVECs was determined using cell-based ELISA. *p < 0.05. This is a representative of three experiments.