Lycopodium Mitigates Oxidative Stress and Inflammation in the Colonic Mucosa of Acetic Acid-Induced Colitis in Rats

Abstract

Inflammatory bowel diseases (IBDs) such as ulcerative colitis (UC) and Crohn's disease (CD) are diseases of the gastrointestinal system involving genetic and environmental factors attributed to oxidative stress and inflammation. Targeting oxidative stress and inflammation by novel dietary compounds of natural origin convincingly appears to be one of the important therapeutic strategies to keep the disease in remission. As there is no permanent cure for IBD except for chronic long-term treatment or surgery, it is therefore imperative to investigate plant-based agents that are receiving attention for their therapeutic benefits to overcome the debilitating clinical conditions of IBD. Lycopodium (LYCO), a plant of tropical and subtropical origin and known by numerous names such as ground pine, club moss, or devil's claw, has been popularly used for centuries in traditional medicine including Chinese and Indian medicines. In the present study, the effect of LYCO has been investigated in an acetic acid (AA)-induced colitis model in Wistar rats. LYCO was orally administered at the dose of 50 mg/kg/day either 3 days before or 30 min after the induction of IBD and continued for 7 days by intrarectal administration of AA. The changes in body weight and macroscopic and microscopic analysis of the colon of rats of different experimental groups were observed on days 0, 2, 4, and 7. The levels of myeloperoxidase (MPO), reduced glutathione (GSH), and malondialdehyde (MDA) were measured. AA caused a significant reduction in body weight and increased macroscopic and microscopic ulcer scores along with a significant decline in antioxidant enzymes, superoxide dismutase (SOD), and catalase and antioxidant substrate, glutathione (GSH). There was a concomitant increased formation of malondialdehyde (MDA), a marker of lipid peroxidation, and raised myeloperoxidase (MPO) activity, a marker of neutrophil activation. Treatment with LYCO significantly improved IBD-induced reduction in body weight, improved histology, inhibited MDA formation, and restored antioxidants along with reduced MPO activity. AA also caused the release of proinflammatory cytokines such as interleukin-1β (IL-1β) and interleukin-23 (IL-23). Furthermore, AA also increased the levels of calprotectin, a protein released by neutrophils under inflammatory conditions of the gastrointestinal tract. LYCO treatment significantly reduced the release of calprotectin and proinflammatory cytokines. The results demonstrate that LYCO treatment has the potential to improve disease activity by inhibiting oxidative stress, lipid peroxidation, and inflammation along with histological preservation of colonic tissues.

Keywords: IBD; colonic inflammation; gut; lycopodium; oxidative stress; plants.

Figure 1

Schematic representation of study design.

Figure 2

Effect of lycopodium on mean body weight. *** p < 0.001 vs. non IBD control group; # p < 0.05, ## p < 0.01 vs. relative IBD control group.

Figure 3

(A) Effect of lycopodium on macroscopic image (a: IBD control, b: post IBD treatment, c: pre IBD treatment) and (B) ulcer score in rat model of IBD. *** p < 0.001 vs. non IBD control group; # p < 0.05, ## p < 0.01 vs. relative IBD control group.

Figure 4

Effect of lycopodium on microscopic images in acetic acid-induced IBD in rats. (A): Naïve (control); (B1–B3): 1: 2-day IBD control–no lycopodium, 2: 2-day IBD, post-treated with lycopodium, 3: 2-day IBD (pre-treated). (C1–C3): 1: 4-day IBD control–no lycopodium, 2: 4-day IBD, post-treated with lycopodium, 3: 4-day IBD (pre-treated). (D1–D3): 1: 7-day IBD control–no lycopodium, 2: 7-day IBD, post-treated with lycopodium, 3: 7-day IBD (pre-treated). (B) Effect of lycopodium on microscopic ulcer score in a rat model of acetic acid-induced IBD. m= mucosa; mm = muscularis mucosae. *** p < 0.001 vs. non IBD control group; # p < 0.05, ## p < 0.01 ### p < 0.001 vs. relative IBD control group.

Figure 4

Effect of lycopodium on microscopic images in acetic acid-induced IBD in rats. (A): Naïve (control); (B1–B3): 1: 2-day IBD control–no lycopodium, 2: 2-day IBD, post-treated with lycopodium, 3: 2-day IBD (pre-treated). (C1–C3): 1: 4-day IBD control–no lycopodium, 2: 4-day IBD, post-treated with lycopodium, 3: 4-day IBD (pre-treated). (D1–D3): 1: 7-day IBD control–no lycopodium, 2: 7-day IBD, post-treated with lycopodium, 3: 7-day IBD (pre-treated). (B) Effect of lycopodium on microscopic ulcer score in a rat model of acetic acid-induced IBD. m= mucosa; mm = muscularis mucosae. *** p < 0.001 vs. non IBD control group; # p < 0.05, ## p < 0.01 ### p < 0.001 vs. relative IBD control group.

Figure 5

Effect of lycopodium on MPO in acetic acid-induced IBD in rats. Results are mean ± SEM; * p < 0.05, *** p < 0.001 vs. non IBD control group; # p < 0.05, ## p < 0.01, ### p < 0.001 vs. relative IBD control group.

Figure 6

Effect of lycopodium on calprotectin levels in acetic acid-induced IBD in rats. Results are mean ± SEM. ** p < 0.01 vs. non IBD control group; # p < 0.05, ## p < 0.01, vs. relative IBD control group.

Figure 7

Effect of lycopodium on proinflammatory cytokines; IL-1β (A), and IL-23 (B) in acetic acid-induced IBD in rats. Results are mean ± SEM. * p < 0.05, *** p < 0.001 vs. non IBD control group; # p < 0.05, ## p < 0.01, ### p < 0.001 vs. relative IBD control group.

Figure 8

Effect of lycopodium on oxidative stress markers; GSH (A), SOD (B), Catalase (C) and MDA (D) in acetic acid-induced IBD in rats. Results are mean ± SEM; * p < 0.05, ** p < 0.01 *** p < 0.001 vs. non IBD control group; # p < 0.05, ## p < 0.01, ### p < 0.001 vs. relative IBD control group.