In the present study the preventative properties of GS leaves extract against experimentally induced model of IBD in Wistar rats were investigated. The histopathological assessment revealed that pretreatment with preserved the functional cytoarchitecture of the entire colonic mucosa, congestion, ulceration, erosions, necrosis and inflammation caused by AA in a dose-dependent manner. Moreover, GS leaves extract markedly protected the colonic mucosal content and prevented oxidative and inflammatory response in the colon of AA induced rats.
Experimentally induced UC by 4% AA is a well-recognized model for IBD. The colonic changes following rectal application of AA to rodents are characterized by mucosal ulceration, hemorrhage and inflammation, which are similar to IBD in human . It also causes infiltration of leukocytes to the damaged area and rupture of colonic barrier, along with an inflammatory mediator’s release, including cytokines and arachidonic acid metabolites as well as release of ROS, leading to oxidative damage [8, 9]. In the current investigation, rectal application of AA significantly increased animals colon weights, which was associated with severe tissue ulceration, necrosis, goblet cell hyperplasia and inflammatory infiltrate as demonstrated in the histopathological screening, which are in accordance with earlier reports using the same animal model [21, 31]. Defects in the colonic mucosal barrier functions are among the etiological factors that characterize IBD . In the current study, the protective colonic mucus content was markedly altered by AA, which is in agreement with the study by Popov et al. 2006 . The mucus layer is well known to enhance the repair of the chemically damaged epithelium .
Several therapies have been used in the management of IBD. However, their adverse effects and toxicity represent major clinical problem . Therefore, naturally occurring alternative options has been suggested along with the conventional therapies . Our previous work demonstrated that GS leaves extract effectively protected against chemically induced gastric ulcers . Results of the present study showed that the increased colon weight after AA administration was significantly reduced by the pretreatment of the animals with GS, indicating a decreased colon inflammation which was demonstrated by histopathological assessments. Pretreatments with GS inhibited colonic wall mucus depletion in the UC rat model, which could be attributed to its anti-inflammatory property, similar to our previous studies showing attenuation of the gastric mucosal damage-induced by absolute ethanol .
Both the reported forms of IBD are multi-factorial intestinal inflammatory disease, however, pro-inflammatory mediators is considered to play a crucial role in the pathogenesis of IBD . They can modulate mucosal immune system, by the alteration of epithelial integrity and colon injury by infiltration of the neutrophils and macrophages . Migration of granulocytes and other leukocytes to the inflamed mucosa and superficial ulcers results in overproduction of pro-inflammatory cytokines [8, 9]. In both IBD forms, levels of pro-inflammatory cytokines, such as IL-1β, TNF-α, and IL-6, were found to be increased [38–40], which suggest that these inflammatory mediators are engaged in determining the severity of the disease. In the present study, pro-inflammatory cytokines including IL-1β, TNF-α, and IL-6 were significantly elevated in colon tissues in AA administered group, suggesting a role of inflammation in the pathogenesis of the disease which is supported by the histopathological results showing epithelial cell necrosis, edema, and neutrophil infiltration in the tissue. Our findings are in agreement with earlier experimental and clinical data reported by others in a number of studies [24, 31, 41, 42]. Next, we found increased colonic levels of PGE2 and NO in AA group of animals, which is in accordance with other investigations [43, 44]. This increase in the levels of inflammatory molecules may be mediated through pro inflammatory cytokines. The anti-inflammatory properties of GS leaves extract were reported previously using various inflammatory animals models [45, 46]. GS leaves extract was found to reduce the level of pro-inflammatory cytokines (IL-1β, IL-6 and TNF-α) in AA colon tissue. The level of prostaglandins and NO is regulated by the cellular enzymes COX-2 and iNOS, respectively. These enzymes are known to be enhanced by inflammatory mediators during the burden of UC [47, 48]. Therefore, we suggest the anti-inflammatory effect of GS might be mediated through inhibiting the level PGE2 and NO in AA model of UC.
Oxidative stress is well known to play the major role in the pathophysiology of IBD [49, 50]. Induction of UC in experimental animals causes oxidative injury due to imbalance between the levels of pro-oxidant and antioxidant systems . UC is characterized by overproduction of reactive oxygen and nitrogen species leading to significant cellular adverse effects such as LPO and damage to tissue proteins and nucleic acids [10, 52]. Furthermore, increased levels of free radicals were found in colonic tissue specimens of patients with UC [53, 54]. Antioxidant enzymes such as SOD and CAT, and the non-enzymatic sulfhydryl groups play the major role in the organism defense against excess free radicals generated under disease conditions . In the present investigation, concentrations of protein and non-protein sulfhydryl groups as well as activities of the antioxidant enzymes such as SOD and CAT were severely reduced in the colon following AA administration, which clearly indicates increased level of oxidative stress which may damage cells by lipid peroxidation of membranes and oxidation of cellular proteins. Indeed, increased levels of TBARS and free radicals found in the study may damage cells as observed by histopathological investigations.
Another damaging effect of oxidative stress have been noted in the present study, is in the alteration in the levels of nucleic acids and proteins in the colon of AA treated animals. These observations were also previously reported by others, which confirm the oxidative damages to cellular macromolecules thereby may weaken epithelial cellular integrity and delay colonic mucosal healing [5, 31]. Thus, it is suggested that those substances that prevent free radicals production or potentiate the endogenous enzymatic or non-enzymatic antioxidant system can have beneficial effects in ulcerative colitis. In agreement with previous studies [56, 57], pretreatment with GS increased antioxidant status and lowered LPO in AA model of UC which suggests its colonic protective effect by enhancing of nucleic acids and proteins levels. It is well known that GS has anti-diabetic properties with antioxidant activity . Previously antioxidant and anti-LPO effects of GS were reported in several animal models [59, 60] and also in an in vitro study by Rachh and colleagues, 2009 . Several pharmacological studies have demonstrated that the antioxidant properties of GS are mainly through the major bioactive constituents in its leaves, which are a group of oleanane type triterpenoid saponins known as gymnemic acids [12, 16], alkaloids, acidic glycosides and anthroquinones and their derivatives . These constituents were also found in our preliminary phytochemical analysis from the GS leaves extract.