# Lingonberry Fruit Ethanol Extract Ameliorates DSS-Induced Ulcerative Colitis In Vivo and In Vitro
## 4 Discussion  
UC is a relapsing inflammatory chronic intestinal malady [23] that is worsened by an imbalanced inflammatory response and oxidative stress [24]. In addition, genetic factors, changes in environment, and an imbalanced immunological function can lead to difficulty in treating the intestinal inflammatory disease [25]. However, various drug treatments, including corticosteroids, immune modulators, and sulfasalazine, have serious side effects, such as poverty of blood, vomiting, and abdominal pain [26]. Therefore, the development of natural original products with few side effects is needed to treat chronic intestinal inflammatory disease. Compared to various UC in vivo models, the DSS-induced UC model is appropriate as it mimics the characteristics of the disorder. Our DSS-induced mouse model exhibited UC symptoms including blood stool, bodyweight loss, and diarrhea [27]. In this study, the administration of DSS worsened the clinical sign in vivo, but the LB treatment regulated the clinical sign of the DSS-induced mice. The administration of LB helped slow the bodyweight loss and decrease the DAI (diarrhea, occult/gross bleeding). Additionally, LB significantly slowed or stopped the colon length shortening caused by DSS (Figure 1). These results indicated that LB attenuated DSS-induced UC symptoms. In inflammatory intestinal conditions, COX-2 leads to production of PG [28]. PGE\({}_{2}\) is over-expressed in UC colon tissue. Both PGE\({}_{2}\) and COX-2 increase in the mucosa [29]. 5-ASA used in UC treatment inhibits COX-2 activation [30]. In our experiment, 5-ASA was also used as the positive control. However, LB 100 mg/kg showed more significant  
Figure 6: (a) NO level was measured by Griess assay. The LB treatment decreased NO production in LPS-stimulated peritoneal mouse macrophages, regulated (b) mRNA expression of iNOS, and attenuated (c) protein expression of iNOS. (d) The bar graph of the relative intensity of the Western blotting band by Image J. Values are represented as the mean \(\pm\) S.D. The data were analyzed by [PERSON]’s post-hoc test. \({}^{*}\)\(p\)\(<\) 0.05 versus normal group and \({}^{**}\)\(p\)\(<\) 0.05 versus LPS stimulation-only group. effects on the regulation of PGE\({}_{2}\) (Figure 3) and COX-2 (Figure 4) activities than 5-ASA. These results suggest that LB extract can be a therapeutic agent for regulating intestinal inflammation. The transcription factor NF-\(\kappa\)B modulates COX-2 synthesis and inflammatory cytokine secretion [31]. In IHC assay, intestinal NF-\(\kappa\)B and iNOS expressions were increased by DSS drinking. However, those were particularly decreased due to LB 100 mg/kg administration. These experimental results suggest that the anti-UC effect of LB might be via COX-2 and NF-\(\kappa\)B pathway. MPO, an enzyme found in neutrophil, is an indicator of neutrophil infiltration and the severity of inflammation [32]. LDH, a ubiquitous enzyme, is released into the injured tissue or bloodstream [33]. The results showed that 100 mg/kg LB regulated MPO and LDH activity in DSS-induced colon tissue injury (Figure 2). The effect of LB administration on MPO and LDH activity also indicates the efficacy of ameliorating acute intestinal inflammation. Under DSS-induced UC condition, crypt damage and inflammatory cell infiltration occurred in inflamed areas and were relieved by LB administration (Figure 2). These results indicate that LB treatment suppressed DSS-induced colon damage. Neutrophils, macrophages, and T cells release inflammatory cytokines (IL-1\(\beta\), IL-6, IFN-\(\gamma\), and TNF-\(\alpha\)) that cause colon inflammation [34; 35]. NO is also recognized as a regulator or mediator that induces inflammation [36]. Regulating inflammatory cytokines and NO is the strategy to remedy UC. In this study, LB suppressed the secretion of IL-1\(\beta\), IL-6, TNF-\(\alpha\), and IFN-\(\gamma\) (Figure 5) and NO production (Figure 6) in LPS-stimulated peritoneal mouse macrophages.