**3. Discussion**

Gastrointestinal disorders are a global health problem affecting millions of people. Inflammation is a defensive biological response to harmful stimuli and infection that promotes the production of inflammatory mediators. Oxidative stress plays a vital role in alterations related to the pathophysiology of inflammation. Modulation of the inflammatory mediators is considered a promising strategy for facing inflammatory disease. Although RI has been traditionally used to treat inflammatory diseases, the underlying molecular mechanism of anti-inflammation properties is still not understood. The study investigated the anti-inflammatory and gastroprotective activity of RI in a model of LPS-induced inflammation in RAW 264.7 cells and HCl/EtOH-induced experimental gastric ulcer.

Antioxidants play an important role in redox mechanisms in a biological system, protecting it against inflammation and apoptosis. Phenolic and flavonoid are the most important plant secondary metabolites and have the strong antioxidant capacity [18,19]. Antioxidants act as oxygen scavenger capable of catalyzing the oxidative process [20]. Significant amounts of phenolic and flavonoid content were found in RI (Table 2) that may be the major donor for the anti-oxidative as well as anti-inflammatory role against gastric damage. For the possible mechanism by which RI protects against LPS-induced macrophage cell damage, these results showed that it may act through its anti-oxidative and anti-inflammatory effects (Figure 1). Treatment with the RI inhibited the LPS-induced intracellular oxidative stress (ROS) and NO production (Figure 2, upper panel). These data strongly suggest that RI could cure various inflammatory symptoms based on its anti-inflammatory properties. Tissue-related macrophages play an important role in the loss of physiological functions of the organ by releasing toxic and inflammatory molecules, such as NO and ROS [21]. The synthesis of these inflammatory molecules is responsible for the progression of various inflammatory diseases, such as gastric ulcer [22,23].


**Table 2.** The nucleotide sequence of the primers for qPCR.

The pathogenesis of ethanol-induced gastric injury is very complex and related to oxidative stress that has been confirmed by recent studies [24,25]. Gastric tissue damage is caused by an imbalance between the damage of the gastric tissue and protective factors. In addition, NO plays a complex role in gastric mucosal integrity and its synthesized independently [26]. NO level declines in patients suffering from gastric distress considerably. By increasing gastric mucosal blood flow, normal production of NO could retain the integrity of gastric mucosa and contribute to the defense and healing of mucosal damage, while preventing chemotaxis and adhesion of inflammatory cells to guard gastric mucosa [27]. Following gastric damage, the gastric tissue may be partially oxidized due to injury. Lipid peroxidation is the result of ROS reaction against cell membranes and produces a significant level of pro-oxidant such as MDA, which leads to oxidative gastric damage [28]. In the present study, RI markedly increased NO and decreased MDA levels in gastric mucosa, demonstrating the anti-inflammatory and antioxidant potential of RI (Figure 2, lower panel). This finding is consistent with an earlier report [27]. In this study, RI attenuated the macroscopic and histopathologic lesions in gastric mucosa and inflammatory cells influx that signifies its prospective anti-gastric ulcer activities (Figure 3), as also shown for the standard compound ranitidine hydrochloride, a histamine-2 receptor

antagonist clinically recommended for the treatment of gastric ulcer [29]. The ethanol-induced gastric lesion is a key experimental model commonly used for likely anti-gastric ulcer activity since ethanol is thought to be a leading cause of gastric ulcer [30,31]. Ethanol has been revealed to cause hemorrhagic gastric lesions characterized by mucosal friability and infiltration of inflammatory cells [32]. Reduction of the infiltration of inflammatory cells (neutrophil) has been considered to be a vital anti-inflammatory mechanism by which effective anti-gastric-ulcer medicine protects against mucosal injuries [33].

Pro-inflammatory cytokines and enzymes such as *TNF-α*, *IL-1β*, *IL-6*, *iNOS* and *COX-2* genes in the tissue may be used as biomarkers of gastric visceral damage. Following inflammatory stimuli, inflammatory mediators have been elevated to prompt deleterious effect in the stomach. In this study, increased production of inflammatory cytokines in ulcerated gastric tissue can be attributed to the damaging effect of ethanol. A high level of cytokines triggers neutrophils, lymphocytes, and monocytes at the inflammatory site; these, in turn, start a different oxidative disturbance, toxic metabolites, and lysosomal enzymes liable for local tissue damage in gastric ulcer [34]. Present study observed that RI remarkably suppressed the pro-inflammatory cytokine production in gastric tissue (Figure 4). The inhibition of NF-κB is a primary mechanism for RI suppression of gastric ulcer since the expression of various pro-inflammatory cytokines including *TNF-α* is mainly regulated by the transcription of NF-κB [35].

Inflammation is a physiological and immunological response triggered by both cell and tissue injury that is primarily controlled by a MAPK cascade signaling pathway [36]. MAPKs are kinases that are responsible for most cellular responses to inflammatory cytokines and external stress signals, and these kinases are essential for the regulation of the production of various inflammation mediators [37,38]. MAPK cascade pathway is activated by inflammatory stimuli such as LPS. MAPK comprising ERK, JNK, and p38, is controlled in response to the triggering of extracellular signal cytoskeletal proteins, nuclear transcription factors and the stabilization of cytokines gene. Experimental results revealed that LPS gradually upregulated the phosphorylation of MAPKs, and these phosphorylated MAPKs was notably inhibited by pretreatment with RI, resulting in the elevation of antioxidant response element of RI regulated phase II enzymes, which are involved in cellular protection mechanism (Figure 6, upper panel). Similarly, COX-2 is an important factor which plays key roles in the pathogenesis of inflammation. The protein expression of COX-2 significantly increased after LPS and HCl/EtOH treatment. However, the expression of COX-2 was decreased markedly by RI treatment (Figure 6, middle and lower panels). The immunohistochemical data showed that RI pretreatment markedly blocked the COX-2 localization in the gastric mucosal epithelium cells of the inflammatory area indicating that COX-2 was involved in the inhibition of inflammatory cells activation and mitigates the oxidative stress, and improve the healing process in the gastric mucosa (Figure 5).

It is known that pro-inflammatory cytokines are regulated by NF-κB signaling pathway [39]. The activation of NF-κB is induced by ROS and NO produced from macrophages following exposure of LPS and it depends on the phosphorylation and degradation of the corresponding upstream factor IκBα. Similarly, gastric damages lead to the production of free radicals that prompt the migration and accumulation of macrophages and leukocytes in the damaged sites and the release of pro-inflammatory mediators [40]. The NF-κB dimers are normally sequestered in the cytosol by binding to the IκB inhibitory protein. The previous study reported that ethanol stimulation activates NF-κB, which leads to phosphorylation of IκBα and p50/p65 heterodimer [27]. These p50/p65 dimers enhanced by phosphorylated IκBα promote gene expression of pro-inflammatory cytokines, which translocate to the nucleus [41]. In the present study, the results showed that RI significantly inhibited phosphorylation of IκBα and NF-κB p65 in RAW 264.7 cells and gastric tissue after LPS and HCl/EtOH stimulation, respectively (Figure 6, middle and lower panels). The study indicates that RI may inhibit early steps of inflammation and modulate upregulation of pro-inflammatory cytokines through suppression of NF-κB translocation.

#### **4. Materials and Methods**

#### *4.1. Chemicals and Antibodies*

LPS, MTT, penicillin/streptomycin, trypsin-EDTA, hematoxylin, eosin, and protease inhibitor were purchased from Sigma-Aldrich (St. Louis, MO, USA). DMEM, FBS, and other cell culture reagents were supplied by Gibco (Carlsbad, CA, USA). DMSO was obtained from Bioshop (Burlington, ON, Canada). RNA extraction kit (RiboEx and Hybrid-R) was bought from Gene All (Seoul, Korea). Griess reagent, cDNA synthesis kit (ReverTra Ace qPCR RT Kit), T-PER, and BCA protein assay kit were purchased from Thermo Scientific (Waltham, MA, USA). SYBR Green qPCR Kit obtained from TOYOBO (Tokyo, Japan). Primary antibodies ERK1/2, JNK, p38, COX-2, IκBα, NF-κB, and β-actin were supplied by Cell Signaling (Danvers, MA, USA). Secondary antibody (goat anti-rabbit immunoglobulin g horseradish peroxidase) was provided by Santa Cruz (Dallas, TX, USA). WESTSAVE gold ECL detection kit was obtained from Abfrontire (Seoul, Korea). TBARS assay kit by Cayman (Ann Arbor, MI, USA). ROS-Glo H2O2 assay kit was supplied by Promega (Madison, WI, USA). Zoletil 50 was bought from Virbac (Carros, France).

#### *4.2. Collection and Preparation of Rabdosia Inflexa Extract*

The aerial part of RI was collected from the Jiri mountain area in the southern part of Korea and was authenticated from its microscopic and macroscopic features by the Korean Institute of Oriental Medicine (KIOM). We prepared RI extract according to the previously described method [42]. Briefly, the aerial parts were chopped and dried completely. The extract was prepared by maceration of the sample with 70% ethanol (twice for 2 h reflux), and then the filtered extract was concentrated under vacuum centrifuge and dehydrated with a lyophilizer. The powder extract was liquefied in dimethyl sulfoxide (DMSO) and was sterilized using a 0.22 μm syringe filter. RI was dissolved in 0.04% DMSO in media for cell culture experiments and 0.1% DMSO in saline for oral gavage. The dried extract was kept at −20 ◦C. The study was conducted using a single batch of extract to avoid batch-to-batch variation and maximize the product constancy.

#### *4.3. Phytochemical Analysis*

Total phenolic and flavonoid content of RI extract was measured using Folin–Ciocalteu (FC) method according to the previously described method [43].

#### *4.4. RAW 264.7 Cells Culture*

Mouse macrophage RAW 264.7 cells were cultured in Dulbecco modified Eagle medium (DMEM) enriched with 10% fetal bovine serum (FBS) and 1% penicillin and streptomycin in a 5% CO2 humidified incubator at 37 ◦C. Cells were maintained as a monolayer and subcultured once cells reached about 90% confluency in the culture flask.

### *4.5. Cell Viability and Morphological Study*

Cell viability was detected using a 3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, following a prescribed method [34]. Briefly, RAW 264.7 cells (1 × 106 cells/mL) were cultured overnight. To determine the cytotoxicity, cells were treated with RI (50, 100, 200, 400, and 800 μg/mL) for 24 h. In contrast, measuring the cell viability, cells were pretreated with RI (100, 200, and 400 μg/mL) for 1 h and co-treated with LPS (0.5 μg/mL) and RI for another 24 h. Moreover, for morphological evaluation of RAW 264.7 cells, the image of the cells was acquired by an inverted microscope (CKX41, Olympus, Tokyo, Japan).
