*2.4. RI Improved the Gross and Histopathology of Gastric Tissue*

The recent study investigated the gastroprotective effect of RI in HCl/EtOH-induced gastric ulcer in mice. HCl/EtOH-induced severe gastric damage, which was notably attenuated by RI pretreatment (Figure 3a, upper panel). In addition, the histological study confirmed that the stomach had the normal structure of mucosa in control group. Besides, in the HCl/EtOH-treated mice, epithelial destruction and inflammatory cells infiltration were found in the mucosa and submucosal area. However, RI and ranitidine-treated groups markedly improved the histopathological changes as compared to HCl/EtOH-treated group (Figure 3a, lower panel). These data are well correlated with the protective abilities of RI against gastric ulcer. Likewise, the gross and histological lesions index of gastric tissue was significantly reduced (*p* < 0.05) by pretreatment with RI and ranitidine treated groups than in HCl/EtOH-induced gastric ulcer mice (Figure 3b,c).

#### *2.5. RI Regulated the NO and MDA Production in Gastric Tissue*

To evaluate the oxidative stress level, the NO and MDA production was measured in gastric tissue. After inducing gastric injury, HCl/EtOH significantly (*p* < 0.05) decreased NO and increased MDA production. Overall, pretreatment with the RI effectively (*p* < 0.05) increased and decreased the NO and MDA production as related to standard drug ranitidine, respectively (Figure 2c,d). Therefore, results evidently reveal that RI reduced the oxidative stress in HCl/EtOH-stimulated gastric ulcer for its strong anti-oxidant and anti-inflammatory capacity.

**Figure 3.** Protective role of RI on HCl/EtOH-induced gastric damage in mice: (**a**) gross lesion (upper panel) and histological lesion (lower panel) (scale bar. 200 μm); (**b**) gross lesion index; and (**c**) histological index. # *p* < 0.05 when compared with the control and \* *p* < 0.05 when compared with HCl/EtOH. Data are expressed as mean ± SEM.

#### *2.6. RI Suppressed the Activation Pro-Inflammatory Cytokines in Gastric Tissue*

Pro-inflammatory cytokines play a fundamental role in various types of inflammation. To elucidate the protective role of RI, the gene expression of pro-inflammatory cytokines was examined in the glandular stomach samples by qPCR analysis. The gene expression level of *TNF-α*, *IL-1β*, *IL-6*, *iNOS*, and *COX-2* were gradually upregulated (*p* < 0.05) in the HCl/EtOH-treated group as compared to the control, whereas pretreatment with RI and ranitidine groups significantly (*p* < 0.05) downregulated the cytokines expression level than in the HCl/EtOH-treated group (Figure 4a–e). Thus, data suggest that RI inhibited the gene expression of pro-inflammatory cytokines in gastric tissue and thereby mitigated the gastric inflammation.

### *2.7. RI Inhibited the COX-2 Expression in Gastric Tissue*

It is recognized that elevated expression of COX-2 plays a vital role in the inflammatory process and previous study has revealed that HCl/EtOH strongly activates COX-2 expression in gastric tissue [17]. COX-2 expression in the gastric mucosal epithelial cells was revealed by immunohistochemical staining analysis. As observed, COX-2 was slightly expressed in the normal control gastric mucosal epithelial cells; in contrast, HCl/EtOH increased the COX-2 expression of gastric mucosal epithelial cells, which was mostly observed in the gastric mucosal inflammatory area (Figure 5a). The expression of COX-2 was markedly (*p* < 0.05) blocked by the pretreatment of RI as related to the standard drug ranitidine (Figure 5b). Thus, RI significantly blocked the activation of COX-2 expression in the gastric mucosal inflammatory area and reduced the inflammatory activity.

**Figure 4.** Protective role of RI on gene expression of pro-inflammatory cytokines in gastric tissue. In HCl/EtOH-treated mice, gene expression level of: (**a**) *TNF-α*; (**b**) *IL-1β*; (**c**) *IL-6*; (**d**) *iNOS*; and (**e**) *COX-2* were significantly upregulated, whereas pretreatment with the RI markedly downregulated the gene expression level as related to ranitidine. # *p* < 0.05 when compared with control and \* *p* < 0.05 when compared with HCl/EtOH. Data are expressed as mean ± SEM.

**Figure 5.** Protective role of RI on COX-2 immunoreactivity in the gastric tissue: (**a**) COX-2 expression in gastric mucosal epithelial cells; and (**b**) COX-2 positive immune-stained cells. Scale bar, 200 μm. # *p* < 0.05 when compared with the control and \* *p* < 0.05 when compared with HCl/EtOH. Data are expressed as mean ± SEM.

#### *2.8. RI Blocked the MAPK Cascade, COX-2, and NF-κB Activation*

To find the possible molecular mechanisms of the anti-inflammatory and gastroprotective role of RI, the protein expression related to anti-inflammation signaling pathways was evaluated. The present data showed that LPS treatment remarkably elevated the phosphorylation of MAPK family protein (ERK1/2, JNK, and p38) in RAW 264.7 cells, whereas RI pretreatment notably (*p* < 0.05) attenuated the phosphorylation of MAPK proteins (Figure 6, upper panel). Meanwhile, LPS and HCl/EtOH treatment increased COX-2 expression in RAW 264.7 cells and gastric tissues were markedly (*p* < 0.05) blocked by RI pretreatment (Figure 6, middle panel). After LPS and HCl/EtOH stimulation, IκBα and NF-κB phosphorylation were noticeably (*p* < 0.05) increased, indicating the activation of NF-κB. However, IκBα phosphorylation and the nuclear translocation of NF-κB (p65) were gradually reduced (*p* < 0.05) by RI pretreatment (Figure 6, middle and lower panels). Moreover, RI alone does not seem to

involve in the signal pathways in vitro study. Together, these results demonstrate that RI significantly inhibited the phosphorylation of MAPK cascade in RAW 264.7 cells as well as activation of COX-2, IκBα, and NF-κB in RAW 264.7 cells and gastric tissues, simultaneously.

**Figure 6.** Protective role of RI on the MAPK cascades, COX-2 expression, and activation of IκBα, NF-κB in RAW 264.7 cells and gastric tissue. Here, upper and middle panels represent the MAPKs (pERK1/2, pJNK, and pp38), COX-2, IκBα and NF-κB expression in RAW 264.7 cells and the lower panel represents the COX-2, IκBα and NF-κB expression in the gastric tissue. The relative band intensity of target protein was measured as compared with total protein and β-actin. LPS-induced the phosphorylation of MAPK cascade, whereas pretreatment with the RI reduced the phosphorylation of MAPK cascade. LPS and HCl/EtOH increased the COX-2 expression, kinetic phosphorylation, and degradation of IκBα and phosphorylation of NF-κB. However, pretreatment with the RI notably decreased the COX-2 expression, IκBα phosphorylation, and degradation, NF-κB translocation as related to standard drug ranitidine. # *p* < 0.05 when compared with the control and \* *p* < 0.05 when compared with LPS and HCl/EtOH. Data are expressed as mean ± SEM.
