3.3. Histopathologic Findings of Gastric Tissues
Intact group: Histopathologic examination of gastric tissues revealed normal histologic structure (Fig. 1B. a)
Indomethacin group: Histopathologic examination of gastric tissues revealed severe erosion and ulceration of the lamina epithelialis in the tunica mucosa, which progressed to the lamina propria. Severe degeneration and necrosis of the epithelium, cell infiltration and hemorrhage foci were observed in the interstitial spaces. Severe edema in the submucosa and severe hyperemia in the vessels were observed (Fig. 1B. b).
Reference group: Histopathologic examination of gastric tissues revealed very mild erosion of the lamina epithelialis, degeneration and necrosis of the epithelium, and moderate hyperemia of the vessels in the lamina propria (Fig. 1B. c).
IND + 100MN group: In histopathologic examination of gastric tissues, moderate erosions in the lamina epithelialis up to the lamina propria, moderate degeneration and necrosis in the epithelium were determined. Severe edema was observed in the submucosa (Fig. 1B. d).
IND + 200MN group: Histopathologic examination of gastric tissues revealed mild erosion of the lamina epithelialis, degeneration and necrosis of the epithelium and moderate hyperemia in the vessels. Mild edema was observed in the submucosa (Fig. 1B. e). A statistically significant difference (p˂0.05) was detected when compared with the indomethacin group.
IND + 400MN group: Histopathologic examination of gastric tissues revealed very mild erosion of the lamina epithelialis, degeneration and necrosis of the epithelium, and mild hyperemia of the vessels (Fig. 1B. f). A statistically significant difference (p˂0.05) was found when compared with the indomethacin group. Histopathologic findings were summarized in Table 2.
Table 2
The scoring of histopathologic findings in gastric tissues
| Erosion ulceration of the lamina epithelium | Epithelium degeneration and necrosis | Hyperemia of the veins | Edema in submucosal |
INTACT | - | - | - | - |
IND | ++++ | ++++ | ++++ | ++++ |
IND + FAM | + | ++ | ++ | - |
IND + 100MN | +++ | +++ | ++++ | ++++ |
IND + 200MN | ++ | ++ | +++ | + |
IND + 400MN | + | + | ++ | - |
IND: Indomethacine; IND + FAM: Indomethacine + Famotidine, IND + 100MN: Indomethacine + 100mg/kg Muscari neglectum; IND + 200MN: Indomethacine + 200mg/kg Muscari neglectum; IND + 400MN: Indomethacine + 400mg/kg Muscari neglectum |
3.4. Antioxidant enzymes and oxidative stress markers
Free oxygen radicals have an important effect on the formation of indomethacin-induced ulcers. For this reason, in the present study, the effect of indomethacin on the antioxidant defense mechanism of MN was determined by GSH, MDA analysis and SOD and CAT enzyme activities (Table 3).
Table 3
SOD, CAT enzyme activities and GSH and MDA levels analyzed in rats gastric tissues.
Groups | Dose (mg/kg) | n | SOD (ng/mg wet tissue) | CAT (ng/ mg wet tissue) | GSH (mg/ g wet tissue) | MDA (nmol/mg wet tissue) |
INTACT | | 6 | 1,26 ± 0,09 b | 16,83 ± 0,14c | 113,16 ± 0,01 b | 0,59 ± 0,11b |
IND | 25 | 6 | 0,58 ± 0,2a | 6,61 ± 0,01 a | 49,81 ± 0,01 a | 1,13 ± 0,11a |
IND + FAM | 40 | 6 | 2,15 ± 0,1 c | 33,99 ± 0,1 e | 137,5 ± 0,1b | 0,55 ± 0,08 b,c |
IND + 100MN IND + 200MN IND + 400MN | 100 | 6 | 1,05 ± 0,15 a,b | 14,71 ± 0,4 b,c | 71,46 ± 0,14a | 1,05 ± 0,17 a |
200 | 6 | 1,13 ± 0,05 a,b | 18,21 ± 0,14c | 124,52 ± 0,11 b | 0,56 ± 0,07b,c |
400 | 6 | 3,22 ± 0,20 d | 26,49 ± 0,08 d | 134,51 ± 0,13 b | 0,31 ± 0,09 c |
Different letters a, b, c, d indicate statistically significant differences (p < 0.001) in the same column. IND: İndometazin, FAM: Famotidin, MN: Muscari neglectum, SOD: Süperoksit dismutase, CAT: Catalase, GSH: Glutathione, MDA: Malonaldehyde |
SOD, CAT enzyme activities, GSH and MDA levels analyzed in rat gastric tissues are given in Table 3 and Fig. 3.
While SOD and CAT activities of the intact group did not show a significant difference when compared with IND + 100MN and IND + 200MN groups (p > 0.05), a significant difference was observed when compared with IND, IND + FAM, IND + 400MN groups (p < 0.0001).
There was no significant difference in the SOD activities of the IND group when compared with IND + 100MN and IND + 200MN groups (p > 0.05), while a significant difference was observed when compared with IND + FAM, IND + 400MN groups (p < 0.0001). When the CAT activities of the IND group were compared with the other groups, a significant difference was observed (p < 0.0001). This showed that the most effective MN dose was 400 mg/kg (p < 0.0001).
No significant difference was observed in the GSH level of the intact group when compared with IND + FAM, IND + 200MN, IND + 400MN (p > 0.05), whereas this difference was significant in IND, IND + 100MN (p < 0.0001). Compared to IND group, IND + FAM IND + 200MN, IND + 400MN increased GSH levels. While 200 and 400 mg/kg doses of MN, whose effect we tested, increased GSH levels (p < 0.0001), it gave similar results with the reference group (p > 0.05).
While there was no significant difference between the MDA levels of the intact group and IND + FAM and IND + 200MN groups (p > 0.05), this difference increased MDA levels in IND, IND + 100MN groups and decreased in IND + 400MN group (p < 0.0001). The 100 mg/kg dose of MN, which we tested the effect of, had no effect on MDA levels, while the 200 and 400 mg/kg doses showed a similar effect with the reference group and decreased MDA levels (p < 0.0001).
SOD and CAT activities showed a significant difference when IND group, IND + FAM, IND + 400MN groups were compared (p < 0.0001).
LC- MS/MS analysis of phenolic compounds of water extracts of above-ground parts of Muscari neglectum used in the experiment is shown in Table 4 and Fig. 4. In the analysis, Fumaric Acid, Protocatechuic Acid, Phloridzindyhrate and Myricetin compounds were found intensively, while Acetohydroxamic Acid, Syringic Acid, Resveratrol, Kaempferol, Gallic Acid, 4-Hydroxybenzoic Acid, Caffeic Acid, Salicylic Acid, Quercetin and Luteolin compounds were also found in certain amounts. It was observed that some of the components found in the studies of MN using different solvents were the same as in our study and some were different. [11, 12].