Balthazar - Rats (2012) 21
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Balthazar - Rats (2012) 21
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Cibotium barometz is a medical herb used traditionally in the Malaysian peninsula for several ailments, including gastric ulcer. The aim of this study was assessment the anti-ulcer effects of C. barometz hair on ethanol-induced stomach hemorrhagic abrasions in animals. Seven groups of Sprague Dawley (SD) rats were administered 10% Tween 20 in the normal control and ulcer control groups, and omeprazole 20 mg/kg and 62.5, 125, 250, and 500 mg/kg of C. barometz hair extract in the experimental groups. After 60 min, the normal control group of rats was orally administered 10% Tween 20, while absolute ethanol was orally administered to the groups of ulcer control, omeprazole and experimental groups. Stomachs of the rats were examined macroscopically and histologically. Homogenates of stomachs were used to evaluate endogenous antioxidant enzyme activities.
Rats pre-fed with plant extract presented a significant decrease in the sore area, increased pH of gastric contents and preserved stomach wall mucus compared to the ulcer group. Histologically, rats pre-fed with C. barometz hair extract showed mild to moderate disruptions of the surface epithelium while animals pre-fed with absolute ethanol showed severe disruptions of the stomach epithelium with edema and leucocyte penetration of the submucosal layer. A Periodic acid Schiff (PAS) staining revealed that each rat pre-treated with the plant extract displayed an intense uptake of stomach epithelial glycoprotein magenta color compared to the ulcer control group. Immunohistochemical analysis revealed that rats pre-fed with the plant extract showed an up-regulation of the heat shock protein 70 (HSP70) and down-regulation of Bax proteins compared to ulcer control rats. Homogenates of the stomach tissue demonstrated significant increases in the endogenous antioxidant enzymatic activity and decreased lipid peroxidation (MDA) in rats pre-treated with C. barometz hair extract compared with the ulcer control rats. In acute toxicity, the liver and kidney revealed no hepatotoxic or nephrotoxic effects histologically.
The results that pre-treated with C. barometz hair ethanol extract of male SD rats shown significant reduced of the ulcer area with compared to the ulcer groups (Table 1, Fig. 1) at four different doses in the pre-treated animals groups (62.5 mg\kg, 125 mg\kg, 250 mg\kg, 500 mg\kg) that were induced by ethanol 95%. However, the significant inhibition percentage of the ulcer area increased in rats pre-treated with C. barometz hair at doses of 62.5 mg\kg, 250 mg\kg, 125 mg\kg, and 500 mg\kg by 68.5%, 74.7%, 75.6%, and 78.5% respectively.
The effect of C. barometz hair on the macroscopic appearance of the stomach mucosa in alcohol-induced stomach mucosal injuries in male SD rats. G1 (Normal control group) exhibited no injuries to the gastric mucosa, G2 (Ulcer control group) had severe injuries to the stomach mucosa, G3 (Omeprazole) showed mild disruptions of the surface epithelium in the gastric mucosa. G4 (62.5 mg/kg), G5 (125 mg/kg), G6 (250 mg/kg), G7 (500 mg/kg) doses of C. barometz hair extract had moderate to mild disruptions of the surface epithelium in the gastric mucosa in a dose-dependent manner. Black arrow points to the hemorrhagic bands
The outcomes that shown in Table 1, the ulcerated SD rat group produced the lowest gastric mucosa mucus content, while animal groups pre-treated with G7 (500 mg/kg) and G6 (250 mg/kg) of C. barometz hair exhibited significant increasing in the mucus weight (g) with respect to G2 (ulcer control rats). However, pre-treatment with C. barometz hair (G4 to G7) produced a significant increase in the pH of the stomach contents compared to the ulcer control rats (G2).
Histology revealed comprehensive damage to the stomach mucosa in the ulcer control rats. Moreover, the ulcerated rat control group had necrotic lesions in the deep gastric mucosa that demonstrated extensive leucocyte infiltration and edema of the submucosal layer, as shown in Fig. 2. Conversely, the animals pre-fed with C. barometz hair extract in the G4 - G7 groups presented relatively enhanced protection of the stomach mucosa with a depression in or lack of infiltration of leucocytes and edema (Fig. 2). C. barometz hair extracts revealed defending effects in a dose-dependent manner and showed remarkably improved protection of the stomach epithelium. The gastric mucosa in the pre-treated experimental groups, depending on the dose, exhibited a gradual increase in PAS staining intensity, indicated by the accumulation of the magenta color in the mucosal cell layer compared to the ulcer control group (Fig. 3). Additionally, this magenta staining was reduced and was not plentiful in the gastric mucosa of the ulcer group where the ulcer was induced with ethanol.
In the gastric mucosa, the expression of the HSP70 protein was down-regulated in the ulcerated group (G2), but significantly up-regulated in the animals pre-treated with omeprazole (G3) and with C. barometz hair extract (G4 to G7), as displayed in Fig. 4. Additionally, the immunohistochemical staining of the Bax proteins (Fig. 5) in the gastric mucosa revealed up-regulation in the ulcerated group while a significant down-regulation was demonstrated in rats pre-treated with C. barometz extract.
A significant reduction was observed in endogenous antioxidants enzymes (SOD, CAT and GPx) activities for the ulcer group of male SD rats. However, the rats pre-treated with C. barometz hair displayed an elevation of all antioxidant activities with respect to the (G2) ulcer group as shown in Figs. 6a, b, and c. The SOD enzyme activities were significantly higher at doses for G5 of C. barometz than G2 (Fig. 6a). The CAT enzyme activities illustrate the significant increases in G4, G5, G6 groups compared to G2 as shown in Fig. 6b. The GPx enzyme activities for gastric mucosal homogenates revealed significant increasing in the rats pre-fed with four doses of C. barometz hair ethanol extract with respect to G2 as shown in Fig. 6c. Additionally, the MDA level of C. barometz hair extract in G4, G5, G6 and G7 were significant lower than in the G2 ulcerated control group as seen in Fig. 6d.
All SD rats that were treated with C. barometz hair ethanol extract demonstrated no mortality and toxic signs in the experiment. There were no signs of hepatotoxic or nephrotoxic effects, which were evaluated histologically and biochemically (Fig. 7). Additionally, there were no body weight variations or abnormal physiological or behavioral changes at 2 g/kg and 5 g/kg doses during the 14 days, compared to the control group that was given 10% Tween 20.
The consequences of the present immunohistochemistry experiments indicate that rats pre-fed with C. barometz hair displayed an up-regulation of HSP70 proteins that contributed to the protection of stomach cells from heat shock, oxidative stress, and down-regulation of the Bax proteins. Earlier studies reported that there was up-regulation of HSP70 protein in animals fed with plant extracts [28, 34]. However, the down-regulation of Heat shock protein expression appeared in the ulcer control animals as a gastric injury marker. This is consistent with the results from other studies [20, 46]. Up-regulation of HSP70 protein in the stomach wall mucosa in animals pre-fed with C. barometz hair extract may stop the initiation of damage to the gastric epithelium upon exposure to absolute alcohol [23, 32]. The HSP70 protein protected mitochondria and delayed a stress-induced apoptotic programmer [47]. Our data suggest that Bax immune-staining confirmed that rats fed with C. barometz hair extract displayed increased an expression of the Bax protein. Thus, these results demonstrate that C. barometz hair extract is a protective agent and prevents alcohol-induced damage to the rat stomach, which is associated with decreased expression of the Bax protein. Similarly, many amendments have been described by many investigators [18, 48]. Animals pre-fed with C. barometz hair extract displayed decreased expression of the Bax protein. On the other hand, up-regulation of Bax was observed in the ulcer control group. Bax is a key protein that is linked to apoptosis during mitochondrial damage and plays a significant role in the disruption of stomach mucosal integrity that is observed after ethanol administration [11, 12].
The results from the present work demonstrated that rats pre-fed with C. barometz displayed an increase in the activity of endogenous antioxidant enzymes (SOD, GPx, CAT) and a decrease in MDA levels in the stomach compared to the ulcer control rats. These findings are consistent with observations from other studies [7, 32]. Therefore, the elevation of SOD and GPx enzymatic activities leads to increased scavenging of superoxides, hydrogen peroxide, hydroxyl and lipid peroxyl radicals, resulting in a reduction in tissue damage [9, 49]. Additionally, increases in CAT enzymatic antioxidant activity rapidly converted the peroxyl radicals into biologically safe substance, such as water [1]. In contrast, the low levels of MDA reduced lipid peroxidation and ROS (reactive oxygen species) which are products of oxidative gastric damage [8]. Taken together, our observations suggest that C. barometz hair facilitates gastric mucosa protection due to its scavenging of free radicals and anti-inflammatory effects [5, 6]. 350c69d7ab
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