الفهرس 
Gastric UlcerationOnly 14 pages are availabe for public view
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Abstract
Peptic ulcer is considered one of the most critical GIT disorders which affect nearly 9-12 % of the world population annually. These kinds of ulcers differentiate mainly into two types; duodenal ulcer (DU) which found in duodenum and gastric ulcer (GU) which found in stomach. It mostly occurs due to internal imbalance between the epithelial defensive factors such as (mucus- prostaglandin-bicarbonate) and the offensive factors such as (acid, pepsin, alcohol, infection and NSAIDs). Among all, NSAIDs use remains one of the predominant causes for developing peptic ulcer disease in addition to Helicobacter pylori (HP) infection. Indomethacin is considered one of the most commonly used NSAIDs to treat pain and inflammation among elder patients. Ironically, it has been associated with a greater risk of developing gastrointestinal complication, ulceration and, in some cases, perforation.
Pinocembrin, a natural flavonoid isolated from honey, propolis and other medicinal plants, has been reported to possess multi-therapeutic effects due to its anti-oxidant, anti-inflammatory, and anti-apoptotic properties. Recent researches indicated its potential biological activities against several disorders such as stroke, cardiac arrhythmia, atherosclerosis, endotoxic shock and cancer. Additionally, pinocembrin has proven to be a possible neuroprotective agent against global cerebral ischemia reperfusion due to its antioxidant, anti-excitotoxic and anti-inflammatory effects which provides cerebral protection. Although extensive research has been carried out on pinocembrin, no single study exists which investigate its effect in treatment of peptic ulcer disease.
Therefore, the potential therapeutic effect of pinocembrin against indomethacin-induced gastric ulcer model in rats was investigated. Moreover, the possible underlying mechanisms were evaluated; the effects on oxidative stress, inflammatory and apoptotic markers. The results were compared to omeprazole, as a reference drug for gastric ulcer treatment worldwide.
Study was divided into two parts;
1) Gastro-protective Study:
Rats were randomly assigned into 4 groups: group 1 (control) received PINO vehicle (2 % DMSO + 98 % PEG-600) for 3 days, followed by a single oral dose of INDO vehicle (2 % gum acacia in distilled water) one hour after the last PINO vehicle administration. group 2 received PINO vehicle (2 % DMSO + 98 % PEG-600) for 3 days, followed by a single oral dose of INDO 48 mg/kg, suspended in 2 % gum acacia in distilled water one hour after the last PINO vehicle administration. group 3 and 4 were pretreated with PINO at doses of 25 and 50 mg/kg, respectively, dissolved in (2 % DMSO + 98 % PEG-600) for 3 days, followed by single oral dose of INDO 48 mg/kg suspended in 2 % gum acacia in distilled water one hour after the last PINO dose.
Animals were sacrificed 6 hrs after treatment. Stomachs were dissected, opened along the greater curvature either for immediate determination of ulcer index or morphological examination. In addition, tissue samples from each group were collected for further histo-pathological examination and mucin content determination to detect the optimum gastro-protective dose of pinocembrin to be used for further mechanistic studies.
Both doses of pinocembrin showed comparable gastro-protective activity against indomethacin-induced gastric ulcer. Therefore, pinocembrin 25 mg/kg for 3 days was chosen for the subsequent investigation of the possible underlying mechanisms of pinocembrin gastro-protective effect. The following parameters were assessed:
Total protein content
Oxidative stress markers:
a) Reduced glutathione (GSH)
b) Lipid peroxidation (MDA)
c) Catalase enzyme (CAT)
Inflammatory markers:
a) Tumor nicrosis factor-α (TNF-α)
b) Interlukin 1β (IL-1β)
c) Nuclear factor kappa-B (NF-B)
Assessment of Apoptotic Enzyme Caspase-3.
Assessment of P38-MAPK.
2) Gastro-therapeutic Study:
Rats were randomly assigned into 7 groups as follow: group 1 (control) received a single oral dose of INDO vehicle (2 % gum acacia in distilled water) then after 1 hr received PINO vehicle (2 % DMSO + 98 % PEG-600) for 3 days. group 2 received single oral dose of INDO 48 mg/kg, suspended in (2 % gum acacia in distilled water) then after 1 hr received PINO vehicle (2 % DMSO + 98 % PEG-600) for 3 days. group 3 and 4 received single oral dose of INDO 48 mg/kg suspended in 2 % gum acacia in distilled water, then after 1 hr treated with PINO at doses of 25 and 50 mg/kg, respectively, dissolved in (2 % DMSO + 98 % PEG-600) for 3 days. group 5 and 6 received single oral dose of INDO 48 mg/kg suspended in 2 % gum acacia in distilled water, then after 1 hr treated with PINO at doses of 25 and 50 mg/kg, respectively, dissolved in (2 % DMSO + 98 % PEG-600) for 5 days. group 7 received a single oral dose of INDO 48 mg/kg suspended in 2% gum acacia in distilled water, then after 1 hr, treated with omeprazole 30 mg/kg, dissolved in (2 % DMSO + 98 % PEG-600) for 3 days.
Animals were sacrificed 6 hrs after treatment. Stomachs were dissected, opened along the greater curvature either for immediate determination of ulcer index or morphological examination. In addition, tissue samples from each group were collected for further histo-pathological examination and mucin content determination to detect the optimum therapeutic dose and duration of pinocembrin treatment to be used for further mechanistic studies.
Treatment with pinocembrin for 3 and 5 days showed ulcer healing activity, which was confirmed with histopathological examination, ulcer index and mucin content detection. There was no difference between treatment with pinocembrin 50 mg/kg for 5 days and pinocembrin 50 mg/kg for 3 days. Moreover, pinocembrin 50 mg/kg/ 3 days treated group showed normal histological structure of gastric tissue, which was confirmed by severe alcian blue reaction as compared to mild alcian blue reaction in case of pinocembrin 25 mg/kg/ 3 days. Therefore, pinocembrin 50 mg/kg for 3 days was chosen to be the most effective therapeutic dose regimen against indomethacin-induced gastric ulcer. Subsequently, an investigation of the possible underlying mechanisms of pinocembrin gastro-therapeutic effect was conducted and the following parameters were assessed:
Total protein content
Oxidative stress markers:
d) Reduced glutathione (GSH)
e) Lipid peroxidation (MDA)
f) Catalase enzyme (CAT)
Inflammatory markers:
d) Tumor nicrosis factor-α (TNF-α)
e) Interlukin 1β (IL-1β)
f) Nuclear factor kappa-B (NF-B)
Assessment of Apoptotic Enzyme Caspase-3.
Assessment of P38-MAPK
Studies have shown the following results:
1) Pretreatment with pinocembrin doses (25 mg/kg and 50 mg/kg) for 3 consecutive days before ulcer induction have shown normal stomach morphology which was confirmed by ulcer index results and mucin content determination.
2) Post-treatment with pinocembrin 25 mg/kg for 3 consecutive days after ulcer induction showed diffuse inflammatory cells infiltration with oedema in submucosal layer while post-treatment with pinocembrin 50 mg/kg showed no histopathological alteration in stomach morphology which was further confirmed by increased mucin content secretion in the mucosal layer proven by the severe reaction of alcian blue stain in pinocembrin treated rats.
3) Post-treatment with both doses of pinocembrin for 5 days after ulcer induction showed normal stomach morphology with was confirmed by mucin content determination.
4) Pre-treatment with pinocembrin 25 mg/kg and post-treatment with pinocembrin 50 mg/kg showed positive anti-oxidant activity which was confirmed by increased levels of GSH, CAT activity and decreased levels of MDA in gastric tissue samples.
5) Treatment with omeprazole 30 mg/kg for 3 days showed anti-oxidant activity which was confirmed by increased GSH level, CAT activity and decreased levels of MDA.
6) Levels of pro-inflammatory cytokines such as TNF-α and IL-1β showed significant elevation in INDO-treated group compared with control group in both studies. Pre-treatment with pinocembrin 25 mg/kg for 3 days showed significant reduction in gastric levels of TNF-α and IL-1β as compared to INDO-treated group.
7) Post-treatment with pinocembrin 50 mg/kg for 3 days significantly decreased levels of TNF-α and IL-1β almost to the control levels as compared to the INDO-treated group.
8) Administration of indomethacin showed significant increase in gastric tissue expression of NF-B as compared to control group in both studies. Pre and post-treatment with pinocembrin 25 and 50 mg/kg respectively showed significant decrease in NF-B expression to a level similar to the corresponding control group as compared to the corresponding INDO-treated group.
9) Treatment with omeprazole showed possible anti-inflammatory effects which were confirmed by significant decrease in gastric tissue levels of TNF-α, IL-1β and NF-B expression level as compared to INDO-treated group.
10) Gastric tissue of INDO-treated group showed elevated mRNA expression level of p38-MAPK as compared to control group in both studies. Interestingly, pre-treatment with pinocembrin 25 mg/kg and post-treatment with pinocembrin 50 mg/kg showed significant reduction in p38-MAPK expression level.
11) Omeprazole-treated group showed significant reduction in p38-MAPK expression level almost to control value as compared to INDO-treated group.
12) Pinocermbrin was effective in opposing indomethacin-induced apoptotic activity via inhibition of caspase-related apoptosis. Pre-treatment with pinocembrin 25 mg/kg and post-treatment with pinocembrin 50 mg/kg showed significant decrease in caspase-3 expression level as compared to the corresponding indomethacin-treated group. Additionally, treatment with omeprazole for 3 days showed anti-apoptotic activity which was confirmed by significant reduction in caspase-3 expression in gastric tissue.