We have confirmed in our laboratory the antiulcer activity of curcumin during the acute chronic phase of gastric ulcer disease at doses of 20, 40 and 80 mg/kg (dissolved in saline solution). In the previous study, the potent effective dose of curcumin was 80 mg/kg that appears a propitious protective effect against gastric ulcer development. Therefore, the comparison between such recommended dose of curcumin and one of the proton pump inhibitors (PPIs) staff is worth-while. Since, the pharmacological control of gastric acid secretion is the main desired goal for gastro-cytoprotection, particularly, the H<sup>+</sup>/K<sup>+</sup>-ATPase (acid proton pump) inhibitors. Nevertheless, several studies have indicated that long-term inhibition of gastric acid secretion results in mucosal hyperplasia and carcinoid tumor development, due to increase circulating gastrin levels. Ulcer and the preventive indexes were scored, mucin, juice volume, total acidity, luminal haemoglobin, total antioxidant and total peroxide were evaluated. The pro-inflammatory cytokine IL-6 and the major angiogenic growth factor VEGF levels were measured. Conclusion, curcumin and omeprazole are potentially preventing gastric lesions development in the gastric wall during the acute phase of gastric ulcer diseases, but curcumin was more potent in its effect. Curcumin promotes gastric ulcer prevention/healing by induction of angiogenesis in the granular tissue of ulcers. That may be via upregulation of VEGF expression as reflected from VEGF level in serum and gastric juice, however, omeprazole might be has no role in this story.
The prevention of gastric ulcer pathogenesis or its recurrence is the main desired goal for the clinical and the experimental studies in the present era. Gastric acid hypersecretion is one of the major pathogenic factors for the induction of gastric ulcer disease. Furthermore, luminal acid interferes with the process of restitution, resulting in the conversion of superficial injury to deeper mucosal lesion and inactivates the acid-labile growth factors important for maintenance of mucosal integrity and repair of superficial injury [
Curcuma longa, has been used in traditional remedy for a wide range of ailments, including wound healing, urinary tract infection, and liver ailments [
Selection of the used doses in this study was carried out by a pioneer experiment using Shay rat model to induce the stomach ulcer in 30 rats. Afterwards the rats were randomly classified to 6 groups (5 rats for each) each group has received a selected dose of Curcumin (0, 5, 10, 20, 40, 80 and 100 mg/kg). Then the rats were scarified and samples were collected for the pioneer evaluation. The number of the ulcerative lesions in the stomach wall was counted, as well as the acid in the gastric juice was determined (Plate 1A). Among all doses 20, 40 and 80 mg/kg of Curcumin have been selected, since, these doses seems to have anti-ulcer activity. Inclusion of the higher doses of Curcumin is due to at high concentrations, Curcumin induce bile acid secretion, which in turn one of the major ulcerogenic effectors.
Cell viability was measured as total DNA by means of the diphenylamine (DPA) assay adapted to 96-well format for robotics (Natarajan et al., 1994). The dose that produced the 50% cell survival (EC50) was extrapolated. Larger EC50 doses indicate less cytotoxicity. Freshly tissue homogenates 20 µg were incubated with DPA as
say, which briefly prepared by adding 80 µL aliquot of a 1:5 mixture of acetaldehyde (0.16%) and 20 µL perchloric acid to100 µL of diphenylamine reagent (4% DPA in glacial acetic acid). After 24 h of incubation at 37˚C, the density of the developed blue/violet color absorbance were measured at 595 nm minus the absorbance at 620 nm (Plate 1B).
Wistar albino male rats weighing 225 ± 25 g were divided into three groups (n = 10) and fasted for 2 days with free access to water. On the third day, the animals were deprived of water for a further 24 h and then underwent surgical operation after general anesthesia with Ketamine/diazepam “according to the rules of the Animal Care and Use Committee (ACUC) of the Alexandria University”, Animals in group one served as a standard ulcerative control group with their pylorus ligated and had only vehicle. The animals in the second group were given curcumin at a dose of 80 mg/kg (dissolved in 0.9% NaCl) after pyloric ligation. In the ulcerated omeprazole-treated therapeutic control group, omeprazole dissolved in DMSO as a single dose was administrated subcutaneously 1 hr before surgery and after pyloric ligation at 20 mg/kg. The dose of omeprazole and administration was carried out according to Warzecha et al. (2001) and Rodriguez et al. (2002), because of the impracticality of its intragastric installation. Since, PPIs are unstable at a low pH; therefore, the oral dosage forms (“delayed release”) are supplied as enteric-coated granules or tablets that dissolve only at the alkaline proximal small intestinal pH to prevent degradation by acid in the oesophagus and stomach.
Inclusion of a reference un-operated control group of animals and another shame-operated control group of animal was not practical since these animals will not be a real mach without pyloric ligation. The later will prevent collection of gastric juice samples and hence investigation of parameters dependent on it. The rats were killed after 19 h by decapitation and the gastric juice was collected and the excised stomach was then filled with 15 mL of 4% formalin. After 24 h, the fixed stomach was opened along the greater curvature, gently rinsed in saline, and then pinned open to expose the gastric mucosa. The haemorrhagic and ulcerative lesions were counted and measured under a light microscope.
Blood samples were collected on EDTA (5 mg/mL) by bleeding through the jugular vein into pyrogen-free polypropylene tubes, samples were mixed and centrifuged for 10 min at 5000 rpm at 4˚C, and were 250 mLaliquotted into Eppindorff’s tubes and stored at −40˚C till used to avoid repeated freeze-thaw cycles in different assays.
After collection of blood, the animal was decapitated and the abdomen was opened. The stomach was opened along its greater curvature. The gastric content was collected in a graduated centrifuge tube. The total volume was recorded and then the juice thorough mixed and 0.5 mL aliquot was separated for total DNA assay and the rest of the juice was cleared of its coarse content by centrifugation at 5000 rpm for 10 min at 4˚C. A total of 250 mL aliquots of the supernatant clear juice were allocated into Eppindorff’s tubes and frozen at −40˚C till used.
Gastric juice samples were back titrated against 0.01 M NaOH using phenolphthalein indicator (1% in absolute ethanol) to the faint pink colored end point. The total acidity expressed as milliequivalents per liter (mEq/L) was calculated using the following equation.
The mucosal surface was washed with cold saline solution (0.9 gm% NaCl). The mucosal ulcerations restricted to the fundic area were rapidly scored. The number of lesion bands of more than 4 mm length was multiplied by the severity factor 2; while lesions of less than 2 mm length had severity factor 1. The ulcer index was calculated as the total number of lesions multiplied by the corresponding severity factor.
The acids in the reagent hydrolyze the complex glycoprotein of the gastric juice mucin to release N-acetylneuraminic acid (NANA) that then reacts with the resorcinol reagent to develop the color that directly proportionates with its concentration [
Luminal hemoglobin of the gastric juice was measured colorometrically using commercial available kit.
Determination of the total peroxide concentration in the gastric juice and plasma were evaluated according to method described by Harma and Erel [
Total antioxidant capacity was measured based on the ability of the test sample to scavenge ABTS+ radical cation was compared to trolox standard [
Determination of the total and fragmented DNA in the gastric juice of the luminal content was measured colorimetrically according to method described by Kurita-Ochiai et al. [
The IL-6 level in plasma and stomach juice was measured by ELISA according to its manufacturer’s recommendations (Quantikine, cat. # PR6000B, R & D Systems, Inc. Minneapolis, MN, USA).
The level of VEGF in the plasma and in the stomach juice was evaluated by ELISA according to its manufacturer’s recommendations (Quantikine Rat VEGF Immunoassay, Cat. # RRV00, R & D Systems, Inc., 614 McKinley Place NE, Minneapolis, MN 55413, USA).
2.4.10.1. Samples preparation for H & E staining
Immediately after killing the rats under diethyl ether anesthesia, the stomach content was cleared, mucosal surface was gently washed with cold saline solution, the specimens of stomach fundic full wall of each of the four studied group were fixed in 10% formalin, embedded in paraffin wax, and paraffin sections of 5 μm in thickness were prepared and stained with H & E for general histological examinations by Light Microscope.
2.4.10.2. Samples preparation for toluidine blue staining
5 - 8 small pieces of 1 × 1 mm size were taken from the stomach fundic wall of the four studied groups, they were fixed in 5% cold buffer glutaraldehyde for one week. The specimens were then washed in phosphate buffer (pH 7.2)—4 changes of 15 min each with slow shaking, post fixed in 1% osmium tetroxide for 2 hrs and washed again with phosphate buffer then were dehydrated using ascending alcoholic series at 50% for 30 min, at 70% overnight, at 95% for 30 min, and finally at 100%, 3 changes of 30 min each. Samples were imbedded in propylene oxide pure for 30 min to remove remnants of alcohol, after those samples were imbedded in propylene oxide plus Epon 812 (1:1, v/v) for 30 minutes and finally they were imbedded in pure Epon 812 for 4 hrs. Samples were finally embedded into capsules containing the embedding mixture and the tissue blocks were polymerized in an oven for 2 days at 60˚C. Semi-thin sections of 0.5 μm thickness were prepared using L.K.B Ultra Microtome and were stained with toluidine blue for histopathological investigations.
The data were expressed as Mean ± SEM. Results were analyzed statistically using column statistics and one way ANOVA with NewmaneKeuls Multiple Comparison Test as a post test using the computer statistics Prism 3.0 package (GraphPad Software, Inc., San Diego, CA, USA). The minimum level of statistical significance was set at P < 0.05.
As shown in Plate 2 (Panel A), On the other hand, administration of curcumin and omeprazole significantly decreased gastric lesion formation (P < 0.001) in the gastric wall from 33.15 ± 3.899 lesions/animal in control group to 4.000 ± 0.6602 and to 8.215 ± 0.8787 lesions/ animal in treated groups with curcumin and omeprazole, respectively. Therefore, the preventive index was recorded 85.75 ± 2.851 and 71.71 ± 2.697% for curcumin and omeprazole treated group, respectively. On the other hand the total gastric juice volume could be considered as a direct indicator for the secretory/anti-secretory ability of the different treatments in the stomach (plate 2, panel B). The ulcerated untreated control animals showed a total gastric juice volume of 7.280 ± 0.7761 mL/animal. Most significant reduction in gastric juice volume was evident after treatment with curcumin into 3.444 ± 0.5556 mL/animal (P < 0.01). Surprisingly, the Omeprazole caused a less but significant juice anti-secretory ability with juice volume of 4.850 ± 0.6747 mL/animal (P < 0.05).
As shown in Plate 2 (Panel C), the ulcerated untreated control animals showed a total gastric juice acidity of 39.60 ± 3.384 mEq/L. In comparison, gastric juice acidity was significantly reduced after treatment with curcumin (80 mg/kg) into 6.400 ± 0.6000 mEq/L (P < 0.001). Omeprazole treatment also caused a significant reduction in total gastric acidity 10.80 ± 1.085. Both, curcumin and omeprazole showed potent anti-secertory abilities against gastric acid output.
The increase in this biomarker is anti-ulcerogenic by being cytoprotective to gastric mucosa (Plate 2, Panel D). As shown in the ulcerated untreated control animals showed a total gastric juice mucin content of 14.410 ± 0.3507 mg/dL. In comparison, mucin content was most significantly increased after treatment with curcumin into 21.42 ± 1.349 mg/dL (P < 0.001), followed by omeprazole that caused a significant increase into 18.73 ± 1.286 mg/dL (P < 0.01). Mucosal bleeding rate was significantly increased from 1.813 ± 0.06263 in control group (figure 1). As a result of the treatment the rate of mucosl bleeding was significantly decline to 0.8032 ± 0.02516 in curcumin treated group and to 0.7136 ± 0.01717 in omeprazole treated group.
As shown in
with curcumin and omeprazole, respectively (
In contrast, the total antioxidant level was significantly lower in plasma of control group (0.7226 ± 0.04026 mM/L) than in treatment groups with curcumin or omeprazole (
The level of the fragmented DNA in the supernatant of the gastric juice was recorded 0.1151±0.01458, 0.02247 ± 0.002274 and 0.05460 ± 0.005243 in the control, curcumin, omeprazole, respectively (
ceiving curcumin (0.3718% ± 0.06264%,) and omeprazole (1.007% ± 0.1458%).
The IL-6 level was significantly higher in plasma of control group (139.9 ± 11.90 pg/mL) than in treatment groups receiving 80 mg/kg curcumin (59.77 ± 5.597 pg/mL) and 20 mg/kg omeprazole (59.77 ± 5.597 pg/mL) (
As shown in figure 6A, the VEGF level was significantly higher in plasma of control group (3.290 ± 0.3659 pg/mL) than in treatment group receiving 80 mg/kg curcumin (11.98 ± 1.956 ng/mL). While, their was no significant difference was recorded in the level of VEGF in omeprazole treated group (5.031 ± 0.2676 ng/mL) when compared with control group. The same event was also recorded at the stomach level, since, their was no significant difference between the level of VEGF in the control group (5.394 ± 0.5998 ng/Ml) and omeprazole treated group (5.412 ± 0.5073 ng/mL) (
significantly increased to 19.88 ± 2.900 ng/mL in comparison with each of control (P < 0.001) and omeprazole (P < 0.001) groups, respectively.
Plate 3(1): a photomicrograph in the stomach fundus of the group I positive ulcerated control “Shay Rats” showing invaded mucosa at one side by multiple lymphoid follicles with discontinuation of the lining epithelium in the affected side (arrow) while the other side looked more or less healthy (H & E × 100). In the insert, a macroscopic photomicrograph of a part of the fresh stomach of positive control animals shows multiple ulcer patches (u) in the fundus, whereas, the body looked free. (2): a photomicrograph in the stomach fundus of the positive ulcerated control “Shay Rats” showing a very large sized lymph follicle extending into the submucosa, with several congested blood capillaries. Note the loss of epithelial lining at the site of invasion (arrow) (H & E × 100). (3): a photomicrograph in the stomach fundus of the group I positive ulcerated control “Shay Rats” shows intact epithelial lining but markedly reduced in its thickness ((Arrow head and intact mucosa (mm) (H & E × 200). (4) a magnified part in the stomach fundus of the group I positive ulcerated control “Shay Rats” showing apoptotic changes (P) affecting the majority of cells in the lamina propria (H & E × 1000).
Plate 3(5): a photomicrograph in the stomach fundus of a protective-treated ulcerated group with Cn showing all layers of the stomach fundus looking healthy (H & E × 100). In the insert there is a macroscopic photomicrograph of a part of the fresh stomach showing the fundus and body both looking healthy. (6): a magnified part in the previous image showing a few scattered inflammatory cells (arrow) in the lamina propria (H & E ×400). (7): a
photomicrograph in the stomach fundus of the group II treated ulcerated “Shay Rats” showing all layers of the stomach fundus look healthy and one blood capillary (bc) in the submucosa (toluidine blue × 200). (8): a magnified part in the previous image showing intact keratin (k), epithelial lining, and muscularis mucosa (mm). The epithelial lining had minimal apoptotic changes in some cells in the form of condensed nuclear chromatin (P) (toluidine blue × 1000).
Plate 3(9): a photomicrograph in the stomach fundus of the group IV omeprazole-treated ulcerated therapeutic control “Shay Rats” showing discontinuation of the epithelial lining associated with lymph follicle aggregation (arrow) (H & E × 100). In the insert, a macroscopic photomicrograph of a part of the fresh stomach with a few ulcer patches (u) observed in the fundus (F), and the body (B) looked healthy. (10): a photomicrograph in the stomach fundus of the group IV omeprazole-treated ulcerated therapeutic control “Shay Rats” showing diffuse lymphocyte infiltration extending into submucosa, where dilated congested blood capillaries were present (bc) (H & E × 100). (11): a photomicrograph in the stomach fundus of the group IV omeprazole-treated ulcerated therapeutic control “Shay Rats” showing sloughing of almost all epithelial surface with wide irregular and congested blood capillary (bc) observed in the lamina propria (toluidine blue × 200). (12): a photomicrograph in the stomach fundus of the group IV omeprazole-treated ulcerated therapeutic control “Shay Rats” showing all layers of fundus looking healthy (H & E × 200).
Several studies have been confirmed the antioxidant gastroprotective properties of omeprazole and strongly suggested omeprazole as a powerful antioxidant drug [
In this study, the gastric acidity, juice volume and the number of lesions in gastric wall of control group were significantly higher in the untreated ulcerated group; in contrast, the level of mucin content in the gastric juice was significantly lower in this group when compared with treated groups with either curcumin or omeprazole (Plate 2). Since, the back-diffusion of acid into the mucosa could directly lead to vascular leakage and aggressive damaging effect in the basement membrane of both epithelial and mucosal cells in the gastric wall, which could inhibit the restitution processes in the injured mucosa and induce a progression of apoptosis to deeper layers of the mucosa. Furthermore, a very brief cessation of mucosal blood flow results in a rapid decrease in the pH within the mucoid cap, which in turn, results in the formation of hemorrhagic erosions [
The massive amounts of peroxide in the control group was significantly increased that may be due to the increased level of IL-6 in the control group compared with the treated groups, particularly at the stomach level. Since, the local inflammatory cytokine IL-6 primarily activates neutrophils, lymphocytes and monocytes/macrophages at the inflammatory site, which in turn initiates different oxidative bursts, toxic metabolites and lysosomal enzymes responsible for local tissue damage in peptic ulcer [37,38]. Thus, the proinflammatory IL6 could predict more precisely the severity and duration of inflammation, particularly in its acute phase, than TNF-α [
Surprisingly, the level of VEGF was significantly increased at the systemic level (in the plasma) and locally (in the stomach) only in treated group with curcumin compared with either control or omeprazole group. In experimental models of acute gastric damage, the expression of VEGF increases during healing [
curcumin exerts its anti-ulcer activity not only by affecting oxidative stress and total antioxidant capacity but also by inhibiting IL-6 secretion and preventing apoptosis. Furthermore, curcumin promotes gastric ulcer prevention/healing by induction of angiogenesis in the granular tissue of ulcers. Further prospective studies are required to investigate the mechanism underlying the upregulation effect of curcumin on VEGF signalling pathways.