| BACKGROUNDNSAIDs are medications used primarily to treat inflammation, pain, and fever. NSAIDs work by blocking the conversion of arachidonic acid to PGs through inhibiting COX activity. There are two isoforms of COX, denoted COX-1 and COX-2. The former is constitutive and essential for the maintenance of normal physiological function. The latter is inductive, participating in some pathological processes such as inflammation, tumor, etc. It was once considered that NSAIDs exerted the function to treat inflammation, pain, and fever via inhibiting COX-2 activity, and brought a series of side effects via inhibiting COX-1 activity. Conventional NSAIDs are nonselective COX inhibitor, inhibiting the activity of both COX-1 and COX-2, the gastrointestinal side effects of which are noticeable. Selective COX-2 inhibitor is a new type of NSAIDs explored in the recent years. It has potent therapeutic efficacy and low gastrointestinal side effects, and is proven effective to some tumors and cardiovascular diseases. But there are still many arguments regarding whether the gastrointestinal safety of selective COX-2 inhibitor is as high as people expect. Some researchers reported lately that it delayed the healing of gastric ulcer as conventional NSAIDs. Studies have shown that the healing of gastric ulcer is closely related to gastric acid secretion, gastric epithelial cell proliferation, angiogenesis at the ulcer base, and activation of ERK signal transduction pathway.AIMSThrough observing the effects of the selective COX-2 inhibitor, celecoxib, on the healing of acetic acid-induced rat gastric ulcer, the study was aimed to: 1) determine whether selective COX-2 inhibitor affects gastric ulcer healing and PGs metabolization in gastric tissue; 2) evaluate the effects of selective COX-2 inhibitor on gastric acid secretion, gastric epithelial cell proliferation, angiogenesis at the ulcer base; 3) explore the effects of selective COX-2 inhibitor on the expression of H% K+ - ATPase, HGF and its receptor c-Met, bFGF and its receptor bFGFR, which are respectively related to gastric acid secretion, gastric epithelial cell proliferation and angiogenesis at the ulcer base, hoping to elucidate the mechanisms of selective COX-2 inhibitor delaying gastric ulcer healing at molecular levels; 4) investigate the effects of selective COX-2 inhibitor on ERK pathway, hoping to elucidate the mechanisms of selective COX-2 inhibitor delaying gastric ulcer healing from the angle of signal transduction.MATERIALS AND METHODS1. Grouping of experimental animals: 48 Wistar rats were randomly divided into model group and sham-operation group with 40 and 8 rats, respectively. Sham-operation group serves as normal control. Model group underwent the operation to produce acetic acid-induced gastric ulcer. Sham-operation group underwent sham operation. 3 days after the operation, all rats of sham-operation group and 8 rats of model group were euthanized. 32 residual rats in model group were divided into celecoxib group and NS group randomly and averagely. Celecoxib group received intragastric perfusion with 0.2% celecoxib solution, and NS group with 0.9% NaCl solution starting 3 days after ulcer induction. Rats in celecoxib group and NS group were euthanized at 6 and 9 days after ulcer induction (8 rats in each group at each time point).2. The gross structure of rat stomach was observered. The ulcer margin was drawn on a cellophane paper to measure the ulcer area using a 1 mm graph paper.The rat gastric content was collected and centrifuged, and the obtained superntant was gastric juice. The titre amount of gastric acid was determined with 0.01 mol/L NaOH, and the total acidity of gastric juice was calculated.3. A 3mm X 5mm tissue strip including the ulcer base and both sides of the ulcer margin was taken and fixed in 4% paraformaldehyde solution to make paraffin sections for H&E staining and for immunohistochemical staining of COX-1, COX-2, PCNA, and vWF. PCNA LI and microvessel number were respectively counted after PCNA and vWF immunohistochemical staining.4. A sample of gastric tissue weighed about 150 mg was taken from the ulcer base and the ulcer margin. A half was used to measure COX activity and PGE2 content by ELISA, and the other half was used to measure the mRNA expression of H+, K+-ATPase, bFGF, bFGFR by RT-PCR as well as the protein expression of COX-1, COX-2, HGF, c-Met, p-raf-1, and p-ERK by Western Blotting.5. A lmmX5mm tissue strip including the ulcer margin was taken from 4 rats in each group, and immediately fixed in 2% Glutaraldehyde solution for the examination under transmission electron microscope to observe the ulreastructure of parietal cell.RESULTS1. Effect of celecoxib on the healing of gastric ulcerThe ulcer area 3 days after ulcer induction was 33.3 + 7.4 mm2. 6 days after ulcer induction, the ulcer area was significantly decreased(versus 3 days) to 24.5 ± 6.4 mm2 in NS group, and slightly but not significantly decreased(versus 3 days) to 28.0 + 5.2 mm2 in celecoxib group. There was no significant difference between the two groups at 6 days after ulcer induction. 9 days after ulcer induction, the ulcer area was respectively 11.9 + 3.1 mm2 and 19.7 + 3.8mm2 in NS group and celecoxib group, which was significantly decreased(versus 3 days) in both groups. However, the ulcer area in celecoxib group was nearly 1.7-fold larger than in NS group (P<0.01) , demonstrating that celecoxib treatment significantly delays gastric ulcerhealing.2. Effects of celecoxib on PGs metabolism in ulcerated rat gastric tissue(1) Changes in the expression of COX-1 and COX-2 after ulcer inductionIn normal rat gastric mucosa, strong COX-1 immunohistochemical staining signal was detected predominantly in chief cells and parietal cells located in the lower 2/3 mucosa. 3 days after ulcer induction, the distribution of COX-1 signal and the number of positive cells in the gastric mucosa at the ulcer margin were similar to that of normal rat. Only faint COX-2 signal was detected in normal rat gastric mucosa. 3 days after ulcer induction, robust COX-2 signal was present in gastric epithelial cells and parietal cells in the gastric mucosa at the ulcer margin. Western Blotting analysis showed that there were relatively high COX-1 and very low COX-2 protein expression in normal rat gastric tissue. 3 days after ulcer induction, COX-2 protein level in ulcerated tissue was increased 10.8-fold, but COX-1 protein level in ulcerated tissue changed little, versus normal rat gastric tissue.(2) Effects of celecoxib on COX activity and PGE2 content3 days after ulcer induction, COX activity and PGE2 content in ulcerated tissue were increased about 4-fold and 6-fold, respectively, versus normal rat gastric tissue. COX activity and PGE2 content in NS group changed little at 6 and 9 days versus 3 days after ulcer induction. COX activity and PGE2 content in celecoxib group were significantly lower than in NS group at both 6 and 9 days after ulcer induction3. Effect of celecoxib on gastric acid secretion in gastric ulcerated rat(1) Effect of celecoxib on total acidity of gastric juiceThe total acidity of gastric juice in normal rat was 14.8 ±3.4 mmol /L. 3 days after ulcer induction, the total acidity of gastric juice was significantly decreased (versus normal rat) to 6.4+1.5 mmol/L. 6 and 9 days after ulcer induction, the total acidity of gastric juice in NS group was similar to 3 days, while that in celecoxib group was significantly increased(versus 3 days) to 10.6±3.1mmol/L and 12.5 + 2.2mmol/L, respectively. The total acidity of gastric juice in celecoxib group was significantly higher than that in NS group at both 6 and 9 days after ulcer induction (P<0.05, P<0.01).(2) Effect of celecoxib on H*, K+-ATPase mRNA expression￡T\ K+-ATPase mRNA level was significantly decreased at 3 days after ulcer induction, only 37% of that in normal rat. 6 and 9 days after ulcer induction, IT1", K+-ATPase mRNA level in NS group was similar to 3 days, while that in celecoxib group was significantly increased (versus 3 days). H+, K+-ATPase mRNA level in celecoxib group was significantly higher than that in NS group at both 6 and 9 days after ulcer induction (P<0.01).(3) Effect of celecoxib on the ultrastructure of parietal cellThere was plenty of mitochondrias and some secretary canaliculuses with a lot of microvilluses in parietal cell cytoplasm of normal rat, indicating the function of gastric acid secretion was active. 3 days after ulcer induction, parietal cell at the ulcer margin had such changes as fewer secretary canaliculuses, sparser microvilluses, and more tubulovesicular systems than in normal rat, indicating the function of gastric acid secretion was inactive. The amount of secretary canaliculus and microvillus were increased {versus 3 days) in both NS group and celecoxib group at 6 and 9 days after ulcer induction, but there weren' t distinct difference between the two groups.4. Effect of celecoxib on gastric epithelial cell proliferation in ulcerated rat(1) Effect of celecoxib on PCNA expressionIn normal rat gastric mucosa, PCNA immunohistochemical staining signal was present mainly in the glandular epithelial cells of gastric pits, and PCNA-positive cells constituted proliferative cell zone. After ulcer induction, the amount of PCNA-positive cells in the gastric mucosa at the ulcer margin was increased, and the proliferative cell zone became broader than that in normal rat. Quantitative analysis showed that the PCNA LI in normal rat was 11.2 + 2.8%. 3 days after ulcer induction,the PCNA LI was significantly increased to 16.4 + 2.3%. 6 and 9 days after ulcer induction, the PCNA LI in NS group was higher than 3 days, while that in celecoxib group was similar to 3 days. The PCNA LI in celecoxib group was significantly lower than that in NS group at both 6 and 9 days after ulcer induction (P<0.05, P<0.01).(2) Effects of celecoxib on protein expression of HGF and c-Met Western Blotting analysis showed that the protein levels of HGF and c-Met in ulcerated tissue were respectively increased 4.5-fold and 1.8-fold (versus normal rat) 3 days after ulcer induction. HGF protein level in both NS group and celecoxib group changed little at 6 and 9 days versus 3 days after ulcer induction, and there was no significantly difference between the two groups at either time point. 6 and 9 days after ulcer induction, the protein level of c-Met in NS group was significantly increased (versus 3 days), while that in celecoxib group was similar to 3 days. The protein level of c-Met in celecoxib group was significantly lower than that in NS group at both 6 and 9 days after ulcer induction (P<0.01). 5. Effect of celecoxib on angiogenesis at the ulcer base(1) Effect of celecoxib on the number of micro vessels at the ulcer baseThe number of microvessels was respectively 28.5±5.9/field and 23.1±5.8/field in NS group and celecoxib group at 6 days after ulcer induction, and there was no significant difference between the two groups. 9 days after ulcer induction, the number of microvessels was respectively 42.9±6.8/field and 30.6 + 6.2/field in NS group and celecoxib group, and the difference between the two groups was significant(P<0.01).(2) Effect of celecoxib on mRNA expression of bFGF and bFGFRLittle expression of bFGF and bFGFR mRNA was detected in normal rat gastric tissue. The mRNA levels of bFGF and bFGFR in ulcerated tissue were respectively increased 6.0-fold and 4.6-fold (versus normal rat) 3 days after ulcer induction. 6 and 9 days after ulcer induction, the mRNA levels of bFGF and bFGFRin NS group were significantly increased (versus 3 days), while those in celecoxib group changed little (versus 3 days). The mRNA levels of bFGF and bFGFR in celecoxib group were significantly lower than in NS group at 9 days after ulcer induction (/><0.01).6. Effects of celecoxib on protein levels of p-raf-1 and p-ERK in ulcerated rat gastric tissueThere were relatively low protein levels of p-raf-1 and p-ERK in normal rat gastric tissue. The protein levels of p-raf-1 and p-ERK in ulcerated tissue were respectively increased 5.4-fold and 7.1-fold (versus normal rat) 3 days after ulcer induction. 6 and 9 days after ulcer induction, the protein levels of p-raf-1 and p-ERK in NS group changed little (versus 3 days), while those in celecoxib group were significantly decreased (versus 3 days). At both 6 and 9 days after ulcer induction, the protein levels of p-raf-1 and p-ERK in celecoxib group were significantly lower than those in NS group. CONCLUSION1. Selective COX-2 inhibitor delays the healing of acetic acid-induced rat gastric ulcer, and decreases PGs content in gastric tissue, demonstrating that COX-2 derived PGs have essentially promotive action on the healing of acetic acid-induced rat gastric ulcer.2. The expression of H+, K+-ATPase mRNA and gastric acid secretion are significantly decreased during the healing of acetic acid-induced rat gastric ulcer. Selective COX-2 inhibitor can intensify gastric acid secretion through upregulating the expression of H+, K+-ATPase mRNA in parietal cell of gastric ulcerated rat, suggesting that strengthening the digestive action on newly formed granulation tissue at the ulcer base through intensifying gastric acid secretion may be one of its mechanisms to delay the healing of acetic acid-induced rat gastric ulcer.3. The protein expression of HGF and its receptor c-Met related to gastric epithelial cell proliferation as well as the mRNA expression of bFGF and its receptorbFGFR related to angiogenesis in gastric tissue are significantly upregulated during the healing of acetic acid-induced rat gastric ulcer. Selective COX-2 inhibitor can inhibit gastric epithelial cell proliferation through downregulating the protein expression of c-Met, inhibit angiogenesis in granulation tissue at ulcer base through downregulating the mRNA expression of bFGF and bFGFR, thereby delay the healing of acetic acid-induced rat gastric ulcer.4. Selective COX-2 inhibitor can significantly decrease the protein levels of p-raf-1 and p-ERK in ulcerated rat gastric tissue, indicating that selective COX-2 inhibitor may delay gastric ulcer healing through inhibiting the activation of raf-1/ERK signal transduction pathway.
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