| Diabetic gastroparesis (DGP) is a common gastrointestinal complication of diabetes. As the morbidity of diabetes rises, the incidence of DGP has been increasing. The pathogenesis is still uncertain and the managements of DGP are far from clinical satisfaction. The life quality of those patients with DGP is severely interfered. It is very important to explore the pathogenesis and corresponding treatment of DGP. Gastric emptying abnormality is an important pathophysiological characteristic of DGP. Accumulative evidences show that arachidonic acid metabolic pathways are related to diabetes and its complications. COX-2 and its metabolites of gastric smooth muscle are associated with gastric emptying. Whether COX-2 in gastric antral smooth muscle is related to the delayed gastric emptying of DGP or not, is unclear.Objective:The present study is to investigate the effect of Celecoxib on gastric emptying and its mechanism in diabetic gastroparesis rats.Methods:Male SD rats were divided into normal control group and experimental group. Diabetes was induced by intraperitoneal injection of streptozotocin (STZ,50 mg/kg) and fed with a high-carbohydrate/high-fat diet. The normal control group (NC group, n=7) were injected with equivalent phosphate buffer solution and fed with standard diet. After 8 weeks, the diabetic rats were randomly divided into two groups, namely diabetic gastroparesis group (DGP group, n=7) and Celecoxib group (CE group, n=7). The diabetic rats were orally administered with Celecoxib [40 mg/(kg·d)] for 21 days. Gastric emptying was detected through semisolid method. The mRNA expression of COX-2 and 5-LOX were measured by quantitative reverse transcription-PCR. COX-2 and 5-LOX protein level were assessed by western blot analysis and immunohistochemical staining. Moreover, prostaglandin E2 (PGE2) was detected by ELISA. The ultrastructural of gastric antral smooth muscle was evaluated by transmission electron microscopy.Results:1. The gastric emptying rate of semisolid paste in DGP group was significantly lower than that of normal control group [(38.5±2.3)% vs (57.8±2.9)%, P<0.01]. The gastric emptying rate of the Celecoxib group was markedly higher than that of DGP group [(49.3±3.5)% vs (38.5±2.3)%, P<0.01].2. Compared with the normal control group, COX-2 mRNA expression in gastric antrum smooth muscle of DGP group significantly elevated (2.075±0.580 vs 1.015±0.191, P<0.01). Expression of mRNA of COX-2 was significantly decreased in Celecoxib group than that in DGP group (1.108±0.210 vs 2.075±0.580, P<0.01).5-LOX mRNA expression in gastric antrum smooth muscle of DGP group significantly elevated when compared with the normal control group (4.414±0.843 vs 1.094±0.542, P<0.05). Expression of mRNA of 5-LOX was significantly higher in Celecoxib group than that in DGP group (10.603±2.764 vs 4.414±0.843, P<0.01).3. The results of immunohistochemistry indicated that the COX-2 protein in antral smooth muscles of stomach was mainly expressed in cytoplasm and cytomembrane. The protein expression of COX-2 in gastric antral smooth muscles of DGP group was much higher than that of the normal control group (0.195±0.014 vs 0.044±0.006, P<0.01). Expression of protein of COX-2 was significantly decreased in Celecoxib group than that in DGP group(0.061±0.008 vs 0.195±0.014, P<0.01).5-LOX was also mainly expressed in cytoplasm and cytomembrane. The protein expression of 5-LOX in gastric antral smooth muscles of DGP group was much higher than that of the normal control group(0.083±0.029 vs 0.033±0.006, P<0.05). Expression of protein of 5-LOX was significantly elevated in celecoxib group than that in DGP group(0.336±0.027 vs 0.083±0.029, P<0.01).4. Western blot analysis results revealed that the protein expression of COX-2 in gastric antral smooth muscles of DGP group was much higher than that of the normal control group (1.048±0.162 vs 0.279±0.059, P<0.01). Expression of protein of COX-2 was significantly decreased in Celecoxib group than that in DGP group (0.411±0.154 vs 1.048±0.162, P<0.01).5-LOX protein expression in gastric antrum smooth muscle of DGP group significantly elevated when compared with the normal control group (0.121±0.009 vs 0.061±0.018, P<0.05). Protein expression of 5-LOX was significantly higher in Celecoxib group than that in DGP group (0.790±0.040 vs 0.121±0.009, P<0.01).5. ELISA analysis results revealed that the PGE2 level of gastric antral smooth muscle in DGP rats was significantly higher than that of the normal control group [(1984.3±163.2) pg/g tissue vs (860.4±95.4) pg/g tissue, P<0.01]. Expression of PGE2 was significantly decreased in Celecoxib group than that in DGP group [(1330.5±182.5) pg/g tissue vs (1984.3±163.2) pg/g tissue,P<0.01].6. The smooth muscle cell of the normal control group was spindle. The nucleus was normal in shape. And, the nucleus membrane was relative flat. Myofilament, dense patch and dense body were visible in cytoplasm which was rich in mitochondria. In contrast, many mitochondria from rats in the DGP group were swollen and vacuolation. Swelling and irregular nucleus, shrinking nuclear membrane were observed. The ultrastructure of smooth muscle improved in Celecoxib rats. Ridges of mitochondria were clearly visible and only several myelin figures emerged in cytoplasm.Conclusions:1. Celecoxib can significantly improve the delayed gastric emptying of the DGP rat.2. Up-regulation of COX-2/PGE2 signal pathway of gastric antrum smooth muscle is related to the pathogenesis of DGP.3. Celecoxib can down-regulate COX-2/PGE2 signal pathway of gastric antrum smooth muscle in DGP rats.4. The ultrastructure of the smooth muscle cells injured in diabetic gastroparesis rats. Celecoxib can ameliorate of gastric antral smooth muscle ultrastructural. |
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