In Vivo Evaluation Of NSAIDs-induced Gastropathy And Therapeutic Medications By Confocal Laser Endomicroscopy

BackgroundNon-steroidal anti-inflammatory drugs (NSAIDs) have been established as valuable among the most widely prescribed drugs for their role in the treatment of hyperpyrexia, inflammation, rheumatism and other musculoskeletal disorders. Unfortunately, increased exposure to NSAIDs remains one of the major etiological factors of gastric mucosal injury.Beside the role of NSAIDs in inhibition of cyclooxygenase (COX) and the related reduction of prostaglandin (PG) synthesis, COX-independent factors also play critical roles in the development of mucosal lesions by NSAIDs. NSAIDs-related gastropathy has become an important medical problem crying out for solution. However, previous works cannot provide a deep insight into the nature of NSAIDs-induced gastric injury, especially in the gastric epithelial barrier dysfunction.Geranylgeranylacetone (GGA, an anti-ulcer drug) and rabeprazole (one of the lastest medication of PPI) both have been widely prescribed in clinical field for their efficient management of gastric lesions, including NSAIDs-induced gastric injury. Recent studies revealed that diverse mechanisms have been proposed for the protective functions of geranylgeranylacetone and rabeprazole in NSAIDs-induced gastropathy, including up-regulation of COX with PG synthesis and stimulating the synthesis of mucin. However, the precise mechanism for geranylgeranylacetone/rabeprazole to protect NSAIDs-related gastropathy, especially their role in protecting gastric mucosal barrier is not very clear and needed to be refined.Confocal laser endomicroscopy (CLE) enables in vivo microscopic evaluation of morphological changes of gastrointestinal tissues. In addition, CLE is well suited to visualize cell shedding process by estimating the leakage of fluorecein, mucosal permeability and finally leads to the determination of mucosal injury severity, which provide gastroenterologist a comprehensive and objective methods to assess mucosal permeability and the severity of gastric mucosal injury. However, the true value of CLE on microscopic features of NSAIDs-induced mucosal lesions remains unclear.AimsThe aim of this study is to investigate changes of mucosal permeability in NSAIDs-induced gastric injury through confocal laser endomicroscopy FIVE1. Additionally, we intend to provide a deep insight into the morphological nature and the molecular mechanism involved in NSAIDs-induced gastric injury and identify how the gastric epithelial barrier was influenced by geranylgeranylacetone and rabeprazole both in vivo and in vitro.Methods1Animal modelsA total of96Wistar rats, were randomly divided into eight groups:normal (N), model (M), geranylgeranylacetone prevention (GP), rabeprazole prevention (RP), treatment control (TC), geranylgeranylacetone treatment (GT), rabeprazole treatment (RT), and double-drug treatment (DT).2Measurement of gross gastric damageThe areas of gastric lesions were measured under decuple-magnifier and the ulcer index (UI) were calculated and accumulated on the basis of Guth standard.3Optiscan FIVE1CLE examinationConfocal laser endomicroscopy FIVE1was used to observe and evaluate morphological changes of gastric mucosa. CLE classification system of NSAIDs-induced gastropathy was established according to the severity of gastric mucosal damage.4Histopathology and immunohistochemistry (IHC)Standard hematoxylin and eosin (H&E) staining was performed for histological examination. Histopathological grading of mucosal injury was established in correspondence with CLE classification. Gastric tissues from different groups were processed for immunohistochemistry to detect the expression of tight junction proteins occludin and ZO-1using widely applied methods.5Scanning electron microscopy (SEM)SEM was used to observe the ultra microstructure of gastric mucosa. Meanwhile, SEM grading of mucasal injury was established in correspondence with CLE classification.6Enzyme-linked immunosorbent assay (ELISA)Blood samples of each rat were used to determine the serum levels of tumor necrosis factor-a (TNF-a) by specific ELISA kits in accordance with the manufacturer’s protocols.7Western blotGastric mucosal samples was used to detect the expression of nuclear factor-κB (NF-κB) and caspase-3in gastric mucasa by western blot.8Statistical analysisStatistical analysis was performed with software package SPSS13.0. All data are expressed as means±SD. The results were evaluated using ANOVA followed by Dunnett’s post hoc test if the variance is homogeneous, otherwise nonparametric Wilcoxon rank sum test was applied for comparisons of multiple groups. Differences were considered as statistically significant if p value is less than0.05.Results1A four grade score system was established according to the severity of NSAIDs-induced gastropathy under CLE.2CLE grading result of mucosal lesions was consistent with macroscopic and histopathological scores. The severity of mucosal injury in NSAIDs-induced gastric mucosal damage group was significantly higher than that in normal group. Macroscopic, histopathological and CLE score were significantly increased in model group compared with that in normal group. However, geranylgeranylacetone and rabeprazole could remarkably prevent and treat mucosal damages evidenced by the gradation of the injury, which was significantly higher in model groups than that in drug prevention and drug treatment group.3Compared with normal group, the TNF-a expression in serum was significantly increased during indomethacin-induced gastric mucosa damage, whereas the level of TNF-a in all prevention and all treatment groups was markedly down regulated than that of model group.4Compared to normal group, NSAIDs administration led to a remarkable increase in caspase-3and NF-κB expression. The mucosal level of caspase-3and NF-κB declined sharply after the prevention or treatment of geranylgeranylacetone and rabeprazole compared to model group or treatment control group.5A distinct disruption and loss of expression of occludin and ZO-1were found inNSAIDs-treated mucosa. However, in the gastric tissues treated with geranylgeranylacetone and rabeprazole, a more regular and abundant distribution of occludin and ZO-1was observed.Conclusions1CLE is objective, real-time, and accurate for assessment of gastric mucosa epithelial injuries and mucosal permeability in NS AIDs-induced gastropathy.2NSAIDs could alter the localization and expression of ZO-1and occludin through up-regulating of TNF-a.3Geranylgeranylacetone and rabeprazole could maintain the integration of mucosal barrier and have effective role in NSIADs-induced gastropathy through the inhibition of TNF-α、caspase-3and NK-κB and up-regulation of ZO-1and occludin.