| BackgroundNon-steroidal anti-inflammatory drugs (NSAIDs) are widely used in clinical practice currently. But they induce different degrees of damage to the mucosa in upper gastrointestinal tract and small intestine when playing therapeutic roles.60%～70%of NSAIDs-related enteropathy patients have no characteristic symptoms. Serious complications such as gastrointestinal bleeding, intestinal stenosis, intestinal perforation are rare, but they are fatal when occured. As we all know, cyclooxygenase plays a key role in NSAIDs-induced gastric mucosal injury. But studies suggested that inhibition of cyclooxygenase didn’t play a major role in the process of NSAIDs-induced intestinal mucosal injury. Currently, the mechanism NSAIDs-related intestinal mucosal injury is inconclusive; the most recognized one is "three hit hypothesis". The hypothesis proposes that integrity of the intestinal epithelial barrier is critical in NSAIDs-induced enteropathy. Intestinal epithelial barrier is composed of epithelial cells and intercellular tight junctions. Rate of epithelial cell regeneration is half the rate of shedding, which determines the existence of gaps after cells shedding. There is a close relationship between the gaps of exfoliated cells and intestinal mucosal barrier. The gap densities of exfoliated cells can be used to predict the severity and recurrence of inflammatory bowel disease (IBD). The aims of the current study were to evaluate the gap density in indomethacin-induced small intestinal lesions using CLE; explore the mechanisms involved; and identify effective drugs to improve the epithelial barrier dysfunction.Materials and Methods Part I An experimental study on the morphology of nonsteroidal anti-inflammatory drugs-induced injury in small intestinal epithelial barrier and the effects of drug interfering Animal modelsA total of96male Wistar rats were randomly divided into eight groups according to body weight:normal group, model group (model established with indomethacin), teprenone prevention group, rabeprazole prevention group, treatment control group, teprenone treatment group, rabeprazole treatment group, and teprenone and rabeprazole conbined group(combined group), six in each group.All rats were anesthetized by intraperitoneal injection of chloral hydrate. A1～2cm longitudinal incision was made along the abdominal median tangent. A1～2cm incision was then made along the mesenteric contralateral longitudinal in the proximal jejunum for CLE observation. Pay attention to protecting the intestinal blood flow. The targeted proximal jejunum lining was cleaned, the confocal probe was gently placed vertically on the mucosal surface and endomicroscopic images were collected1min after intracardiac injection of fluorescein sodium. The observation time is controlled within20min. After CLE examination, five good-quality endomicroscopic images were selected. The gap density was calculated as the number of gaps divided by the number of epithelial cells counted, normalized to a count of1,000cells. The upper jejunum tissues (about1cm) were washed with PBS and chymotrypsin and immediately fixed in10%neutral buffered formalin for12h. The tissues were then taken out and opened along the mesenteric side, fixed on sheets with pins to flatten the bowels, and then fixed in formalin for another12h to avoid damaging the small intestinal villi. The embedded tissues were cut into4u m-thick sections and underwent routine deparaffinization and rehydration. Hematoxylin and eosin (H&E) staining was performed according to the standard protocol. Other upper jejunum tissues of the model group were washed and viewed using a Hitachi H-600scanning electron microscope. All data wre expressed as mean±SEM. SPSS13.0statistical software was used to assess the significance of differences. The LSD-t test or Hamhane’s T2test were used for statistical analysis. Differences were considered to be significant at values of P<0.05.Part Ⅱ An experimental study on the molecular mechanisms of nonsteroidal anti-inflammatory drugs-induced injury in small intestinal epithelial barrier and the effects of drug interferingA total of96male Wistar rats were randomly divided into eight groups according to body weight:normal group, model group(model established with indomethacin), teprenone prevention group, rabeprazole prevention group, treatment control group, teprenone treatment group, rabeprazole treatment group, and teprenone and rabeprazole conbined group(combined group), six in each group. All rats were anesthetized by intraperitoneal injection of chloral hydrate. Blood samples were taken by cardiac puncture and centrifuged. Serum was stored at-80℃for determining TNF-α levels by ELISA kit. The upper jejunum tissues were taken out and the mucosal layer was striped with microstructure forceps. Total protein was extracted from the samples. Using Western blot method, the contents of caspase-3, NF-κB, occludin and zonula occludens protein1(ZO-1) were measured. All data were expressed as mean±SEM. SPSS13.0statistical software was used to assess the significance of differences. The LSD-t test or Hamhane’s T2test were used for statistical analysis. Differences were considered to be significant at values of P<0.05.Result1. Gap Density in CLEGaps were smaller than absorption cells, partially visible as jet-like signs. The gap densities in the jejunum tissue of teprenone prevention group and rabeprazole prevention group were (57.43±24.55)/1000and (59.80±21.14)/1000. Compared with the model group (110.93±50.58)/1000, the gap densities of both groups were significantly decreased (t=53.50,54.13, P<0.01, respectively). Compared with the treatment control group (40.53±15.39)/1000, the gap density of combined group (93.80±40.65)/1000, was significantly increased (t=44.27, P<0.01).2. Gaps in HE and SEMAfter histopathologic fixation and staining, we found that cells shed in a budding form. The shed cells were fragmented and decomposed. Tapered gaps eventually formed. Under the scanning electron microscope, the gaps were like deep holes. There were normal intestinal epithelial cells around the gaps, similar to that observed by CLE.3. Serum Concentration of TNF-αThe levels of serum TNF-α in teprenone prevention group and rabeprazole prevention group were (25.80±8.97) and (22.74±7.15) ng/L. Compared with the model group (44.48±7.42) ng/L, the levels of serum TNF-α in both groups were significantly decreased (t=18.68,21.74, P<0.01, respectively). Compared with the treatment control group (13.66±4.98) ng/L, the level of serum TNF-α in combined group (24.67±6.70) ng/L was significantly decreased (t=9.02, P<0.01).4. The Contents of Caspase-3and NK-κB in TissuesThe levels of caspase-3in teprenone prevention group and rabeprazole prevention group were (1.47±0.35) and (1.58±0.34) ng/L. The levels of NF-κB in teprenone prevention group and rabeprazole prevention group were (1.27±0.14) and (1.21±0.10). Compared with the model group (2.44±0.45) and (1.69±0.13), the levels of both groups were significantly decreased (t=0.97,0.86,0.42,0.48, P<0.01, respectively). Compared with the treatment control group, the levels of caspase-3and NF-κB in combined group (0.66±0.06) and (0.44±0.21) were significantly decreased (t=0.34,0.56, P<0.01, respectively).5. The Expression of Occludin and ZO-1in IHCThe levels of occludin in teprenone prevention group and rabeprazole prevention group were0.69±0.16and0.74±0.11. The levels of ZO-1in teprenone prevention group and rabeprazole prevention group were0.81±0.08and0.84±0.12. Compared with the model group, they were significantly increased(t=0.24,0.29,0.17,0.21, P<0.01, respectively). The levels of occluding and ZO-1in combined group were2.50±0.46and1.76±0.18. Compared with the treatment control group, they were significantly increased (t=1.50, t=0.76, P<0.01, respectively)Conclusions1. Gaps could be clearly observed and the gap density could be measured by confocal laser endomicroscopy. Gap density was expected to be an indicator of evaluating intestinal epithelial barrier function and therapeutic effects.2. There were damages of intestinal epithelial barrier, and the expressions of occluding and ZO-1were decreased. One of the mechanisms was increase in TNF-a accelerated apoptosis necrosis by NF-κB-caspase-3pathway.3. Teprenone and rabeprazole could inhibit the expression of TNF-α expression, improve the decreased expression of cell tight junction proteins and reduce the intestinal epithelial barrier injury. By the NF-KB-caspase-3pathway, teprenone and rabeprazole could achieve the prevention and treatment of indomethacin-induced intestinal lesion.SignificanceThis study demonstrated that gaps could be clearly observed and the gap density could be measured in vivo by confocal laser endomicroscopy, thus the lesions of indomethacin-induced intestinal mucosal injury could be objectively evaluated. Also the therapeutic effect of medicines on indomethacin-induced enteropathy could be evaluated. We found that there was a correlation between the increased epithelial cells gaps and the increased epithelial permeability. Through the research of gaps, we confirmed that teprenone and rabeprazole played positive effects by improving the intestinal epithelial barrier function and its permeability. In the future, it is expected to be applied to the clinical, for detecting the severity of intestinal inflammation. Fluorescein leakage is hopeful to apply in the assessment of the intestinal permeability. It will become a new method to detect the permeability of the intestinal epithelial cells in vivo, a new means to evaluate the efficacy of medicines. |
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