| Part1:Autophagy of Small Intestinal Epithelium in Sepsis RatsObjective To reserch autophagy in small intestinal epithelial in sepsis rats induced by cecal ligation and puncture(CLP), and explore the relationship between autophagy and septic intestinal epithelial injury.Methods Sixty male SD rats were randomly divided into six groups as the control group, CLP4h group, CLP8h group, CLP12h group, CLP16h group and CLP20h group (n=10). After building the cecal ligation and puncture rat modles, they were sacrificed at each time point randomly. Then, we collected serum and small intestinal, and scraped intestinal epithelial mucosa at low temperature for inspection. Enzyme-linked immunosorbent assay was used to detect cytokines change of serum and mucosal tissues. Western-blot method and Real time-PCR method were used to detect expression level of autophagy-associated protein LC3. Besides, we analysis micro cell structure changes of8h and20h after sepsis, and obeserved morphology of autophagic vacuoles by electron microscopy, and calculated the number of autophagosome and autolysosome in rat intestinal epithelial.Results The level of TNF-a and MCP-lin serum and mucosal tissues of CLP-induced sepsis rats were significantly higher at4h (P<0.05). The expression of autophagy-associated protein LC3-II/LC3were significantly higher at8h after sepsis (1.255±0.088vs1.470±0.295, P=0.038, n=4), and declined at12h. And compared with the control group, the level of LC3gene transcription was increased (0.120±0.069vs3.104±1.191, P=0.00, n=10), followed by a downward trend. Observed intestinal epithelial cells at8h and20h after CLP, we found that mitochondrial damage was significantly severe, that swelling dissolved and endoplasmic reticulum vacuoles, the number of autophagosome and autolysosome significantly increased at CLP8h (19.33±2.16vs9.33±1.86,37.67±3.27vs19.50±1.87, P=0.00, n=3), we also found within larger autolysosomes in CLP20h rats.Conclusion TNF-a in systemic and local intestinal increased and reached peaking earlier in sepsis rats. The expression level of autophagy-associated protein LC3in small intestinal rised both in protein and gene levels in the early phase, then falled down. Under the sepsis pathology environment, autophagy was affected by inflammatory cytokines, and positively correlated with TNF-a level. Part2:Ghrelin Regulates Autophagy of Small Intestinal Epithelium in Sepsis RatsObjective To explore the regulation of ghrelin in intestinal epithelium autophagy of sepsis rats, and investigate the protective effect in regulating autophagy by ghrelin in sepsis rats.Methods Forty male SD rats were randomly divided into the normal group, the sham operation group, the CLP20h group and the ghrelin intervention group. We established animal sepsis models by cecal ligation and puncture. Then continuous infusion ghrelin for20h by tail vein. After that animals in each group were sacrificed to collect serum and intestinal tissues, fixed parts of the small intestine segment in glutaraldehyde or formalin for pathological analysis and electron microscopy analysis, and scraped the other parts of intestinal epithelial mucosa at low temperature for the molecular biology detection. Pathological changes and micro-structural changes of the intestinal epithelium in each group were observed by microscopy, and the number of autophagic vacuole was counted by electron microscopy. The level of GFP-LC3dots in the intestinal epithelial were observed by Immunofluorescence. Western-blot and Real-time PCR detected the autophagy-associated protein LC3expression of small intestinal, the change of inflammatory cytokines of serum and mucosal in each group were detected by enzyme-linked immunosorbent assay, and the differences between groups were analyzed.Results (1) Compared with the normal group and the sham group, pathological damages of intestinal epithelial were obvious in CLP20h rats. The level of TNF-a in serum and mucosal (154.57±19.54vs88.58±8.19.96.08±9.93,74.95±4.89vs19.94±1.10.22.44±4.18, P=0.00, n=10) and MCP-1(96.33±4.89vs61.66±6.94.69.16±10.42,66.74±4.80vs41.74±1.78.49.24±4.32, P=0.00, n=10) increased. The expression of LC3protein increased (1.52±0.03vs0.98±0.18、1.25±0.09, P=0.00, n=4), which also paralleled with the increased level of gene transcription (0.42±0.16vs0.13±0.11.0.13±0.12, P=0.00, n=10). The expression level of GFP-LC3in intestinal epithelial of septic rat increased compared with the sham group (210±18.03vs168±17.66, P=0.003, n=5). The number of autophagosome and autolysosome in intestinal epithelial cells compared with the sham group increased (36.83±3.76vs9.33±1.86,71.83±5.27vs 19.50±1.87, P=0.00, n=3).(2) Compared with the sepsis group, the intestinal mucosal epithelial capillary congestion was alleviated. The level of TNF-a (154.57±19.54vs192.75±21.91,74.95±4.89vs96.85±15.35, P=0.00, n=10) and MCP-1(96.33±4.89vs189.93±14.59,66.74±4.80vs99.08±7.47, P=0.00, n=10) in serum and mucosal tissues decreased. The level of LC3protein expression (1.52±0.03vs1.35±0.06, P=0.035, n=4) and gene transcription (0.42±0.16vs0.14±0.10, P=0.00, n=10) were elevated. The level of GFP-LC3increased in intestinal epithelial (210±18.03vs177.80±17.66, P=0.014, n=5). The number of autolysosome significantly increased in the ghrelin group (36.83±3.76vs19.33±2.16,71.83±5.27vs37.67±3.27, P=0.00, n=3).Conclusion Ghrelin significantly alleviated the pathological damage of intestinal epithelial in septic rats, reduced the level of systemic and local inflammatory cytokines, and promoted the expression and transcription of autophagy-associated protein LC3in intestinal epithelial cells. It increased the number of autophagosome and autolysosome. In a word, ghrelin could regulate autophagy of intestinal epithelial cells in sepsis rats. |
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