| Objective:To explore the effect of remote ischemic preconditioning(RIPC)on nuclear factorκB(NF-κB)expression in gastric mucosal tissues of rats with gastric ischemia-reperfusion injury(GI-RI).Methods1.Model preparation and grouping of gastric ischemic reperfusion ratsThe rates were injected intraperitoneally with 10%chloral hydrate(300~350mg)for the purpose of anesthesia.With linea alba being median incision,rats’celiac arteries,the branches of the abdominal aorta,were separated and clamped by small clamp for 30 min.After the removal of the clamp,the rates’celiac arteries were reperfused for 0 h,1 h,3 h,6 h,12 h and 24 h respectively.In sham group,rats’celiac arteries were separated without clamp;In RIPC group,before the gastric ischemic reperfusion,hepatic artery and portal vein were clamped for 5 min and then reperfused for 10 min,which was repeated one time,and the following steps were done as what were done in the I/R group.The successful criteria of rats’gastric ischemic reperfusion:After the celiac arteries were clamped,it could be observed that stomachs turned dark purple from scarlet,when the clamp was removed,perfusion was recovered and it could be seen that stomachs immediately reversed to their original color,turning scarlet from dark purple.2.Observation of pathological slices and index determination of gastric mucosal damage1)After anesthesia,rats’abdominal cavities were opened and cut along greater curvature of stomach,and then rinsed by cold normal saline.The rats were then stretched on an ice tray and placed on pane-counting plate(1 mm~2 per pane),and scores were determined cumulatively according to pin-point erosions,ulcer and the length of hemorrhagic focus within gastric epithelium:Normal,0 point;Damage length≤1 mm,1point;>1 mm and≤2 mm,2 points;>2 mm and≤3 mm,3 points;The following was done in the same way and when the damage length exceeded1 mm,the scores were doubled.The average score of each group was drawn to be the GMDI of the group2)TdT-mediated UTP Nick-End Labeling(TUNEL)method was adapted to detect gastric mucosal cells apoptosis.Under the fluorescence microscope,visual field was selected randomly to observe and count positive apoptotic cells and the total cells,and the apoptosis rate was calculated with positive apoptotic cells/total cells×100%.3.Gastric superoxide dismutase activity(SOD),malondialdehyde expression was detected(MDA)content and the protein nuclear factorκB p651)After the experiment,rats’stomachs were taken out immediately,incised along lesser curvature of stomach and rinsed by normal saline.About 30 ul homogenate was taken in each specimen to detect total SOD and Cu-Zn SOD activity by using SOD detection kit,with all the operation strictly following the manufacturers’instructions.Gastric mucosa was taken in the same way as above.Gastric mucosa was made to be 10%homogenate with cold normal saline.About 200μl homogenate was taken in each specimen to detect gastric mucosal MDA content by MDA detection kit,with all the operation strictly following the manufacturers’instructions.2)Immunohistochemical method was used to detect the expression of nuclear factor kappa B p65 protein in gastric mucosa:colloid or cell nucleus brown granule cells were positive under fluorescence microscope.The percentage of positive cells in the positive cells was scaled up,and the3 samples were randomly sampled in each section,and the average percentage was obtained from 10 randomly selected fields.3)Protein expression detection of NF-κB p65 by Western blot:Ladder analysis was conducted by Gel-Pro analyzer 4.0 image analysis software,and the protein expression of NF-κB p65 was determined by the glyceraldehyde-3-phosphate dehydrogenase(GAPDH)gray value ratio of target protein and interior reference.Results1.Model preparation and grouping of gastric ischemic reperfusion rats was successfully preparedAll the rats had good appetite and mental status.And no rat died during or after the operation,causing 100%success rate of ischemic reperfusion operation.2.Observation of pathological slices and index determination of gastric mucosal damage1)In sham group,smooth gastric mucosa,integrated epithelium and glands in order were observed.In I/R group,it was seen that there were eosinophils and a few neutrophils between parts of muscular layer of gastric mucosa and glands,dilatation and hyperemia in interstitial blood vessel and a little hemorrhage in intestinal mucosa.In RIPC group,it was observed that there were a few eosinophils and few neutrophils between parts of gastric mucosa and glands as well as dilatation and hyperemia in interstitial blood vessel2)Statistical analysis revealed that there were significantly higher GMDIs of 0 h,1 h,3 h,6 h,12 h and 24 h reperfusion in I/R group than in sham group(all P<0.05).In I/R group,the GMDI was highest in T1sub-group and then decreased sustainably to the lowest in T24 sub-group,which was,still,higher than sham group;And GMDIs of 0 h,1 h,3 h,6 h,12 h and 24 h reperfusion decreased obviously in RIPC group compared to those in I/R group(all P<0.05).3)Changes of gastric mucosal cells apoptosis in T6 sub-group:In sham group,there were some positive apoptotic cells scattering on epithelial layer,submucosa and glands.In I/R group,apoptosis rate of gastric mucosal cells increased significantly,and the apoptotic cells were densely distributed on mucosa epithelium and glands;Compared to I/R group,there was a significantly decreased apoptosis rate of gastric mucosal cells and smaller range of positive cells in RIPC group.3.Changes of MDA contents and SOD activity in each group and expression of nuclear factorκB p65 protein1)Compared to sham group,the MDA activity increased and SOD activity decreased of 0 h,1 h,3 h,6 h,12 h and 24 h reperfusion in I/R group and RIPC group(all P<0.05).Compared to I/R group,MDA activity decreased and SOD activity increased in RIPC group;In I/R group,compared to T0 sub-group,MDA activity increased to the highest in T6sub-group(P<0.05),and SOD activity started to decrease after T3sub-group(P<0.05).In RIPC group,MDA activity increased after T3sub-group and decreased in T24 sub-group(P<0.05),and SOD activity started to decrease in T1 sub-group and to the lowest in T6 sub-group(P<0.05).2)Compared to sham group,there was a significantly increased positive cells rate of 0 h,1 h,3 h,6 h,12 h and 24 h reperfusion in I/R group and RIPC group(all P<0.05).Compared to I/R group,obviously decreased positive cells rate of 0 h,1 h,3 h,6 h,12 h and 24 h reperfusion in RIPC group was shown(all P<0.05).And in I/R and RIPC groups,compared to T0 sub-group,positive cells rate was highest in T1 and lowest in T24 sub-group(both P<0.05).3)Electrophoretogram of each group was analyzed by image analysis software and was examined area integral optical density.Compared to sham group,the protein level of NF-κB p65 of 0 h,1 h,3 h,6 h,12 h and24 h reperfusion in I/R group and RIPC group were significantly increased(all P<0.05);Compared to I/R group,decreased protein level of NF-κB p65 of 0 h,1 h,3 h,6 h,12 h and 24 h reperfusion was observed in RIPC group;In I/R and RIPC groups,compared to T0,protein level of NF-κB p65 was highest in T1 sub-group and lowest in T24 sub-group averagely(P<0.05).ConclusionBy reducing oxygen free radicals(OFR)production,infiltration of neutrophils and inhibition of NF-κB activity,RIPC may effectively relieve the early reperfusion injury of gastric mucosa. |
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