| IntroductionSevere infection is a common cause of gastrointestinal dysfunction in children's life-threatening diseases, the mechanism of which is closely related to en-dotoxemia and impairment of gut mucosal barrier function, Apoptosis is a basic physiological process in gastrointestinal epithelia which proliferated actively. The integrity of gastrointestinal mucosa and its normal function are strictly depended on the well synchronization of epithelia' s apoptotic and proliferating speed. So the increasing apoptosis or decreasing proliferation what ever resulted from any cause could lead to the impairment of tight junction of gastrointestinal epithelia and gastrointestinal barrier, thus result in gastrointestinal dysfunction. It is short of investigation on the relationship of impairment of gastrointestinal barrier function and abnormally enterocyte apoptosis and proliferation, as well as apoptosis conducting way and its regulating genes during endotoxemia that resulted from severe infection in children's life-threatening diseases. It also lacks of report about the medical intervention, which could prevent or treat the above dysfunction.Caspase family is one of apoptosis conducting ways and Caspase-3 is a most important common medium of apoptosis. The activation of Caspease determined by specific stimulus and cells and it exits many apoptotic conducting ways during the activation. It is under investigation that whether the impairment of gastrointestinal barrier has relation to the changes of Caspase family members during en-dotoxemia.The family of genes Bcl-2 is a family of genes that regulating apoptosis of gastrointestinal tract. Its members include Bcl-2, Bcl-x, Bax, Bak, and Bad etc. Bcl-2 and Bcl-x suppress apoptosis, while Bax, Bak and Bad accelerate apoptosis via making Bcl-2 or Bcl-x to get together to dimmer and suppressing their function. It needs to testify that what change would take place about the expression of Bcl-2 family members and whether the impairment of gastrointestinal barrier has relation to them during endotoxemia.Proliferating cell nuclear antigen (PCNA) , also called cyclin, is an inter-nucleus protein, and it is also an assistant protein of DNA polymerase that is necessary for cell proliferation. It is usually taken as a marker of cell proliferation and it takes part in the regulation of cell cycle itself. It has no report about the change of PCNA in small intestine of endotoxemic rats.Glutamine ( Gin) is an important energy source of intestinal tract, and it is proved that glutamine could attenuate the structural impairment of intestine mu-cosa during pathologic condition and improve the function of gut mucosal barrier. The mechanism of which is that it provides energy for intestine epithelia regeneration as well as reinforces ability of intestine tissue to against oxidative injury. Since oxidative stress is one of inductive factors of apoptosis, it is postulated that glutamine may play a rule on prevention of apoptosis and improve intestinal barrier function, so as to accelerate the rehabilitation of gastrointestinal dysfunction.Materials and Methods1. MaterialsThe model of endotoxemia in 18 days rats was made by intraperitoneal injection of either saline ( sham) or endotoxin (4mg/kg of O55B5 Escherichia coli lipopolysaccharide) , the glutamine group was made by intraperitoneal injection of N ( 2 ) -L-alanyl-L-glutamine ( 2g/kg) and endotoxin simultaneously. 4cm lower ileum and several pieces of 1 mm3 end ileum were collected immediately (control only) after injection or within 10 minutes after dead (endotoxin grouponly) , 2h, 4h, 6h, 24h, 72h after injection respectively, and threw into 40g/ L formaldehydum polymerisatum dispensing with 0. lmol/L PBS and 2. 5% glut-aral respectively, fixed and preserved. The entire ileum was collected 2h, 4h, 6h, 24h, 72h after injection respectively and shrew into no-ribonuclease test tube as soon as possible, then froze in liquid nitrogen and preserved in - 80TI. 8 rats were sacrificed at one time point in each group.2. MethodsThe pathologic changes of small intestine villus were observed by hematoxy-lin-eosin ( HE) staining and transmission electron microscope (TEM). Apoptotic cells were detected by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) staining. Proliferating cell nuclear antigen ( PCNA) and Caspase-3 expression were measured by immunohistochemistry staining. Expression of Bcl-2 and Bax mRNA was performed by semi-quantitative RT-PCR method. Statistical analysis was performed with SPSS 11.0 statistical package, and the data was expressed as x ± s, the comparison between groups was made by analysis of variance as well as SLD method, student t-test, and corrective t-test ( when variance equality was not accepted).ResultsPart one: Changes of ultrastructure and expression of PCNA in enterocyte of endotoxemic baby ratsVarious kinds of apoptotic morphology of intestinal epithelia in endotoxin group were shown under transmission electron microscope. Apoptotic activity in each endotoxin group was significantly higher than that of sham group (P <0. 01). PCNA expression in each endotoxin group was significantly lower than that of corresponding sham group ( P <0. 05 ). The significantly decreased PCNA expression started from 2h (30.2 ± 8.3 vs 1'4.6 ± 16.2, t = 6. 874, P <0.01) after injection of endotoxin, reached the lowest level at 4h (21.4 ± 6. 7 vs 74.6 ± 18.7, t =7.566, P<0.01) and 6h (23.9±14.7 vs73.6±13.7, t =6.999, P<0.01) , retrieved to the level of 2h at 24h (35.8 ±11.4 vs 76.3 ±11.8, t = 7.010, P<0.01) , and did not get normal at 72h (59.6 ±18.0 vs78.7±16.9, t=2.185, P=0.046 <0.05). Apoptosis index (33.9 ±6. 1) and PC-NA expression (20.2 ±9. 2) in dead group were closed to those of 2h and 4h after injection of endotoxin respectively. Comparison was made by the time point between ratios of PCNA expression and apoptosis index in the same hours. It was found that the ratio of indotoxin group (0. 8 ±0.3, 0. 5 ±0.1, 0. 5 ±0. 3 , 0. 6 ±0.2, 1.3 ±0. 3, and 0.6 ±0.2 respectively) was significantly lower than normal [ (3.2 ±0.8) x 103 - (3.7 ±0.7) x 103) ] (P<0.01), and that the ratio of dead group was close to those of 4, 6, 24h after injection of endotoxin, and it was approximately the lowest level.Part two; Effect of glutamine on expression of enterocyte apoptosis and Caspase-3 during endotoxemia in baby ratsIt was showed that apoptotic activity (37.435 ±7. 138 vs 0. 021 ±0. 005, 44.112 ±9.349 vs 0. 020 ±0. 005,46. 618 ±8. 242 vs 0. 020 ±0. 005,54. 726 ± 8.999 vs 0.020 ±0.005,45.264 ±7.168 vs 0.020 ± 0.005 by the time order) and Caspase-3 expression (3.4±1.0vsl.7±0.5,3.7±0.5vsl.7±0. 3,5.1 ±0.5 vs 1.6 ±0.5,4.6 ±0.7 vs 1.7 ±0.5,4. 1 ±1.4 vs 1.7 ±0.5 by the time order) in each endotoxin subgroup were both significantly higher than the sham group accordingly ( all p <0.001). They are both significant between each endotoxin subgroup ( F = 4. 527,p = 0. 005 and F = 5. 026,p = 0. 003 respectively ). The markedly increased apoptosis and Caspase-3 both started from 2h after injection of endotoxin, and peaked at 24h and 6h respectively, retrieved approximately to the level of 4h at 72h. Apoptotic activity (7.725 ±3.063,11. 444 ±2.742,15.067 ±4.801,26.272 ±5.975,31.608 ±6.691 by the time order) in each glutamine subgroup was significantly lower than that of endotoxin group accordingly but it was still significantly higher than control (all p < 0. 01). Caspase-3 expression (1.9 ±0.5, 2. 8 ±0. 7,3. 6 ±0. 9,2. 0 ±0. 3,2. 7 ±0. 7 by the time order) in each glutamine subgroup was significantly lower than that of endotoxin group accordingly ( all p < 0. 05) and it was higher or closed to normal.Part three; Role of Glutamine on regulation to Bcl-2 and Bax expression in small intestine of endotoximic baby ratsThe result showed that the negative Bcl-2 mRNA expression in small intes-tine was found at each time point both in control and endotoxemia groups. The up-regulation of Bcl-2 mRNA expression was found in glutamine group. The lighted positive expression of Bax mRNA was found in control group, while significantly positive expression of Bax mRNA was found at each time point in endotoxemia group, but the positive expression of Bax mRNA in glutamine group was close to that of control group except that it was weaker in 2h subgroup. The ratio of Bax and Bcl-2 mRNA expression at each time point of endotoxemia group was significantly higher than that of control group. The changing trend of the ratio was that; it increased significantly at 2h (5.08 ±0.58) after endotoxin injection, peaked at 4h (6. 00 ± 0. 72) , and retrieved at 6 ~ 24h (5. 12 ± 0. 93 and 4. 89 ±0.76) , retrieved more at 72h (3.72 ±0. 63) , but it was still higher than normal. The ratio of Bax and Bcl-2 mRNA expression in glutamine group (2h: 0.37 ±0.12; 4h: 1.02 ±0. 23; 6h: 1.00 ±0.30; 24h: 0.97 ±0.13; 72h: 0.99 ±0. 17) was significantly lower than those of control and endotoxin group, especially that of 2h subgroup.Conclusion1. Enterocyte apoptotic activity is increased and its ability of proliferation is decreased during endotoxemia in baby rats, and imbalance between apoptosis and proliferation is one of pathologic mechanisms of gut barrier impairment during severe infection.2. Enterocyte apoptotic activity is increased during endotoxemia in baby rats, and one of its conducting pathways is Caspase-3. Glutamine made enterocyte apoptosis decreased through lowering Caspase-3 expression and played a protective role on intestinal construction.3. Endotoxemia made no positive expression of gene Bcl-2 in small intestine , but it made the expression of gene Bax and the ratio of Bax and Bcl-2 mRNA expression markedly up-regulated and significantly higher than normal. Glutamine made the expression of gene Bcl-2 in small intestine up-regulated and markedly higher than normal during endotoxemia. It also made the expression of gene Bax in small intestine down-regulated to nearly normal during endotoxemia,...
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