The Role Of PTEN Inhibitor In Intestinal Injury-repair Model Of Newborn Rats

Establish intestine injury-repair model of newborn Rats and intervene the intestinal injury-repair process of newborn Rats by administration of PTEN inhibitor-phen. Evaluate intestine injury severity according to the Nadler standard assessment scores by histopathological examination. Then observe the changes of the expression of Lgr5、β-catenin and PTEN mRNAs and proteins in intestine recovery process. Initially explore the role of PTEN inhibitor-phen in intestine recovery after injury.Part1、Evaluate the histopathological changes in the intestine injury-repair model of newborn Rats with PTEN inhibitor interventionMethod:Establish intestine injury-repair model of newborn Rats by hypoxia and cold stimulation. Newborn rats(clean SD Rats、1day old) were divided into normal control group (N group) experiment control group (C group) and intervention group (E group). All the Rats were breast fed by their mother Rats. N group Rats were left with their mother with no stimulation. C and E group Rats were given hypoxia(N2with6%O2,30min) and cold (4℃,20min)stimulation twice(at8am and8pm) every day,by3days. E group Rats were given phen1.6μmol/kg/d (dissolved in normal saline) by intraperitoneal injection after the first stimulation. However, C group was injected with same volume of normal saline according to weight. The items of response to stimulation、breast feeding、activities、abdominal distension、diarrhea and gastric retention of3groups’Rats were observed. Compare the weights before first and after last stimulation.5Rats of3groups were sacrificed at24h (1d、48h (2d)%72h (3d) and120h(5d) after the last stimulation (N1、N2、N3、N5; C1、C2、C3、C5and E1、 E2、E3、E5). Each group has5, altogether for60. Histopathological examination was made to understand intestinal damage and changes in villus structure. Use Nadler standard assessment scores to evaluate intestinal injury.Results:1. The C and E groups appeared different levels of activity decrease, eating down, mild to moderate diarrhea and abdominal, gastric retention. 2. The mean weights of3groups are7.36±0.1g、7.59±0.19g、7.60±0.11g before stimulation, with no statistical difference, P=0.407. After the last stimulation, the mean weights were11.17±0.15g、10.38±0.31g.10.75±0.14g. The mean weight of C group is significantly lower than the that of N group, with statistical difference, P=0.032. E group’s mean weight is lower than N group’s but higher than C group’s, with no statistical difference with the other two, PEvsN=0.05、PEvsC=0.295.3. There is turbid or even hemorrhagic abdominal effusion and the intestine is ruddy、 edema and congestion to varying degree and patchy bleeding or elasticity loss during samples-collection process in C and E groups.4. HE stain:In N group, villus structural is integrity and in alignment; Intestinal gland is integrity and clear in structure, mucosa and epithelial integrity and continuity; No vessel dilation、congestion、and inflammatory cellular infiltration are found in intestinal lamina propria. There is different level of inflammatory cellular infiltration in intestinal lamina propria and patchy、partial or even completely necrosis in C and E groups. C and E groups’ intestine damage is significantly worse than N groups’,scored between1and3levels, with statistical difference, P=0.000. E groups’ histopathlogical scores are lower than C groups’, although there is no statistical difference between C and E groups’ histopathlogical scores, P=0.468.Conclusion:1. Intestine injury-repair model of newborn Rats, established by hypoxia(N2with6%O2,30min) and cold (4℃,20min) stimulation, change significantly in appearance、body weight changes、and intestine histological changes. Moreover, these Rats can live longer for histological changes examination.2. The severest damage showed at the2nd or3rd day after the last stimulation and improvement at the5th day can illustrate intestine self-recovery process after damage.3. The roles of PTEN inhibitor in intestine injury-repair model show as improvement in appearance and weight of rats, even alleviation in histopathlogical damage.Part2、Observation of the effects of PTEN inhibitor in transcription and translation of Lgr5、β-catenin and PTEN in intestinal injury-repair processMethod: Establish intestine injury-repair model of newborn Rats by hypoxia and cold stimulation (as part1) and intervene by administration of PTEN inhibitor-phen. Newborn Rats were divided into normal control group (N)、experimental control group (C) and intervention group (E). Ileocecus of3groups were collected for test at24h(1d)、48h(2d、72h(3d) after the last stimulation, Each group has5, altogether for45. Detect the transcriptional and translational level of Lgr5、β-catenin and PTEN in intestine by Real-time PCR and Western blot techniques. Initially explore the role of inhibition of PTEN in intestine repair after injury.Results:1. Lgr5mRNA and protein’s expression levels:C and E groups’ Lgr5mRNA and protein expression levels are higher than N group, and E is further higher than C. Lgr5mRNA expression level is higher in C1(compared with N1, P=0.031)、 El(compared with C1, P=0.032;E2(compared with C2,P=0.005),with statistic difference. Lgr5mRNA expression level is higher in C2(compared with N2, P=0.264)、C3(compared with N3, P=0.146)、E3(compared with C3,P=0.081), but with no statistic difference. Lgr5protein expression level is higher in C1(compared with Nl, P=0.045)、El(compared with C1, P=0.036),with statistic difference. Lgr5protein expression level is higher in C2(compared with N2, P=0.062;C3(compared with N3, P=0.059)、E2(compared with C2, P=0.093)、 E3(compared with C3,P=0.177), but with no statistic difference.2.β-catenin mRNA and protein’s expression levels:C and E groups’ β-catenin mRNA and protein expression levels are higher than N group, and E is further higher than C. β-catenin mRNA expression level is higher in C1(compared with N1, P=0.039)、E1(compared with Cl, P=0.008)、E2(compared with C2,P=0.010),with statistic difference.β-catenin mRNA expression level is higher in C2(compared with N2, P=0.060). C3(compared with N3, P=0.200)、E3(compared with C3,P=0.077), but with no statistic difference.p-β-catenin protein expression level is higher in C1(compared with N1, P=0.013)、C2(compared with N2, P=0.007)、E1(compared with C1, P=0.043),with statistic difference. P-β-catenin protein expression level is higher in C3(compared with N3,P=0.052)、 E2(compared with C2, P=0.109)、E3(compared with C3, P=0.094), but with no statistic difference.3. PTEN mRNA and protein’s expression levels:C and E groups’ PTEN mRNA and protein expression levels are lower than N group, and E is further lower than C. PTEN mRNA expression level is lower in C1(compared with N1, P=0.038)、 E1(compared with C1, P=0.003)、E2(compared with C2,P=0.030)、 E3(compared with C3, P=0.030),with statistic difference. PTEN mRNA expression level is lower in C2(compared with N2, P=0.134)、C3(compared with N3, P=0.679), but with no statistic difference. PTEN protein expression level is lower in C1(compared with N1,P=0.037)、E2(compared with C2, P=0.008)-. E3(compared with C3, P=0.048),with statistic difference. PTEN protein expression level is lower in C2(compared with N2, P=0.123)、C3(compared with N3,P=0.522)、E1(compared with C1,P=0.093)、but with no statistic difference.4. A positive correlation is found between Lgr5and P-catenin mRNA’s expression levels, r=0.885,P=0.000. And a negative one between Lgr5and PTEN mRNA’s, r=-0.877、P=0.000. Lgr5、p-β-catenin and PTEN proteins’expression levels and trends coincide with their corresponding mRNA expression levels. A positive correlation is found between Lgr5and β-catenin protein’s expression, r=0.789,P=0.000, and a negative one between Lgr5and PTEN protein’s, r=-0.823, P=0.000.5. No statistical difference is found in Lgr5、β-catenin and PTEN mRNAs or proteins’ expression levels at different times in each groups except for the comparison between C1and C3(P=0.022)Conclusion:1. The higher transcription and translation levels of Lgr5and β-catenin levels reflect a corresponding accelerating rate of intestine stem cells proliferation and differentiation. PTEN mRNA and protein expression levels’ increase maybe due to the organism’s negative feedback regulation.2. PTEN inhibitor can accelerate intestine stem cells proliferation and differentiation so as to promote intestine recovery from injury by down-regulation of PTEN mRNA and protein levels, and up-regulation of Lgr5and β-catenin mRNA and protein Levels.3. PTEN inhibitor’s promotion in intestine recovery from injury to some extent may be the mechanism that it enhances PI3K/Akt signaling pathway.