Protective Effect Of Pim-3 On Intestinal Epithelia Cells Damaged By Lipopolysaccharide

Background and Research StrategyThe gut is the biggest reservoir for pathogenic bacteria and endotoxin.But under normal condition,the intestinal mucosa possesses barrier function to protect against the escape of intraluminal microorganisms and endotoxin through the intestinal mucosa into the body.When following severe trauma,burn,hemorrhagic shock and infection,intestinal barrier is damage,Intestinally derived bacteria or endotoxin serve as triggers to initiate,perpetuate,or exacerbate the septic state and thereby promote the development of Multiple organ system failure(MOSF).so intestinal barrier function is one of an important index for serverve patient.At present,people have done many research work about pathogeny and mechanism of enterogenic infection.It is rare report about burning and endotoxin intestinal barrier and gene therapy.So,how to protect intestinal barrier function is an hot spot.Previously people only research enteral nutrition and microorganisms environment.It is rare report how to use oncogene gene to cure intestinal mucosa damage.After the rats were inflicted with 30%TBSA full-thickness skin scald,endotoxin rat model,they were sacrificed and samples were taken.Then record intestinal barrier and gene dynamic state,epithelioid cell cultures from rat small intestine.There are still some problems to be further explored.we want to observe oncogene gene how to therap endotoxin intestinal epithelial cell.Oncogene Pim-3 manipulate cell growth,they can promote cell proliferation.When intestinal mucisawound,intestinal enterocyte cell apoptosis.Oncogene can inhibit cell apoptosis,they can promote intestinal mucosa repair.Oncogene Pim-3 and protein production are not only related with the cellular growth and differention,but also participate cellular message deliver and energy metabolism.They are essential for life.Above all,it is close relation about Pim-3 and intestinal tract damage repair.All of these studies showed that Pim-3 provided an important role in intestinal cell damage repair.We hypothesized that Pim-3 play an important role in intestinal barrier damage in acute post-bum rats,and act as an endogenous protective substance against the intestinal mucosa damage.We conducted the researches as follows:At first,the degree of intestinal tract damage and the changes of Pim-3 were examined with Occludin,ICAM-1,TNF-α,IL-6,pathology analysis,and the level of Pim-3 mRNA and protein in intestinal tract in well-described 30%TBSAⅢ°burn rats model and endotoxin rats model in vivo.It is provide evidence in vitro.Then,according to research result,small intestinal were isolated and culture under endotoxin stress model to mimic effects of in post-burn rats,evaluate the degree of injury.Transfect Pim-3 into intestinal cell to protect endotoxin intestine epithelial cell damage.Using RT-PCR method to detect changes in the level of intestinal mucosa Occludin,ICAM-1,IL-6,TNF-α.Using Flow cytometer to detect cell aptopois condition.The purpose was to study whether Pim-3 directly protect against the intestinal mucosa injury in post-burn rats on cellular and molecular level.Partâ… (In vivo)Changes of intestinal barrier damage and Pim-3 expression in acute post-burn ratsObjective To understand the pattern of intestinal barrier damage in severe burn and endotoxin treatment.changes of Occludin,ICAM-1,IL-6,TNF-α,pathology analysis were examined during 48 h follow severe burn and endotoxin treatment. Some molecular biology methods such as reverse transcription-polymerase chain reaction(RT-PCR)and western blotting so on were used to detect the changes of Pim-3 in intestine.The purpose was to investigate the variation pattern of intestinal barrier injury,.The variation of Pim-3 in post-burn rat and endotoxin rat in vivo.Methods Rats were randomized into A burn group(n=30);B endotoxin group(n=30);control group(n=6),followed a procedure to construct a well-described 30%TBSAⅢ°burn rats model.Changes of Occludin,ICAM-1,IL-6,TNF-α,HE stain was used to intestinal mucosa and pathological section in optical microscope and were analyzed at 3h,6h,12h,24h and 48h in post-burn and endotoxin treatment with rats sacrifice.And the level of Pim-3 mRNA and protein was detected with RT-PCR and Western blotting method in intestine,respectively.Result 1.Histological changes of intestinal mucosa:Samples taken postburn and made slides,stained by HE,showed infiltration of inflammatory cells and villi became edema,interstitial edema,tissue gap widen,became much more series than the control group.The changes became serious at 3PBH,reached peak at 12 PBH. Burn group and endotoxin group have the same change.Control group has no change.2.The level of Pim-3:The quantity of Pim-3 mRNA and protein was very low in control group rats.At 3h,6h,12h,24h,48h post-burn in burn group rats,The burn group quantity of Pim-3 mRNA was 12.25±1.24,21.13±1.78,14.21±1.12,2.12±0.11, 1.08±0.97 fold of that in intestine in control group rats,respectively.At 3h,6h,12h, 24h,48h,The endotoxin group quantity of Pim-3mRNA was 15.08±2.07,25.24±2.11, 16.32±1.23,2.15±0.23,1.10±0.87 fold of that in intestine in control group rats, respectively.The quantity of Pim-3 mRNA endotoxin group and burn group began to increase at 3h(p＜0.05),significantly increased at 6h,and achieved highest at 6h (p＜0.01),Compared with control group.There was no significantly different between burn group and endotoxint group rats(p＞0.05)The quantity of Pim-3 protein increased at 3h post-burn,and significantly at 6h.3.intestine barrier damage:In burn group,Western blotting assay showed that the level of occluding protein increased At 3 PBH and 6 PBH.RT-PCR assay demonstrated that the level of occludin mRNA was enhanced.At 3h,6h,12h,24h,48h,The burn group quantity of Occludin mRNA was3.25±0.26,6.65±0.24.,3.10±0.8,1.55±0.9,1.01±0.22 fold of that in intestine in control group rats,respectively.The endotoxin group quantity of occludin mRNA was 3.15±0.55,6.25±0.56,2.52±0.78,1.42±0.3,1.05±0.2 fold of that in intestine in control group rats,respectively.At 12 PBH,the level reached peak(P＜0.01),The level at 48 PBH increased gradually to normal.There was no significantly different between burn group and endotoxint group rats(p＞0.05).4. ICAM-1:RT-PCR assay demonstrated that the level of ICAM-1 mRNA was enhance. The level ICAM-1mRNA were 55.12±5.32,82.16±5.68,88.31±7.41,58.16±4.56, 56.12±3.89 times that of the control group,endotoxin group,At 3h,6h,12h, 24h,48h,,the levels were 37.81±4.48,89.68±6.67,58.43±5.67,53.24±4.36, 88.12±6.21 times that of the control group.The levels at 6 PBH and 12 PBH were higher than that of the control group,have statistical significance(P＜0.01).No significant difference(P＞0.05)between burn group and endotoxint group rats.5.The level of TNF-αmRNA and IL-6mRNA:There were no express in normal intestine.Burn and LPS enhanced the activation of TNF-αand IL-6(,p＜0.01),and induced the high expression of TNF-αtinintestine(reach,p＜0.01).Burn and LPs promoted the enhancement of on the activation of IL-6 and inducement of TNF-α.At 3h,6h,12h,24h,48h,The activity TNF-αof reached 30.12±4.87,76.12±6.87, 63.27±5.91,59.45±4.65,55.36±3.63 fold of that in control,and increased significantly than that of LPS group and burn group.Conclusions 1.Pim-3 play an important role in intestinal barrier damage in acute post-burn rats,and act as an endogenous protective substance against the intestinal mucosa damage.2.The level of Pim-3,occludin,ICAM-1,IL-6,TNF-αin intestinal epithelium is increased after severe burn injury and endotoxin treatment. 3.The damages of tight junction in intestinal epithelium are closely associated with increased level of occludin and decreased level of IL-6,TNF-αhave up-regulated.Partâ…¡The investigation of protection Mechanisms of Pim-3 against intestine cell injury on LPS stressObjective To observe the protection mechanism of pim-3 in intestine cell on LPS stress.Methods The primary neonatal rat intestine was culture in DMEM with 0.5ug/ml LPS,to construct a LPS stress cell model.LPS was dissolved in DMEM medium before.Cells were randomized intoâ‘ control group+LPS;â‘¡pEGFP-N₂+LPS group;â‘¢pEGFP-N₂/pim3+LPS group;â‘£control group;⑤pEGFP-N₂ group;â‘¥pEGFP-N₂/pim3 group.LPS the concentration was 0.5ug/ml.The degree of cell injury was evaluated by measure of apoptosis rate in flow cytometrr.The protection mechanism of Pim-3 on the intestine cell injury caused by LPS stress was investigated by measure of the level of occludin,ICAM-1,IL-6,TNF-αwith RT-PCR.Results 1.Intestine cell damage:The level of cell apoptosis,occludin mRNA,ICAM-1 mRNA have significant difference betweenâ‘¢group andâ‘¡group,â‘ group (p＜0.05).The apoptosis rate decreased,occludin mRNA reached high level and ICAM-1 mRNA inâ‘¢group.The apoptosis rate decreased,occludin mRNA reached high level and ICAM-1 mRNA inâ‘¢group.The level of cell apoptosis,occludin mRNA,ICAM-1 mRNA have no significant difference between⑤group andâ‘£group,;â‘¥group(p＞0.05).The level of occludin mRNA and ICAM-1 mRNA have no significant difference betweenâ‘£group,⑤group andâ‘¥group.The level of ICAM-1 mRNA have significant difference betweenâ‘ â‘¡â‘¢and④⑤⑥. 2.IL-6 and TNF-α:The level of TNF-αand IL-6 have no express in④⑤⑥group. The level of TNF-αand IL-6 have significant difference betweenâ‘¢group and â‘¡group,â‘ group(p＜0.05).The level of TNF-αand IL-6 have significant difference betweenâ‘ â‘¡â‘¢and④⑤⑥.Conclusions Oncogene Pim3 is not only inhibite intestine cell aptoposis but also ehance the function of Occuldin,reduce inflammatory factor.