The Study Of The Role Of JNK/c-Jun And PI3K/Akt Signal Transduction Pathway And Protection Mechanism Of The Glutamine In Small Intestinal Injury Of Endotoxemia Baby Rats

BackgroundThe serious infection is one of the common causes of gastrointestinal dysfunction in the pediatrics' critically dangerous illnesses.Its pathogenesis is connected closely with the endotoxemia and the destruction of intestinal barrier function.The endotoxin is the cell wall ingredient of Gram-negative bacteria.Its main biological activity ingredient is the lipopolysacchcride.More and more importance is attached to gastrointestinal dysfunction in critically dangerous illness,which is considered as one of initial factors of multi-organs dysfunction.Dysfunction and failure of children's gastrointestinal tract usually indicates poor prognosis and aggravated condition.when the body is infected by Gram-negative bacteria,LPS affects Cell membrane acceptors and mediates the genesis and development of the cell injury.It changes the gene expression through the signal transmission cascade,causes the host cytokine's expression out of control and the change of the cell's metabolism and shape and so on. So LPS-mediated signal transduction mechanism has become a wide field of study and much progress has been made in recent years.MAPK Signal transduction pathway is the important messenger from the cell surface to the internal nucleus.It has identified four MAPK signal transduction pathways:ERK pathway,JNK pathway,P38 pathway and ERK5 pathway.JNK signal transduction pathway is an important pathway of LPS signal transduction pathways.C-jun is the direct substrate of JNK downstream.Except for growth factors,JNK pathway can be activated by LPS,TNF-α,L-1,ultraviolet ray, ray,thermal shock,and hypertonic activation of extra cellular,DNA denaturizing agent and so on.JNK pathway's activation is related with pro-apoptotic effect of the multi-systems.Researches have reported that JNK in Kupffer cells is an important signaling molecule of LPS signal transduction pathway.The JNK activation possibly participates in the damage of neuron that caused by brain ischemia and the occurrence and the development of the brain low oxygen pre-adaptation.It's also discovered that the abnormal activation of the JNK/C-Jun signal transduction pathway in the intestinal ischemia-reperfusion injury rats model of IL-10 gene deletion.However,the change of JNK/c-Jun pathway caused by gastrointestinal mucosal injury after serious infection was reported little at home and abroad.PI3K is an important molecular of the signal Wansduction process of growth factor super family.Akt is Serine/threonine kinase encoded by proto-oncogene c- Akt expression.It is a direct target protein of PI3K.PI3K is mainly activated through binding with growth factor receptor,which has anti-apoptotic effect and promotes the cell proliferation and differentiation.It relates with many kinds of biology event,Such as Vesicle transportation,cytoskeleton reconstruction,cell survival,tumor genesis and cell apoptosis and so on.PI3K signal transduction pathway as a key cell survival pathway is important to the enterocyte survival,proliferation and reconstruction.Some research indicates that PI3K is the upstream way of activation NF-κB and the NF-κB signaling pathway is the most important downstream pathway in the signal transduction pathway mediated by LPS.The activation of NF-κB may induce the expression of multitudinous special Inflammation-related genes like TNF,L-6,and L-8 and so on. However,the research has little yet been reported on LPS and PI3K/AKT signal transduction pathway.We have not seen the Domestic report.Gln is the most abundant amino acid of the blood circulation and free amino acids pool within the organization.Gin rather than glucose is the main energy origin of the intestines mucosa and other rapid proliferate cell.Many animal experiments and clinical researches show that Gln can play a role in the protection of the intestinal barrier function through maintenance intestines mucous barrier,adjustment of intestines immunity function as well as the protection of intestinal tract oxidation damage.Parenteral nutrition and enteral nutrition of appropriate dose Gln can increase intestinal mucosal surface area,illus height and crypt depth,Promote crypt cell mitosis, accelerate the renewal of enterocyte,enhance healing capabilities,promote the synthesis of mucus,strengthen the close link among the intestinal cells,reduce epithelia cell apoptosis,stop intestinal mucus's atrophy and increase the permeability induced by inflammation.At present,improvement of the growth and repair function of Gin to various pathological states of intestinal mucous has been fully confirmed,especially in the burn injury,infection,surgery,radiotherapy and chemotherapy of cancer patients, which has achieved certain results.But its mechanism is not fully understood.In brief,LPS,through affecting the cell membrane acceptor,activating a number of intracellular signal transduction systems and formation of a complex signal transduction network,finally causes the occurrences of endotoxin shock,the systemic inflammation responsive syndrome and multi-organs function failure.Especially in children,because of their immune function and intestinal barrier dysfunction, gastrointestinal dysfunction caused by severe infections often evokes other organs' dysfunction.The treatment has certain difficulty.Once in late stage,mortality is extremely high.And the mechanism of gastrointestinal dysfunction in children is not fully clear.This research,studying on the JNK/c-Jun and the PI3K/Akt messenger transduction pathway of small intestine mucosa of endotoxemia and Gln treatment,is for the purpose of discussing the cell signal transduction mechanism related with the small intestine mucosa damage caused by LPS and Gln protective function in order to explore the effective prevention and the treatment way.Materials and methods 1.Materials18-day-old rat model of endotoxemia was made by intraperitoneal injection of endotoxin(4mg/kg E.coli polysaccharide O55:B5).No adding any processing factors was served as normal control group(8 rats).The false-injection control group was defined as intraperitoneal injection of normal saline.Intraperitoneal injection of endotoxin was administered as lipopolysacchcride group,at the same time, intraperitoneal injection of endotoxin and glutamine(2g/kg)was administered as glutamine treatment group.Each group consisted of 40 rats.8 rats' final parts of ileum were taken for 4cm at the time point of 2,4,6,24 and 72 hours after the beginning of the experiment,respectively.They were fixed in 10%neutral formaldehyde,embedded in paraffin;they were preserved in 2.5%glutaric dialdehydes,examined by electron microscope ion;they were frozen in liquid nitrogen,stored in the refrigerator-76 and assayed of protein and nucleic acid.2.MethodsUnder the light microscope and the transmission electron microscope,the small intestine mucosa pathology changes were observed.The expression of small intestine mucosa proliferation cell nucleus antigen(PCNA)was detected by Immunohistochemical method.Small intestine enterocyte apoptosis was examined by TUNEL(TdT—mediated dUTP Nick-End Labeling)method,the expression of JNK, c-Jun,PI3K and the AKT mRNA was examined by the RT-PCR method,the expression of the PI3K total protein and JNK,c-Jun and the AKT phosphorylation protein was examined by Western Blot blot analysis.SPSS 10.0 software was used for statistical analysis.Results1.Morphology change in small intestineUnder the light microscope,the small intestine had not obvious pathology change. Under the electron microscope,the endotoxin group appeared varying degree of apoptosis morphology change,pathological change degree of the Gln group corresponding to various time points had reduced.2.Proliferation and apoptosis in small Intestinal enterocyteThere were lots of intestinal epithelial PCNA-positive cells in the control group. PCNA expression intensity in the endotoxin group was lower than that of the control group obviously(P＜0.01),the expression intensity reduced obviously at hour 2, achieved its minimum value at hour 4-6,and did not restore the normal level at hour 72.PCNA expression in the Gln group was higher than that of the endotoxin group obviously(P＜0.01),but was lower than that of the control group(P＜0.01).Compared with the endotoxin group,the expression at the time point of 2h,4h and 24h ascended remarkable,with the lowest point expressing at hour 6.The apoptosis cells in the control group was very few,the apoptosis index of the endotoxin group was higher than that of the control group obviously(P＜0.01),the apoptosis index namely ascended at hour 2,achieved the peak at hour 24,and did not restore normally at hour 72.Although compared with the control group,the apoptosis index of the Gln group was high(P＜0.01),expression of various time points compared with the endotoxin group was descendent(P=0.01).The apoptosis index of the Gln group increased along gradually in the progression,showing no descending trend at hour 72.3.The analysis of the expression of JNK,c-Jun mRNA and protein in small intestineJNK mRNA expression of endotoxin group compared with the control group was obviously enhancement(P＜0.01),began to elevate obviously at hour 2,achieved the peak at hour 6,did not restore normally at hour 72.The expression tendency of c-Jun mRNA and the JNK mRNA's were the same.The expression of JNK and c-Jun mRNA of Gln group compared with the endotoxin group had the drop tendency.However,it did not have statistic significance. The p-JNK protein expression of endotoxin group compared with the control group was obviously ascended(P＜0.01),began to elevate at hour 2,achieved the peak at hour 4-6,and did not restore normally at hour 72.Although p-JNK expression of the Gln group compared with the control group was high(P＜0.01),it compared with the endotoxin group had drop tendency obviously(P=0.01).Dropping tendency was remarkable at the time point of 4h and the 6h,the peak was located at the time point of 6h.The expression tendency of p-c-Jun protein and p-JNK were similar.Difference was that p-c-Jun protein peak of the Gln group was located at the time point of 4h.4.The analysis of PI3K,Akt mRNA and protein expression in Small intestineThe PI3K mRNA expression of Endotoxin group compared with the control group was strengthen(P=0.05),began to elevate obviously at hour 4,achieved the peak at hour 6.Expression of other time were not obvious different.The expression of Gln group is higher than that of the endotoxin group obviously(P＜0.05).Akt mRNA of endotoxin group compared with the control group elevated lessly,the expression of the Gln group compared with the endotoxin group also had not elevated obviously.The expression of PI3K total protein and PI3K mRNA were similar.The expression tendency of p-Akt protein differed from that of the PI3K protein and Akt mRNA,the expression of the endotoxin group p-Akt protein compared with the control group was obvious increased(p＜0.01),began to elevate obviously at 4h,then reached the peak at hour 24.The expression level at hour 72 had not remarkable difference with that of the control group.The p-AKT protein expression of Gln group was higher than the endotoxin group obviously(P＜0.01),began to elevate obviously at hour 2,and reached the peak at hour 4,which was nearly same as the endotoxin group after hour 6.Conclusions 1.The increasing of the apoptosis of intestinal enterocyte of Endotoxemia rats, declining of proliferation and destroy of the balance between cell proliferation and apoptosis are ones of the mechanisms of the intestinal barrier in endotoxemia.2.At the early stage of the endotoxemia,the baby rats small intestinal JNK/c-Jun signal transduction pathway is activated,which result from increasing of apoptosis of enterocyte and the aggravation of the damage;in the late period,PI3K/Akt signal passage is activated,participates the survival,the proliferation and the reconstruction of the small intestine enterocyte.3.The glutamine,possibly through suppressing the endotoxemia rats small intestine JNK/c-Jun signal transduction pathway's activation,promotes the PI3K/Akt signal passage activation in early time,reduces apoptosis,promotes cell proliferation, repairs damaged intestines mucosa,thus reduces the destruction of the intestines mucosa barrier and displays the protective function to the small intestine enterocyte.