The Study Of Glutamine On The Protection Of Cerebral Injury Of Young Rats Induced By Endotoxin

ObjectiveThe infection is a serious kind of diseases which threatens to the health of children. The incidence and case fatality rate of it are very high. The infectious cerebral injury affects the life span and living quality. It is necessary to look for a new way to prevent and treat this kind of illness. The study on the pathogene-sis and mechanism of cerebral injury as well as the application of glutamine attracts more and more attention.At present, the study on the cerebral injury caused by endotoxin indicated the pathological process are cerebral edema. It eventually develops to diffused degeneration and necrosis of neuron in cortex and hypomylination. It is now thought that the infectious cerebral injury is associated with the necrosis of neuron and oligodendrocyte injury. Its pathological process is a continuous procedure. The advanced stage generates from the activation in the early stage of inflammation. It leads to the occurrence and maintenance of cytokine chain reaction. The cytokines involved in cerebral injury induced by endotoxin are IL -1β, IL - 4, IL - 8, MIP - 1, TNF - a which participate in local injury and inflammatory reaction and PDGF, TGF - a, bFGF which take part in tissue recovery. The expression of cytokines are controlled by special reversed factors. A-mong them NF - kB is involved in gene controls of many cytokine and inflammatory mediators. It plays a main role in the network of inflammatory cytokines. Now more and more attention has been given to the relationship between NF -kB and cerebral injury.NF - kB exists in neuron. CNS, especially the neurons in the cerebral cor-tex and hippocampus contain complicated active NF - kB. These NF - kB may be involved in regulation of antioxidative system of the neurons with active metabolism. NF - kB has been found in neuron, density layer indicating that the proce signal has been transferred into nucleus. The study has shown that inactive NF - kB exists in glio, such as astrocyte, mecroglia. The main signals which can activate NF - kB in CNS are (1) the signals exist in the peripheral tissues, including inflammatory cytokines (TNF - a, IL - 1) , oxidation, stress, ultraviolet radiation, products of bacteria and virus. (2) special signals, including depolarization , neurotransmi tters (glutamic acid, opium - like substance) , neuro-growth factors and several different neurotoxic stimuli, such as concentrated glutamic acid0At the normal conditions HSP (heat stock protein, HSP) 70 mRNA is low in the cellular level. The amount of synthesis of it is also very little. HSP70 can be induced to synthesize in arterial endothelial cells by pretreatment of heat shock. And can also inhibit the degradation in arterial endothelial cells caused by TNF - a. Cerebral ischemia may induce the HSP70 expression of neural cells.PDGF ( platelet - derived growth factor, PDGF) is a kind of protein in the human platelets with the promotion of growth. It possesses extensive biological effects. It can maintain the growth, differentiation, and living of neuron. It is one of the main neuronutritional factors which can induce the development of brain. PDGF is one of main factors to accelerate the recovery or neurological system. It can not only protect the effective neuron, but also promote the continuous growth of nerve fibers.STAT3 is another kind of DNA binding protein which consists of 750 - 795 amino acid. It is extensively expressed in different types of cells and tissues, participating in physiological control of cellular growth, degeneration and apop-tosis. It has been proved recently that STAT3 is associated with diseases in the central nervous system, tumors and cardiovascular system.After the binding of ligand and receptor of growth factor, to form homogenous or heterogenous receptor complex, they combine JAKs in cytoplasm and make STAT3 activation, provoke tyrosine phosphatase activation of itself andJAK. Activated STAT3 left from receptor, interaction between SH2 (Src - ho-mology 2 domain) and Y701 and Y705, to form homogenous or heterogenous dimmers in cytoplasm. And then penetrat into nucleus, combines with special gene starters, activate gene transmission. STAT3 changes the temporal cytoplasm signal into long - term gene expression in order to control multifunctions of cells.Glutamine is free amino acid in cells with great amount. It is the precursor of biological molecules, such as purin, pyridine, nucleic acid and protein. It is also the precursor of ATP. It can promote the protein synthesis and inhibit protein degradation.Glutamine is also an important precursor of antioxidative — reductive GSH. It plays a main role in growth, keeping the integrity of cells, anti - inflammation and immune regulation. It provides a new idea for the prevention and treatment of endotoxic cerebral injury.It has not been reported recently on the role of glutamine in the treatment of endotoxic cerebral injury. In this study, animals models were established by the administration of LPS and the interference of glutamine intraperitoneally with normal diet. After the study of cerebral tissue on NF - kB , HSP70, PDGF - B, PDGFR - 3 and the expression of STAT3 from 10 days old rats, to investigate the function of glutamine in the protection of brain by the regulation of HSP70, NF - kB , PDGF and other transmission signals in order to provide the theoretical and practical sources for the prevention and treatment of endotoxic cerebral injury.Material and MethodAnimals10 days old Wistar rats were randomized devided into four groups by injection intraperitoneally different agonts. 1. Normal saline control. 1. LPS group. 3. Gin group A (Gln,lh later LPS). 4. Gin group B (Gin and LPS at the same time). There were five time points in each group (2h,6h, 12h,24h,72h). Samples were taken from eight rats at each time point. The brain tissue of themwere kept for the examination.To observe the pathological changes with optic microscope, including the neuron marked with NF ( neurofilament) and marked by astrocyte GFAP ( glial fibeill aryacidic protein ). To study the ultrastructure of them by Transmission E-lectron Microscope.To test NF - kB , HSP70, PDGF - B by immunohistochemistry and the distribution and expression of receptor ￡ of PDGF, STAT3 in brain. If the stain is brown in neurocytoplasm or intranucleus, or the brown stain on the cell membrane, it is positive.Western blot was used to measure HSP70, PDGF - B, PDGFR—(3, STAT3.RNA was extracted from the brain by guanidine isothiocyanate - phenol -chloroform method. PDGF' — B and PDGFR - (3 mRNA expression are measured by RT-PCR.ResultThe expression of NF and GFAP proteins in brainNeuron were marked by NF, astrocyte was marked by GFAP. The NF positive neuron reduced obviously at 6 h in LPS group. It was even less on the whole slide at 72 h. Positive stain of astrocyte increased. The NF positive neuron in Gin A group increased greatly compared with LPS group at 72 h. The NF positive neuron in GLn groups B increased to some extent. The GFAP positive stain increased largely with the Gin interference, especially at 24 h.2. The ultrastructure of neuron and astrocyte in brainThe ultrastructure of neuron in LPS group changed from 2 h to 72 h. The membrane of nucleus is not integrity. Vacuole deformity occurred in mitochondria with the lack of ridge. The density on the basement reduced. The arrangement of endoplasmic reticulum was irregular. Granule dropped from ribosome. Astrocyte swollen in LPS group. Nucleus increased, especially at 72 h. On the other hand neurons were interfered by Gin in two groups, at 24 h, the nucleus membrane and nucleolus cam be seen clearly, mitochondria, rough endoplasmicreticulum and Golgi complex can be seen in cytoplasm.3. The protein expression of NF - kB in brainThe nuclei of neuron in cerebral cortex in LPS group at 2 h, and obviously clear at 6 h and it lasted to 12 h. The positive stain of nuclei in Gin group A and Gin group B at 2 h can not be seen.4. The protein expression of HSP70 in brainThe stain of nuclei in cerebral cortex was weakened in LPS group at 2 h by immunohistochemistry. The amount of protein expression decreased with the measurement of Western blot, especially at 24 h. The stain of nuclei in neuron in Gin group A at 12 h increased. It also showed the amount of protein expression increased in Western blot.5. The protein of PDGF - B and its mRNA expression in brain tissue Few neuron PDGF - B positive stain were observed in LPS group at 72 h.In the two groups interfered by Gin at 72 h, positive stain of neuron in cortex could be seen. The analysis of Western bolt showed that the amount of PDGF -B protein expression reduced in LPS group at 12 and 72 h. The expression of PDGF - B mRNA at 12 h reduced. The expression of PDGF - B mRNA in Gin group B decreased after 2 h, and nearly reached to the normal level and maintained to 24 h.6. The protein of PDGFR - ￡ and its mRNA expression in brain tissue The stain on the cellular membrane of was weak positive in LPS group at 12h. The protein expression of PDGFR - (3 greatly reduced with the semiquantitive method from 12 h. The expression of PDGFR - |3 mRNA decreased at 12 h. The stain on the membrane of neuron increased in Gin group A at 72 h, the amount of protein expression increased obviously. The expression at 6, 12 h decreased and increased at 24 h in Gin group A. Weak stain of neuron membrane is positive at 72 h in Gin group B. The expression of PDGFR - (3 mRNA increased continuously.7. The expression of STAT3 protein;The STAT3 stain on the neuron of cerebral cortex was increased at 6 h in LPS group. The STAT3 stain of astrocyte increased at 2 h, and stain decreased at 24 h. The amount of protein increased at 12 h by the method of western blot.The stain on the neuron and astrocyte was positive and increased at 24 h in Gin group A. The protein content increased at from 6 h to 72 h. The neuron stain increased with the positive signal at 24 h in Gin group B. The stain on astrocyte also increased.Conclusion■- - ? ■■ ■ ' ■ ■.■-■ , > ■ .TJie cerebral neuron, was ruptured in the model of infectious cerebral injury induced by .LPS. The'reaction, of astrocyte.increased, Gin can reduce the injury. It showed that it is possible for Gin to protect the cerebral injury caused by the infection. The administration of Gin can reduce the patholodical alterations.In the model of cerebral injury induced by LPS, it was NF - kB that first shifted from cytoplasm to nucleus in neuron. That means it can regulate, the reg-ulation of cellular metabolism. Gin can inhibit the movement of NF - kB from cytoplasm to nucleus.The expression of HSP70 decreased dramatically in the model of cerebral injury induced by LPS. It may indicate that the low expression of HSP70 may be the molecular basis of cerebral neuron. Gin may promote its expression in order' to prevent the brain from damage.The low expression of PDGF - B, PDGFR - (3 and their mRNA in the cerebral injury induced by LPS was increased after the interference by Gin.' Gin may promote their expression in order to recover the neuron. . .'The expression of STAT3 increased in a short term in the model of cerebral injury induced by LPS. Gin can promote its expression.