Study Of The Molecular Mechanism And The Effects Of PDS On The Endotoxic Shock In Rats Brain

21st century is a "Brain's Era". For a long time , it is only a wonderful dream for human being to explain the brain clearly because of the complexity of the cerebral structures and functions. In 1996,the Japanese scientists presented the slogan that "understanding brain""protecting brain""developing brain". The Noble Prize winner Watson Crick said:"There is no more important research than the study on the brain for our human being."IBRO(International Brain Research Organization) proposed that the 21st century is a "Brain's Era". In general, brain research has become one of the most popular topics in this century. Lipopolysaccharide(LPS) is the main component of endotoxin(ET) which is released from Gram negative bacteria after death or during the rapid growing course. As a pathogen, LPS exists extensively in the nature. After LPS entering the body, many pathophysiological reactions can take place, such as fever, dilation of blood vessels, vascular permeation increase, leukocytosis, complement activation, blood pressure decrease and etc. In some serious cases, LPS can also lead to disseminated intravascular coagulation(DIC), fatal shock and multiple organ dysfunction syndrome(MODS). Above all, LPS plays a very important role on the pathogenesis of Gram negative bacterial infection. With the development of basic medicine and clinical research, people have some insight of the structure, function, and action mechanism of LPS. But now the most study of the diseases caused by LPS is focused and limited on peripheral organs such as lung, heart, liver, kidney, and et.al. The corresponding knowledge about brain is little known by now. There are many suspending problems and many different views in the nervous system diseases because of the existence of brain barrier and the complicated structures. Scientists always think that endotoxin is too large to pass through the brain barrier, but some scientists believe that big dose of LPS injection from peripheral blood can come into the brain by passing through the brain barrier. At present , we still can not deny the possibility when the brain barrier is destroyed by diseases or big dose of LPS can enter hypothalamus and act on the neuronal cells. The recent research demonstrates that peripheral LPS injection can cause a series of intracerebral changes such as iNOS activity increase in the astrocytes which involved in the pathological process of CNS bacterial infection. At the same time, LPS can also stimulate the astrocytes to secrete IL-1,TNF-αand IL-6 which enhanced the inflammation injury of the brain. Furthermore there are still manydifferent even paradoxical results. Based on such backgrounds, this experiment has explored the oxidization injury of endotoxin shock rats cerebral cortex, endotoxin signal transduction pathway and the changes of NF-κB and glutamate transporter expression. Through the study of the LPS action mechanism on central nervous system, we aimed to look for the effective target medicine for treating endotoxin disease . On such background, we duplicated the animal models of endotoxic shock rats to reveal the molecular mechanism of cerebral injury by LPS and observe the Panaxadiol(PDS) effects as well. The results of this experimental research are as follows: 1. Endotoxic shock model: One hundred Wistar rats were divided into 5 groups randomly the experimental control group (Control group); the LPS endotoxic shock model group (LPS group); Dexamethasone administered group (LPS+DEX group); PDS low-dose group (LPS+PDSL group, 22.5mg/Kg); PDS mid-dose group (LPS+PDSM group,45.0mg/kg); PDS high-dose group (LPS+PDSH group,90.0mg/kg), and each group was divided into 2 groups: killed at 60min and killed at 240min after shock. LPS (4mg/kg) was sublingually administered intravenously to build up the animal models of endotoxic shock. In addition, among the PDS-treated groups, PDS was administered before the use of LPS. When blood pressure fell to 2/3 of the baseline it was regarded as the start point of shock. The mean arterial blood pressure (MABP) was monitored dynamically by the RM6000 4-lead physiologic recording apparatus. Meanwhile, the blood viscosity was determined, andhistomorphological changes of brain tissue was observed as well. (1) MABP dynamic observation: Injection of LPS, rats MABP fell to 2/3 of the baseline in all groups after 5 minutes and 10 minutes later, MABP fell progressively. MABP had an compensatory restoration at 20min of shock and at 30min reached the summit. From then on, the above index come down constantly again until the models got close to death at 240min. Though the other groups treated with PDS took on the similar effects after injection of LPS , 30-60 minutes later they had steady and constant effect of increasing MABP which is not like that of the groups treated with only LPS. The statistical analysis demonstrated that MABP in the groups treated with PDS was significantly higher than that of LPS groups at the time of 60min, 120min, 180min and 240min after shock (p<0.05,p<0.01,p<0.001). At the same time, there was no significance of MABP between PDS groups and Dex groups(p>0.05). The LPS endotoxic shock model duplicated successfully on the evidence that the decreasing percentage of MABP at the different time is accordance to what of general MABP. This gave off the individual variance interference and set up a good basis for further study. (2) Observation on pathological alternation of cerebral tissue. In LPS group brain tissue we observed that inflammatory cells infiltration, peri-capillary space increase, partial neuronalvacuolar degeneration, karyopyknosis, obvious contraction of Nissl's body and decrease of neurone amount. The tendency is dependant on stimulation time. Otherwise in LPS+Dex group, LPS+PDSL group, LPS+PDSM group and LPS+PDSH group, the degree of this kind of injury alternation was less relatively. (3) Microcirculation change is one of most important indicators reflecting shock state. Some people even thought shock is almost a hema-rheological phenomenon. : the blood viscosity in PDS groups are all significantly higher under various shear rates than that in other groups(p<0.05). It suggested that shock had been in out of compensatory and irreversible stage. (4) Result of the determination of LPO and NO contents, activity of SOD and NOS: The activity of SODs in Control group, LPS+Dex group, LPS+PDSLgroup and LPS+PDSM is significantly higher than that in LPS group(p<0.05) after 4 hours of shock. However LPO contents in former groups are significantly lower than that in LPS group. At the same time, the activity of NOSs and NO contents in Control group, LPS+Dex group, LPS+PDSL group and LPS+PDSM are significantly lower than those in LPS group(p<0.05) after 4 hours of shock. 2.The molecular mechanism of brain injury caused by LPS-induced endotoxic shock and the protective effects of PDS The precondition to express LPS biological effect is LPS binding corresponding receptors on the membrane. CD14 and Toll-like receptors(TLRs) are two very important model-recognizing receptors which play a important role on resistance of various pathogen.(1) The expression of CD14 mRNA and protein on cerebral cortex: In normal situation, the expression of CD14 mRNA and protein on cerebral cortex is not very strong. By the time of the LPS stimulation, results of CD14 mRNA expression and the expression of CD14 protein with western blot showed that in LPS group is significantly higher than that in control group after 4 hours of shock( p<0.05). While in DEX group and PDS groups, the expression of CD14 mRNA decreased significantly(p<0.05 vs LPS group). (2) The TLRs family mRNA expression changes on cerebral cortex: The experiment showed that, with the LPS stimulation time, TLR4 and TLR2mRNA expression increased constantly and significantly. While in LPS group, TLR9mRNA expression increased but had no significance versus Control group( p<0.05). However PDS could inhibit TLR4/TLR2mRNA expression. (3) IL-18 changes on cerebral cortex of LPS-induced endotoxic shock rats: The experimental results demonstrated that IL-18mRNA with RT-PCR and IL-18 protein expression with Western blot in LPS groups were significantly higher than those in Control group at the time of 1h and 4h after shock(p>0.05). But Dex and PDS could lessen the expression of them at some degree. 3.The expression of NF-κB/IκB on cerebral cortex In this study, we utilized EMSA method to detect NF-κB binding activity with DNA, use Western Blot to determine the expression of NF-kB subunit protein and use immunochemical method to observe the nuclear translocation process of NF-κB. Most current data showsthat NF-κB will be activated by stimulation after 30min, and reach peak after 1h.Thereafter, we kill the rats at the time of 1h and 4h to observe the NF-κB change. EMSA result showed that NF-κB binding activity with DNA significantly strengthened after LPS injection. In this experiment we extracted nuclear protein and cytosolic protein to detect the NF-κB subunit protein expression. Contrast to Control group, p65 of nuclear protein expression with Western blot was strengthened after 1h and 4h LPS injection(p<0.05)and the same result happened to p50 protein. In LPS+Dex group, LPS+PDSL group and LPS+PDSM group, p65 protein expression decreased significantly but p50 protein expression decreased not obviously. These results was accordance to that of EMSA. The research also showed that before and after LPS injection , the cytosol p50 and p65 protein expressed and not decreased significantly because of the p65/p50 nuclear translocation among different groups (p>0.05). The immunochemistry results demonstrated that the protein of p65 and p50 for Control group expressed mainly in cytoplasm of neuron. However they translocated into nucleus after LPS stimulation, and the immunochemistry showed the process clearly. In this study, we proved that the NF-kB was mediated by binding with I-kB and in LPS group I-kB mRNA expression was less than Control group obviously(p<0.05). 4.EAAT expression on cerebral cortex: In contrast to Control group after LPS injection, EAAT2mRNA expression decreased at 1h and 4h(p<0.05); But EAAT3mRNA onlydecreased a little without significance at 1h (p>0.05) whereas decreased obviously at 4h (p<0.05). The further Western blot results showed the same changing tendency of EAAT2 and EAAT3 protein expression. There were no obvious changes of EAAT1和EAAT4mRNA expression after LPS stimulation (p>0.05). in addition, EAAT3 protein expression increased in LPS+Dex group, LPS+PDSL group and LPS+PDSM group at 4h. The immunochemistry results demonstrated EAAT2 located on astrocytic membrane while EAAT3 expressed on neuronal membrane. In conclusion, during endotoxic shock, LPS provoked LPS/CD14 complex expression, and then activated TLR4/TLR2,in turn affected a series of subsequent protein. After phosphorylation of IkB which induced the translocation of NF-kB to the nucleus, NF-kB associated with specific DNA binding sites initiating gene transcription. It induced the expression of mRNA of a variety of pro-inflammatory mediators including iNOS/NO ,and IL-18. This process finally becomes the major cause to the brain tissue injury. The brain as an integrating nervous regulating organ, its functional alternation and structural injury will affect other organ's response to cytokines which canconfer that LPS may regulate the LPS/CD14/TLR4/NF-κB expression and some inflammatory cytokines release to cause cerebral injury and shock condition. In addition, other study shows that oxygen free radicals also can be some kinds of signal molecules to mediate NF-kB signaling pathway which involves the cell damage. Our results proved the brain EAATmRNA expression decrease after LPS injection associated with the free...