Studies Of Apoptosis And The Signal Transduction Molecules In Bacterial Endotoxin Tolerant Mice

Bacteria endotoxin lipopolysaccharide (LPS), a major component of the outer surface of Gram-negative bacteria, is a complex glycolipid composed of a hydrophilic polysaccharide moiety and a hydrophobic domain known as lipid A. Lipid A is the most conservation section in the LPS, and has no species-specificity. The toxicity of LPS was associated with integrity structure of lipid A. The LPS molecules is not toxic when it is incorporated into the bacterial outer membrane, but after release from the bacterial wall, its toxic moiety, lipid A, is exposed to immune cells, thus evoking an inflammatory response. LPS and other cell wall constituents are released from the bacterial cells when they multiply but also when bacteria die or lyse. Various endogenous factors like complement and bactericidal proteins can cause disintegration of bacteria, resulting in the release of LPS. LPS is the most important pathogen associated molecular patterns (PAMPs), which not only the main causative agent of Gram-negative bacteria infection (such as sepsis and septic shock), but also closed relationship of many human disease. Followed the immunodepressan, chemo-cytotoxic and broad-spectrum antibiotic generally applied, morbidity of sepsis and septic shock have gradually raise tendency since last century 50 era, the sepsis and septic shock had caused ~ 200,000 deaths annually in the United States. Case fatality of sepsis was 10%—15% in children and 40% in adult. There have not related report in our country. The pathogenic mechanisms of LPS are LPS activates monocytes and macrophages to produce proinflammatory cytokines such as tumor necrosis factor-α (TNF-α), interleukin (IL)-1, IL-6, IL-8, and IL—12. Macrophages also secrete, in response to LPS, a wide variety of other biological response mediators including platelet-activating factor, prostaglandins, enzymes, and free radicals, such as nitric oxide. Production of these inflammatory cytokines and mediators by monocytes/macrophages contributes to the efficient control of growth and dissemination of invading pathogens. However, excessive and uncontrolled production of these inflammatory cytokines and mediators may lead to serious systemic complications such as microcirculatory dysfunction, tissue damage, and septic shock with a high mortality.As soon as bacteria enter the body, host always defense bacteria attack to relief the damage, and at last, was died caused by cell apoptosis. Butexperimental animals and human become refractory to a subset of endotoxin-driven responses after an initial sublethal exposure to endotoxin. This phenomenon, termed endotoxin tolerance (also called LPS desensitization, hyporesponsiveness, or refractoriness) is thought to be a host adaptation, limiting deleterious inflammatory damage due to excessive activation of monocytes and macrophages by LPS and preventing development of septic shock.Endotoxin tolerance has been realized a century ago, but the mechanisms of endotoxin tolerance have not well known. It is also a considerable attention problem weather this phenomenon is associated with suppression of cell apoptosis. In this study, we, first, induced experimental animals to appearance endotoxin tolerance. Then, detected weather appeared apoptotic reaction in the liver of LPS-induced tolerant mouse. We, next, investigate the relationship between the enotoxin tolerance and the cell apoptosis, and the change of signal molecule both in the apoptotic reaction pathway and LPS signal transduction pathway.1. Establishment of endotoxin tolerant mouse modelWe use LPS that from Salmonella abortus equi according to the literature and Kunming mice, which were innate insensitivity to LPS, for experiment model in this study. The semi-lethal dose of LPS which we extracted is 66.78mg/kg, so we used D-GalN as sensitizer in this study. After combined with D-GalN, the lethal dose of LPS only need 2 u g. To found LPS-induced tolerant model, we first injected sublethel dose of LPS (0. lug) to mice, after different time points, re-injected lethal dose LPS in combination with D-GalN to induce endtoxin tolerance appeared.Experiment design: The experimental mice were divided into three groups randomly. In control group, mice were injected with LPS (0. 1 |ig), D-GalN or normal saline, respectively. In sensitive group, mice were challenged with LPS in combination with D-GalN directly. While in LPS-induced tolerance group, mice were pretreated with a minute amount of LPS (0. 1 P g) in different time points (3h, Id, 2d, 7d), and subsequently challenged with LPS in combination with D-GalN.2. Survival rate in endotoxin tolerance mouseThe survival rate of mice that pretreatment with sub-lethal LPS increased substantially, the survival rate is 100% after pretreatment of LPS 3h and Id,and then it gradually descended with the tolerant time prolong. At pretreated of LPS 7d, the survival rate dropped to 70%, still higher than that of sensitive group (K0.01). In the control group, mice were all alive. And on the contrary, mice were all dead in Id in sensitive group. The activities of ALT and AST in sera from the LPS-induced tolerance group were lower than those of sensitive and gradually recvered followed tolerance time prlong. It recovered at 38% and 35% of sensitive, respectively, at pretreated LPS 7d. The differences were significant (P < 0.01).The results of HE staining showed that there were no pathologically changes in tolerance 3h and Id mice livers. Followed the tolerance time prolong, it appeared some inflammatory in mouse liver but the structure of liver have no obviously changed. On the contrary, it has serious damage in the sensitive mouse liver, and lead to mouse dead at last. 3. Suppression of apoptosis in endotoxin tolerant miceAfter extracted from experimental mouse liver and detected by agar gel electrophoresis, genome DNA were shown the typical apoptotic characteristic— DNA laddering in sensitive group mouse, while it did not found in tolerant and control groups. The similar data were also obtained from TUNEL detection. In sensitive group mouse liver, the apoptotic reaction was shown evidently with strong color reaction (shown in deep brown). And that was not detected in LPS-induced tolerant and control groups, except individual liver cells in tolerance 7d were seen visible apoptotic phenomenon. So we conclude that cell apoptosis that caused by endtoxin is paralleled with host death, and endtoxin tolerance can suppress both cell apoptosis and host death.To approach the mechanism of suppression of apoptosis, we found that the Caspase-3 and DFF40, proteins in the liver, activities changed conspicuously. Their activities are fairly higher in the sensitive group mouse, while obviously down-regulated in tolerance and control group. And the activities of these two molecules had a gradually recovered tendency. The result of Caspase-3 activity shown that the Caspase-3 activity returned to 54% as that of sensitive group when pretreated LPS 7d. And in this time point, the transcribe activity of Caspase-3 also recovered at 53% as that of sensitive group. The similar result also obtained in DFF40. The DFF40 activity in tolerance 7d were at 45% of sensitive group in protein expression, and 54% of sensitive in transcriptionlevel. This indicted that the suppression of apoptosis in LPS-induced tolerant mice were caused by the molecules which associated with apoptosis down-regulated. And these molecules activities were gradually recovered followed tolerant time prolong.4. Alteration of signaling molecules in endotoxin tolerant miceIn present, the relationship between the change of signaling molecules in LPS signal transduction pathway and the mechanism of endotoxin tolerance was still not well known. In our study, we mainly investigated the expression of some key signaling molecules in LPS signal transduction pathway. Respond mediated by TLR-4 was the main reaction to LPS stimulation. In our study, Western-blotting results shown, TLR-4 and IRAK-1 expression were degraded after the endotoxin tolerance appeared. But there had not visibly changed in TRAF-60 And it showed the same results of RT-PCR as Western blotting. The transcription level of TLR-4 and IRAK-1 also displayed down-regulated in LPS-induced tolerant group. And molecules TRAF-6, TLR-2 and MD-2 unchanged in transcription level yet. So it indicated that TLR-4 and IRAK-1 may play impotent role in endotoxin tolerance.After LPS stimulated, NF-kB will be activated at last and lead to a serial of inflammatory reaction and apoptotic reaction. Our research found, the expression of I k B was obviously higher in tolerance group than that of in sensitive group. On the contrary, the subunit of NF-kB —p65, its expression was lower in tolerance group than in sensitive . During tolerant Id period, the expression of p65 was 25% of sensitive, just similar as the normal saline. And the expression of p65 showed recovery followed the tolerant time prolong. This suggested that NF- k B activity was suppressed after endotoxin tolerance which caused by degraded expression of p65. On the other hand, the high expression of I * B also suppressed NF-kB nuclear shifting and subsequent effectiveness.5. The role of LPS in different organs of mouseLPS can activate immunocyte to produce a variety of inflammatory cytokines and other biological response mediators. However, excessive and uncontrolled production of these inflammatory cytokines and mediators may lead to serious tissue damage and septic shock with a high mortality. So in this study, we researched whether there was inflammatory reaction and apoptotic reaction inother organs of mouse after LPS stimulated. The results shown, apoptosis appeared not only in liver but also in intestinal and thymus cell after LPS in combination with D-Gal challenged. The results of RT-PCR indicted that TLR-4 and MD-2 may play different role in different organs. TLR-4 had higher expressed in thymus than in other organs. MD-2 expression was higher in kidney than in others, but had not detected in gut, thymus and brain. However, TLR-2 may play unimportant role in LPS stimulated. What lead to these difference is the problem we will study.In this study, we investigated the alteration in cell apoptosis and host survival rate after LPS-induced tolerance. The expression difference of signal molecules, which associated with apoptosis and LPS signal transduction pathway, was also detected in LPS-induced tolerant and sensitive mice. In previous studies, mouse peritoneal macrophage or human peripheral blood monocyte always be applied in endotoxin research. In our study, we founded an intravital LPS-induced tolerance model. This model can imitate complicated internal environment influential factor of human, and provide a platform to human endotoxin tolerance research.