The Study On The Role Of Growth Factor Independence 1 In Endotoxin Tolerance

Gram-negative bacteria is one of the most common pathogenic microorganisms. The endotoxin also named as lipopolysaccharide(LPS) in the outer membrane of Gram-negative bacteria is the primary constituent of pathopoiesis. LPS which has extensive biological activity can induce severe inflammation in cascade and lead to sepsis,systemic inflammatory response syndromes(SIRS),multiple system organ failure(MSOF). Although it is nearly one century from Richrd Pfeiffe first reported it, endotoxemia is still a big chanllenged for doctors as its high morbility and mortality. There is no effective means to endotoxin now.Endotoxin tolerance was first described in the 1960-1970s. It was observed that animals receiving a low dose preexposure to bacterial endotoxin or lipopolysaccharide (LPS) had a markedly reduced mortality when rechallenged with a lethal injection of endotoxin. A partial explanation for the improved survival of endotoxin-tolerant animals maybe the reduction of inflammatory cytokines, such as tumor necrosis factor (TNF) and interleukin (IL)-1．In vitro studies of macrophages show that pretreatment with low-dose LPS produces significant inhibition of TNF in response to subsequent LPS stimulation. The mechanism of endotoxin tolerance is not clearly so far.Growth Factor Independence 1 (Gfi1) is a 55-kD nuclear zinc finger protein The protein has six C-terminal C₂H₂-type zinc-finger domains and a characteristic stretch of 20 amino acids, called the SNAG-domain, at its N-terminus. Expression of Gfi1 ranges from the hematopoietic and lymphoid system, to sensory epithelia, lung and parts of the CNS. Gfi1 as a transcriptional repressor can repress transcription by enhancing histone deacetylation. Gfi1 is essential for the development of granulocytes and plays a role in T-cell differentiation. Gfi1-deficient mice are severely neutropenic.Recent studies proved that Gfi1 represents a novel factor limiting the inflammatory immune response, which mediated by TLR4．Gfi1ˉ/ˉmice showed a very strong systemic response to the endotoxin/LPS and died of septic shock within 36 h. After LPS treatment, lungs of Gfi1ˉ/ˉmice showed a rapid accumulation of mononuclear cells and a significant overproduction of inflammatory cytokines such as TNF, IL-1βand IL-6．In vitro, alveolar macrophages can express Gfi1 induced by LPS. Alveolar macrophages but not airway epithelial cells from Gfi1ˉ/ˉmice were found to be responsible for the enhanced cytokine production. It proposed that Gfi1 exerts its regulatory function in alveolar macrophages downstream of the LPS receptor (TLR4) and upstream of TNF.So we hypothesized that Gfi1 may overexpress in endotoxin tolerance after rechallenged with LPS, which relate with inhibition of some inflammatory cytokines. In this study, we detected the expression of Gfi1 in endotoxin tolerant mice and macrophages in vitro to investigate the role of Gfi1 in endotoxin tolerance.Partâ… The Study on the Role of Growth Factor Independence 1 in the Lungs of Endotoxin Tolerance MiceObjective: To observe the Growth Factor Independence 1 (Gfi1) expression in the lung of endotoxin tolerance mice before and after challenged with high dose LPS.Methods: Seventy-four female C57BL/6 mice were randomized into normal control (NC) group and endotoxin tolerance (ET) group. Endotoxin tolerance models of C57BL/6 mice were induced by four daily intraperitoneal injections of 1mg.kg^-1 LPS. Normal control mice received intraperitoneal injections of the same volume saline. On the fifth day, mice were injected with 10 mg.kg^-1 LPS to induce endotoxemia. The general situation and survival rates were compared . Before (0h) and 2h,6h,12h,24h after mice were challenged with high dose of LPS, the pathology of lungs was observed, the protein production of TNF-αin lungs was measured by ELISA, the gene expression of Gfi1 in lungs was measured by semi-quantitative RT-PCR and the protein expression of Gfi1 in lungs was measured by immuno-histochemistry.Results: (1) In ET group , the survival rate was higher; the pulmonary inflammatory response was mild and the protein production of TNF-αwas not increased. The endotoxin tolerance models were made successfully.(2) 2h after mice challenged high dose of LPS in NC group, pulmonary pathological study showed inflammatory cells accumulated in lung and exudates. At 6h and 12h time point the pulmonary inflammatory response was severe, while at 24h point mild. In ET group the lesion was much mild than that in NC group at 6h and 12h, while at 2h and 24h was similar.(3) In NC group the level of Gfi1 mRNA expression before (0h) and 2h,6h,12h, 24h after challenged with high dose of LPS was 0．2271±0．1252,0．1716±0．1474,0．2151±0．0950,0．4259±0．2550,0．4120±0．1618 respectively. In ET group, those were 0．5618±0．2222,0．6305±0．2362,0．9046±0．2914,0．5335±0．2928,0．3552±0．1468 respectively. Compared with NC group, the expression of Gfi1 mRNA in ET group was upregulated significantly at 0h,2h,6h (0h p<0．05,2h,6h p<0．01) .There were no statistically significant changes at 12h and 24h (p>0．05) .(4) The location of Gfi1 proteins was mainly in alveolar epithelial cells and alveolar macrophages. The immuno-histochemistry positive index of Gfi1 protein in NC group before (0h) and 2h,6h,12h,24h after challenged with high dose of LPS was 14725．81±2311．01,18599．45±2681．13,18400．85±2631．86,14101．41±2734．11,20462．01±3468．31 respectively. In ET group, it was 18429．09±2315．09,17710．28±3153．19,22791．63±2273．45,23184．17±2811．97,20219．83±2574．17 respectively. The expression of Gfi1 proteins was upregulated significantly after challenged with LPS. Compared with NC group, the expression of Gfi1 proteins in ET group was upregulated significantly at 0h,6h,12h (p＜0．01) .There were no statistically significant changes at 2h and 24h (p>0．05) .(5) The level of TNF-αsecretion in NC group before (0h) and 6h after challenged with high dose of LPS was 55．12±12．25,94．00±17．38pg/ml respectively. In ET group, there were 105．93±26．14 pg/ml,51．10±10．08 pg/ml respectively. Compared with NC group, the level of TNF-αsecretion in ET group was upregulated significantly at 0h (p<0．01) . Compared with that at 0h in the same group, the level of TNF-αwas increased significantly at 6h in NC group (p<0．01) , while decreased significantly at 6h in ET group (p<0．01) .Conclusion: The induction of endotoxin tolerance is associated with a profoundly high level of Gfi1 in lung tissue when challenged with high dose of LPS. Partâ…¡The Study on the Role of Growth Factor Independence 1 in Endotoxin Tolerant Macrophages in VitroObjective: To observe the Growth factor independence 1 (Gfi1) expression in mouse endotoxin tolerant macrophage cell line RAW264．7 cells and thioglycolate-elicited mouse peritoneal macrophages cells before and after stimulated with high dose of LPS. To study on the role of Gfi1 in endotoxin tolerant macrophages in vitro.Methods: (1) Macrophages were pretreated with 100 ng/ml LPS for 20h to induce endotoxin tolerance, then were stimulated with 1000 ng/ml LPS . The control group macrophages were stimulated with 1000 ng/ml LPS directly. (2) Real-time PCR was performed to detect the expression of Gfi1 mRNA in RAW264．7 cells and thioglycolate-elicited mouse peritoneal macrophages cells before (0h) and 2h,6h,12h,24h after macrophages were stimulated with high dose of LPS. (3) Western blot method was performed to observe the expression of Gfi1 protein in RAW 264．7 cells before (0h) and 2h,6h,12h,24h after macrophages were stimulated with high dose of LPS. (4) Specific capture enzyme-linked immunosorbent assay (ELISA) was performed to quantify the level of TNF-αand IL-6 before (0h) and 2h,6h,12h,24h after macrophages were stimulated with high dose of LPS in RAW264．7 cells, and the level of TNF-αwas quantified in thioglycolate-elicited mouse peritoneal macrophages.Result: (1) 6h after macrophages were stimulated with 1000 ng/ml LPS, the level of TNF-αin control group was significantly increased than before stimulation, while that in endotoxin tolerance group decreased significantly. The endotoxin tolerance in vitro models was made successfully.(2) For RAW264．7 cells, the expression of Gfi1 mRNA at 0h and 6h time point in endotoxin tolerance group was higher than that in control group (p<0．05) . There were no statistically significant changes at 2h,6h,12h and 24h time point (p>0．05) .(3) For thioglycolate-elicited mouse peritoneal macrophages, the expression of Gfi1 mRNA at 0h,2h and 6h time point in endotoxin tolerance group was higher than that in control group (0h,2h p<0．05, 6h p<0．01) . There were no statistically significant changes at 12h and 24h time point (p>0．05) . (4) For RAW264．7 cells, the expression of Gfi1 protein at 0h,6h,12h time point in endotoxin tolerance group was upregulated than that in control group (0h,6h p<0．01, 12h p<0．05) . There were no statistically significant changes at 2h and 24h time point (p>0．05) .(5) For RAW264．7 cells, the level of TNF-αat 0h time point in endotoxin tolerance group was increased than that in control group (p<0．01) and at 6h,12h and 24h it was decreased than that in control group (6h,12h p<0．01, 24h p<0．05) . There was no statistically significant changes at 2h time point (p>0．05) .(6) For thioglycolate-elicited mouse peritoneal macrophages, the level of TNF-αat 0h time point in endotoxin tolerance group was increased than that in control group (p<0．01) and at 6h,12h it was decreased than that in control group (6h p<0．05, 12h p<0．01) . There was no statistically significant change at 2h and 24h time point (p>0．05) .(7) For RAW264．7 cells, the level of IL-6 at 0h and 2h time points in endotoxin tolerance group was increased than that in control group (0h p<0．01, 2h p<0．05) and at 6h,12h and 24h was decreased than that in control group (p<0．01) .Conclusion: In endotoxin tolerance in vitro models, the expression of Gfi1 mRNA and Gfi1 protein in mouse peritoneal macrophages were up-regulated after high dose of LPS stimulated than that in control groups. Gfi1 maybe join in the mechanism of endotoxin tolerance.