The Effects Of SNPs In HSP90 Gene On MODSafter Severe Trauma And The Mechanisms Of Its Inhibitor In Inflammation

With the progress of the society and the development of advantaged tools of transportation, severe trauma and its complication, multiple organ dysfunction, are undoubtedly the most common critical diseases in clinic medicine. Especially for multiple organ dysfunction syndrome (MODS), it is the important cause of the death. In recent years, with the growing recognition of genome researcher has found lots of differences in genetic background of many diseases including trauma. So we can explain why the same degree of trauma (equal ISS score) leads to various consequence,some retrieved rapidly and the others continued to deteriorate accepted the same treatment.Heat shock protein 90 (HSP90) as a conservational chaperone widely exists in cells from bacteria to eukaryotic cells and its expression increases under trauma, infection, surgery, hypoxia, heavy metals, radiation and other stress conditions in order to maintain the steady-state of internal environment. The clients of HSP90 are various, including glucocorticoid receptor (GR) which is a major dowmstream molecular of the hypothalamus-pituitary-adrenal axis (HPA) and toll-like receprot 4 (TLR4) signaling pathway related molecules which induce inflammatory reaction. In view of the important role of HSP90 in the inflammatory response, the relationship between the HSP90βgene SNPs and inflammation and the damage in multiple organs and how to improve the effect of HSP90 on anti-inflammation are the focus of this topic.Therefore, the purpose of this study is to examine, analysis and comparison the SNPs in part of the healthy population in Chongqing region. Furthermore, in vitro experiments the expression of HSP90βmRNA in peripheral blood leucocyte with different genotypes and the expression of inflammatory factor TNF-αin U937 cells after LPS stimulation were detected to confirm the differenct effect of various genotype on inflammtion. In additionally, the transcriptional activity of HSP90βpromoter with various genotypes was examined through construction of luciferase expression vector containing the different HSP90β promoters. Moreover, the allele and the genotype frequency in the patients with severe trauma were tested and the association analysis was done to clarify the relationship between the SNPs in HSP90βgene and the severe extent of dysfunction of multiple organs.In addition, recent studies showed that HSP90 inhibitors also had a protective effect and reduced the mortality in septic mice, which is contrary to the generally accepted view of HSP90's protection effect. How to correctly regulate HSP90 and make it play a protective role of inhibiting inflammation? This project intended to study the seemingly contradictory role of HSP90 and its antagonist to clarify the mechanism of HSP90 inhibitor in protective effect and their conditions. This study deployed the model of acute lung injury induced by cecal ligation peferation (CLP) and LPS-induced cell model to test the role of 17-AAG, a kind of HSP90 inhibitor, at vious doses and time points. In advance, the mainly anti-inflammatory GC-GR pathway, the proinflammatory TLR4 signaling pathway and the interaction between them was examined to explore the underlying molecular mechanism. This research will provide theoretical and experimental basis for development of anti-inflammatory drugs.The main results and conclusions were summarized as follows:1. The -741G/C,-509C/T,-427A/C,-398 C/G and -257G/A polymorphisms within the promoter of HSP90βgene were closely linkaged in Chongqingnese Han population, so we selected -741G/C SNP as a genetic biomarker. Additionally, linkage disequilibrium analysis showed that among the -144C/A, -141 T/C and -741G/C there were strong linkage disequilibrium. All genotypes distribution was tested for departure from Hardy-Weinberg equilibrium by chisquare analysis.2. Experimental data of in vitro transcription activity showed that the mutant -144A promoter had a high transcription activity compared to -144C wild-type and caused more transcription of target genes. In response to ex vivo LPS stimulation, HSP90βmRNA expression was significantly higher and TNF-αmRNA expression was lower on peripheral leukocytes of AA homozygous genotype for -144 allele compared to homozygous CC and heterozygous CA. As indicated -144 SNP was a functional polymorphism and regulated the transcriptional activity of HSP90βgene.3. Clinical association analysis revealed that MOD scores in trauma patients with -144A allele was significantly lower than -144C allele, in other words,trauma patients carrying -144A allele was injured at a slighter extent compared to -144C carriers. So we presumed the mRNA expression differences resulting from -144 SNP within HSP90βgene were one of the most important factors lead to various inflammation and functional damages in multiple organs. Thus, it had important implications in the prediction and the judgement of the complication of MODS.4. It was confirmed that low doses of 17-AAG, a kind of HSP90 inhibitors, induced heat shock reaction characterized by elevation of heat shock protein in animals model of CLP and ex vivo experiments. In addition, the mRNA expression of TFN-αwere decreaed following the increase of HSP90 when with low doses of inhibitor. Although high doses of inhibitor increased the HSP90 level, the function of the protein was blocked and showed a damaged effect。5. In animal models, the low (0.5mg/kg) and high (2.5mg/kg) doses of 17-AAG improved the protein level of P-HSF1, which rapidly promoted the expression of HSP90, both at 3h and 24h time points. In addition, the low doses of 17-AAG decreased the protein level of TLR4 and the high doses of 17-AAG had no effect on the TLR4 expression.6. Ex vivo experiments revealed low (0.1μM) and high (0.5μM) concentrations of 17-AAG improved the expression of P-HSF1. Further more, low concentration of the drug enhanced the transcriptional activity of HSP90βgene and the protein expression via a dose-dependent mode. It was demonstrated that the low doses of 17-AAG alleviated inflammation through dowm-regulated the lipocortin 1, a downstream molecular of GC-GR pathway in U937 cells induced by lipopolysaccharide (LPS). On the contrary, high doses of 17-AAG inhibited the production of the lipocortin 1 via the GC-GR pathway and aggravated the inflammation.7. Data indicated both 17-AAG (0.1μM) and 50nM dexamethasone (DEX) decreased the expression of TLR4. More over, it was interesting that combined application of 17-AAG with DEX, a common glucocorticoid, significantly decreased the expression of TLR4 than the solely stimulation. The existed data demonstrated that HSP90 promoted the GR effects through raising the nuclear translocation of GR. So we speculated that DEX-induced GR effects inhibited the TLR4 signal pathway and low doses of 17-AAG indirectly decreased the TLR4 expression via prompting the GR activity by improved the HSP90 level without the inhibition of its function. Together, the idea of HSP90s as protectional moleculars is extensively accepted. Low dose of 17-AAG plays an important role in alleviation of inflammation via GC-GR pathway because of its induction of heat shock reaction. Moreover, it is obvious that synergistic effect of 17-AAG and DEX lead to a better anti-inflammatory effect because 17-AAG indirectly inhibited the TLR4 pathway. Therefore, the low dose of HSP90 inhibitor has a significant protective effect in severe inflammatory response. The synergy of with DEX can enhance the anti-inflammatory effect, while reducing the side effects of large doses of DEX, so 17-AAG is an ideal potential anti-inflammatory drug.