Expression Of Heat Shock Protein90and Damage Mechanism Of The Tissues And Cells In Acute Cold Stressed Rats

Background:Severe cold stress responses were always induced in human bodiesin the presence of extreme climates such as a heavy snow and freezing. Cold stressresponse has been considered as a new hot in the recent decades. According to ourknowledge, the level of neuroendocrine hormone changed during the cold stress,resulting in the release of stress hormones including catechol amine (CA) andglucocorticoid (GC) that could trigger changes of internal environments andfunctional disorders. The up-regulation of these hormones was biomarkers of stressresponses. Also, they could activate the immunocytes and endotheliocytes involved ininflammatory and immune reactions. Heat shock protein90(HSP90), an importantmember of the heat shock protein family quickly synthesized in response to stress,played a pivotal role in the early stage of cold stress and protein synthesis (includingassemble, folding, transmission, and transfer) as well as the stability of cytoskeleton.Enhanced expression of HSP90demonstrated organism adaptation and protectiveeffects of the human bodies. Meanwhile, the up-regulation of HSP90may causeinjuries to the cell functions. In addition, enhanced expression of HSP90may be abiomarker for evaluating the cellular injures under stress conditions. As the molecularchaperons of GR and its receptor, HSP90could affect the interaction between GR andits receptors. However, few reports were performed to investigate the injuries inducedby cold stress through detecting the expression of HSP90.Under stress states, extracellular HSPs could function as a danger-associatedmolecular pattern molecule (DAMP) and induce inflammatory and immune response,resulted in secondary injury. Previous studies indicated that enhanced expression ofHSP70was noted in the presence of increased GC expression to protect the organsagainst the injuries induced by decreased glucocorticoid receptor (GR). However,significant secondary injuries were always induced due to inflammatory disorders. Todate, it is not well defined whether enhanced expression of HSP90and decreasedexpression of GR in aortic tissues are involved in the stress associated injuries undercold stress. Another study indicated extracellular HSP70might be a danger signal forthe immune system, inducing microenvironment changes. HSP70could induceimmune response through the interaction with antigen presenting cells. Also, it could activate the innate immune response through binding the Toll like receptor2(TLR2)and TLR4, leading to the secretion of inflammatory reaction associated factors, i.e.tumor necrosis factor a (TNF-a), IL-6and IL-1.As is known to all, HSP70and HSP90are endogenic ligands of TLR4whichexpressed in the membranes of immunocytes and endothelial cells. Their interactionwill trigger the signal pathways associated with inflammatory reactions and inducethe innate immune response. In the presence of lipopolysaccharide, significantincrease of TLR4was noted in the endothelial cells, which can induce the release ofNF-κB, ICAM-1and TNF-α, activate the inflammatory reactions, and result in theinflammatory and immune injuries in the vascular endothelial cells. Moreover, HSPcould induce antoimmunity through binding with major histocompatibility complex Iand/or II. Previous studies indicated that HSPs could induce the structural changes ofT lymphocytes subpopulation CD28in coronary atherosclerotic plaque, resulting inthe proliferation of CD4~+CD28~-T lymphocytes. As CD4~+CD28~-T lymphocytes arenatural killer cells with long life span, it is of great clinical significance to identifytheir antigens. To date, the association between HSP90and inflammatory andimmune injuries was not well defined in the rat aortic endothelial cells after coldstress.In this study, the expression of HSP90in rat aorta, spleen, myocardial tissue andlymphocytes well as its serum concentration of HSP90HSP90was investigated atdifferent time points after cold stress. The adaptation to the cold stress and injuryinduced by cold stress was illustrated.We aim to illustrate the role of HSP90inmediating the innate immunity initiated by TLR-4and its function in the injuriesinduced by cold stress. Additionally, the role of HSP90in inducing proliferation ofCD4~+CD28~-T cells through antigen presentation, the activation of acquired immunityas well as its role in endothelial cell injuries induced by acute cold stress wereinvestigated in this study.Methods:Epinephrine,Norepinephrine,ACTH,cortisone,ET-1,NO and,LDH weredeteeted by ELISA. IFN-γ,TNF-a,IL-2,Grazm-BmRNA were deteeted by real-timeRT-PCR.Expression of HSP90,TLR4and GR were deteeted by western-blotting.DCsand T lymphocyte were extracted in vitro from rats spleen observed themorphological changes;detected the variation of phenotype DC and T lymphocytesubtype by flow cytometric analysis;the stimulating capacity determined in allogenicmixed lymphocyte reaction(MLR) by3H-TdR Proliferation Assay;ELISA was used to analyze the level of cytokines secreted by mDCs and stimulated lymphocyte in themedium of MLR.(ConA) antigen stimulate spleen T lymphocyte proliferation by3H-TdR Proliferation Assay.Resultes:The cold stress has the important influence on neuroendocrine functionsuch as: cortisone,ACTH. The ELISA showed that the E and NE got up at6hour,peak value at12h (9.11±2.19ng/ml;11.27±2.01pg/ml) then down at24hourafter-4℃cold exposure.The cortisone(Cor) retained in the higher level at more partstime of cold exposure, and obviously high at12hour (112.2±1.12ng/ml) and24hourafter end cold exposure.The cortisone (Cor) retained in the higher level at more partstime of cold exposure,and obviously high at12hour and24hour after end coldexposure. This study discovered that animal Cor concentration increase did notdepend on HPA axial regulation,but not indicated that the glucocorticoid effect wasenhanced.That may be concerned with glucocorticoid receptors decreased andcatecholamine increased,and HSP90expression enhanced.From cold environment into ordinary temperature,equal to exert a new stress stimulus,so ACTH and Cor will goup quickly in a short time. The catecholamine,ACTH and Cor are sensitive to coldstimulus.They can as the supply indexes evaluating cold stress.This article detected HSP90express amounts in cold stress rats histiocyte usingwestern-blotting method.At cold environment, The myocardium was sensitive to coldstress reaction, The HSP90levels changed in a bigger extent and emerged highervalue after cold exposure6hour and12hour (p<0.01).The The HSP90went upquickly at1hour after ending cold expose,afterward tend gradually to normal levelsthrough wave up and down.The change of aorta HSP90levels increased obviously at6hour,12hour and18hour (p<0.01).Then HSP90levels went up again at12hour and24hour after ending cold exposure(p<0.01). The spleen HSP90was basic normal inperiod of cold exposure,but appeared higher at12and24hour after cold exposeending(p<0.01). The lymphocyte HSP90express levels increased quickly andretained high level.It went up obviously again at12hour and24hour after coldexposure ending.The research discovered, at cold environment,the faster lymphocyteHSP90expression,and the longer HSP90keep in high levels.That having somethingto do with body facilitates subsequence stress.The high expressed of HSP90in different tissue cells has the physiological foundation respectively.The resultsdemonstrated that it play a part in protective cell function,on the other hand, denotedthat histiocyte function was placed in the urgent condition.According neuroendocrine hormone and different expression amount of HSP90in histiocyte in cold stress rats,which model established0,12and24hour after coldexpose12hour ending were used as indexes to study.The aortic tissue was collectedfor pathologic study. Plasma level of ET-1,NO and LDH were detected by ELISA.Plasma level of NO were lower and level of ET-1and LDH were higher..pathological observation showed that12h and24h groups could significant thedamage of the intima of the aorta.Results By TEM demonstrated desquamation ofendothelial cells, chromatin condensation, vacuolated cytoplasm.The results are described as:The expression of co-stimulating factor CD86wassignificantly up-regulated in12h and24h groups (p<0.01),Which T lymphocytesco-cultured (MLR) secreted higher levels of pro-inflammation cytokines(IFN-γ)(p<0.01).The percentage of CD4~+CD28~-T lymphocytes was significantly higher in12h and24h group(p<0.01).Research in vitro:the peripheral blood mononuclear cells(PBMC) are obtainedby separation technology,the CD4~+T lymphocytes are obtained by magnetic activatedcell sorting technology,which are co-cultured with HSP90-DCs in vitro.Using flowand ELISA to investigate at whether the HSP90can promote the cells from PBMC ofhuman source to the mature DCs and whether the HSP90-loaded DCs can reducedCD28molecule expression and IFN-γ secretion of CD4~+T lymphocytes.Then detectedexpression of CD4~+CD28~-T lymphocytes by Real-Time RT-PCR.The results are described as:Detection of CD4~+CD28~-T lymphocytes percentagein10ug/LHSP90-DC and20ug/LHSP90-DC were significantly higher than controlgroup(p<0.01),the concentrations of supernatant IFN-γ(14.3±2.26;15.4±41.5)ng/Lwere significant higher than control group(4.5±0.6)ng/L(p<0.01).CD4~+CD28null Tcells are terminally differentiated and have pro-inflammatory functions characterizedby the expression of high levels of interferon-g (IFN-γ),tumour necrosis factor-a(TNF-a),andIL-2mRNA(p<0.01).CD4~+CD28~-T could efficiently inhibited endothelialcells proliferation(p<0.01),Atorvastatin inhibited the preration of CD4T lymphocyte (p<0.01).DCs and T lymphocyte were extracted in vitro from rats spleen, MTT was usedto assay the ability of DCs from each group rats spleen to stimulate autologous T cellproliferation and cytotoxic T lymphocytes(CTL) mediated lysis HSP90-loadedendothelial cells the and CFSE-flow was used to detected the prolifieration of HSP90loaded endothelial cells;ELISA was used to assay supernate of interferin-γ and LDH.The results are described as:Capacity of DCs from12h and24h group andHSP90-DC mixed with autologous T cell proliferation(19.5±9.5;32±5.4)%significantly higher than control group(2.8±0.5%()p<0.01,which could efficiently killendothelial cells loading HSP90(p<0.01).Atorvastatin could inhibited the function ofCTL on endothelial cells loading HSP90,and the concentrations of supernatant LDH(125±16.5)IU/L in Ato group significantly lower than control group(p<0.01).Conclusion:Under the cold stress12h, the plasma level of catecholamine and hormone riseto peak, after stoping cold stimulation, the level of cortical still remain at high levels.It went up obviously again at12hour and24hour after cold exposureending,which the high expressed of HSP90in different tissue cells.12h and24h groups after cold exposure ending,could significant the damage ofthe intima of the aorta,which Plasma level of NO were lower and level of ET-1andLDH were higher.In the12h and24h group after rats cold exposuring12h of spleen DC functionmature and T lymphocytes sensitization,The percentage of CD4~+CD28~-T lymphocytessignificantly increase;DCs from the12h and24h group rats spleen to stimulateautologous T lymphocytes co-cultured (MLR),IFN-γ increasing secretion, which mayplay an important role in directly lyse endothelial endothelial cells loadingHSP90,which can be inhibited by Atorvastatin.In the vitro,HSP90–DC could reduced CD4~+T lymphocytes to express CD28molecules,resulting in CD4~+CD28~-Tlymphocyte proliferation, CD4~+CD28~-T cells areterminally differentiated and have pro-inflammatory functions characterized byexpression of high levels of interferon-γ(IFN-γ),tumour necrosis factor-a (TNF-a),and IL-2mRNA.HSP90as a self-antigen related to CD4~+CD28~-T lymphocyte proliferation and IFN-γ increasing secretion, which can be inhibited by AtorvastatinHSP90in mediating the innate immunity initiated by TLR-4and its function inthe injuries induced by cold stress. Additionally, the role of HSP90in inducingproliferation of CD4~+CD28~-T cells through antigen presentation, the activation ofacquired immunity as well as its role in endothelial cell injuries induced by acute coldstress were investigated in this study.