Research On The Molecular Regulation Mechanisms Of Cold Shock Protein Which Involved In Neuroprotective Effect

Background Mild hypothermia has been widely used in surgical operation of heart,central nervous system and organ transplant, for the temperature is lowered to reducethe metabolic rate of the tissues and organs, which could make a significant protectiveeffect on the heart, brain, liver and other organs. But it is still not very clear that howmild hypothermia could specifically produce a protective role. This stage of theexperiment mainly further investigated the state of molecular mechanisms of CIRPwhich involved in neuroprotection with mild hypothermia, by dealing rat neuronswith mild hypothermia, overexpression of CIRP and RNA interference. The prophasestudy had found that the elevated CIRP expression by treating with mild hypothermiacould inhibit H2O2-induced apoptosis in rat neurons, so on this basis we did furtherstudy to investigate possible CIRP-mediated apoptosis related signal transductionpathways with mild hypothermia.Objective Through research on CIRP-mediated apoptosis related signal transductionpathway, then we further investigated the molecular mechanisms of cold shockprotein which involved in neuroprotective effect with mild hypothermia, meanwhile itcould provide certain basis for the mechanisms of mild hypothermia treatment inbrain injury.Methods We dissociated, inoculated and cultured rat cortical neurons in vitro andthen researched the change of relevant factors on CIRP-mediated apoptosis relatedsignal transduction pathway with mild hypothermia from the cell, gene and protein levels. The experiment was accomplished with applications of the Annexin V-FITC/PIon neuronal apoptosis detected by flow cytometry, the ultrastructure of neuronsscaned by transmission electron microscope (TEM), infecting neurons with CIRPoverexpression and CIRP shRNA interference lentiviral plasmid vector, RT2profilerPCR arrays pathway analysis by quantitative real-time PCR and Western blotting.Results Our study found that the number of neuronal apoptosis was declined and theintracellular ultrastructures remained intact after mild hypothermia treatment throughapoptosis detection with flow cytometry and transmission electron microscopyscanning. RT2profiler PCR arrays pathway analysis by real-time quantitative PCRamong84apoptosis signal transduction pathway related factors showed:32℃12htreatment group versus normal control group,12genes were upregulated,38geneswere downregulated, and other34genes were no significant changed; CIRPoverexpressed group versus normal control group,15genes were upregulated,46genes were downregulated, and other23genes were no significant changed; CIRPsilence expressed and then32℃12h treatment group versus normal control group,9genes were upregulated,40genes were downregulated, and other35genes were nosignificant changed. After integrating all datas, we noticed that there were somecommon changed factors with singly CIRP overexpressed or mild hypothermia32℃12h treatment, moreover a part of these factors were no significant changed after RNAinterference CIRP expressed and then32℃12h treatment. After a careful analysis ofthis part of the factors, we found their distribution and changes were in accordancewith the mitochondrial apoptotic pathway, including Bcl-2, Bax, Bad, Bak, Cycs,Apaf-1, Caspase-9, Caspase-3and so on. The results of Western blotting analysisshowed that changes in the protein level of each factor were consistented with thegene level changes. The target proteins CIRP and RBM3expression was significantlyincreased after mild hypothermia32℃12h treatment, and Bcl-2, Akt, p-ERK1/2expression levels were also increased, Meanwhile Bax, Bad, Bak, Cycs, Apaf-1,Caspase-9and Caspase-3expression were reduced in different extents. The changetrends of each factor with CIRP overexpressed treatment were consistented with mild hypothermia32℃12h treatment. These factors were no significant changed after RNAinterference CIRP expressed and then32℃12h treatment.Conclusion The cold shock protein expression were increased after mild hypothermia,then activated CIRP-mediated anti-apoptotic signal transduction pathway, inhibittedneuronal apoptosis, accordingly play a protective role. These results indicated thatcold shock protein may be involved in neuroprotection with mild hypothermia, andplayed an important role in the molecular regulation mechanisms, simutaneouslyprovided certain basis for the mechanisms of mild hypothermia treatment in braininjury. Background In recent years, a number of foreign scholars have found cold shockprotein high expressed in many tumor that may be involved in tumorigenesis,proliferation, aggravation and many other aspects. There is no relevant clearlyreported about tumors of the nervous system, so at this stage, I pay more attention toastrocytoma which is the most common malignant tumors in the nervous system, andto take the differential expression studies of cold shock protein in low-grade (WHOgrade I-II), high-grade astrocytomas (WHO grade III-IV) and normal brain tissues.The detections were performed from mRNA and protein levels at the same time. Thepurpose of the study is considered from another point to explore its role in neuralprotection by observing its impact on the nervous system tumors, especially otherpossible mechanisms.Objective To investigate the expression of cold shock protein (CIRP and RBM3) inlow-grade, high-grade astrocytomas and normal brain tissues. To explore therelationship between the differential expression and tumor malignant degree.Methods The cold shock protein expression of mRNA and protein levels in normalbrain tissues and astrocytomas of different grades was detected by RT-PCR,quantitative real-time PCR, immunohistochemistry and Western blotting.Results The results of quantitative real-time PCR detection suggested that CIRPmRNA expression was no significant difference in each grade astrocytomas andnormal brain tissues, but RBM3mRNA expression was significantly higher inastrocytomas than in normal brain tissues, also there were significant differences between high-level and low-grade astrocytomas, especially in the glioma cell lineU251and U87. Successively detected by immunohistochemistry and Western blotting,it was found that CIRP and RBM3were located in the nucleus in normal brain tissuesand astrocytomas, and CIRP expression was no significant difference in each gradeastrocytomas and normal brain tissues, but RBM3expression was significantly higherin astrocytomas than in normal brain tissues, meanwhile its expression was graduallyincreased with the increased malignancy degree of astrocytomas.Conclusion CIRP expression was no significant difference in each gradeastrocytomas and normal brain tissues, but another gene RBM3of the same family interms of the level of mRNA or protein expression was significantly higher inastrocytomas than in normal brain tissues, also there were significant differencesbetween high-level and low-grade astrocytomas. These data suggested that RBM3overexpression may be closely related to the malignancy degree of astrocytomas.Moreover RBM3may play the role of a certain degree in the development andprogression of astrocytomas.