| BackgroundIntracerebral hemorrhage (ICH) is the most common disease of neurology. The fatal rate is as high as50%, and40%of the survivors left severely disabled. So it’s one of the most common diseases to threat the health of the elderly, and in recent years the age of onset is getting younger and younger. At present, there is still no effective method to treat the neurological deficit caused by ICH. Previous studies have shown that a large number of inflammatory cells exist surrounding the hematoma in rat model of ICH, and the inflammatory response is an important mechanism of secondary nerve damage after ICH. Nuclear factor-κB (NF-κB) plays a central role in the inflammatory response, and various inflammatory cytokines such as TNF-α, IL-1β are its downstream factors. Also it is closely related to the proliferation of the microglia cells and apoptosis of nerve cells. Until now, there is still no in-depth study devoted to the expression of inflammatory factors in perihematomal brain tissue in human and its relationship with apoptosis. In this study, we selected53cases with ICH hospitalized in the Emergency Surgery of Shandong University Qilu Hospital, studied their clinical data and brain tissue around the hematoma, and analyzed the expression pattern of NF-κB-mediated inflammatory cytokines and its relationship with apoptosis.Materials and Methods1. Clinical data and grouping53patients with basal ganglia hemorrhage, who admitted Emergency Surgery of Shandong University Qilu Hospital from November2010to November2012, were selected as the experimental cases. For all the53cases, the hematoma volume was40-80ml, and hematoma evacuation operation was carried out along the non-functional cortex. It included33males (62.26%) and20females (37.74%); the age arranged from30to70years old, with an average of56.7; the preoperative GCS score was13-14points for6cases,9-12points for32cases, and4-8points for15 cases. According to the time interval between onset and specimen collection, the53cases were divided into6experimental groups:8cases in≤6hours group (group A),14cases in7-12hours group (group B),12cases in13-24hours group (group C),8cases in25-48hours group (group D),6cases in49-96hours group (group E) and5cases in>96hours group (group F).8patients with benign disease and needed neuroendoscope operation were selected as the control group (Ctrl), including5males and3females, aged30-69years with an average of54.3.The study was written consented by the patients’ families and the hospital ethics committee. Exclusion criteria:rule out all factors which may influence the study, such as bleeding, inflammation, trauma, surgery, using drugs influencing the immune system, and suffering with underlying diseases within the previous month.2. Specimen collection:The brain tissue1cm away from the hematoma was collected from the experimental groups, and that at the junction of the gray and white matter was collected from the control group. Each brain tissue was divided into two parts, one of which was quickly fixed with the10%formalin for HE staining, immunohistochemistry, and detection of apoptosis, and the other was rapidly dropped into the liquid nitrogen and transferred into-80℃refrigerator within half an hour for RT-PCR and Western Blot detection.3. RT-PCR:Total RNA of the brain tissue was extracted and the expression of NF-κB p65, IL-1β, TNF-α mRNA was detected with RT-PCR method. Primer sequences:human NF-κB p65(321bp):F:5’-TGCTGTGCGGCTCTGCTTCC-3’, R:5’-AGGCTGGGGTCTGCGTAGGG-3’; human IL-1β (442bp):F:5’-ATAAGCCCA CTCTACAGCT-3’, and R:5’-ATTGGCCCTGA AAGGAGAGA-3’; human TNF-α (325bp):F:5’-CAGAGGGAAGAGTTCCCCAG-5’, and R:5’-CCTTGGTCTGGTA GGAGACG-3’; β-actin (539bp):F:5’-GTGGGGCGCCCCAGGCACCA-3’, R:5’-CT CCTTAATGTCACGCACGATTTC-3’.4. Western Blot:The nucleoprotein and cytoplasmic protein were extracted from the brain tissue by using the NE-PER Nuclear and Cytoplasmic Extraction Kit (Thermo Fisher Scientific Inc., USA). In the pre-experiment, HSP90and HDAC-1were selected as the internal references of cytoplasmic protein and nucleoprotein respectively to determine the purity of the extracts. The results suggested that the purity of the extracts was high. Then the nucleoprotein was extracted and the expression of NF-κB p65in the nucleus was detected with the Western Blot method, to evaluate the level of NF-κB activation. The cytoplasmic protein was extracted, and the expression of IL-1β of TNF-α was detected with the Western Blot method. Used primary antibody: anti-NF-κB p65antibody (B7162rabbit polyclonal, ANBO, USA), anti-IL-1β antibody (AP8531c rabbit polyclonal, ABGENT, USA), anti-TNF-a antibody (ab9579mouse monoclonal, Abcam, USA); secondary antibody:Peroxidase AffiniPure Goat Anti-Rabbit IgG (H+L)(Jackson ImmunoResearch, USA), Peroxidase AffiniPure Goat Anti-Mouse IgG (H+L)(Jackson ImmunoResearch, USA); protein concentrations were determined by using a Micro BCA Protein Assay Kit (Thermo Fisher Scientific Inc., USA).5. Immunohistochemical detection:The experiment was performed according to the instruction of the two-step immunohistochemistry kit. The sections were observed under the ordinary microscope with400x high magnification. Five no-repeat fields were randomly selected, the nucleus NF-κB p65positive cells, IL-1β and TNF-a positive cells were counted, and the numbers of positive cells in the five fields were added up as the results. Used primary antibody:same with Western Blot; secondary antibody:non-biotin rabbit hypersensitive two-step secondary antibody (PV-9001, GBI, USA), non-biotin mice hypersensitive two-step secondary antibody (PV-9002, GBI, USA).6. Apoptosis detection:Apoptosis was detected with the terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling (TUNEL) assay, and the experiment was performed according to the instruction of the TUNEL kit (Roche, Germany). The sections were observed under the fluorescence microscope (Olympus BX51) and then under the ordinary microscope after DAB color development with400x high magnification. Five no-repeat fields were randomly selected, and the numbers of positive cells in the five fields were counted and added up as the results.7. Statistical analysis:DP Controller and DP Manager were used to analyze the pathological sections. SPSS19.0statistical analysis software and Excel2003were used in the statistical analysis and charting. The variables were shown as mean±standard deviation. Student’s t-test was used to evaluate the results, and Pearson’s correlation coefficient was used to evaluate the linear relationship between two variables. Significance was set at P<0.05.Results1. Time-dependent changes of NF-κB p65, IL-1β and TNF-α at the mRNA and protein levels1.1Expression of NF-κB p65, IL-1β and TNF-α at the mRNA level detected with RT-PCRThe expressions of NF-κB p65, IL-1β, TNF-α at the mRNA level in each experimental group were all significantly different from the control group (P<0.001). The expression of NF-κB p65mRNA gradually increased over time after ICH, and reached the highest level at the13-24hours group, compared with which, the7-12and25-48hours groups did not show significant differences (PB/C, PC/D>0.05), and all the other experimental groups showed significant differences (PA/C, PC/E<0.001), suggesting that the expression peak hours were7-48hours. The highest level of the IL-1β mRNA expression was at the0-6hours group, compared with which,the7-12hours group did not show a significant difference (PA/B>0.05), and all the other experimental groups showed significant differences (PA/C<0.05, PA/F<0.001), suggesting that the expression peak hours were0-12hours. The expression of TNF-α mRNA gradually increased over time after ICH, and reached the highest level at the7-12hours group, compared with which, the13-24hours group did not show a significant difference (PB/C>0.05), and all the other experimental groups showed significant differences (PA/B, PB/D<0.001), suggesting that the expression peak hours were7-24hours.1.2Expression of nucleoprotein NF-κB p65, IL-1β and TNF-α at the protein level detected with Western BlotThe expressions of NF-κB p65in nucleoprotein, IL-1β and TNF-α in cytoplasmic protein in each experimental group were all significantly different from the control group (P<0.001). The expression of nucleoprotein NF-κB p65gradually increased over time after ICH, and reached the highest level at the25-48hours group, compared with which, the13-24hours group did not show a significant difference (PC/D>0.05), and all the other experimental groups showed significant differences (PB/D<0.05, PA/D, PD/E<0.01), suggesting that the expression peak hours were13-48hours. The highest level of the IL-1β expression in cytoplasmic protein was at the0-6hours group, compared with which, the7-12and12-24hours groups did not show significant differences (PA/B, PA/C>0.05), and all the other experimental groups showed significant differences (PA/C<0.05, PA/F<0.001), suggesting that the expression peak hours were0-24hours. The expression of TNF-α in cytoplasmic protein gradually increased over time after ICH, and reached the highest level at the13-24hours group, compared with which, the25-48hours group did not show a significant difference (PC/D>0.05), and all the other experimental groups showed significant differences (PA/C, PB/C<0.05, PC/E<0.005), suggesting that the expression peak hours were13-48hours.1.3Immunohistochemistry detection of nucleus NF-κB p65positive cells, IL-1β and TNF-α positive cellsImmunohistochemical detection of the experimental groups showed that NF-κB p65expressed in the nucleus of neurons and glial cells, which suggested that NF-κB was activated and migrated into the nucleus. There were significant differences between each experiment group and the control group (P<0.001). Nucleus NF-κB p65positive cells gradually increased over time after ICH, and reached the highest level at the13-24hours group, compared with which, the25-48hours group did not show a significant difference (PC/D>0.05), and all the other experimental groups showed significant differences (PB/C<0.05, PA/C, PC/e<0.001), suggesting that the expression peak hours were13-48hours. IL-1β expressed in the cytoplasm of neurons and glial cells, and there were significant differences between each experiment group and the control group (P<0.001). The number of IL-1β positive cells reached the highest level at the0-6hours group, compared with which, the7-12and12-24hours groups did not show significant differences (PA/B, PA/C>0.05), and all the other experimental groups showed significant differences (PA/D<0.05, PA/E<0.01), suggesting that the expression peak hours were0-24hours. TNF-α expressed in the cytoplasm of neurons and glial cells, and there were significant differences between each experiment group and the control group (P<0.001). TNF-α positive cells gradually increased over time after ICH, and reached the highest level at the25-48hours group, compared with which, the13-24hours group did not show a significant difference (PC/D>0.05), and all the other experimental groups showed significant differences (PB/D<0.05, PA/D, PD/E<0.01), suggesting that the expression peak hours were13-48hours.2. Time-dependent changes of apoptosis Apoptosis was detected with the TUNEL assay. The sections were observed under the fluorescence microscope (Olympus BX51)and then under the ordinary light microscope after DAB color development. A large number of TUNEL positive cells were seen for the experimental group. There were significant differences between each experimental group and the control group (P<0.001). The number of apoptotic cells gradually increased over time after ICH, and reached the highest level at the25-48hours group, compared with which, the13-24hours group did not show a significant difference (PC/D>0.05), and all the other experimental groups showed significant differences (PD/E<0.05, PB/D,PD/F<0.001), suggesting that the peak hours of apoptosis were13-48hours.3. Relationship between apoptosis and activation of NF-κBImmunohistochemistry detection demonstrated that the activation peak hours of NF-κB, the expression peak hours of IL-1β and TNF-α at the protein level were13-48hours,0-24hours and13-48hours respectively, in agreement with the Western Blot results, and the peak hours of apoptosis were13-48hours, which suggested that there was a consistent variation between apoptosis and NF-κB activation. Pearson’s correlation analysis revealed strong linear correlation between apoptosis and activation of NF-κB (P<0.0001).Conelusions1. The activation peak hours of NF-κB and the expression peak hours of IL-1β and TNF-a at the protein level were13-48h,0-24h, and13-48h respectively. The peak hours of apoptosis were13-48h, and correlation analysis revealed a close relationship between apoptosis and NF-κB activation.2. The results above were similar with the results obtained in experiments on ICH animal models.3. It is feasible to treat ICH by intervening the NF-κB activation, which can relieve the inflammation reaction, reduce apoptosis and improve the outcome of ICH patients. |
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