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Objective To observe the effect of the nuclear factor (NF)-κB inhibitor on the inflammatory injury and the secondary remote damage in remote areas of the CA1 region in the right hippocampus of the focal cerebral ischemic/reperfusion rats,and the NF-κB essential modifier binding domain (NBD) peptide was used to inhibit the activation of NF-κB signaling pathway to explore the function and mechanism of the NBD peptide in restraining inflammatory injury and reducing secondary remote damage in the hippocampus CA1 region.Methods According to the random number table,the male Sprague-Dawley (SD) rats were randomly divided into a sham group (n =24),an ischemia/reperfusion (I/R)group (n =38),a NBD group (n =38) and a modified type peptide (MT-NBD) group (n =38),then at the time point of 24 h and 7 d after reperfusion,the above 4 groups were divided into 2 subgroups.The experimental models were made by middle cerebral artery occlusion (modified line plug method) for 2 hours.The NBD peptide and the MT-NBD peptide were respectively injected into the right hippocampus of the experimental groups.The injury of neurons was examined by the methods of H&E and Fluoro-Jade B-(FJB)staining.The levels of interleukin-1β (IL-1β) and IL-1Ra were detected by using enzyme-linked immunosorbent assay.The protein expressions of NF-κB p65 and IκBα were analyzed by Western blotting and the double-labelling immunofluorescence.Results Compared with the NBD group (24 h 0.206 ±0.013,7 d 0.090 ±0.012) and the sham group (24 h 0.120 ±0.007,7 d 0.100 ±0.014),the NF-κB p65 protein expression in the I/R group (24 h 2.340 ± 0.101,7 d 2.440 ± 0.081) was increased significantly (q =64.431,66.704,67.747,56.624,all P ＜ 0.05).The level of IL-1β was remarkably increased in the I/R group (24 h (1.850 ±0.192) ng/ml,7 d (1.000 ±0.178) ng/ml) compared with the NBD group (24 h (1.250 ± 0.211) ng/ml,7 d (0.560 ± 0.183) ng/ml,q =10.730,9.710,P ＜0.05).The percent of survival neurons was significantly lower in the I/R group (24 h 27.50％ ± 3.59％,7 d 28.10％ ±4.46％) and the MT-NBD group (24 h 27.30％ ±4.53％,7 d 26.30％ ±5.03％)than the NBD group (24 h 58.90％ ± 3.46％,7 d 68.40％ ±4.20％,q =19.949,19.731,2.139,22.249,all P ＜0.05).The FJB staining showed that the number of neuron degeneration in the I/R group (24 h 28.10 ±2.13,7 d 29.50 ±2.45) was higher than the NBD group (24 h 12.50 ±2.41,7 d 9.30 ±2.52,q =3.211,4.521,P ＜ 0.05).Compared with the other three groups (sham group:24 h 0.130 ± 0.008,7 d 0.150 ±0.010;I/R group:24 h 1.340 ±0.213,7 d 1.750 ±0.119;MT-NBD group:24 h 1.250 ±0.114,7 d 1.620 ±0.097),the IκBα protein expression in the NBD group (24 h 1.680 ±0.148,7 d 2.010 ±0.085) was significantly increased (q =6.348,9.139,9.414,1.711,5.277,5.555,all P ＜0.05).Compared with the I/R group (24 h (0.570 ± 0.028) ng/ml,7 d (0.430 ± 0.039) ng/ml) and the MT-NBD group (24 h (0.490 ± 0.042) ng/ml,7 d (0.380 ± 0.018) ng/ml),the level of IL-1Ra in the NBD group (24 h (1.390 ± 0.055) ng/ml,7 d (1.250 ± 0.043) ng/ml) was remarkably increased (q =4.577,6.205,9.683,6.389,all P ＜ 0.05).The results between the I/R group and the MT-NBD group were not significantly different.Conclusions The research shows that NBD peptide treatment contributes to altering the NF-κB p65/IκBα expression in nucleus effectively.And it directly regulates the NF-κB activation to alleviate the inflammatory injury in the hippocampus CA1 region after the secondary remote damage.
ABSTRACT
Objective:To investigate the expression and mechanism of NF-κB signal pathway in murine lupus nephritis.Methods:The BXSB mice as well as C57BL/6 of 16 weeks were used.Transmission electron microscope and PAS were used to detect the pathological change of renal tissue.RT-PCR and ELISA were used to detect the expression of HMGB1 mRNA and protein.The expression of HMGB1,p- NF-κB,RAGE,IκB and PCNA protein was detected by immunohistochemical stain,FCM and Western blot.Results:The level of BUN in serum and Micro-albumin in urine of BXSB mice was higher than that in C57BL/6 mice.The expression of HMGB1 mRNA and HMGB1 protein level in peripheral blood increased significantly in BXSB group.Compared with those in control group,electron microscopy and PAS revealed the thickness of glomerular basement membrane(GBM),fusion of foot processes partly of epithelial dell and subepithelial electron-dense deposits in the renal tissue of BXSBA mice.Compared with that of control group,expression of PCNA was higher in glomeruli of BXSB mouse.HMGB1 protein over-expression localized in cytoplasm and extracellular milieu,especially in proliferative glomeruli in BXSB group,while the HMGB1 protein primarily confined to the nuclear of tubule in control group.In BXSB group,the expression of p-NF-κB and RAGE increased,while the expression of IκB decreased.There were positive correlation between the expression of HMGB1,RAGE and p-NF-κB protein (r=0.833,0.621,0.848,P＜0.01),while the expression of p-NF-κB protein negatively correlated with that of IκB.Conclusion:HMGB1 could activate NF-κB through combining with its receptor-RAGE,induce the form of proliferative glomerulonephritis by promoting the proliferation of inherent cell of glomeruli,which may play an important role in the murine lupus nephritis.
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Aim To study the interfering effects of picrosideⅡ on the expressions of nuclear transcription factor kappaB(NF-κB)and inhibitor of NF-κB(I-κB)after cerebral ischemic reperfusion in rats.Methods Intraluminal thread methods were applied to establish the middle cerebral artery occlusion reperfusion models in rats.PicrosideⅡ(10 mg·kg~(-1))and salvianic acid A sodium(10 mg·kg~(-1))were injected from the tail vein for treatment.TUNEL positive cells were counted by immunofluorescence assay.The expressions of NF-κB and I-κB were determined by immunohistochemical assay,and the concentration of NF-κB and I-κB in brain tissue was determined by ELISA.Results The exprssions of NF-κB and I-κB were weakly and the apoptotic cells were scattering at cortex,striatum and hippocampus in the sham operative group.In the negative control group,the number of TUNEL positive cells and the expressions of NF-κB and I-κB increased,the absorption(A)values and the concentration were significantly higher than those in the sham operative group(P<0.05).While in the positive control and picroside groups,the expressions(A values)and concentration of NF-κB and I-κB and the number of TUNEL positive cells were significantly lower than those in the negative control group(P<0.05).There was no significant difference between the positive control group and picroside group(P>0.05).Conclusion Picroside Ⅱ might downregulate the expressions of NF-κB and I-κB to inhibit neuronal apoptosis induced by inflammation after cerebral ischemia reperfusion injury in rats.
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Purpose: In periprosthetic osteolysis, cytokines, which are secreted from macrophages by the stimulation of particles, up-regulate the signaling for osteoclast activation through RANKL (Receptor activator of Nuclear Factor Kappa-B Ligand). This study compared the reaction to the particles and RANKL in the macrophages by examining the changes in the pro-inflammatory signals. In addition, because erythromycin has an anti-inflammatory effect, the effect of erythromycin on the pro-inflammatory signals by particles and RANKL was also analyzed to clarify the mechanism for the anti-resorptive effect with particles. Materials and Methods: The Raw 264.7 cell line (murine macrophage cell line) was used for these experiments. The particles were made from PMMA (poly-methyl-meth-acrylate) and UHMWPE (ultra high molecular weight polyethylene) to enhance their stimulatory effects. Under the same culture conditions used for macrophages, the cells were treated with either particles or RANKL. The differences in the production of TNF-α, activities of MAP kinase, I-κB and reactive oxygen species (ROS) between the particle and RANKL treated macrophages were examined. The influence of erythromycin on these models was also observed. Results: Erythromycin inhibited ERK and p38 phosphorylation in both models, and suppressed the increase in H2O2 production in the particle-treated macrophages. However, erythromycin inhibited neither the production of TNF- in both models nor the production of H2O2 in the RANKL-treated macrophages. In addition, erythromycin reversed the suppression of I-κB by the particles. Conclusion: For the response of macrophages, erythromycin mainly suppresses the particle induced ROS and NF-κB activation compared with RANKL-induced osteoclastogenesis signaling. Erythromycin might suppress particle-induced osteolysis through these anti-inflammatory effects. Therefore, further studies on the downstream signals of osteoclastogenesis will be needed.