Objective:To investigate the expression of nuclear factor-?B(NF-?B)subunits P50 and c-Rel protein in primary cortical neurons of Wistar rats at different time points of oxygen glucose deprivation/reoxygenation(OGD/R).Methods:The neurons dissociated from the cortex of the neonatal rats were primary cultured and were identified by immunocytochemistry.OGD/R model was established.The study was divided into 6 groups according to different processing methods,including normal group,OGD 4 h treated,OGD 4 h/R 2 h treated,OGD 4 h/R 6 h treated,OGD 4 h/R 12 h treated and OGD 4 h/R 24 h treated groups.The expression of NF-?B P50 and c-Rel protein in neurons was examined by immunocytochemistry method and Western blotting.Results:(1)Immunocytochemistry detection targeting neuron specific enolase(NSE)and beta-Ⅲ tubulin confirmed that the cultured cells were neurons.(2)The expression of NF-?B P50 protein was significantly higher in OGD 4 h group than in control group(P

Objective To study the inhibitory effects of circular dumbbell decoy oligodeoxynucleotides targeting NF-?B on TNF-? expression in cortical neurons under the oxygen glucose deprivation/reoxygenation(OGD/R).Methods The cultured neurons dissociated from the cortex of neonatal rats were observed on the 7th day after culture.To make the ischemia/reperfusion model in vitro,the neurons were exposed to OGD 4 h before cultured in normal culture media.The neurons were divided into 5 groups according to different processing methods 2 h before exposure to OGD,including normal group,OGD4 h/R6 h treated,NF-?B decoy ODNs treated,scrambled decoy ODNs treated and TransfastFastTM Transfection Reagent treated groups.The protein level of NF-?B P65 in primary neurons was detected by Western blotting.The protein and mRNA levels of TNF-? were detected by immunocytochemical method and RT-PCR,respectively.Results NF-?B decoy ODNs could effectively inhibit the expression levels of NF-?B P65 protein,TNF-? mRNA and protein in neurons(P

Objective To investigate the effect of electrical stimulation to cerebellar fastigial nucleus on expression of nuclear factor-kappa B (NF-кB) P50, tumor necrosis factor-α (TNF-α) and Bcl-xL mRNA in rats brain after cerebral ischemia-reperfusion. Methods Sprague-Dawley rats were randomly divided into normal control group (NC group), cerebral ischemia-reperfusion group (I/R group), fastigial nucleus stimulation (FNS) group, and fastigial nucleus lesion (FNL) group. A focal cerebral ischemia-reperfusion model was established with middle cerebral artery occlusion (MCAO). 7 and 14 days after operation, the infarct volume was measured, and the protein of NF-кB P50 in rats brain was detected with Western blotting; the expression of TNF-α and Bcl-xL mRNA was detected with RT-PCR. Results Compared with I/R group, the expression of NF-кB P50 protein increased in FNS group (P<0.05), with the decrease of expression of TNF-α mRNA (P<0.01) and increase of Bcl-xL mRNA (P<0.05), while the infarct size decreased (P<0.01). There was no significant difference between FNL group and I/R group for all the measurements (P>0.05). Conclusion FNS could induce the expression of P50 protein and Bcl-xL mRNA, and inhibit the expression of TNF-α mRNA, and reduce infarct size, which may associated with the neuroprotection of central nervous system from injury.

Objective To investigate the effect of pioglitazone (PIO) on the expression of peroxisome proliferators-activated receptor γ (PPARγ),nuclear factor-κB (NF-κB) c-Rel and anti-apoptosis factor Bcl-xL in cultured Wistar rat cortical neurons after oxygen-glucose deprivation/reoxygenation (OGD/R).Methods The rat cerebral cortical neurons were primarily cultured in vitro and a model of OGD/R was induced.The rats were divided into 7 groups:normal,OGD 4 h/R 6 h,OGD 4 h/R 12 h,OGD 4 h/R 6 h + PIO,OGD 4 h/R 6 h + PPARγ inhibitor T0070907 (TIO),OGD 4 h/R 12 h + PIO,and OGD 4 h/R 12 h + TIO groups.Western blot was used to detect the expression of PPARγ and NF-κB c-Rel protein in neurons.Reverse transcriptionpolymerase chain reaction assay was used to detect the expression of Bcl-xL mRNA in neurons.Results Western blot analysis showed that the expression levels of PPARγ and NF-κB c-Rel proteins after OGD/R were increased significantly compared to those of the normal group (P ＜0.01).After giving PIO,the expression levels of PPARγ and NF-κB c-Rel proteins were further increased,and they were significantly higher than those in the OGD 4 h/R 6 h and OGD 4 h/R 12 h groups (P ＜0.01),and after giving TIO,the expression levels of PPARγ and NF-κB c-Rel proteins were decreased,and they were significantly lower than those in the OGD/R group (P ＜0.05) and the corresponding PIO group (P ＜0.01).Reverse transcription-polymerase chain reaction analysis showed that the expression level of Bcl-xL mRNA in the OGD 4 h/R 6 h group was significantly higher than that in the normal group (P ＜0.01).The expression level of Bcl-xL mRNA in the PIO group was significantly higher than that in the OGD 4 h/R 6 h and OGD 4 h/R 12 h groups (P ＜0.01).After giving TIO,the expression of Bcl-xL mRNA was decreased.It was significantly lower than that in the OGD/R and corresponding PIO groups (P ＜ 0.05 and P ＜ 0.01).Conclusions PIO can upregulate the expression of PPARγ protein in cultured cortical neurons after OGD/R,and then increase the expression of anti-apoptotic factor Bcl-xL mRNA of NF-κB c-Rel regulation.This may be the part mechanism of PPARγ neuroprotective effect.

Objective To investigate neuroprotective mechanisms of vinpocetine by observing the effects of vinpocetine injection on the expressions of peroxisome proliferators-activated receptor γ(PPARγ),nuclear factor (NF)-κB p65,cyclooxygenase-2 (COX-2) in the ischemic cortex,and infarct volume after focal cerebral ischemia-reperfusion in rats.Methods A focal cerebral ischemia-reperfusion injury model was induced by suture method.The rats were randomly divided into a normal control,a cerebral ischemiareperfusion and a vinpocetine groups.They were also divided into either a day 7 subgroup or a day 14 subgroup (n =6 in each subgroup) according to the reperfusion time.Western blot was used to detect the expression levels of PPARγand NF-κB P65 in the ischemic cortex.Triphenyl tetrazolium staining was used to detect the volume of cerebral infarction.Results Western blot showed that at day 7 and 14 after cerebral ischemia-reperfusion,expression levels of PPARκ (all P＜0.001) and NF-κB p65 (all P＜0.001) in the cerebral ischemia-reperfusion group were significantly higher than those in the sham operation group,the expression levels of PPARκ (all P ＜0.05) in the vinpocetine group were significantly higher than those in the cerebral ischemia-reperfusion group,but the expression levels of NF-κB p65 (all P ＜0.05) were significantly lower than those in the cerebral ischemia-reperfusion group.Reverse transcription polymerase chain reaction showed that COX-2 mRNA expression levels were upregulated significantly at day 7 and 14 after cerebral ischemia-reperfusion compared with the sham operation group (all P ＜ 0.001),the expression levels of COX-2 mRNA in the vinpocetine group were significantly downregulated compared with the cerebral ischemia-reperfusion group (all P＜ 0.05).The infarct volumes at day 7 (134.308± 9.954 mm3vs.185.543 ± 9.100 mm3;q=10.659,P＜0.001) and at day 14 (137.865 ± 9.094 mm3vs.183.210±4.368 mm3;q=11.166,P＜0.001) in the vinpocetine group were significantly less than those in the cerebral ischemia-reperfusion group.Conclusions Vimpocetine significantly reduces infarct vohme after focal cerebral ischemia-reperfusion,its mechanism may be associated with upreguhtion of PPARγexpression and downreguhtion of the expressions of NF-κB p65 and COX-2.

BACKGROUND:Some studies have shown that more copy number variations are present in early passage human induced pluripotent stem cells than later passage human human induced pluripotent stem cells, their parental somatic fibroblasts or human embryonic stem cells. OBJECTIVE:To investigate whether the reprogramming process itself compromises genomic stability and further explore the efficiency of induced pluripotent stem cellestablishment. METHODS:Using high-resolution Affymetrix CytoScan HD array, we compared copy number variations and loss of heterozygosity in early passage induced pluripotent stem cells with their fibroblast cellorigins from genetic epilepsy patients. RESULTS AND CONCLUSION:Compared with somatic fibroblasts from genetic epilepsy patient, there was no difference in the loss of heterozygosity between the two types of cells, but more copy number variations were present in early passage human induced pluripotent stem cells which were characterized as microduplication and involved oncogenic genes. Results demonstrate the dynamic nature of genomic abnormalities during reprogramming process and the necessity of frequent monitoring human induced pluripotent stem cells to assure their genomic stability and clinical safety.

Objective To study the value of MR diffusion weighted imaging(DWI)and apparent diffusion coefficient(ADC)in differentiating benign and malignant lesions of prostate.Methods Twenty-two patients with prostate cancer and 17 patients with benign prostatic hyperplasia confirmed by pathology or biopsy,and 20 healthy volunteers were underwent prostate plain MRI,DWI and enhanced MRI,and measuring the value of ADC in the regions of interest in the workstation,and the ADC values of prostate cancer,benign prostatic hyperplasia,normal prostate were analyzed statistically.Results The ADC of prostate cancer,benign prostatic hyperplasia,normal prostate in central gland and peripheral zone were 1.08 ±0.23,1.43 ±0.27,1.51 ±0.26 and 1.26 ±0.47,1.72 ±0.40,1.75 ± 0.28,respectively,the ADC of prostate cancer was significantly lower than that of benign prostatic hyperplasia and normal prostate,and there was significant difference(P＜ 0.05),but there was no significant difference between benign prostatic hyperplasia and normal prostate(P ＞0.05).Looking the ADC was 1.30 and 1.60 in central gland and peripheral zone as the threshold to distinguish benign and malignant lesions of prostate,had high sensitivity and specificity.Conclusion The application of DWI combined with ADC,has improved the capability of diagnosis and differential diagnosis in benign and malignant lesions of prostate greatly.

AIM:This study was performed to investigate the feasibility and efficiency of exogenous mesenchymal stem cells(MSCs) transplantation on post-infarction ventricular remodeling and heart function in rats and compare the effects between adult rat MSCs and neonate rat MSCs transplantation.METHODS:1-2 hours after left coronary artery ligation,MSCs cultured in ex vivo,marked with BrdU,were injected directly into the border of infarcts in exogenous rats.6 weeks after transplantation,rat' heart function,ventricular remodeling and pathological results were measured.RESULTS:MSCs transplantation decreased LV end-diastolic diameter and end-systolic diameter,limited LV chamber dilatation and reduced collagen content significantly.The numbers of blood vessels and cardiomyocytes were increased.BrdU-labelled MSCs with oval nucleus were widely distributed.There were no significant difference between adult rat MSCs and neonate rat MSCs transplanted groups.CONCLUSION:MSCs can survive and home in exogenous host infarct hearts without addition of any immunosuppressant.MSCs transplantation has benificial effects on remodeling processes and contributes to improvement of cardiac function,which may be related with the reduction of the amount of the collagen,promotion of myogenesis and angiogenesis.

Objective To further investigate direct effects of dipeptidyl peptidase-4(DPP4) on calcification and to identify responsible signaling pathways in human vascular smooth muscle cells (HVSMC). Methods The effect of DPP-4 on calcification of HVSMC was observed by alizarin red, and Western blot was used to detect whether DPP4 induced calcification-related protein expressions through extracellular signal-regulated kinases 1/2 (ERK1/2) pathway. Results The Alizarin red staining results showed that calcified nodules in DPP4 group were significantly increased as compared with control group, similar to calcification group.The protein expressions of osteoprotegerin (OPG), osteopontin (OPN), Runt-related transcription factor 2 (RUNX2), and bone morphogenetic protein 2 (BMP2) were stimulated by DPP4 in a concentration- and time-dependent manner. The phosphorylation level of ERK1/2 was significantly increased after DPP4 incubation for 15 min (P<0.05). PD98059, an ERK1/2 inhibitor, significantly lowered DPP4-stimulated expressions of calcification-related proteins (P<0.05). Conclusion DPP4 may promote the calcification of HVSMC through ERK1/2 signaling pathways.

Objective To investigate does intracellular protein degradation pathway play an important role in decrease of endothelial nitric oxide synthase (eNOS) in human umbilical vein endothelial cells (HUVECs).MethodsTo establish a primary HUVECs culture methods,the HUVECs were incubated with concentration gradient group of TNF-α(0.01,0.1,1 and 10 ng/mL) in different time periods (24,48 and 72 h).The HUVECs were pretreated with NH4Cl or treated with caspase inhibitor or MG-132 1.5 h prior to incubation for an additional 24 h with TNF-α.The expression of eNOS was detected via Western blot assay.Results Treatment of the HUVECs with TNF-α(0.01-10 ng/mL) led to a dose-dependent reduction of eNOS expression.And treatment with TNF-α(1 ng/mL) reduced the eNOS expression in a time-depended manner.Compared with the TNF-α group,the protein expression level of eNOS was obviously increased in the co-working group of MG-123 and TNF-α.Conclusions TNF-α induces degradation of eNOS through a ubiquitin-proteasome pathway.