Objective To study the effect of advanced glycosylation end products (AGEs) on human retinal pigment epithelium (RPE) cells. Methods Human primary RPE cells were cultured in basal and different concentrations of AGEs with different times. The cells were divided into several groups as follows: group C (control): bovine serum albumin 0.1 g/L, 24 hours (C1) and 48 hours (C2); group NC (normal control): basal culture medium with 5.6 mmol/L of glucose, 24 hours (NC1) and 48 hours (NC2); group A (AGEs): 0.1 g/L, 24 hours and 48 hours, A1 and A4; 0.2 g/L, 24 hours and 48 hours, A2 and A5; 0.4 g/L, 24 hours and 48 hours, A3 and A6. Immunohistochemistry analysis was used to study the protein expression of receptor for AGEs (RAGE), peroxisome proliferativeactivated receptor-gamma coactivator-1 alpha (PCG -1α) and vascular endothelial growth factor (VEGF) protein. The activation of nuclear factor-kappa B (NF-κB) was detected by confocal microscope. Software IPP6.0 and SPSS 17.0 were used to analyze the quantitation data. Results Immunohistochemistry analysis showed that RAGE protein, PGC-1α protein and VEGF protein were basally secreted in RPE cells, but AGEs can obviously increases the expression level of these proteins (F=294.5, 228.3, 241.5; P＜0.05). Confocal microscope demonstrated that AGEs increased the activation of NF-κB significantly. Conclusion Accumulation of AGEs can stimulate the expression of RAGE protein, PGC-1α protein and VEGF protein, activation of NF-κB and induce apoptosis of RPE cells.
Objective To investigate the expression of nuclear factor (NF)-κB and intercellular adhesion molecule (ICAM)-1 in rat′s retina injured by ischemia-reperfusion, and the effect of pyrrolidine dithiocarbamate (PDTC) on the expression of NF-κB and ICAM-1. Method The model of retinal ischemia-reperfusion was set up in 60 SD rats, which were divided into two groups with 30 rats in each: ischemia-reperfusion group and ischemia-reperfussion with injection of PDTC group. The left cephalic artery of each rat was ligated, and the right side was the control. Every group was subdivided into group 1 hour, 6, 12, 24, 48, and 72 hours after ischemia-reperfusion injury, and with 5 rats in each group. mRNA of NF-κB and ICAM-1 mRNA was measured by in situ hybridization (ISH) method in rat′s retina. Every rat underwent electroretinography (ERG) at the corresponding time before executed by neck breaking. Results In ischemia-reperfusion group, expression of NF-κB and ICAM-1 was detected at the 6th hour after ischemia-reperfusion, reached the highest level at the 24th hour, and weakened gradually later. In ischemia-reperfusion with injection of PDTC group, expression of NF-κB and ICAM-1 was detected at the 12th hour after ischemia-reperfusion, and reached the highest level at the 24th hour but lower than that in ischemia-reperfusion group. No expression of NF-κB and ICAM-1 was found in the control group. The relative recovery rate of ERG a and b wave amplitude in ischemia-reperfusion groups was lower than that in ischemia-reperfusion with injection of PDTC group at every stage(P＜0.01 ). The lowest relative recovery rate of ERG a and b wave amplitude in different stages in both of the 2 groups was at the 24th hour(P＜0.01). Conclusions NF-κB and ICAM-1 may play an important role in retinal ischemia-reperfusion injury, as the inhibitor of NF-κB, PDTC may relieve the retinal ischemia-reperfusion injury. (Chin J Ocul Fundus Dis，2004，20：175-178)
ObjectiveTo observe the effect of tert-Butylhydroquinone (tBHQ) on the expression of nuclear factor erythroid 2-related factor 2 (Nrf2), heme oxygenase (HO)-1 and phosphatidylinositol 3-kinase (PI3K) in high glucose cultured retinal Müller cells; and to investigate the anti-oxidative stress and anti-apoptotic effects of tBHQ.MethodsRetinal Müller cells were divided into normal glucose group (5.5 mmol/L, N group), high glucose group (45 mmol/L, HG group) and tBHQ intervention group (HG+tBHQ group). After retinal Müller cells were cultured with high glucose for 48 hours, the pretreatment with tBHQ (20 μmol/L) induced the expressions of nuclear factor erythroid 2-related factor 2 (Nrf2) and HO-1. The Müller cells were identified by immunofluorescence staining. The expressions of Nrf2, HO-1, PI3K, B-cell lymphoma-2 (Bcl-2) and Bax were detected by Western blot and real-time fluorescence quantitative PCR. Flow cytometry was used to detect the apoptosis of retinal Müller cells in rats.ResultsMüller cytoplasm and nucleus GS showed strong positive, large cell body, abundant cytoplasm, uniform green fluorescence; nuclear DAPI staining round or oval, clear boundary. The expression of Nrf2 protein (t=4.114, P=0.006), HO-1 protein (t=9.275, P=0.000), Nrf2 mRNA (t=7.292, P=0.000) and HO-1 mRNA (t=15.014, P=0.000) in the HG group were higher than those in the N group. The expressions of Nrf2 protein (t=7.847, P=0.000) ,HO-1 protein (t=7.947, P=0.000), PI3K protein (t=5.397, P=0.002), Bcl-2 protein (t=6.825, P=0.000), Nrf2 mRNA (t=18.046, P=0.000), HO-1 mRNA (t=39.458, P=0.000), PI3K mRNA (t=4.979, P=0.003) and Bcl-2 mRNA (t=9.535, P=0.000) in the HG+tBHQ group were significantly higher than those in the HG group. The protein and mRNA expressions of Bax protein in the HG+tBHQ group were significantly lower than those in the HG group (t=14.998, 16.520; P=0.000, 0.000). Flow cytometry showed that the apoptosis rate of Müller cells in the HG group was significantly higher than that in the N group (t=39.905, P=0.000). The apoptosis rate of Müller cells in the HG+tBHQ group was significantly lower than that in the HG group (t=21.083, P=0.000).ConclusiontBHQ can inhibit the apoptosis of retinal Müller cells by up-regulating the expression of Nrf2, HO-1 and PI3K.
Objective To explore the difference between the hemorheology levels and the expression of hypoxia inducible factor 1α/2α (HIF-1α/2α) in the peripheral blood mononuclear cells of the Tibetan and Han patients with obstructive sleep apnea hypopnea syndrome (OSAHS). Methods This research recruited 30 high-risk Tibetan and Han patients with OSAHS, and 30 Tibetan and Han healthy volunteers at the same period. The whole blood viscometer was used to detect the high shear rate of whole blood viscosity, low shear rate of whole blood viscosity, plasma viscosity ratio, red blood cell aggregation index, and hematocrit in each group. RT-qPCR and Western blot assays were used to detect the mRNA and protein levels of phosphoinositide 3-kinase (PI3K), serine/threonine kinase (AKT), nuclear factor-κB (NF-κB) p65, HIF-1α and HIF-2α in peripheral blood mononuclear cells. Results The hemorheology level of Tibetan OSAHS patients was significantly higher than that of healthy Tibetans and Han OSAHS patients (P<0.05), and the hemorheology level of Han OSAHS patients was significantly higher than that of Han healthy people (P<0.05) . The mRNA and protein levels of PI3K, AKT, NF-κB p65 and HIF-1α in the peripheral blood mononuclear cells of Tibetan OSAHS patients were significantly higher than those of the healthy Tibetans or Han people, and these indexes of the Han OSAHS patients were significantly higher than those of the Han healthy people (all P<0.05), while HIF-2α mRNA and protein levels were significantly lower than those of healthy Han people (all P<0.05). Conclusion The upregulation of HIF-1α level and downregulation of HIF-2α expression in peripheral blood mononuclear cells of OSAHS patients depend on the activation of the PI3K/AKT/NF-κB p65 signaling pathway, and the hemorheological level of Tibetan OSAHS patients is higher than that of Han OSAHS patients.
Objective To investigate the effects of knocking down Rac1 gene (ras-related C3 botulinum toxin substrate 1) by small hairpin RNA (shRNA) on retinal neovascularization in a mouse model of oxygen-induced retinopathy (OIR). Methods One hundred and eight 7-day-old C57BL/6J mice were divided into three groups randomly.The OIR was induced by Smith protocol in 2 groups. OIR mice received an intravitreal injection of Rac1-shRNA plasmid or the nonsense plasmid in the geneintervention group and control group respectively at the age of postnatal day 11 (P11). Non-OIR mice also received an intravitreal injection of Rac1-shRNA plasmid at P11 as the blankintervention group which lived in the normoxic environment.Retinal neovascularization was investigated on flat-mounts after fluorescence angiography at P15 and P17. Endothelial cell nuclei breaking through the internal limiting membrane were counted on pathological section at P17.The expression of Rac1 and NF-kappa;B p65 subunit was measured by immuohistochemistry, Western blot, real-time polymerase chain reaction (RT-PCR) and in situ hybridization. Results Compared with the blank-control group,the level of Rac1 mRNA in the gene-intervention group decreased obviously（t=4.500，P=0.001）;the retinal non-perfusion areas,fluorescence leakage, neovascularization and the number of endothelial cell nuclei breaking through the internal limiting membrane were reduced significantly（t=6.521,P＜0.001）; the level of NF-kappa;B p65 nuclear translocation decreased（t=16.008,P＜0.001）while the expression of NF-kappa;B p65 mRNA was reduced obviously（t=3.354,P=0.006）, which was positively correlated with the expression of Rac1-mRNA (P=0.012).Conclusion Intravitreal injection of Rac1-shRNA with liposome in mice can effectively inhibit the expression of Rac1,and inhibit the retinal neovascularization under relative hypoxia via blocking the ROS-NF-kappa;B pathway.
ObjectiveTo investigate the association between TNF-α gene -308G/A polymorphism and the risk of coronary atherosclerotic heart disease (CHD) in Chinese population.MethodsWe searched PubMed, Web of Science, CNKI, WanFang Data and VIP Databases from inception to February 2017, to collect case-control studies about the association between TNF-α gene -308G/A polymorphism and risk of CHD in Chinese population. Two reviewers independently screened literature, extracted data and assessed the risk of bias of the included studies. Meta-analysis was performed by Stata 12.0 software.ResultsA total of 10 case-control studies were included. The results of meta-analysis showed a significant association between the TNF-α gene -308G/A polymorphism and CHD risk in Chinese population (A vs. G: OR=1.13, 95%CI 1.02 to 1.26, P=0.020; AA vs. GA/GG: OR=1.47, 95%CI 1.02 to 2.12, P=0.038; AA vs. GG: OR=1.50, 95%CI 1.04 to 2.16, P=0.029).ConclusionThe current evidence shows that the TNF-α gene -308G/A polymorphism may be associated with CHD risk in Chinese population and A allele may be a risk factor. Due to the limited quantity and quality of included studies, more high quality studies are needed to verify the above conclusion.
Objective To investigate the cellular viability and mitochondrial reactive oxygen species (ROS) production of the Müller cells under high glucose condition, and explore the protection role of the 5,6-dihydrocyclopenta-1, 2-dithiole-3-thione (CPDT) on Müller cells. Methods Müller cells from Sprague Dawley rats were divided into 5 groups randomly, including 25 mmol/L normal glucose group (group A) and 65 mmol/L high glucose group (group B). High glucose group with 45, 60, 70 μmol/L CPDT and cultured them 72 hour was set as group C, D and E. Water soluble tetrazolium salt (WST)-8 was used to measure the cellular viability. Flow cytometry was used to measure the active oxygen and apoptosis index. The expression of nuclear factor erythroid 2-related factor 2 (Nrf2), hemeoxygenase-1 (HO-1), Bcl-2 and Bax protein were measured by Western blot. Results Compared with group A, the WST-8 showed that the viability of Müller cells apparently decreased in group B (t=39.59,P＜0.05). Compared with the group B, the viability of Müller cells had changes in group C (t=0.97,P＞0.05), but recovered in group D and E (t=−4.17, −7.52;P＜0.05). Compared with group A, the FCM showed that the mitochondrial ROS levels was higher in group B (t=−30.99,P＜0.05). Compared with group B, the mitochondrial ROS levels were decreased in group D (t=27.68,P＜0.05). Compared with group A, Bax, Nrf2 and HO-1 increased (t=–11.03, –63.17, –11.44;P＜0.05), while the bcl-2 decreased in group B (t=7.861,P＜0.05). Compared with the group B, Nrf2, HO-1 and Bax decreased (t=15.11, 26.59, 6.27;P＜0.05), while the bcl-2 increased in group D (t=−6.53,P＜0.05). Conclusions Under the high glucose, CPDT may reduce the mitochondrial ROS levels and the expression of Nrf2, HO-1 and Bax protein of Müller cells. It may inhibit apoptosis through activating the Nrf2/HO-1 pathway and balancing of level of Bcl-2 protein and mitochondrial ROS.
ObjectiveTo analyze the expression and significance of NF-κBp65 and autophagy-related proteins Beclin1 and p62 in patients with papillary thyroid carcinoma (PTC).MethodsOne hundred and sixty cases of PTC patients' tumor tissue specimens and paracancerous tissue specimens in our hospital from March 2013 to February 2015 were collected, and 90 cases of cervical lymph node metastasis tissue specimens of the above patients were collected. The expressions of NF-κBp65, Beclin1 and p62 in PTC tissues, metastatic lymph node tissues and paracancerous tissues were detected by immunohistochemical method, and the relationship between the above indexes and the clinicopathological characteristics and prognosis of PTC patients was analyzed.ResultsThe positive rates of expression of NF-kappa Bp65 and p62 in PTC tissues and metastatic lymph node tissues were higher than those in paracancerous tissues (P<0.05). The expression rate of Beclin1 in PTC tissues and metastatic lymph node tissues was lower than that in paracancerous tissues (P<0.05). The positive rate of NF-κBp65 expression in PTC tissues was not related to the clinicopathological characteristics of patients (P>0.05). The expression of p62 decreased with the increase of tumor differentiation (P<0.05). The expression of Beclin1 in patients with stage Ⅲ+Ⅳ and lymph node metastasis were lower than those in patients with stage Ⅰ+Ⅱ and without lymph node metastasis (P<0.05), while the expression of p62 was opposite. Spearman correlation analysis showed that the expression of Beclin1 and p62 in PTC tissues was negatively correlated (r=–0.656, P<0.01). In metastatic lymph node tissues, the expression of Beclin1 and p62 was also negatively correlated (r=–0.562, P<0.01). The 3-year survival rates of patients with positive expression of p62 and NF-κBp65 in PTC tissues were lower than that of patients with negative expression (P<0.05). The 3-year survival rate of patients with positive expression of Becrin1 was higher than that of negative expression (P<0.05). TNM stage, lymph node metastasis, NF-κBp65 and p62 were independent risk factors for PTC prognosis, and Beclin1 was protective factor.ConclusionsNF-κBp65 and p62 are highly expressed in PTC tissues and lymph node metastasis tissues, while Beclin1 is poorly expressed, which could be used as independent prognostic factors for PTC patients. In addition, Beclin1 and p62 are related to PTC biological behavior and may become potential indicators for PTC diagnosis.
Objective To investigate the influence of diammonium glycyrrhizinate (DG) on the expression of NF-κB and neuron apoptosis after spinal cord ischemia-reperfusion injury in rats. Methods Fourty-eight healthy SD male rats, weighing 220-270 g, were randomly divided into the experimental group and the control group, with 24 rats in each group. A model of spinal cord ischemia-reperfusion injury was completed by intercepting the rats’ abdominal aorta between right and left renal arteries for 30 minunts. In the experimental group, each rat was injected 20 mg/kg DG via subl ingual vein 10 minutes before ischemia occurred. Equal qual ities of physiological sal ine were injected into the rats in the control group. The two groups were observed at 3, 24, 72 and 168 hours after ischemia-reperfusion, respectively. Lumbar myeloid tissues were prepared at the different times, respectively. The expression of NF-κB p65 in lumbar myeloidtissues was analyzed by immunohistochemistry and the apoptosis of neurons was examined by TUNEL reaction. Meanwhile, histological changes of spinal cord were observed by HE staining. Then the correlation between NF-κB and neuron apoptosis was analyzed. Results HE staining showed obvious histological changes of spinal cord of the two groups. In the control group, myeloid tissue edema and normal neurons were observed at 3 hours; there were more histological changes at 24 hours and 72 hours; vacuolus in gray matters and some survived neurons were seen at 168 hours. The histological changes at each time in the experimental group were fewer than those in the control group. The immunohistochemical staining showed that the expression of NF-κB p65 was observed. After ischemia-reperfusion, the expression strengthened at 3 hours, reached the peak at 24 hours and then weakened slowly. At 3, 24, 72 and 168 hours after ischemia-reperfusion, the absorbency (A) value of NF-κB p65 in the experimental group was 0.306 0 ± 0.024 4, 0.396 4 ± 0.022 7, 0.296 6 ± 0.021 1 and 0.267 9 ± 0.015 3, respectively, and that in the control group was 0.361 1 ± 0.017 7, 0.496 6 ± 0.020 1, 0.356 3 ± 0.021 0 and 0.301 4 ± 0.018 1, respectively. There were significant differences between the two groups (P lt; 0.05). The inhabitation ratio of NF- κB p65 expression by DG was 15.40%, 20.17%, 19.28% and 11.11% at 3, 24, 72 and 168 hours after ischemia-reperfusion, respectively. Neuron apoptosis was observed, which strengthened at 3 hours and was the most serious at 24 and 168 hours after ischemia-reperfusion. At 3, 24, 72 and 168 hours after ischemia-reperfusion, the A value of neuron apoptosis in the experimental group was 0.171 0 ± 0.029 1, 0.175 5 ± 0.031 1, 0.175 1 ± 0.027 9 and 0.183 2 ± 0.023 7, respectively, and that in the control group was 0.236 8 ± 0.063 6, 0.241 2 ± 0.042 6, 0.201 5 ± 0.049 8 and 0.250 1 ± 0.048 4, respectively. There were significant differences between the two groups (P lt; 0.05). The inhabitation ratio of neuron apoptosis by DG was 27.79%, 27.23%, 13.08% and 26.74% at 3, 24, 72 and 168 hours after ischemia-reperfusion, respectively. The expression of NF-κB in myeloid tissues was positively correlated with neurons apoptosis in the two groups (r = 0.838, P lt; 0.01). Conclusion Spinal cord ischemia-reperfusion injury may cause a marked expression of NF-κB and notable evidence of neurons apoptosis. DGcan reduce neurons apoptosis by inhibiting the expression of NF-κB.
ObjectiveTo investigate the protective effect and the regulation mechanism of oxaloacetate (OAA) on myocardial ischemia reperfusion injury in rats. MethodsSixty rats, weight ranged from 200 to 250 grams, were randomly divided into 6 groups:a negative control group, a sham operation control group, a model control group, an OAA pretreatment myocardial ischemia-reperfusion model group (three subgroups:15 mg/kg, 60 mg/kg, 240 mg/kg). We established the model of myocardial ischemia reperfusion of rats and recorded the internal pressure of left ventricle (LVSP), the maximal rate of left ventricular pressure change (±dp/dtmax) and left ventricular end diastolic pressure (LVEDP). We restored reperfusion 180 minutes after ligating the left anterior descending coronary artery 30 minutes and determinated cardiac troponin Ⅰ (cTn-I), lactate dehydrogenase (LDH), superoxide dismutase (SOD), glutathione peroxidase (GSH-Px). We took out heart tissues, stained it and calculated the infarcted size. We used the Western blot to detect the expression of NF-E2 related factor 2 (Nrf2), Kelch-like ECH-associated protein-1 (Keap1) and heme oxygenase-1 (HO-1). ResultsCompared with the sham operation group, heart function indexes in the negative control group had no significant difference (P>0.05). But in the model control group there was a decrease (P<0.05) And the serum levels of LDH, cTn-I, and myocardial infarcted size were significantly increased (P<0.01). Compared with the model control group, heart function indexes in the OAA pretreatment groups improved, the serum LDH, cTn-I activity, and infarct size decreased (P<0.05), SOD and GSH-Px activity increased (P<0.05). And these results were statistically different (P<0.01) in the high dose OAA pretreatment groups. Compared with the model control group, the expression of Keap1 in the OAA pretreatment group was down-regulated (P<0.001) while total Nrf2, nucleus Nrf2 and its downstream HO-1 was up-regulated (P<0.001), which suggested that OAA enhanced antioxidant capacity by (at least in part) Keap1-Nrf2 pathway, resulting in reducing myocardial damage and protecting myocardium after acute myocardial ischemia reperfusion injury. ConclusionOxaloacetate can provide protective effects on myocardial ischemia reperfusion injury through down-regulating the expression of Keap1 and up-regulating the expression of Nrf2 and its downstream peroxiredoxins to improve antioxidant capacity.