Objective To investigate the effect of recombinant human erythropoietin (rhEPO) on the expression of glutamine synthetase (GS) of cultured rat retinal Müller cells in high glucose environment in vitro. Methods Müller cells were isolated from retinas of 10 Sprague-Dawley rats at postnatal day three to five by trypsin digestion, and were randomly divided into six groups, including normal control group, high glucose group, high glucose +5 U/ml rhEPO group, high glucose+10 U/ml rhEPO group, high glucose +20 U/ml rhEPO group, high glucose+40 U/ml rhEPO groups. After 48 hours, the apoptosis of retinal Müller cells were assayed by terminal transferase-mediated DNA end labelling assay, and the expression levels of GS protein were detected with semi-quantitative immunocytochemistry. Results Compared with the normal control group, the cell viability and GS protein were reduced while the cell death increased in Müller cells cultured in high glucose, the difference was statistically significant (t=27.4,P＜0.01). Compared with the high glucose group, rhEPO treatment reduced the apoptotic Müller cells (t=857.2, 2 374.6, 2 473.2, 2 537.7;P＜0.01), induced the expression of GS proteins (t=3.2, 18.0, 22.5, 26.4; P＜0.05). Conclusions rhEPO can protect Müller cells from apoptosis under high glucose condition. The mechanism may be related to its function to up-regulate the GS protein expression, promote glutamic acid cycle, and reduce the excitotoxicity effects of high concentration of glutamate.
Objective To investigate the effect of pigment epitheliumderived factor (PEDF）on the expression of glutamine synthetase in retinal Muuml;ller cells of diabetic rats.Methods Diabetic rats were induced with streptozotocin injection.Before and after injection of 10 mu;l (0.1 mu;g/mu;l) PEDF (experimental group) or 10 mu;l PBS (control group) into the vitreous cavities of diabetic rats respectively for 48 hours,the expressions of GS and IL-1beta; in retina were analyzed by immunohistochemistry and real time RTPCR techniques. After being treated with 100 ng/ml PEDF for 24 hours in high glucose conditions,the expressions of GS and IL-1beta; in cultured Muuml;ller cells were studied by western blot and real time RT-PCR techniques. Apoptosis was analyzed by flow cytometry after Annexin V fluorescein isothiocyanate/Propidium idoium (Annexin V-FITC/PI) staining.Results By immunohistochemistry (the protein level) and real time RT-PCR (the mRNA level),it was found that the expression of GS decreased and the expression of IL-1beta; increased obviously (real time RT-PCR：GS:t=4.23,P＜0.01；IL-1beta;:t=16.73,P＜0.01；immunohistochemistry：GS: t=5.13,P＜0.01；IL-1beta;:t=9.32,P＜0.01) in diabetic rats. After injection of 10 mu;l (0.1 mu;g/mu;l) PEDF into the vitreous cavities of diabetic rats for 48 hours,it was found that the expression of GS increased and the expression of IL-1beta; decreased significantly(RT-PCR GS:t=3.87,P＜0.01IL-1beta;:t=3.61,P＜0.05;immunohistochemistry:GS:t=3.32, P＜0.05；IL-1beta;: t=2.63,P＜0.05). Under high glucose conditions, 100 ng/ml PEDF induced decreasing expression of IL-1beta; and increasing expression of GS significantly (RT-PCR：GS: t=2.89, P＜0.05；IL-1beta;: t=3.37,P＜0.05；Western blot：GS:t=2.66,P＜0.05;IL-1beta;:t=3.23,P＜0.05).Apoptosis of Muuml;ller cells under high glucose conditions was inhibited significantly by the treatment with 100 nmol/ml PEDF (t=3.21,P＜0.05). Conclusions In diabetic rats,PEDF may decrease expression of IL-1beta; in rat retinal Muuml;ller cells, which may result in increasing expression of GS.To some degree,it inhibits possibly the death of retinal ganglion cells.
ObjectiveTo observe the expression of glutamate (Glu) andγ-aminobutyric acid (GABA) in the retina of diabetic rats which were intervened later by insulin intensive therapy, and to investigate the mechanism of metabolic memory of hyperglycemia which induced the retina neuropathy in diabetic rats. Methods60 Brown Norway rats were randomly divided into normal control (NC) group, diabetes mellitus (DM) group (6 weeks at DM1, 12 weeks at DM2) and metabolic memory (MM) group, 15 rats in each group. Diabetes was induced by intraperitoneal injection of streptozocin. After 6 weeks, MM group was treated with insulin intensive therapy for 6 weeks. DM1 group was sacrificed at the end of 6 weeks and other groups were sacrificed at the end of 12 weeks. High performance liquid chromatography was used to detect the amount of Glu and GABA in the rat retina. Real-time polymerase chain reaction was applied to quantify the mRNA expressions of Glutamate decarboxylase (GAD). TdT mediated dUTP nick ending labelling was used to detect cell apoptosis. ResultsThe concentration of Glu (t=6.963), GABA (t=4.385) and the ratio of Glu/GABA (t=4.163) in MM group were significantly higher than DM1 group, but the concentration of Glu (t=3.411) and GABA (t=3.709) were significantly lower than DM2 group (P < 0.05). And there was no significant difference in the ratio of Glu/GABA between MM and DM2 groups (t=1.199, P > 0.05). The level of expressions of GAD mRNA in MM group was significantly lower than DM1 group (t=3.496, P < 0.05), but higher than DM2 group (t=8.613, P < 0.05). The number of nerve cells apoptosis in MM group was significantly higher than DM1 group (t=2.584, P < 0.05), but lower than DM2 group (t=3.531, P < 0.05). ConclusionsIntensive therapy later by insulin can partially reduce the content of Glu and GABA and the rate of nerve cells apoptosis, which cannot return to normal levels, and has no effect on the rise in the ratio of Glu/GABA caused by the hyperglycemia. The disorders of Glu and GABA may participate in the metabolic memory of hyperglycemia.
Objective To discuss the correlation between glutamate receptor 5 (GLUR5) and the pathogenesis of intractable temporal lobe epilepsy (ITLE), through detecting the GLUR5 expression in human with ITLE and Coriaria lactone-induced rhesus monkey temporal lobe epilepsy model. Methods Fifty-four patients with ITLE treated in West China Hospital between January 2007 and December 2015 were regarded as clinical case group in this study. The other 43 patients who underwent temporal lobe removal decompression surgery in the same time period due to trauma, tumor or large area cerebral hemorrhage complicated with cerebral hernia were designated as the clinical control group. Quantitive polymerase chain reaction (PCR) and Western blot methods were used to detect mRNA and protein levels of GLUR5. Western blot was also used to detect the GLUR5 protein level in the hippocampus and temporal lobe tissues of Coriaria lactone-induced rhesus monkey epilepsy model, and the result was compared with that of animal controls. Results Quantitive PCR results showed that the expression ratio (R value) of GLUR5 in the temporal lobe of the clinical case group to the clinical control group was 0.262, without significant difference (P>0.05), while theR value in the hippocampus was 4.896, with a significant difference (P<0.05). The amplification curve showed that the GLUR5 level in the hippocampus of the clinical case group was higher than that of the clinical control group, but the GLUR5 mRNA level in the temporal lobe tissue was not significantly changed. GLUR5 PCR amplified product electrophoresis showed that the amplified fragment was 161 bp. Western blot analysis showed that the GLUR5/actin value of the temporal lobe tissue in the clinical case group was 2.172±0.063, while the value in the clinical control group was 2.142±0.060, and the difference was not statistically significant (P>0.05). The GLUR5/actin value of the hippocampus in the clinial case group was 2.548±0.509, while it was 1.584±0.415 in the clinial control group, and the difference was statistically significant (P<0.05). The GLUR5/actin value of the hippocampus of the rhesus monkey model of epilepsy was 1.007±0.034, and it was 1.001±0.032 in the animal control group, and the difference was not statistically significant (P>0.05). The GLUR5/actin value of the temporal lobe tissue in the animal experimental group of rhesus model of epilepsy was 0.763±0.026, and it was 0.742±0.034 in the animal control group, and the difference was not statistically significant (P>0.05). The target protein bands showed that GLUR5 protein expression in the temporal lobe tissue and hippocampus of the rhesus model of epilepsy and animal controls was not significantly different (P>0.05). Conclusions GLUR5 participates in the pathogenesis of human ITLE by acting on the hippocampus. The expression of GLUR5 in human ITLE is abnormal, but the expression of GLUR5 is not changed in the rhesus model of epilepsy. The abnormal expression of GLUR5 may play a role in the pathogenesis of ITLE.
ObjectiveTo observe the expression of probucol on high glucose-induced specificity protein 1(SP1), kelchlike ECH associated protein1 (Keap1), NF-E2-related factor 2 (Nrf2) and glutamate-cysteine ligase catalytic (GCLC) in the cultured human müller cells and preliminary study the antioxidation of the probucol on müller cells.MethodsPrimary cultured human müller cells were randomly divided into four groups: normoglycaemia group (5.5 mmol/L glucose), normoglycaemia with probucol group (5.5 mmol/L glucose+100 μmol/L probucol), hyperglycemia group (25.0 mmol/L glucose), hyperglycemia with probucol group (25.0 mmol/L glucose + 100 μmol/L probucol). Immunofluorescence staining was used to assess distribution of SP1, Keap1, Nrf2, GCLC in human Müller cells. SP1, Keap1, Nrf2 and GCLC messenger RNA (mRNA) expression was evaluated by quantitative real-time RT-PCR (qRT-PCR). Independent sample t test was used to compare the data between the two groups.ResultsAll müller cells expressed glutamine synthetase (＞95%), which confirmed the cultured cells in vitro were the purification of generations of müller cells. The expressions of SP1, Keap1, Nrf2, and GCLC protein were positive in human müller cells. qRT-PCR indicated that SP1 (t=28.30, P＜0.000), Keap1 (t=5.369, P=0.006), and Nrf2 (t=10.59, P=0.001) mRNA in the hyperglycemia group increased obviously compared with the normoglycaemia group; GCLC (t=4.633, P=0.010) mRNA in the hyperglycemia group decreased significantly compared with the normoglycaemia group. However, SP1 (t=12.60, P=0.000) and Keap1 (t=4.076, P=0.015) in the hyperglycemia with probucol group decreased significantly compared with the hyperglycemia group; Nrf2 (t=12.90, P=0.000) and GCLC (t=15.96, P＜0.000) mRNA in the hyperglycemia with probucol group increased obviously compared with with the hyperglycemia group.ConclusionProbucol plays an antioxidant role by inhibiting the expression of SP1, Keap1 and up-regulating the expression of Nrf2, GCLC in müller cells induced by high glucose.
ObjectiveExplore the mechanism of action of Kruppel-like factor 4 (KLF4) in the oxidative damage model of hippocampal neurons in mice induced by glutamate. MethodsTo clarify the role of KLF4 and glutamate in the oxidative toxicity of epilepsy, the mouse hippocampal neuron cell line (HT22) was adopted, and a neuronal death excitotoxicity cell model was formed by induction with glutamate as the in vitro epilepsy experimental model. The expression level of KLF4 was detected by Real-Time PCR. HT22 cells were transfected with KLF4-specific siRNA, and the experiments were grouped as follows: Ctrl group, Glu group, Glu + siKLF4-1 group, and Glu + siKLF4-2 group. The cell viability of each group was detected by the CCK8 method. ResultsKLF4 was significantly increased in the epilepsy model of HT22 cells induced by glutamate, while downregulation of KLF4 improved the proliferation and viability of neurons in the epilepsy model of HT22 cells induced by glutamate. ConclusionIn the hippocampal neuron cells of epileptic mice, KLF4 is highly expressed. The downregulation of KLF4 improves the proliferation function and vitality of glutamate-induced HT22 cells, indicating that KLF4 may contribute to the occurrence and development of epilepsy by participating in the regulation of oxidative stress responses.