Objective To investigate the alteration of protein kinase C (PKC) and endothelin system in early diabetic rats, and the effect of specific PKC inhibitor on the expression of retinal endothelin-1 (ET-1). Methods The rats model with streptozotocin(STZ)-induced diabetes were set up. The expression of retinal PKC was detected by enzyme-linked immunoabsorbent assay (ELISA). The expression of retinal ET-1, ET-3, ET-A and ET-B receptor mRNA was determined by semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR). The alteration of retinal ET-1 mRNA after intravitreal injection of PKC inhibitor GF109203X in diabetic rats was also observed. Results The activities of membranous PKC were significantly increased in 2-week diabetic rats compared with that in normal rats（t=3.296 , P=0.008）, while activities of cytosolic PKC were unchangeable（t=0.138, P=0.894）. The expression of retinal ET-1 mRNA was significantly increased（P=0.008）, while no change was found in expression of ET-3, ET-A and ET-B mRNA（P=0.918，P=0.889，P=0.500）. After intravitreal in jection of 10-5、10-6、10-7 mol/L PKC inhibitor GF109203X in diabetic rats, the expression of retinal ET-1 mRNA was decreased in a dose-dependent manner compared with the control rats. Conclusion Activation of PKC and increased expression of ET-1 could be found in the retina of early diabetic rats, and PKC inhibitor could inhibit the expression of retinal ET-1. (Chin J Ocul Fundus Dis，2004，20：168-171)
Objective To investigate the response of retinal ganglion cells （RGC）in detached and reattached retina in adult rats, and the effect of IL-1beta antibody and IL-1Ra on the loss of RGC. Methods A total of 73 Sprague-Dawley (SD) rats were subretinally injected with healon GV（1.4% hyaluronate）and retrograde labeled with fluorogold (FG), and 10 ng IL-1 Ra and 500 ng IL-1beta antibody were injected into the subretinal space combined with healon GV. The retinal flakes were observed under the fluoroscope and the number of RGC was counted 2 hours, 1, 3, 5, 7, 10, 20, 50, and 90 days after deta chment; 10 days after detachment and 30 days after reattachement; 90 days after detachment and 20 days after reattachement, and 1 and 10 days after injection with IL-1beta antibody and IL-1Ra,respectively. And the control group was only developed an intraocular injection of the same valume of healon GV. Result Two hours after detachment, the RGC loss was found, reached the peak at first day, and decreased gradually. RGC loss was also found in the non-detached area. The reattachment 10 days after detachment (early reattachment) stopped the loss of RGC, and the reattachment 90 days after detachment (late reattachment) promoted the loss, which rested on a certain level. Subretinal space injection of IL-1Ra and IL-1beta antibody decreased the loss of RGCs in the detached retina. Conclusion The RGCs loss were found both in the detached and attached retina. Early reattachment may stop the loss of RGC, and late reattachment may promote the loss. Both IL-1beta antibody and IL-1Ra have neuro protective effect on RGC. (Chin J Ocul Fundus Dis，2004，20：233-236)
ObjectiveTo observe the serum vascular endothelial growth factor (VEGF), apelin and heme oxygenase-1 (HO-1) levels in patients with type 2 diabetes mellitus (T2DM) and to explore their their relationship with diabetic retinopathy (DR).MethodsA total of 208 patients with T2DM and 50 healthy subjects (control group) from the Central Hospital of Western Hainan during January 2014 and December 2017 were selected in this study. Vision, slit lamp microscope, indirect ophthalmoscope and FFA examinations were performed on all the subjects. According to the results of the examinations combined with the DR clinical staging criteria, the patients were divided into non-DR (NDR) group, non-proliferative DR (NPDR) group, and proliferative DR (PDR) group, with 72, 76 and 60 patients in each, respectively. The clinical data of each group were recorded, and the levels of fasting blood glucose (FPG), HbA1c, total cholesterol (TC), three acylglycerol (TG), high density lipoprotein (HDL-C), low density lipoprotein (LDL-C), VEGF, apelin and HO-1 were detected in each group. The receiver operating characteristic curve (ROC) were used to analyze the value of VEGF, apelin and HO-1 in predicting the occurrence of PDR. Correlation analysis of serum VEGF, Apelin and HO-1 with clinical parameters in PDR patients by Pearson correlation analysis.ResultsThe level of VEGF (56.82±10.16 vs 91.74±22.83, 140.15±36.40, 195.28±42.26 pg/ml) and apelin (2.95±0.53 vs 4.68±0.74, 7.25±1.13, 10.16±1.35 ng/ml) in PDR group were significantly higher than those in NPDR, NDR and control groups (F=17.306, 21.814; P＜0.05). The level of HO-1 (50.37±10.14 vs 43.58±8.16, 30.25±6.28, 22.60±4.72 mmol/L) in PDR group was significantly lower than those in NPDR, NDR and control groups (F=15.827, P＜0.05). The ROC curve analysis showed that the best cut-off values of serum VEGF, apelin and HO-1 were 162.50 pg/ml, 8.30 ng/ml, 27.13 mmol/L, and the three combined to predict PDR of AUC (95%CI) was 0.906 (0.849−0.962), and their sensitivity (90.3%) and specificity (83%) were better. The correlation analysis showed that the VEGF, apelin and HO-1 of PDR patients were correlated with the course of diabetes (r=0.382, 0.416, −0.36; P＜0.05), FPG (r=0.438, 0.460, −0.397; P＜0.05) and HbAlc (r=0.375, 0.478, −0.405; P＜0.05), and the serum VEGF were correlated with apelin and HO-1 (r=0.793, −0.594; P＜0.01).ConclusionElevated serum VEGF and apelin levels and reduced HO-1 levels are associated with the progression of DR, and the three combination helps predict the occurrence of PDR.
ObjectiveTo observe the changes of eotaxin-1(CCL11), eotaxin-2(CCL24)and eotaxin-3(CCL26)in ranibizumab treated light-injured human retinal pigment epithelium (RPE) cells ARPE-19 and investigate the effects of vascular endothelial growth factor (VEGF) antagonist to the expressions of eotaxins. MethodsCultured human RPE cells(8th-12th generations)were divided into light-injured group, ranibizumab treated group and normal control group. Cells of the three groups were exposed to the blue light at the intensity of(600±100) Lux for 12 h to establish the light injured model, while cell culture dishes of the normal control group were wrapped with double-layer foil. The cells of ranibizumab treated group were treated with VEGF-A antagonist(ranibizumab)at the final concentration of 0.125 mg/ml for 24 hours directly after the illumination. The mRNA and protein of VEGF-A, eotaxin-1, eotaxin-2, eotaxin-3, NF-κB were determined by Real time-PCR, enzyme-linked immunosorbent assay, Western blot, immunohistochemical staining at 0, 3, 6, 12, 24 hours after light damage. ResultsThe mRNA and protein level of VEGF-A, eotaxin-1, eotaxin-2, eotaxin-3, NF-κB in the light-injuried group increased significantly compared to that in normal control group (P < 0.05). After treating with ranibizumab, the expression of eotaxin-1, eotaxin-2, eotaxin-3, NF-κB were significantly suppressed (P < 0.05). ConclusionThe suppression of over-expression of VEGF in human RPE may down-regulate the expression of eotaxins, via the suppression of NF-κB.
ObjectiveTo observe the effect of complement receptor 1 (CR1) on barrier of cultured human retinal epithelial cells (hRPE) under complement-activated oxidative stress. MethodsThe third to fifth passage of hRPE cultured on Transwell insert were used to establish a stable hRPE monolayer barrier. The hRPE monolayer barrier was exposed to 500 μmol/L ten-butyl hydroperoxide and 10% normal human serum to establish the hRPE monolayer barrier model of complement-activated oxidative stress in vitro. hRPE monolayer barriers under complement-activated oxidative stress were divided into two groups including model group and CR1 treatment (1 μg/ml) group. Model group and CR1 treatment group were treated with 1 μl phosphate buffer solution (PBS) or CR1 for 4 hours. Normal hRPE monolayer barrier were used as control in transepithelial resistance (TER) measurement experiment. TER was measured to evaluate the barrier function of hRPE. The hRPE-secreted vascular endothelial growth factor (VEGF) and chemokine (C-C Motif) Ligand 2 (CCL2), together with complement bioactive fragments (C3a, C5a) and membrane-attack complex (MAC) in the supernatant were detected by enzyme-linked immune sorbent assay. ResultsStable hRPE monolayer barrier was established 3 weeks after hRPE seeded on Transwell insert. Complement-activated oxidative stress resulted in a sharp decrease of TER to 54.51% compared with normal hRPE barrier. CR1 treatment could significantly improve TER of barrier under complement-activated oxidative stress to 63.48% compared with normal hRPE barrier(t=21.60, P < 0.05). Compared with model group, CR1 treatment could significantly decrease the concentration of VEGF and CCL2 by 11.48% and 23.47% secreted by hRPE under complement-activated oxidative stress (t=3.26, 2.43; P < 0.05). Compared with model group, CR1 treatment could also decreased the concentration of C3a, C5a and MAC by 24.00%, 27.87%, 22.44%.The difference were statistically significant (t=9.86, 2.63, 6.94; P < 0.05). ConclusionsCR1 could protect the barrier function of hRPE cells against complement-activated oxidative stress. The underlying mechanism may involve inhibiting complement activation and down-regulating the expression of VEGF and CCL2.
As most patients of central serous retinopathy (CSC), the symptoms of acute onset will alleviate by oneself after 4-6 months. About 30%-50% of patients with CSC experience chronic or recurrent cases. Resulting in persistent neurosensory detachments and subretinal fluid, causing significant vision loss. Mineralocorticoid receptor (MR) is a kind of nuclear hormone receptors, plays a role in theregulation of water and electrolyte balance. Excessive MR signaling is associated with many diseases. Study found that MR antagonists decreased the thickness of the retina and improved in vision, there was no serious adverse reactions during the period of treatment for chronic CSC. Initial dose of MR antagonists was 25 mg per day, 1 week later, dosage was increased to 50 mg per day, and treatment for about 3 months. There is no conclusive effective treatment and the dosage are still unknown. MR antagonists may be a safe and effective way to treat chronic CSC, though evidence is scant. Prospective, multicenter, large-scale trials is required.
Objective To evaluate the inhibiting effect of adenosine on rat retinal ganglion cells (RGC) death induced by P2X7 and N-methyl-D-aspartate (NMDA) receptor. Methods (1) Long-Evan neonatal rats were back labeled with aminostilbamidine to identify RGC. The viability of RGC affected by P2X7 excitomotor BzATP (50 mu;mol/L), glutamate receptor excitomotor NMDA (100 mu;mol/L) and adenosine (300 mu;mol/L) was detected. (2) RGC from the retinae of unlabeled neonatal rats were cultured in vitro. After labeled with Fura-2 methyl acetate, an intracellular calcium indicator, the effect of BzATP, NMDA and adenosine on intracellular Ca2+ level was detected byCa2+ imaging system. Results Both BzATP (50 mu;mol/L) and NMDA(100 mu;mol/L) could kill about 30% of the RGC. Cell death was prevented by adenosine (300 mu;mol/L) with the cell viability increased from (68.9plusmn;2.3)% and (69.9plusmn;3.2)% to (91.2plusmn;3.5)% (P＜0.001) and (102.1plusmn;3.9)% (P＜0.001), respectively. BzATP (50 mu;mol/L) led to a large, sustained increase of intracellular Ca2+ concentration to (1183plusmn;109) nmol/L. After the adenosine intervened, Ca2+ concentration increased slightly to (314plusmn;64) nmol/L (P＜0.001). Conclusion Adenosine may prevent RGC death and increase of intracellular Ca2+ concentration from P2X7and NMDA receptor stimulation. (Chin J Ocul Fundus Dis, 2007, 23: 133-136)
Neovascularization is a characteristic manifestation of a variety of retinal diseases. Vascular endothelial growth factor (VEGF) mainly regulates the proliferation and migration of endothelial cells. VEGF receptor 2 (VEGFR2) is the main receptor to mediate this effect. The activation of downstream signals requires the binding of VEGF and VEGFR2, followed by receptor dimerization and autophosphorylation. Blocking this process and inhibiting neovascularization is very attractive treatment ideas. Monoclonal antibodies and fusion protein drugs currently used in ophthalmology can bind free VEGF. In addition, there are also macromolecular antibodies binding VEGFR2 and small molecule tyrosine kinase inhibitors, which is expected to further expand into the field of ophthalmology. Although anti-VEGFR2 therapy is a revolutionary method to inhibit neovascularization, there are no sufficient clinical evidences at present. In-depth understanding of the application status and progress of anti-VEGFR2 in the treatment of retinal neovascular diseases has important clinical significance.
Objective To investigate the expression of eotaxin-1, eotaxin-2 and eotaxin-3 in ARPE-19 human RPE cells after exposure to light. Methods Cultured human RPE cells (5th~10th generations) were divided into lightinduced group and control group. Cells light-induced group were exposed to the blue light at the intensity of (600plusmn;100) Lux for 12 h to establish the light damaged model. Eotaxin-1, eotaxin-2 and eotaxin-3 mRNA and protein were determined by real time polymerase chain reaction and Western blot at 0, 3, 6, 12, 24 hours after light-induced. Results In light-induced groups, mRNA levels of eotaxin-1 and eotaxin-2 were increased at 0 h (t1=6.05.t2=12.561) and 3 h （t1=2.95.t2=3.67） significantly(P＜0.05), but the mRNA level of eotaxin-3 had not changed （t3=1.57 and 1.00 respectively，P＞0.05） at that time. At 6 h （t1=4.73，t2=18.64，t3=28.48）, 12 h （t1=3.11，t2=20.62，t3=18.50）, 24 h （t1=8.25，t2=38.27，t3=18.60）, mRNA levels of eotaxin-1, 2, 3 were increased significantly （P＜0.05）. Except for the eotaxin-3 protein had not changed at 3 h （t3=1.28，P＞0.05）, protein expression of eotaxin-1, 2, 3 were increased significantly （P＜0.05） at 0 h （t1=4.85，t2=5.45，t3=6..21）, 3 h （t1=5.64，t2=4.55）, 6 h （t1=31.60，t2=6.63，t3=7.15）, 12 h （t1=14.09，t2=18.22，t3=15.76）, 24 h （t1=6.96，t2=10.47，t3=12.85）. Conclusion Eotaxin-1, eotaxin-2 and eotaxin-3 expression were increased after Light-damage, corresponding to the time after light exposure. Eotaxin-3 was the most prominent isoform.
The mineralocorticoid receptor (MR) belongs to the nuclear receptor superfamily and is expressed in the retina and choroid. MR antagonist (MRA) has a long history of application in non-ophthalmic clinical practice. Various cellular and animal models indicated that inappropriate activation of MR participated in pathological angiogenesis, oxidative stress, inflammation, disturbance of ion/water homeostasis and neurodegenerative changes, while the application of MRA can reduce or reverse these pathological processes. After using MRA in central serous chorioretinopathy (CSC) patients, improved visual function, less subretinal fluid and reduced sub-foveal choroidal thickness were observed. Single nucleotide polymorphisms in MR and plasma aldosterone levels were significantly different between chronic CSC patients and CSC patients with spontaneous remission. Novel formulation for sustained-release MRA and the mechanisms involving inflammation may become the new focus of MR study. This review summarizes the research status of MR and MRA in order to provide a reference for future basic research and clinical treatment.