Objective To observe and preliminarily explore the effects of Deferasirox (DFX) on lipid peroxidation and ferroptosis in human retinal endothelial cells (HREC). MethodsA cell experimental study. Divided the in vitro cultured HREC into normal glucose (NG) group, high glucose (HG) group, NG+DFX group, HG+DFX group, NG+DFX+ferric ammonium citrate (FAC) group, and HG+DFX+FAC group. Light microscope was used to observe the morphology of the cells; cell proliferation was detected by Cell Counting Kit-8 assay, and Calcein-AM staining was used to detect the unstable iron pool (LIP) content; enzyme-linked immunosorbent assay reader was used to detect the reactive oxygen species (ROS), malondialdehyde (MDA), glutathione (GSH), and oxidized glutathione (GSSG); Western blot was used to detect the relative protein expression of Glutathione Peroxidase 4 (GPX4) and Solute Carrier Family 7 Member 11 (SLC7A11). Two-tailed Student t test was used for comparison between the two groups; one-way ANOVA was used for comparison between multiple groups. ResultsCompared with the HG group and the HG+DFX+FAC group, the cell proliferation rate and the contents of GSH and the relative protein expression of GPX4, and SLC7A11 in the HG+DFX group were significantly increased, and the differences were statistically significant (F=150.70, 21.02, 26.09, 52.62; P＜0.001). The contents of LIP, ROS, MDA, and GSSG were significantly decreased, and the differences were statistically significant (F=807.20, 16.94, 31.62, 19.21; P＜0.001). ConclusionsHigh glucose significantly induces an increase in LIP, lipid peroxidation, and ferroptosis in HREC. Deferasirox inhibits lipid peroxidation and ferroptosis in HREC by downregulating LIP levels.
Objective To investigate the effects of lights with different wavelength on the retina of rd12 and C57BL/6J mice. Methods Thirty two rd12 mice and C57BL/6J mice were randomly divided into the control group, white light group, midwavelength light (505 nm) group and shortwavelength light (405 nm) group, with eight mice in each group. Besides the control group, other groups were exposed to cycle illuminations ［12 hours dark, 12 hours (800plusmn;130) Lux］ for seven days to establish the model of retinal light damage. Electroretinogram (ERG) responses of all mice were recorded at the day before illumination and 1st, 4th and 7th days after illumination. The eyes were enucleated at 7th days after illumination to assess levels of reactive oxygen species (ROS), expression of peroxiredoxin 6 (PRDX6), and activity of caspase-3. Results ERG amplitudes of all groups declined gradually in C57BL/6J mice, and the most significant effects was found in the short-wavelength light group. The amplitudes of photopic b-wave were significantly different at 1st, 4th and 7th days (F=4.412, 5.082, 9.980；P＜0.01). The amplitudes of cone b-wave of the four groups decreased to (85plusmn;10) %, (70plusmn;19) %, (57plusmn;22) % and (46plusmn;19) % at 7th days, respectively, and were significantly different between white light group and short-wavelength light group(t=3.19,P＜0.01). The levels of ROS were significantly different in rd12 mice (F=16.08,P＜0.01), and elevated obviously in shortwavelength light group. The expressions of PRDX6 of retina were significantly different in rd12 mice (F=7.214,P＜0.05), and were decreased obviously in short-wavelength light group. The caspase-3 relative activity was significantly different in rd12 retina (F=7.530,P＜0.05); but there was no significant difference in C57BL/6J mice (F=3.625, 1.993, 1.133; P＞0.05).The caspase-3 relative activity were significant different between rd12 mice and C57BL/6J mice in short wavelength light group (t=5.474,P＜0.05). Conclusions Short-wavelength light can induce retinal damage of mouse retina, especially in rd12 mouse. The retinal light damage possibly relates to the oxidative damage.
Objective To investigate the protective effects of estrogen on rabbit retinal damages induced by chronic ocular hypertension.Methods A total of 18 white New Zealand female rabbits were randomly divided into ovariectomized (OV) group and sham OV (SOV) group. Bilateral ovaries were remove in OV group while only the adipose tissue around ovarian were remove in SOV group. Chronic ocular hypertension was induced by anterior chamber injection of carbomer. Retinal cell apoptosis was measured by terminal deoxynucleotidyl transferasemediated dUTP nick end labeling (TUNEL), the expression of bcl-2, bax were detected by immunohistochemistry. The images were captured under microscope and analyzed with computer-image-analysis system. Results Four, six and eight weeks after ocular hypertension modeling, the OV retinas have less retinal ganglion cells, thinner optic nerve fiber layer and inner nuclear layer and more TUNEL positive cells (t=3.285，4.012，3.624；P＜0.01). The OV retinas also have less bcl-2 expression (t=4.256，3.867，3.459；P＜0.01), more bax positive cells (t=3.211，3.625，3.253；P＜0.01). Bcl-2 expression was negatively correlated with TUNEL positive cells indicating bcl-2 can inhibit apoptosis. Bax expression was positively correlated with TUNEL positive cells indicating bax induce apoptosis.ConclusionEstrogen has a neuroprotection role to rabbit retina under chronic ocular hypertension by reducing apoptosis.
Objective To observe the effects of immunologic cytokines or anti-angiogenesis gene transfer mediated by electroporation for choroidal melanoma cells.Methods The human embryo kidney cells and malignant choroidal melanoma cells were transfected with plasmids pNGVL-mIL2, pNGVL-mIL12, pCI-sFLK-1, pCR3.1-antiVEGF121,pCI-ExTek. Then the expression of mIL2, mIL12, sFLK-1, VEGF and ExTek were detected by enzymelinked immunosorbentassay (ELISA) and Western blot. Nude mice models of malignant choroidal melanoma were established and they were divided into four groups randomly. Each group was treated with 30 mu;l of 0.9% NaCl, 30 mu;g pNGVL, 30 mu;g antiVEGF121+sFLK-1+ExTek and 30 mu;g mIL2+mIL12 respectively by electroporation. Seven，14, 21, 28, 35 and 42 days after treatment, the tumor volumes were measured to calculate the tumor inhibition rate. Results ELISA and Western blot showed that mIL2，mIL12，sFLK-1 and ExTek were expressed after electroporation，VEGF expression was decreased remarkably. After treatment，the tumors of mIL2+mIL12 group were greatly inhibited with a tumor inhibition rate of 97.33%，while the tumors of antiVEGF121+sFLK-1+ExTek and pNGVL group were partially inhibited with tumor inhibition rates of 53.33% and 36.33% respectively.Conclusions Immunologic cytokines transfer mediated by electroporation can inhibit the growth of melanoma，but anti-angiogenesis only have a mild effects.
Objective To observe the influence of the expression of CD18 on the neutrophile and the leukocyte adhesion to retinal vascular endothelium by hypoxia-inducible factor-1 alpha (HIF-1alpha;) in early diabetic retinopathy rats. Methods Male Sprague-Dawley rats received intraperitoneal injection of streptozotocin to induce diabetes model. 18 diabetic rats were divided into 3 groups randomly after 2 months of diabetes induction, including diabetic group (group B), HIF-1alpha; anti-sense oligonucleotides (ASODN) injection group (group C) and HIF-1alpha; sense oligonucleotides (SODN) injection group (group D), the age and weigh matched health rats were chosen as control group (group A), with 6 rats in each group. Then group A and B rats received 5% glucose solution caudalis veins injection, group C and group D rats received HIF-1alpha; ASODN and HIF-1alpha; SODN caudalis veins injection, respectively(025 mg/kg).The level of CD18 on the neutrophil isolated from the peripheral blood was measured by flow cytometry. Retinal leukostasis was quantified with acridine orange leukocyte fluorography. Results The percentage of CD18 positive neutrophil cell was（44.93plusmn;3.60）% in group B，（18.66plusmn;1.52）% in group A，（31.66plusmn;4.72）% in group C，（51.00plusmn;5.66）% in group D. Compared with each other groups，the differences are statistically significant （F=42.46, Plt;0.001）. The number of positive staining cells of retinal leukocyte was （46.16plusmn;10.68）in group A，（133.83plusmn;20.43）in group B,（99.83plusmn;9.28）in group C，（121.33plusmn;10.23） in group C. Compared group B with group C，the number of positive staining cells raised about 2.89 times；compared group B with group C and D，the differences are statistically significant （P=0.12，95% confidence interval -3.69~28.69）. Conclusions In vivo, HIF-1alpha; can decreased the expression of CD18 on neutrophils from diabetic ratsprime; peripheral blood and the collection of retinal leukostasis in the diabetic animals. HIF-1alpha; may serve as a therapeutic target for the treatment and/or prevention of early diabetic retinopathy. （Chin J Ocul Fundus Dis，2008，24：268-271）
Objective To observe the effect of Fufang XueShuanTong (FXST) on prevention for retinal microangiopathy of diabetic rats. Methods Take the normal male Wistar rats as normal control group; take the streptozotocin (STZ) Wistar rats as diabetic model group. And then the diabetic model group was divided into two groups: diabetic control group (without other treatment) and FXST treatment group (with FXST at dose 900 mg/kg, by the way of given medicine from esophagus to stomach, 1 time/day, experimental period was 20 weeks). When all the animals had been raised for 20 weeks, not only retinal digesting preparations were used, the endothelium/pericyte ratio (E/P ratio) and micro-vascular changes were observed by microscope, vascular relative area were measured by image system，but also the thickness of capillary basement membrane, the ultrastructural changes of endothelium and pericyte were observed by transmission electron microscope. Results On the 20th week, retinal digesting preparations showed that acellular capillaries, irregular vessel nets, segmental expansion, segmental stricture even occlusion, pericyte number decreased obviously, E/P ratio increased, vascular relative area increased and ghosts of pericytes etc in diabetic control group. Compared to diabetic control group, the retinal changes of FXST treatment group was lighter, the E/P ratio and vascular relative area were closer to normal control group. Transmission electron microscopy results showed that thickness of basement membrane was increased in DM group, vascular changes was light in FXST treated group. Conclusions FXST can prevent the changes of micrangium in diabetic rats effectively. （Chin J Ocul Fundus Dis，2008，24：272-275）
From the results of this experiment, it showed that the implanted tendon was gradually extruded from the tibia hole and attached to the periosteum. The dominant breeding of tissue cells, cytodynamics, the perimeter ratio of tendon/bone and the effect of revascularization were discussed in detail.
Objective To investigate the effects and mechanism of curcumin on the retinal neovasularization in mice with oxygeninduced retinopathy (OIR). Methods A total of 72 C57BL/6J mice were divided into normal, OIR model, vehicle control ［dimethyl sulphoxide (DMSO)］, and curcumin group (100, 50, and 10 mg). The mice in normal group lived in normoxia condition; OIR model was set up according to standard methods in the literature. Five days after OIR establishment, the mice in curcumin group received an intraperitoneal (IP) injection of 0.1 ml curcumin (100, 50, and 10 mg), and the mice in DMSO group received an IP injection of 0.1 ml 1permil; DMSO. All of the mice were executed at the age of postnatal day 17 (P17) and the eyeballs were collected. Endothelial cell nuclei breaking through the internal limiting membrane were counted after stained with hematoxylin and eosin (HE). The expression of vascular endothelial growth factor-A (VEGF-A), vascular endothelial growth factor receptor-2 (VEGFR-2), endostatin (ES), and phosphorylated p38 mitogen-activated protein kinase (p-p38MAPK) in the retina in each group were measured by real-time polymerase chain reaction (RT-PCR) and Western blot methods.Results Compared with the normal group, retinal neovascularization was found in OIR model group (P＜0.05). The number of endothelial cell nuclei was 46.00plusmn;16.00 in OIR model group and 0.17plusmn;0.41 in normal group (P＜0.05). The expression of VEGF-A, ES, and p-p38MAPK in 100 mg curcumin group differed statistically from which in 50 and 10 mg curcumin group (P＜0.05). The expression of VEGFR-2 was same in the three curcumin groups (P＞0.05). Conclusion Curcumin can inhibit the formation of retinal neovascularization; the mechanism may be associated with inhibiting the expression of VEGFA and VEGFR-2, increasing the expression of ES, and inhibiting the p38MAPK signal transduction pathway.
ObjectiveTo observe the effect of Crocin on structure and the expression of tumor necrosis factor-alpha; (TNF-alpha;) and interleukin-1beta; (IL-1beta;) in rat retina after injury by ischemia-reperfusion. Methods A total of 80 Sprague-Dawley male rats at the age of 8 -10 weeks were divided into control group, model group, low-dose Crocin group and high-dose Crocin group, with 20 rats in each group. The rats of control group were not treated. The rats in model, low-dose Crocin and high-dose Crocin group were induced with normal saline by anterior chamber perfusion creating a retinal ischemia-reperfusion (RIR) model. The rats of the low-dose Crocin and highdose Crocin group received intraperitoneal injection with different doses of Crocin solution (5 mg/kg, or 50 mg/kg) 30 minutes prior to ischemic injury and one time per day after successful RIR. Optical microscopy was used to observe the retinal structure. Enzymelinked immunosorbent assay (ELISA) was used to measure the expression of TNF-alpha; and IL-1beta; 6, 12, 24 and 48 hours after RIR. ResultsThe retinal structure of control group was normal. Pathological changes were found in the RIR model and low-dose Crocin group, such as retinal edema, disorganized structure and loosely packed cells. The degree of pathological changes in lowdose Crocin group was less than the RIR model group. The retinal structure of high-dose Crocin group was similar to the control group. The expression of TNF-alpha; was the highest at 24 hours after modeling, while the expression of IL-1beta; was the highest at 12 and 48 hours after RIR modeling. Six, 12, 24 and 48 hours after RIR modeling, compared with the control group, the TNF-alpha; expression of model (t=5.42, 7.94, 9.32, 9.18;P＜0.05 ), low-dose Crocin (t=3.94, 4.12, 4.98, 3.84;P＜0.05) and high-dose Crocin group (t=2.13, 2.34, 2.96, 2.78;P＞0.05) were increased. Compared with the RIR model group, the TNF-alpha; expression of low-dose Crocin (t=3.95, 4.56, 4.01, 5.12) and high-dose Crocin group (t=5.23, 7.65, 7.74, 7.63) was decreased. Compared with the control group, the IL-1beta; expression of model (t=7.23, 7.87, 7.15, 15.60), low-dose Crocin (t=5.65, 5.10, 5.54, 6.87;P＜0.05) and high-dose Crocin group (t=4.38, 5.21, 4.56, 4.75) was increased (P＜0.05). Compared with the model group, the IL-1beta; expression of low.dose Crocin group was decreased significantly 48 hours after RIR modeling (t=7.56,P＜0.05); but it decreased significantly at each time point in high-dose Crocin group (t=6.94, 5.36, 6.05, 10.50;P＜0.05). Conclusion Crocin can improve the retinal pathologic changes, while down-regulating TNF-alpha; and IL-1beta; expression in RIR rats.