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Objective: To investigate the protective effect of safflower yellow pigment (SYP) on diabetic (DM) retinal ganglion cells (RGC) by regulating p38 mitogen-activated protein kinase (MAPK) signaling pathway. Methods: Selected 50 rats and 40 of them were used to establish DM rat model by single intraperitoneal injection of streptozotocin. They were randomly divided into model group and safflower yellow pigment low, medium and high dose groups, and the remaining 10 rats was control group. Safflower yellow pigment low, medium and high dose groups were intraperitoneally injected with 20, 40, and 80 mg/kg of SYP. Model group and control group were intraperitoneally injected with the same amount of physiological saline solution for six weeks. The general state of the rats was observed, and the morphological changes of RGC were observed by hematoxylin-eosin (HE) staining, and the number of RGC cells was counted. RGC apoptosis rate was detected by in situ apoptosis (TUNEL) method. Real-time quantitative polymerase chain reaction (RT-PCR) and Western blotting were used to detect the mRNA and protein expressions of p38 mitogen-activated protein kinase (MAPK), phosphorylated p38MAPK (p-p38MAPK) and cysteine-containing aspartate proteolytic enzyme-3 (Caspasae-3), vascular endothelial growth factor polyclonal antibody (VEGF), and protein expression ratio of p-p38/p38MAPK was compared. Results: All the rats survived during the experiment. There were no abnormalities in the rats in the control group. The blood glucose in the model group was higher, the diet and urine volume were larger, and the body weight was reduced. The safflower yellow pigment low, medium and high dose groups were better than the model group. Pathological observation showed that there were no abnormalities in the cells of the retina of the control group, while the RGC cells in the model group were disordered arranged and the nucleus were sparse, and the number of cells in the bipolar cell layer and the photoreceptor layer was reduced, and the arrangement was sparse. The abnormalities of RGC cells and outer bipolar cells in the safflower yellow pigment low, medium and high dose groups were significantly lower than those in the model group, and the high dose groups was the most obvious. There was significant difference in the number of RGC between groups (P < 0.05). Compared with the control group, the number of RGC in the model group was significantly decreased (P < 0.05), the apoptotic rate was significantly increased (P < 0.05), the levels of Caspase-3 and VEGF in the retina were significantly increased (P < 0.05), and the values of p-p38MAPK/p38MAPK were significantly increased (P < 0.05). Compared with the model group, the number of RGC was increased significantly (P < 0.05), the apoptotic rate of RGC was decreased significantly (P < 0.05), the expression levels of Caspase-3, VEGF and protein in retina were decreased significantly (P < 0.05), and the value of p-p38MAPK/p38MAPK was decreased significantly (P < 0.05), and the differences among the dose groups were significant (P < 0.05). Conclusion: Safflower yellow pigment can protect RGC of DM rats by inhibiting p38MAPK signaling pathway, and reduce RGC apoptosis. The 80 mg/kg of SYP has the best protective effect.
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Objective To investigate the effect of silment information regulator factor related enzymes 1 (SIRT1) on the apoptosis of retinal ganglion cells (RGCs) in rats with diabetic retinopathy and its downstream molecular mechanisms.Methods Together 60 healthy male Sprague-Dawley rats were collected and randomly divided into normal group,diabetic group,SIRTI activator-resveratrol treatment group (treatment group),and diabetic rat model was induced by intraperitoneal injection of streptozotocin at 60 mg · kg-1 in the latter two group rats,while the normal group was injected with sodium citrate buffer at 60 mg · kg-1.Then,after 72 h,rats with blood glucose ＞ 16.7 mmol · L-1 were designated as diabetic rats by blood glucose test.Then each rat in the treatment group was treated with SIRT1 activator-resveratrol at 20 g · kg-1 once a day at the 2nd day after the success of the model,and the normal group and diabetic group were given methylene chloride.Finally,after immunohistochemical staining for retina,TUNEL assay was used to evaluate the apoptosis of RGCs,while the expression of SIRTI,p38 MAPK and Caspase-3 protein was detected by Western blot.Results The apoptotic index of RGCs in the normal group,diabetic group and treatment group was (0.848+0.131)％,(19.038 + 1.327)％,(10.461 + 1.089)％ respectively at 8 weeks,and the difference among the three groups was statistically significant (F =670.497,P =0.000),while the differences between each two groups were also statistically significant (all P =0.000).Furthermore,when compared with the normal group (0.132 ± 0.043),the expression of SIRT1 protein in the diabetic group (0.060 ± 0.028) and the treatment group (0.073 ± 0.026) was significantly decreased,and the overall difference among the three groups was statistically significant (F =1 310.663,P =0.000),while the differences between each two groups were also statistically significant (all P =0.000).The expression levels of p38 MAPK and Caspase-3 were increased in diabetic group (1.121 ± 0.082,0.266 ± 0.005) and treatment group (0.574 ± 0.012,0.190 ±0.060) respectively,and the overall difference and pairwise comparison in the three groups approached statistically significance (all P =0.000,0.000).Conelusion Up-regulation of SIRT1,can inhibit the apoptosis of RGCs,and protect RGCs against apoptosis in rat model of diabetic retinopathy,which may be correlated with the downregulation of p38 MAPK signal pathway.
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Objective To investigate the effect of silment information regulator factor related enzymes 1 (SIRT1) on the apoptosis of retinal ganglion cells (RGCs) in rats with diabetic retinopathy and its downstream molecular mechanisms.Methods Together 60 healthy male Sprague-Dawley rats were collected and randomly divided into normal group,diabetic group,SIRTI activator-resveratrol treatment group (treatment group),and diabetic rat model was induced by intraperitoneal injection of streptozotocin at 60 mg · kg-1 in the latter two group rats,while the normal group was injected with sodium citrate buffer at 60 mg · kg-1.Then,after 72 h,rats with blood glucose ＞ 16.7 mmol · L-1 were designated as diabetic rats by blood glucose test.Then each rat in the treatment group was treated with SIRT1 activator-resveratrol at 20 g · kg-1 once a day at the 2nd day after the success of the model,and the normal group and diabetic group were given methylene chloride.Finally,after immunohistochemical staining for retina,TUNEL assay was used to evaluate the apoptosis of RGCs,while the expression of SIRTI,p38 MAPK and Caspase-3 protein was detected by Western blot.Results The apoptotic index of RGCs in the normal group,diabetic group and treatment group was (0.848+0.131)％,(19.038 + 1.327)％,(10.461 + 1.089)％ respectively at 8 weeks,and the difference among the three groups was statistically significant (F =670.497,P =0.000),while the differences between each two groups were also statistically significant (all P =0.000).Furthermore,when compared with the normal group (0.132 ± 0.043),the expression of SIRT1 protein in the diabetic group (0.060 ± 0.028) and the treatment group (0.073 ± 0.026) was significantly decreased,and the overall difference among the three groups was statistically significant (F =1 310.663,P =0.000),while the differences between each two groups were also statistically significant (all P =0.000).The expression levels of p38 MAPK and Caspase-3 were increased in diabetic group (1.121 ± 0.082,0.266 ± 0.005) and treatment group (0.574 ± 0.012,0.190 ±0.060) respectively,and the overall difference and pairwise comparison in the three groups approached statistically significance (all P =0.000,0.000).Conelusion Up-regulation of SIRT1,can inhibit the apoptosis of RGCs,and protect RGCs against apoptosis in rat model of diabetic retinopathy,which may be correlated with the downregulation of p38 MAPK signal pathway.
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Background Recently,the study on the cause of optic nerve damage induced by glaucoma is of concern in ophthalmology.Some research showed that the immune system is associated with glaucoma-induced optic neuropathy.Acute ischemia-reperfusion is an ideal model of studying optic neuropathy.ObjectiveThe present study investigates the effect of T and B lymphocyte deficiency on the retinal neurocytes of mice with acute intraocular hypertension.Methods Sixteen SPF CB-17/Icr.Cg-Prkdc~(scid)Lyst~(bg)/CrlVR mice 6-8 week-old (severe combined immunodeficiency mouse,SCID) were used in this study and 16 age-matched SPF wild type (C57BL/6) mice served as controls.The ischemia-reperfusion injury models were induced in the right eyes of 10 SCID mice and 10 C57BL/6 mice through intra-anterior chamber infusion of balanced saline solution for 45minutes to increase the intraocular pressure to 104mmHg,and the left eyes served as model controls.The other 6 SCID mice and 6 C57BL/6 mice served as normal control group.10g/L (2μL) of FlouroGold was injected into the brains of the mice for the labeling of surviving retinal ganglion cells 21 days after ischemia-reperfusion.The thickness of retinal inner nuclear layer was measured by H&E staining under the fluorescent microscope 21 days after ischemic insult.The use of the animals followed the Standard of Association for Research in Vision and Ophthalmology.Results In normal control mice,the morphology of retinal ganglion cells (RGCs) and retinal structure were similar between SCID mice and C57BL/6 mice.The differences in the numbers of RGCs and retinal thickness were insignificant between the two types of mice(P＞0.05).In the experimental mice,the surviving RGCs were strikingly increased in SCID mice (91%±5%) compared with C57BL/6 mice(78%±5%)(P=0.003).The thickness of the retinal inner nuclear layer was obviously thinner in the model eyes (22.44±1.70μm) compared to model control eyes (31.06±3.75μm) in C57BL/6 mice(P=0.004),but no statistically significant difference was found between the model eyes and model control eyes in SCID mice (33.52±2.13μm vs 34.06±3.00μm) 21 days after ischemia-reperfusion injury(P＞0.05).Conclusion T and B lymphocytes deficient mice show a better tolerance to acute intraocular hypertension than the wild type C57BL/6 mice.
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To investigate the neuroprotective effect of melatonin (MT) on retinal ganglion cells (RGCs) in rats with ischemia reperfusion injury (RIR), 24 healthy SD rats were randomly divided into two groups:group A and group B. RIR model was induced in the left eyes by increasing the pressure of the anterior chamber. Group A was treated with 10 % alcohol- normal saline (1 mL/kg/d, ip), while group B was treated with 0.5 % MT (1 mL/kg/d, ip). On the basis of the time interval between the left eyes RIR and the sacrifice, rats in both group A and group B were further divided into 3 subgroups: groups A1 and B1 (days 7), groups A2 and B2 (days 14), groups A3 and B3 (days 30), with4 rats in each subgroup. 7 day before the sacrifice, 3 % fluorogold was bilaterally injected into superior colliculi and geniculate body. The eyes were enucleated after being sacrificed, and mounting of the retina from both eyes was performed on a slide and observed under a fluorescence microscope. Four photos were taken from each of the four quadrants of the retina.The labeled-RGCs were counted by using a computerized image analyzer. The rate of the labeledRGCs was used for statistical analysis. Our results showed that, in group A, the rate of the labeled-RGCs was (77. 16±6.35) %, (65.53±7.01) %, (53.85±4.38) % on day 7, 14 and 30.In group B, the rate of the labeled-RGCs was (81.33±9.27) %, (79.80±8.36) %, (80.34±11.05) % on day 7, 14 and 30. In group B, which was treated with MT after RIR, the rate of labeled-RGCs was significantly higher than that of group A on day 14 and day 30 (P＜0.05). It is concluded that, in the RIR rats, MT therapy could increase the survival rate of the RGCs and could rescue and restore the injured RGCs.
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AIM: To investigate the differentiation of neural stem cells (NSCs) after transplanted into vitreous and the effects on the regeneration of retina ganglion cells (RGCs) after optic nerve microcrushed.METHODS: After optic nerve microcrushed in adult rat, 2?104/2 ?L NSCs or 2 ?L 0.1 mol/L PBS was injected into vitreous. Animals were divided into control group (MC group, MC+PBS group) and experiment group (MC+NSCs). Animals in each group were allowed to survive for 3, 4, 5 weeks, respectively. The regenerating RGCs were labeled retrogradely with granular blue, and the numbers of regenerating RGCs in each retina were observed under fluorescent microscope. In addition after 5 animals in MC+NSCs group survived for 4 weeks, rat eyeballs were removed and prepared as freezing microtome sections for observing the migration of NSCs and NF, GFAP, CNP immumodetection.RESULTS: Compared the mean numbers of regenerating RGCs between experiment group and control group at 3, 4, 5 weeks, the difference was significant (P
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Objective To study methylprednisolone's effect of up-regulation of Hsp27 on provocation and enhancement of self-protection and self-repair to injured central nerves,and if the injured nerves can get protection in early period,and therefore benefit injured central nerves'survival and regeneration. Methods Optic nerve axotomy was used in the experiment.The animals survived for 4,7,14,21,28 days respectively after surgery with and without MP treatment.The retinas were taken out and cut,then the number and morphological changes of RGCs with Nissl staining and the expression of Hsp27(optic density) in ganglion cell layers with immunohistochemical staining were observed respectively.The data were analysed with SAS soft ware. Results The quantity of the surviving retina ganglion cells increased in the group with high-dose intravenous methylprednisolone treatment at 7th and 14th days(P