| Background:Visual impairment is increasing as a global public health problem and solutions for the impairment are still needed. In spite of the progress made in surgical techniques in recent years, many retinal diseases remain incurable. Classic ocular drugs consist of anti-infective, anti-inflammatory and anti-allergic agents, such as antibiotics, glucocorticoids and ETC. However, these drugs, with their similar effectiveness and side effects, are double-edged swords for patients. In addition, the progress of drug development for eye diseases remain stagnant. Comparing to the drugs mentioned above, Chinese medicine is rich in resources with few side effects. Chrysanthemum is a traditional Chinese medicine for protecting vision. Numerous studies, in ancient and modern China, have reported that chrysanthemum is helpful in many eye diseases, including age-related macular degeneration (AMD), arteriosclerosis of the retina, diabetic retinopathy and some drug-related retinal toxicity. However, few scientific studies have focused on how chrysanthemum benefits our eyes. Therefore, we aim to investigate the role and mechanism of chrysanthemum cytoprotection in the retina.Methods.1. Human retinal pigment epithelium cells (ARPE-19) were treated with varying concentrations of ADR and diosmetin (DIO), viable cells were determined using an MTT assay. Sprague-Dawley (SD) male rats were used to receive 5μL intravitreal injections in the right eye with ADR through a Hamilton syringe with a 30-gauge needle into the inferotemporal part of the eye, and were euthanized after 5 days injection. The eyeballs were surgically excised and fixed in phosphate-buffered 10% formaldehyde. The fixed eyes were sectioned at the pars plana area and the posterior segment was dissected into tissue samples for further analysis.2. ARPE-19 cells were treated with ADR and diosmetin (DIO). Cell apoptosis were detected by DAPI staining, Annexin V/PI staining and Western-blot. SD rats were used to receive 5μL intravitreal injections of DIO and ADR. The fixed eyes were sectioned at the pars plana area and the posterior segment was dissected into tissue samples. The retinal damage were analyzed by H&E, TUNEL staining and Western-blot.3. ARPE-19 cells were treated with ADR and diosmetin (DIO). DNA damages were analyzed by y-H2AX staining and Western-blot. SD rats were used to receive 5μL intravitreal injections of DIO and ADR. The fixed eyes were sectioned at the pars plana area and the posterior segment was dissected into tissue samples. DNA damage were detected by y-H2AX expresion.4. ARPE-19 cells were treated with ADR and diosmetin (DIO). Oxidative stresses were analyzed by H2DCFDA staining and GSH Enzymatic cycling methods. And mitochondria dysfunctions were detected by JC-1 staining and Western-blot.Results:1. DIO Protects against ADR-Induced Retinal Injury in vivo and in vitro.DIO treatment could significantly decrease the ADR-induced cytotoxicity in ARPE-19 cells even at a high concentration. After a single injection of ADR per eye, slices of retina exhibit atrophy and degeneration of the pigment epithelium with detachment and widespread apoptotic cells. By contrast, the damage was largely attenuated by DIO.2. DIO Attenuates ADR-Induced Human Retinal Pigment Epithelium Cell ApoptosisThe extensive nuclear condensation and fragmentation in ARPE-19 cells treated with ADR, which resulted in a decrease by co-treatment with DIO. Meanwhile, DIO showed a significant decrease of the apoptosis rate in ARPE-19 cells exposed to ADR by Annexin V/PI staining. In addition, the number of TUNEL-positive cells in retina increased with ADR was attenuated with DIO. Moreover, the increase in cleaved caspase-3 and PARP fragments expression from ADR treatment, were blocked by treatment with DIO both in vivo and in vitro.3. DIO Prevents ADR-Triggered DNA Damage in RPE Cells.γ-H2AX foci, indicating that the breaks were induced with ADR exposure and abated by DIO through immunofluorescence. Corroborating this observation, western blot analysis of drug-treated cell ly sates also confirmed the expression of γ-H2AX in vivo and in vitro. In addition, it was confirmed the high expression of p53 induced by ADR, was significantly inhibited by DIO treatment in ARPE-19 cells.4. DIO Inhibits ADR-Induced Oxidative Stress and Mitochondria Dysfunction in RPE Cells.DIO resulted in much lower levels of ADR-induced intracellular production of ROS in ARPE-19 cells by H2DCFDA staining. Meanwhile, ADR-induced GSH reduction was increased with DIO treatment. In addition, was observed that DIO preprocessing could antagonize the loss of mitochondrial membrane potential caused by ADR through JC-1 staining. It was also observed that the anti-apoptotic protein Bcl-2, on the mitochondrial membrane, was up-regulated in the DIO+ADR group compared to the ADR group.Conclusion:In conclusion, this is the first time to identify a definite extract, DIO, from chrysanthemum, which possesses a retinal-protective effect through restoring oxidative stress and DNA injury in the RPE layer. The results strongly justify the role of DIO as a potential retinal-protective drug candidate to alleviate the severity of eye diseases in the clinic and provide evidence that it should be developed as a complementary medicine and undergo clinical evaluation. |
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