Chrysanthemum water extraction
Chrysanthemum produced in Hangzhou, China was used in this study, and the petals were the main parts used for water extraction. Chrysanthemum petals were soaked in water at a weight eight times that of the petals and then boiled for 0.5 h. The liquid was stored in bottles, and the residue was boiled again. This process was repeated three times, and all the liquids obtained were combined. The combined liquid was concentrated to low and high concentrations of extract (1 g of chrysanthemum extract was obtained from 4.255 g of material) . The total content of flavonoids in the chrysanthemum reached an effective concentration of 5.0%, according to technical specifications for inspection and evaluation of healthy food.
ARPE-19 cells (Beijing Dingguo Biotechnology, Beijing, China) were cultured in DMEM/F12 (HyClone, USA) containing 10% foetal bovine serum, 100 U/mL penicillin, and 100 mg/mL streptomycin and incubated in 5% CO2 at a constant temperature of 37 °C. The cells were grown to the exponential phase before use in the experiments.
Toxicity of the chrysanthemum extract to the ARPE-19 cells
The concentration of the chrysanthemum extract that was safe and non-toxic to the ARPE-19 cells was assessed using the MTT assay. The ARPE-19 cells (approximately 10 × 103 cells/well) were plated in 96-well plates at 100 μL/well. After the cells were grown to the exponential phase, the chrysanthemum extracts at concentrations of 0.2, 0.4, 0.8, 1.0, 1.2, 1.5, 2.00, and 5.00 mg/mL were added to the 96-well plates and incubated for 24 h. At the end of the culture period, 20 μL MTT (5 mg/mL) solution was added to the cells, followed by incubation in a CO2 incubator for 4 h. The MTT solution was discarded, and 150 μL of DMSO solution was added. After the cells were fully dissolved, the cell activity was evaluated by measuring the absorbance (A value) at a wavelength of 492 nm. Based on the results of this assay, a concentration deemed safe was determined and used in the subsequent experiments.
Light-induced damage to the RPE cells
The cells were digested with trypsin to prepare a suspension and then plated on a culture plate. They were irradiated directly with LED white light with an intensity of 2500 ± 500 lx for 24 h in the light-damaged group (LD group), low-dose chrysanthemum extract group (LC group), and high-dose chrysanthemum extract group (HC group) during the exponential growth phase .
Measurement of ROS levels in the four groups
Intracellular ROS levels were measured using DCFH-DA probes (Gibco, USA). A suspension of ARPE-19 cells (1.5 × 105/well) was seeded into a 6-well plate and cultured for 24 h. The cell culture medium was replaced with DCFH-DA, and the cells were cultured at 37 °C for 20 min. The ImageJ software (National Institutes of Health, https://imagej.en.softonic.com/) was used to analyse the fluorescence intensity.
Group division and chrysanthemum extract treatment
Male C57BL/6 J mice aged 8 weeks (n = 32) were obtained from SPF Biotechnology Co., Ltd. (Beijing, China). The mice were raised in the following conditions: 12/12 h light/dark cycles, temperature of 23 ± 2 °C, and relative humidity of 55%, at the Tianjin Eye Institute; food and water were provided freely. They were randomly divided into four groups: control (n = 8), LD (n = 8), LC (n = 8), and HC groups (n = 8). After the chrysanthemum extract was concentrated and dried, the obtained solid was crushed into a powder. The powder was suspended in 0.9% saline for intragastric administration, which was conducted via insertion of an intragastric tube with a diameter of 1.25 mm and a length of no more than 3 cm into the digestive tract. The effective concentration ranged from 0.23 (low dose) to 0.38 g/kg/day (high dose). Intragastric administration (0.2 mL/day) was performed continuously for 8 weeks. The dose and concentration induced no obvious uncomfortable response in our experiments, which was based on the study by Lumeng et al. on the protective effect of chrysanthemum in mice with Parkinson’s disease .
Light damage induction
After 8 weeks of intragastric administration, the light injury mode was established as follows: the mice in the LD, LC, and HC groups were treated with continuous white light at 10000 lx for 7 days under the condition of dilating pupils using 0.5% tropicamide phenylephrine eye drops (Mydrin, Santen, Japan) 4 h/day for 28 h in total. Each mouse was separated by a transparent box during light exposure to avoid injury caused by crowding.
After 12 h of dark adaptation, the mice were anaesthetised via inhalation of 2% isoflurane and placed on an experimental table at temperature of 40 °C. Both eyes were dilated, and an eye gel was used to keep the cornea transparent. Three types of electrode were used. The reference electrode was connected to the head, annular corneal electrode to the eyeball, and ground electrode to the tail of the mouse . ERG was used to record the a-, b-, and Ops waves after a flash stimulus of 0.01 cds/m2 and 3.0 cds/m2 in scotopic adaptation. After 10 minutes of photopic adaptation, a- and b-waves were evoked by a flash of 3.0 cds/m2 in the photopic mode (RETI-Port 21, Roland, Germany).
Optical coherence tomography (OCT)
The mice were placed on the animal experimental platform after being anaesthetised via inhalation of 2% isoflurane; 0.5% tropicamide phenylephrine eye drops (Mydrin, Santen) were used to dilate the pupils, and a carbomer eye gel was used to keep the corneas transparent. Phoenix eye testing equipment for animals (model: Micron IV, Phoenix Research Labs) was used to scan the retina in vivo and obtain morphological images of each layer. Furthermore, the retinal thickness of the inner nuclear layer (INL) and outer nuclear layer (ONL) of the four groups was measured using a software and compared manually from an area of − 600 to an area of 600 μm from the optic nerve.
Fluorescein fundus angiography (FFA)
Approximately 2 mL of 2% sodium fluorescein was intraperitoneally injected at the end of OCT . The arterial, venous, and arteriovenous phases were clearly observed under the same gain value. The AngioTool (version 0.6a; https://ccrod cancer.gov/confluence/display/ROB2/Downloads), a free-to-download software, was used to analyse the parameters consisting of the vessel area, vessel area percentage, and total number of junctions. This application can be easily used, and the detailed method has been described by Zudaire et al. .
ERG, OCT, and FFA were performed step by step when the pupil was dilated enough. ERG was first conducted as soon as possible, and repeat examination was avoided because this would make the outcome incorrect. The ERG room was dark and absolutely quiet. When ERG was finished, the same mouse was transferred to the OCT and FFA rooms. The two examinations were completed using a Phoenix eye testing equipment for animals (model: Micron IV, Phoenix Research Labs) . All abovementioned progress was measured in vivo; the mice were kept alive; the cornea was specifically evaluated; a carbomer eye gel was continuously added to the surface of the eye to prevent opacities from occurring; and no fewer than three experienced operators were needed.
Haematoxylin and eosin (HE) and TUNEL staining
To obtain blood only after 3 days when sodium fluorescein was excreted completely, we sacrificed all the mice via inhalation of isoflurane, collected the ophthalmic blood vessels, and removed the eyeballs simultaneously. Retinal tissue could be a better option for measuring SOD, CAT, and GSH-Px activities; however, this will require too many mice to obtain enough materials, so blood from the optic artery was considered an economical sample. After 12 h of fixation of the eyeballs in formalin, part of the cornea was cut off and fixed in fresh formalin for another 8 h. After dehydration and paraffin liquid immersion at 40 °C for 6 h, the eyeballs were completely embedded in paraffin blocks. The paraffin blocks were cut into 4-μm-thick parasagittal sections. The sections were then subjected to HE staining, TUNEL immunofluorescence staining (TUNEL fluorescence kit provided by Dalian Meilun Biotechnology Co., Ltd., Dalian, China), and other immunofluorescence techniques [18, 19]. In each group, four eyeballs from different mice were analysed to count the apoptotic cells. At least four sections were counted from each eye; thus, 16 sections were used in each group and subjected to TUNEL immunostaining. To calculate the apoptosis rate, the tunel (+) cells in the retina were counted manually with Image J software (National Institutes of Health, https://imagej.en.softonic.com/).
Immunofluorescence staining of the retina
The paraffin slices (4 μm) were performed with antigen repair and blocked with 5% BSA for 1 h. Then the slices were incubated with the antibodies of NF-κB (1:200, SAB4300554, Sigma-Aldrich LLC, Germany) and TNF-α (1:200, 17,590-1-AP, Proteintech Group, Inc., Wuhan, China) at 4 °C overnight. The secondary antibodies coupled with Alexa Fluor 488 (1: 1000, SA00013-2, Proteintech Group, Inc.) were used to incubated with slices for 2 h. The nucleus was stained with DAPI for 5 min. Finally, the slices were imaged by Leica SP8 confocal microscope with 20× objective. The fluorescence intensity was detected using the ImageJ software.
Antioxidant enzyme assays
All the groups of blood collected from the ophthalmic artery were centrifuged at 4 °C, 3000 r/min for 10 min to obtain the serum. They were kept at − 80 °C to measure the enzymatic activities of SOD, CAT, and GSH-Px. The method of examination followed the instructions of the SOD, CAT, and GSH-Px assay kits (WST-1, Nanjing Jiancheng Bioengineering Institute).
All parameters, including ROS expression, cell viability, a- and b-wave amplitude on ERG, retinal thickness, vessel area, vessel percentage, total number of junctions, apoptosis rate, antioxidant enzyme (SOD, CAT, and GSH-Px) content in the blood, and NF-κB and TNF-α expression density, were presented as x ± s. One-way ANOVA and the SPSS software (version 22.0, IBM Corp., Chicago, USA) were used to evaluate all parameters. Differences with a P-value of < 0.05 were considered significant. P-values of < 0.01 indicated a more obvious difference.