2.1. agents and cell lines
PA (purity ≥ 98%, PU0510-0025) was purchased from Push Biotechnology. PA used for animal experiments was purchased from Nanjing Jin Yibai Biological Technology Co. Ltd. SI used in animals was purchased from Shanghai Macklin Biochemical Co., Ltd. The hydrogen peroxide solution is purchased from Guangdong Hengjian Pharmaceutical Co., Ltd. Antibodies anti-Nrf2 (16396-1-AP), anti-lysis caspase-3, anti-HO-1, anti-Bcl-2 and anti-Bax were obtained from Proteintech. ML385 and ZNPP were purchased from AbMole bioscience, and si was purchased from Thermofisher, Human RPE cell line (ARPE-19) was taken from the American Typical Culture Collection (ATCC, Mantissa, VA).
Cells were cultured in a complete medium ( DMEM medium + 10% FBS + 1% antibiotics) at 37°C in a humidified atmosphere of 5% CO2.
2.2. cell viability assay
An MTT kit (Sevier G4101-1000T) was used to assess the cell viability of different groups. ARPE-19 cells were seeded in 96-well plates for 24 h with 2×104 cells in each well, then, stimulate with H2O2 (100–800 µmol/L) for 24 h. As in PA treated group, cells were pretreated with PA of different concentrations (10, 20, 40, 80 µmol/L), before stimulation with H2O2 (400 µmol/L) for 24 h. In the end, the cells were incubated with MTT (20 µL) for 4 hours, remove the supernatant afterward, and lysed by DMSO, the absorbance of MTT in cells was measured at 570 nm. The ZNPP or ML385 was used to confirm the effect of PA.
2.3. flow cytometry
The cells of each group were collected and stained with annexin V/PI after different treatments (Yeasen Biotechnology Co., Ltd, Shanghai), the fluorescence was measured by flow cytometry, and the apoptosis rate was calculated according to the status of co-staining. All procedures were carried out following the manufacturer’s instruction
2.4. mitochondrial membrane potential measurement
To measure the potential damage to the mitochondria, mitochondrial membrane potential (MMP) was measured using a JC-1 fluorescent probe (Servicebio). After different treatments, RPE cells were subjected to 1 mL of JC-1 solution and incubated for 30 min at 37°C protected from light. The ratio between red and green fluorescence was determined by fluorescence microscopy.
2.5. determine the intracellular ROS level
DCFH-DA (2,7-dichlorofluorescein diacetate, Beyotime) was used to determine the level of intracellular reactive oxygen species (ROS), RPE cells of each group were incubated with 10 µM detecting compound for 30 min at 37°C protected from light. Afterwards, the level of ROS in ARPE cells is observed under fluorescence microscopy (OLYMPUS).
2.6. western blot
The cells of different groups were lysed and the proteins were extracted by RIPA (Best Bio) buffer and PMSF and their concentration was determined with the BCA Protein Assay Kit (Beyotime). After electrophoresis and transferring protein on a blotting membrane, the blotting membrane was incubated with the primary antibody overnight at 4°C, and the excess primary antibody was washed off and incubated with the secondary antibody for 1 h. The intensity representing the expression level of the proteins was measured by ImageJ software. The following antibodies were used: anti-HO-1, anti-Nrf2 (Proteintech 16396-1-AP), anti-lysis caspase-3, Bax and Bcl-2 as primary antibodies.
2.7. animal
Healthy male C57BL/6J mice (body weight 20–25 g). Mice were accessed freely to standard food and water at room temperature at 22–24°C with 12 h light/dark cycles. The animals were adapted for at least 1 week before the experiment. All procedures followed the Ethics Committee of Liyang Traditional Chinese Medicine Hospital (No.2023LY-1-20-01).
24 mice are randomly divided into three groups: (1) Control group (n = 8): Intraperitoneal injection of normal saline (NS) containing 0.1% DMSO once daily for 1 week, then tail intravenous injection of NS without SI once, continue intraperitoneal injection of NS containing 0.1% DMSO once daily until mice are sacrificed. (2) Si group (n = 8): NS containing 0.1% DMSO is injected intraperitoneally once daily for 1 week, followed by tail intravenous injection of NS containing SI (40 mg/kg) once, followed by continued intraperitoneal injection of NS containing 0.1% DMSO once daily until mice are sacrificed. (3) SI + PA group (n = 8): intraperitoneal injection of NS containing 0.1% DMSO and PA (10 mg/kg) once daily for 1 week, followed by intravenous injection of NS containing SI (40 mg/kg) once by tail, then continued intraperitoneal injection of NS containing 0.1% DMSO and PA (10 mg/kg) once daily for 4 weeks until mice are sacrificed.
2.8. Micron IV imaging
On day 28 of treatment, mice are anesthetized by intraperitoneal injection of Tribromoethanol (400 mg/kg). Then, the retinal of five mice (both eyes) in each group were observed by Micron IV retinal imaging camera system. Mice are subjected to optical coherence tomography (OCT) to observe retinal morphology, and retinal thickness is measured using ImageJ software.
2.9. HE and TUNEL staining
On day 28 after SI injection, the eyes mouse are freshly harvested and fixed overnight with eyeball fixative solution, dewaxed vertically (5 µm thick), then stained with hematoxylin and eosin. the Pathological changes were observed with light microscopy, the retinal layer thickness was measured using ImageJ software
An in situ cell death detection kit (Servicebio) the apoptosis in the mouse retina was detected by the use of. Samples fixed in 4% paraformaldehyde were infiltrated with diluted Triton X-100 sodium citrate buffer for 2 min, and subjected to terminal dUTP notched end labeling (TUNEL) staining and visualized with diaminobenzidine (DAB) kit. The nucleus was counterstained with hematoxylin. The total number of apoptotic cells was counted as nuclei stained by TUNEL in three fields of view randomly selected for each tissue, and the apoptosis rate was presented as the ratio of TUNEL-positive nuclei to total nuclei/field of view.
2.10. immunofluorescence
Fixed section samples were permeabilized for 30 min and blocked (3% BSA) for 1 h at room temperature. a specific primary antibody was added for incubation overnight at 4°C with anti- Nrf2 and HO-1 (Servicebio). Then recovered at 37°C for one hour. The secondary antibodies were incubated for 1 h after washing off the excess primary antibody, and the nuclei were stained by DAPI for extra 10 min. The specific fluorescence was observed by fluorescence microscope (Nikon Eclipse C1).
2.11. detection of SOD, CAT and GSH-px
Blood samples of mice from different groups were collected by Eye removal into the Eppendorf tube, and centrifuged at 4,000 r/min for 10 min at 4°C after resting at room temperature for 1 h to collect serum samples. On the other hand, RPE cells are lysed by ultrasound and centrifuged to collect the supernatant according to the manufacturer's instructions.
After mice were sacrificed, a certain amount of tissue samples were added with cold PBS and homogenized in an ice bath, followed by centrifugation at 4°C for 10 min. The BCA kit (Beyotime) is used to detect protein concentration in the supernatant. Subsequently, the activity of (GSH-px, ml011256-J), catalase (CAT, ml095231-J), and superoxide dismutase (SOD, ml095231-J) was performed with the corresponding biochemical kit purchased from Shanghai Enzyme-linked Biotechnology Co., Ltd. as previously described in our laboratory.
2.12. statistics
The GraphPad Prism 9.0 statistical software (La Jolla, CA, USA) was used for data analysis. Data are expressed as mean ± S.D. for a minimum of three independent experiments. One-way ANOVA and the LSD test were used to analyze significant differences in multiple and single comparisons, respectively. Values of p<0.05 were considered statistically significant.