The GSPE with over 98% proanthocyanindin was purchased from Rongsheng Biotechnology Co., Ltd. (Xi'an, China) and dissolved in drinking pure water for in-vivo study and in DMSO for in-vitro treatment. NAMPT inhibitor, Fk866, and SIRT1 inhibitor, EX-527, were purchased from Sigma (Sigma-Aldrich, USA). The NAD + precursor, NMN, was purchased from APExBIO (APExBIO, USA).
All animal experiments were conducted in accordance with the Association for Research in Vision and Ophthalmology Statement for use of Animals in Ophthalmic and Vision Research. The animal protocol was approved by the Animal Ethics Committee of Zhengzhou University. Both male and female C57BL/6J mice of different age groups were obtained from a commercial vendor (Sipuer-Bike Laboratory, Shanghai). The mice would be housed under a pathogen-free environment with a 12:12 h light/dark cycle and free access to laboratory chow and water. Animal rooms were maintained at 20–22°C with 30–70% relative humidity. Blood samples collections and RPE isolations were conducted in accordance with the previous studies in different time points. The obtained animal samples were stored in -80 ℃ until advanced experiments. GSPE treatment would be conducted through added GSPE in drinking water and GSPE treatment conducted in the 15 months aged mice. After three months GSPE treatment, the 18 months aged mice would be used in the following experiments.
The ARPE19 cells were purchased from American type culture collection (ATCC) and cultured with Dulbecco’s modified Eagle medium (DMEM) (HyClone, USA) supplemented with 10% heated-inactivated fetal bovine serum (FBS), 100 U/ml penicillin, and 100 µg/ml streptomycin (Gibco, USA). The ARPE-19 cells would be maintained at 37°C in a humidified chamber with 5% CO2 and 1:3 subcultures were conducted using trypsin-EDTA solution.
RNA extraction and real-time PCR
The extraction of total RNA of both animal examples and RPE cells were conducted with a Trizol reagent (Invitrogen, USA) following the manufacturer’s protocol. cDNA synthesis was conducted using Transcriptor First Strand cDNA Synthesis Kit (Thermo Fisher Scientific, USA). RT-PCR was performed with Applied Biosystem™ SYBR™ using the TaqMan Multiplex Real-Time PCR Solution according to the manufacturer’s instructions. Relative mRNA expressions would be calculated using the ΔΔCt method. The primers used in this study were listed in Table 1. Three independent experiments were conducted for each sample and data were used for advanced analyses.
NAD + assay
The NAD + contents in both animal samples and cellular homogenates were detected using a commercial NAD + assay kit (Sigma-Aldrich, USA). All the experiments were conducted according to the manufacturer's instructions.
In-vitro aging model
The in-vitro aging model of RPE cells were conducted according to a protocol as previously described in our lab  with slight modifications. In general, the APRE-19 cells were placed in petri dishes and then exposed to 800 µM H2O2 for 2 hours at a temperature of 37°C from day one for five continuous days. Fresh complete media would be replaced after H2O2 treatment. The aging RPE cells would be collected in the other day after last H2O2 treatment and this aging in-vitro model would be used in the following studies.
The cell viability was examined in this study with MTT assay. MTT dye (Solarbio, China) was added to the 96-well plate with ARPE-19 cells in different groups and incubated for four hours at 37°C. After replacing the culture media with 150 µL of DMSO to each well, the 96-cell plates were shocked thoroughly until the crystals dissolved. Absorbance was measured at a wavelength of 490 nm using the Thermo Scientific Fluoroskan microplate reader (Thermo Fisher Scientific, USA). Three independent experiments were conducted for each group and data were used for advanced analyses.
Cellular senescence marker, beta-galactosidase (β-Gal), was used in the measurement of senescent status in different cell models and the assay was performed with a commercia assay kit (Beyotime Biotechnology, China) according to the manufacturer's protocols. The view and counting would be conducted under the brightfield in Leica Microscope (Leica Biosystems, Germany). The senescent cells expressing SA β-gal were positive cells and stained blue. The positive cells rates were recorded in each group and the data would be used in advanced analyses.
mtDNA lesions measurement
As NAD + was an important contributor in mtDNA damage repairment, the mtDNA lesion measurement was conducted in each group. The detection of mtDNA was performed using a well-validated QPCR based detection assay, as previously described . The DNA extraction was conducted using a genomic-tip kit (Qiagen, CA) and the extracted DNA would be stored in -80 ℃ before experiments. The principle of mtDNA lesion measurement was the that any lesion on a DNA template would block the function of thermostable polymerase and therefore only the DNA templates without any DNA damages could be amplified. After DNA templates quality control, the QPCR would be conducted with a High-Stability PCR Kit (Genscript, China). The β-globin product was used as an inner-control for the detection of relative mtDNA lesion as described previously. Three independent experiments were conducted with three repetitions.
Mitochondrial membrane potential
The JC-1 dye accumulated in the mitochondria in a potential-dependent manner and could be used to detect the membrane potential of cells, tissues or purified mitochondria. JC-1 was an ideal fluorescent probe widely used to detect the △Ψm position of mitochondrial membrane electricity with a JC-1 assay kit (Beyotime Biotechnology, China). After aspirate culture media in the 6-well plate and washing the cells with PBS, 1ml JC-1 dyeing working solution was added each well. After incubating the mixed working system for 20 minutes at 37°C, the cells would be washed with the JC-1 Buffer (1×) and placed on ice. The cells would be observed under a confocal microscope (Leica, Germany). The polymer emits strong red fluorescence (Ex = 585 nm, Em = 590 nm) and the monomers produced green fluorescence (Ex = 514 nm, Em = 529 nm). The fluorescence rate of monomers/polymer was detected and analyzed.
Trans-epithelial cell resistance measurement
Trans-epithelial cell resistance (TEER) was used to identify the cellular permeability and it was conducted with TEER24 (Applied Biophysics, USA). ARPE-19 cells were inoculated on the bottom membrane of the upper layer of the transwell chamber (0.3×104/well), and 1.2 ml of complete cell culture medium was added to the chamber. The TEER was measured after the cell was confluent. After debugging the TEER24 epithelial volt ohmmeter and wiping the electrode pads with alcohol, the electrode sheets were immersed into the inner and outer culture medium, respectively. The resistance of the blank control group (without any cells inoculated) and the experimental group was measured vertically in sequence. The measurement was repeated three times for each hole, and three multiple holes were set for each group. Calculation of TEER value was conducted as following: TEER=(resistance value experimental group-resistance blank control) ×0.3 and the unit is Ω cm2.
After plating the ARPE-19 cells into six-well plates and generating cells grown on coverslips, the cover slides were washed once with PBS buffer and 1 mL of immunostaining fixative was fixed for 20 min at room temperature. To break the membrane, 0.2% Triton X-100 was added after three times washes with PBS. Incubation with blocking solution (PBS containing 10% goat serum) was conducted for 2h at 4°C and then the cells would be incubated with dilute primary antibody (anti-ZO1 and anti-NLRP3) for immunostaining in a cold room overnight. The cells were washed three times with a PBS and then the secondary antibody was added incubate with the cells for 1h in the dark. Drop the DAPI & anti-quencher mixture onto the glass slide and flip the cover glass. Observation and record of the immunofluorescence photographs were conducted with a Zeiss confocal microscope (Leica Biosystems, Germany) under dark.
After the total protein extracted from tissues and cellular samples with RIPA buffer with protease inhibitors, BCA quantitative measurement of protein concentration was conducted. The relative expression of the target protein was normalized to β-actin. A total of 20 µg protein for each sample was separated by SDS-PAGE and transferred onto PVDF membrane (Millipore, USA). After incubating with the milk in TBS for blocking, the membrane would be incubated the corresponding primary antibody, including anti-p16INK4a, p21Waf/Cip1, SIRT1, NLRP3, apoptosis-associated speck-like protein (ASC), pro-caspase-1, caspase1 and β-actin, at 4°C overnight. On the second day, the membrane was washed three times with TBST and incubated with the secondary antibody for 1h. The density of the bands was quantified using Labworks image acquisition software (UVP, USA) and were quantitated using ImageJ software (NIH, USA).
IL-1β and IL18 production in the supernatant in ARPE-19 cells of different groups were measured using IL-1β and IL-18 ELISA kit (BioPioneer Tech, China) according to the manufacturer’s instructions. Samples were quantified with a corrected value of 450 and 570nm, reading the absorbance measurements in the Thermo Scientific Fluoroskan microplate reader (Thermo Fisher Scientific, USA). Three replications for all the samples were adopted and the data of three independent studies were used in final analyses.
The data was expressed as a mean ± stand difference (SD) in this study. The difference between groups was analyzed using non-paired t test and the difference between over two groups were analyzed using one-way analysis of variance (ANOVA) followed by the newman-keuls method for multiple comparison analysis test. The P value < 0.05 denotes statistical significance.