Cell culture and treatment

HCECs (Purchased from BNCC, Beijing, China) were cultured in MEM medium (Basal Media, Shanghai, China) supplemented with 10% fetal bovine serum (FBS, Gibco, USA) and Penicillin–Streptomycin Solution (10 kU/ml Penicillin, 10 mg/ml Streptomycin, Procell, Wuhan, China) in a humidified incubator at 37 °C containing 5% CO2. According to the groups, D-Glucose (Procell, Wuhan, China) was dilated to different concentration. The whole research procedure was approved by the Ethics Committee of Chongqing Medical University and was performed following the Declaration of Helsinki.

Cell proliferation assay

The capacity of cell proliferation was evaluated by CCK-8 assay (Shanghai Yi Sheng Biotechnology Co., Ltd., Shanghai, China). This commercially available kit was used following the manufacturer’s instructions. In brief, HCECs (1 × 104 cells per well) were seeded in 96 well plates. The culture medium was replaced after the cells adhered to the wall. The culture medium was replaced with 5.5, 10, 25, 30, 40, and 50 mM glucose for 48 h and then 10 μl CCK-8 solution was added into the microplate. Two hours after incubating, the microplate reader was used to detect the absorbance at 450 nm. According to the results of cell proliferation, 25 mM glucose was used in following HG stimulation. The treating concentration of ALA was determined by the capacity of proliferation. In brief, HCECs (1 × 104 cells per well) were seeded in 96 well plates, the culture medium was replaced after the cells adhered to the wall. The medium was replaced with various concentration of ALA (25, 50, 125, 250, 500 μM, MCE, China) for 24 h at 37 °C in an atmosphere containing 5% CO2. According to the cell viability, 25 and 50 μM ALA were used in the following experiment. In order to avoid the influence of osmotic pressure changes caused by high glucose on experimental results, we set 5.5 mM glucose + 22.5 mM mannitol as mannitol hypertonic control. After the HCECs adhered to the wall, the medium was, respectively, replaced with 5.5 mM glucose, 5.5 mM glucose + 22.5 mM mannitol, 25 mM glucose, 25 mM glucose + 25 μM ALA and 25 mM glucose + 50 μM ALA for 24 h at 37 °C in an atmosphere containing 5% CO2. 10 μl CCK-8 solution was added into the microplate, 2 h after incubating, the microplate reader was used to detect the absorbance at 450 nm.

Measurement of intracellular ROS

HCECs were cultured in a 96-well plate with the density of 2 × 104 cells per well. After the cells adhered to the wall, according to the groups, the culture medium was, respectively, replaced with 5.5 mM glucose, 5.5 mM glucose + 22.5 mM mannitol, 25 mM glucose and 25 mM glucose + 25 μM ALA for 24 and 48 h. The production of intracellular ROS was measured with an ROS assay kit (Beyotime Biotechnology, Shanghai, China) using 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA) (10 Um) as a fluorescence probe in dark. After incubation with 200 μl per wall diluted DCFH-DA for 30 min at 37 °C, cells were washed three times with PBS. The fluorescence released was detected using a fluorescence microplate reader (Molecular Devices, Sunnyvale, CA, USA) at an excitation and emission wave length of 480 nm and 520 nm. A fluorescence microscope (Leica Microsystems, Wetzlar, Germany) was used to obtain the images. The percentage increase in fluorescence per well was calculated by the formula 【(Ft30-Ft0)/Ft0 × 100】, where Ft30 = fluorescence at time 30 min and Ft0 = fluorescence at time 0 min 13.

Cell apoptosis assay

HCECs were seeded in a six-well plate according to the groups. When the cells adhered to the wall, the culture medium was, respectively, replaced according to the groups. After incubating for 24 and 48 h, cells were harvested and suspended in 1 × Annexin V Bingding Buffer. Then, cells were stained with Annexin V-FITC and propidium iodide (PI) using a cell apoptosis detection kit (Procell Life Scinece & Technology Co., Ltd, China). After incubating for 20 min, cell apoptosis was analyzed by a flow cytometry (BD Biosciences, NJ, USA). The percentage of apoptotic cells was analyzed using FlowJo software (Becton–Dickinson-San Jose, CA, USA). TUNEL cells apoptosis staining was detected by commercial TUNEL cell apoptosis detection Kit (Beyotime, Shanghai, China).

Protein expression

The protein expression of RAGE, CAT, SOD2, TLR4, NLRP3, Cleaved caspase-3,IL-1β and IL-18 was evaluated by Western blotting (WB). GAPDH was considered as control. In brief, the medium was removed from the six-well plates and cells were washed with iced PBS for three times. The RIPA lysis buffer (Beyotime, Shanghai, China) was used to extract total proteins. Then, a BCA Kit (Beyotime, Shanghai, China) was executed for detecting the concentrations of proteins. A total of 20 μg protein samples in cell lysates were separated in SDS-PAGE gels (FuturePAGETM4%-20%, ACE Biotechnology, Nanjing, China) followed by transferring to polyvinylidene difluoride membranes (Millipore, Bedford, MA, USA). The unspecific bands were blocked with QuickBlock™ Blocking Buffer (Beyotime, Shanghai, China) for 15 min. Then, the membranes were washed with TBST for 15 min. Subsequently, these membranes were, respectively, probed with primary antibodies and stored in 4℃ overnight (RAGE Rabbit pAb, Catalase Rabbit pAb, SOD2 Rabbit mAb, TLR4 Rabbit pAb, NLRP3 Rabbit pAb, Cleaved caspase-3, IL1 beta Rabbit pAb, IL18 Rabbit pAb, GAPDH Rabbit mAb, AB clonal, China). After having been washed with TBST for three times, these membranes were incubated with HRP-conjugated Goat Anti-Rabbit IgG (AB clonal, Wuhan, China). The bands were visualized using an Odyssey Infrared Imaging Scanner (LI-COR Biosciences). Intensity of bands was examined using Image-J software (National Institutes of Health, Bethesda, MA, USA). The protein expression was normalized to GAPDH levels. The cells were lysed with RIPA and the supernatant was collected for detecting the concentration of AGEs by commercially available Enzyme-linked immunosorbent assay (Glucose-derived AGEs ELISA Kit) (Shanghai Xitang Biotechnology Co., Ltd., Shanghai, China).

Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) analysis

Total RNA was extracted from cells using Trizol reagent (Invitrogen). Then, first-strand cDNA was synthesized from RNA using a Sensiscript RT kit (Takara Biotechnology Co., Ltd., Japan) following the manufacturer’s recommendations. Then, qPCR was conducted with SYBR Green Supermix (Bio-Rad Laboratories, Inc.) on the ABI 7500 system (Applied Biosystems; Thermo Fisher Scientific, Inc.). Relative gene expression was normalized to GAPDH. The relative primers were described in Table 1.

Table 1 Nucleotide sequence of specific primers used for polymerase chain reaction amplification (human)Immunofluorescence

HCECs were seeded in 24-well plates according to the groups. When the cells adhered to the wall, the culture medium was, respectively, replaced according to the groups. After incubating for 48 h, the HCECs were fixed by 4% PFA for 10 min, rinsed with PBS to remove PFA, then incubated for 12 min in 1% Triton X-100 (Aladdin, Shanghai, China). After being washed, cells were incubated in 3% BSA for blocking. The permeabilized cells were incubated with primary antibody overnight at 4 °C. Then, after being served with PBS again, cells were incubated with goat anti-rabbit IgG (H + L), fluorescein Isothiocyanate conjugate (TransGen, Beijing, China) for 1 h at room temperature and DAPI for 7 min. Cover the cell slides on the micro slides with Mounting Medium, antifading, and around the cell slides with neutral balsam to prevent drying. Specimens were examined with a Leica SP8 Laser Scanning confocal microscope (Leica, Wetzlar, Germany).

Statistical analysis

Representative data from three independent experiments are presented as means ± standard deviation (SD). A significant difference between two groups using Mann–Whitney U test/Student’s test and more than two groups by One-way ANOVA followed by Bonferroni’s multiple comparison test using Graph pad prism 8 (Graphpad Holdings, LLC, San Diego, CA, USA). Statistical significance was defined as p < 0.05.