Non-obese diabetic (NOD) mice

Female NOD mice (n = 20 for each group) were purchased from Hunan SJA Laboratory Animal Co., Ltd. For early intervention, mice were administrated with UC (Sigma-Aldrich, Burlington, MA, USA) at 50 mg/kg or vehicle through gavage every day for 4 weeks. For late intervention, mice were administrated with UC or vehicle for 10 weeks. The weight, food intake and blood sugar of mice were weekly measured. Mice were sacrificed after a month, and the pancreas were isolated. Blood was collected, and serum was harvested after centrifugation. Mice were randomly grouped (Random allocation was performed using the table of random number), and experiments involving mice were conducted in a blind manner. The investigator was blinded to the group allocation during experiments. Our study was approved by the Animal Care and Use Committee of the Second Xiangya Hospital of Central South University and conducted in compliance with the Guide for the Care and Use of Laboratory Animals. Gpower was conducted to choose the sample size.

Diabetes incidence

Diabetes incidence was monitored weekly from 10 to 30 weeks. Levels of blood glucose were measured using Accu-Chek Advantage II Strips (Roche, Basel, Switzerland), and mice with two consecutive glucose levels above 250 mg/dL were considered diabetic.

Hematoxylin and eosin (H&E) staining, insulitis score and immunohistochemistry (IHC) staining

NOD mice were euthanized, and the pancreas was gently collected from mice in early and late intervention groups. The pancreas was fixed in fresh-prepared 4% paraformaldehyde solution overnight and processed for embedding in paraffin and sectioning at 5 µm. Sections were stained with H&E (Beyotime, Shanghai, China). Insulitis was scored 0–4 following these categories: 0 = clear (no infiltration), 1 = <25% infiltration, 2 = 25~50% infiltration, 3 = 50~75% infiltration and 4 = >75% infiltration. The percentage of each score of insulitis/total pancreas islet in each group was analyzed. For IHC staining, sections were rehydrated, and antigen was retrieved in pH 6.0 antigen retrieval citrate buffer (Abcam, Cambridge, UK). Subsequently, sections were blocked and incubated with an insulin antibody (1:10,000, ab282459, Abcam) overnight and then incubated with a goat anti-rabbit IgG HRP (1:1000, ab6721, Abcam) for 1 h. The signal was visualized by 3,3’-Diaminobenzidine (Solarbio, Beijing, China). The quantitative analysis of insulin expression was performed by using ImageJ software. Relative insulin levels were calculated by the normalization method.

Cell culture and transfection

Murine pancreatic β-cell line MIN6 was purchased from American Type Culture Collection (ATCC, Rockville, MD, USA) and maintained in DMEM containing 10% FBS (Thermo Fisher Scientific, Waltham, MA, USA). Cells were authenticated by STR profiling and tested for mycoplasma contamination. The siRNA against Nrf2 (si-Nrf2) and scramble siRNA (si-NC) were purchased from GenePharma (Shanghai, China). Cells were treated with a mixture of cytokines (cytomix: 10 ng/mL IFN-γ, 5 ng/mL IL-1β and 5 ng/mL TNF-α. PeproTech, Cranbury, NJ, USA) for 24 h. For UC and verapamil treatment, MIN6 cells were treated with UC at 0, 2.5, 5, 15, 25, 50 or 100 μM or verapamil (Sigma-Aldrich) at 50 µM for 24 h. MIN6 cells were transfected with si-Nrf2 or si-NC via Lipofectamine RNAiMAX (Thermo Fisher Scientific) following the manuals. After 72 h, cells were collected for cytokines and UC treatment.

Glucose-stimulated insulin secretion (GSIS)

For in vitro GSIS, MIN6 cells were treated with glucose at 2.5 mM (normal glucose) or 20 mM (high glucose) for 1 h. Supernatants were collected and stored at −80 °C. Protein concentration was examined using BCA kit (Beyotime), and total protein was used as a normalization control for insulin. For in vivo GSIS, NOD mice treated with vehicle or UC were administrated with glucose at 2 g/kg through oral gavage. After 30 min, tail blood was collected to measure serum insulin levels. Insulin was measured using Mouse Insulin ELISA (Mercodia, Uppsala, Sweden). Blood glucose was examined using a Medisense glucometer.

Oral glucose tolerance test (OGTT)

NOD mice treated with vehicle or UC were fasting for 6 h, weighed and administrated with glucose at 2 g/kg through oral gavage. Blood was collected at various time points, and glucose was examined using a Medisense glucometer. The Area Under the Curve (AUC) was calculated.

Cell counting kit-8 (CCK-8)

MIN6 cells were treated as indicated, and culture medium was removed. CCK-8 (Beyotime) was pre-mixed with fresh culture medium at 1:10, and 100 µL of the mixture was added and incubated for 2 h. The absorbance (450 nm) was examined.

Cell apoptosis

Annexin V-FITC Apoptosis Detection Kit (CA1020) was provided by Solarbio. Briefly, MIN6 cells were treated as indicated, detached and resuspended in binding buffer. Subsequently, Annexin V- FITC (5 µL) was added and incubated for 5 min protected from light. PI (5 µL) was added, and 400 µL of PBS was added. Cells were analyzed using a flow cytometer immediately.

Immunofluorescence (IF) staining

MIN6 cells were plated on coverslips and treated with cytokines or cytokines + UC. Cells were fixed in 4% paraformaldehyde and permeabilized in 0.2% Triton X-100 for 15 min. Cells were blocked in 5% normal goat serum and incubated with a Nrf2 antibody (PA5-27882, 1:1000, Thermo Fisher Scientific) overnight. Cells were washed and incubated with a Goat Anti-Rabbit IgG H&L (Alexa Fluor 594, ab150080, Abcam) for 1 h. For IF staining in the pancreas, the pancreas was removed and fixed overnight. Next day, the pancreas was embedded in paraffin and cut into 5-µm sections. Sections were dewaxed and permeabilized in 0.5% Triton X-100 for 30 min. After block, sections were incubated with a Nrf2 antibody (PA5-27882, 1:100, Thermo Fisher Scientific) and an insulin antibody (1:1000, 14-9769-82, Thermo Fisher Scientific) overnight. Subsequently, sections were incubated with a Goat Anti-Rabbit IgG H&L (Alexa Fluor 594, ab150080, Abcam) and a Goat Anti-Mouse IgG H&L (Alexa Fluor 488, ab150113, Abcam) for 1 h. Nuclei were counter-stained with DAPI (Beyotime) followed by mounting and imaging. The quantitative analysis of insulin expression was performed by using ImageJ software.

Enzyme-linked immunosorbent assay (ELISA)

Insulin secretion was determined using Mouse Insulin ELISA (Mercodia). Briefly, 5 µL of samples were added, and 100 µL of 1× enzyme conjugate solution was added and incubated for 2 h on a shaker. Plates were washed, and 200 µL of TMB was added and incubated for 30 min. Stop solution (5 µL) was added, and the absorbance (450 nm) was measured.

EdU incorporation assay

MIN6 cells (1 × 104) were plated and treated with cytokines, cytokines + UC or cytokines + verapamil for 24 h. EdU (10 µM) was supplemented into the medium. Cells were fixed in 4% formaldehyde for 15 min. Triton X-100 (0.3%) solution was added to permeabilize cells. The reaction cocktail was prepared, added into cells and incubated for 30 min. DAPI was used to stain DNA, and cells were imaged.

Comet assay

Comet Assay Kit was provided by Abcam, and DNA damage in MIN6 cells was analyzed using Comet assay following the manual. In brief, MIN6 cells were treated with cytokines, cytokines + UC or cytokines + verapamil for 24 h. Cells were suspended in low-melting agarose and mixed thoroughly at 37 °C. The mixture of cells and agarose was plated on comet slides (Abcam) and gelled. Slides were immersed in pre-chilled lysis solution for 1 h and subjected to electrophoresis. Finally, slides were stained with GelRed Nucleic Acid Gel Stain (Biotium, Fremont, CA, USA).

Western blot

Islets were isolated from NOD mouse pancreas as previously described [24]. MIN6 cells were treated as indicated, and isolated islets were lysed in lysis buffer, and protein was quantified using BCA kit (Abcam). Nuclear and cytoplasmic fractions were separated using Nuclear/Cytosol Fractionation Kit (BioVision, Milpitas, CA, USA). Subsequently, protein (30 µg per lane) was electrophoresed and transferred. Membranes were blocked and incubated with anti-Keap1 (1:2000, ab227828), anti-Nrf2 (1:500, PA5-27882), anti-HO-1 (1:2000, ab52947), anti-NQO1 (1:1000, ab80588) or anti-β-actin (1:5000, ab8227). After washing, membranes were incubated with the HRP-conjugated secondary antibody (1:10,000, ab6721), and bands were visualized by applying ECL substrate.

Statistical analysis

All data were analyzed using GraphPad Prism 9 (GraphPad Software, San Diego, CA, USA). Results were shown as mean ± standard deviation (SD). Two-sided Student’s t-test between two groups and one-way analysis of variance (ANOVA) followed by Tukey’s post hoc test among multiple groups were conducted to analyze the difference comparison. The correlation of blood glucose or insulin and Keap1, Nrf2, HO-1 and NQO1 was analyzed through the Pearson correlation. The variance is similar between the groups that are being statistically compared. P < 0.05 was statistically significant.