The EUG treatment effectively alleviated symptoms associated with T1DM mice.

(A) The schematic diagram depicts the progress of animal experiments with EUG in STZ-induced T1DM mice. Different colors indicate different treatments for mice. (B) The fasting weight levels of mice was measured weekly in each group (n = 3). (C) The fasting blood glucose levels of mice was measured weekly in each group (n = 3). (D) The water intake/cage in each group of mice. (E) The food intake/cage in each group of mice. (F) Urine ketones in each group were detected by ELISA (n = 3). (G) The urine glucose levels of mice was measured by biochemical test in each group (n = 3). (H) The curve graph of oral glucose tolerance test (OGTT) from 0 min to 120 min at week 1, week 3, week 5, and week 10 (n = 3). (I) The quantitative results of OGTT at week 1, week 3, week 5, and week 10 (n = 3). (J) The curve graph of insulin tolerance test (ITT) from 0 min to 90 min at week 10 (n = 3). Mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001 indicate significant differences, and ns > 0.05 means no significance difference.

EUG improved the pancreas islet structure and function in T1DM mice.

(A) The detection of Insulin expression in different groups using western blot. (B) The quantification of western blot gel bands in different groups (n = 3). (C) The gene levels of Ins1 in different groups (n = 3). (D) ELISA analysis of serum fasting insulin levels at different time points (n = 3). (E) The representative H&E staining images of pancreatic paraffin sections in each group of mice. Scale bars 50 µm, 20 µm. (F) The representative immumohistochemical staining of Insulin in pancreas islet in each group of mice. Scale bars 50 µm, 20 µm. (G) The quantitative analysis of immumohistochemical staining (n = 3). Mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001 indicate significant differences, and ns > 0.05 means no significance difference.

EUG decreased the expression level of γH2AX in T1DM mice.

(A) The detection of γH2AX expression in different groups using western blot. (B) The quantification of western blot gel bands in different groups (n = 3). (C) The representative immumohistochemical staining of γH2AX in pancreas islet in each group of mice. Scale bars 100 µm, 25 µm. (D) The quantitative analysis of immumohistochemical staining (n = 3). Mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001 indicate significant differences, and ns > 0.05 means no significance difference.

EUG reduced apoptosis of pancreatic β cells in T1DM mice.

(A) The detection of BCL2, BAX, Cleaved Caspase-3 expression in different groups using western blot. (B) The quantification of western blot gel bands in different groups (n = 3). (C) The gene levels of Bax, Bcl2, and Bcl2/Bax in different groups (n = 3). (D) The immumohistochemical staining of TUNEL in pancreas islet in each group of mice. Scale bars 50 µm, 20 µm. (E) The quantification results of TUNEL staining in different groups (n = 3). Mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001 indicate significant differences, and ns > 0.05 means no significance difference.

EUG attenuated excessive oxidative stress through activating NRF2 signaling pathway in T1DM mice.

(A) The detection of T-NRF2, N-NRF2 expression in different groups using western blot. (B) The quantification of western blot gel bands in different groups (n = 3). (C) The gene levels of Nrf2 in different groups (n = 3). (D) The detection of KEAP1, HO-1, and NQO-1 expression in different groups using western blot. (E) The quantification of western blot gel bands in different groups (n = 3). (F) The gene levels of Ho-1 in different groups (n = 3). (G) The levels of serum biochemical indicators (MDA, SOD, CAT, and GSH-Px) in each group of mice. Mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001 indicate significant differences, and ns > 0.05 means no significance difference.

EUG improved STZ-induced MIN6 cells insulin secretion by facilitating NRF2 nuclear translocation in vitro.

(A) The detection of Insulin expression in different groups using western blot. (B) The quantification of western blot gel bands in different groups (n = 3). (C) The gene levels of Ins1 in different groups (n = 3). (D) ELISA analysis of serum insulin levels of MIN6 cell in different groups (n = 3). (E) The detection of T-NRF2, N-NRF2 expression in different groups using western blot. (F) The quantification of western blot gel bands in different groups (n = 3). (G) The gene levels of Nrf2 in different groups (n = 3). (H) The representative immunofluorescence staining images of NRF2 (green) in each group of MIN6 cells. Nuclei were stained with DAPI (blue). Scale bar 10 μm. (I) The quantification of immunofluorescence staining in different groups (n = 3). Mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001 indicate significant differences, and ns > 0.05 means no significance difference.

EUG promoted the expression of NRF2 signaling pathway related proteins to reduce intracellular ROS level.

(A) The detection of KEAP1, HO-1, and NQO-1 expression in different groups using western blot. (B) The quantification of western blot gel bands in different groups (n = 3). (C) The gene levels of Ho-1 in different groups (n = 3). (D) The representative immunofluorescence staining images of HO-1 (green) in each group of MIN6 cells. Nuclei were stained with DAPI (blue). Scale bar 10 μm. (E) The quantification of immunofluorescence staining in different groups (n = 3). (F) The generation of mitochondrial ROS in each group was detected by MitoSOX (red) and DAPI (blue) staining. Scale bar 100 μm. (G) The quantitative analysis of immunofluorescence staining in different groups (n = 3). Mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001 indicate significant differences, and ns > 0.05 means no significance difference.

EUG attenuated γH2AX expression in STZ-induced MIN6 cells in vitro.

(A) The detection of γH2AX expression in different groups using western blot. (B) The quantification of western blot gel bands in different groups (n = 3). (C) The representative immunofluorescence staining images of γH2AX (red) in each group of MIN6 cells. Nuclei were stained with DAPI (blue). Scale bar 50 μm. (D) The quantitative analysis of γH2AX positive cells in different groups (n = 3) Mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001 indicate significant differences, and ns > 0.05 means no significance difference.

EUG exerted protection of STZ-induced MIN6 cells through inhibition of the apoptosis in vitro.

(A) The detection of BCL2, BAX, Cleaved Caspase-3 expression in different groups using western blot. (B) The quantification of western blot gel bands in different groups (n = 3). (C) The gene levels of Bax, Bcl2,and Bcl2/Bax in different groups (n = 3). (D) The detection of MIN6 cells apoptosis in each group using TUNEL staining. The cells with red fluorescence represents apoptosis. Scale bar 100 μm. (E) The quantitative analysis of TUNEL positive cells in different groups (n = 3). (F) The apoptosis in each group was analyzed using flow cytometry after Annexin V FITC and PI co-staining. (G) The quantitative analysis of flow cytometry after Annexin V FITC and PI co-staining in different groups (n = 3). Mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001 indicate significant differences, and ns > 0.05 means no significance difference.

Diagram of the underlying mechanisms involved in the protective effects of EUG on T1DM.

EUG alleviated the related complications in T1DM mice.

(A) The changes of urine wetting area of bedding material of mice in each group at different time points were recorded. (B) External appearance and body transformation in each group of mice (n = 3). Scale bar 2 cm. (C) The qualitative determination of urine glucose in each group of mice (n = 3). The shade of orange represents the concentration of glucose in the urine. (D) The PAS staining images of glomerulus paraffin sections in each group of mice. Mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001 indicate significant differences, and ns > 0.05 means no significance difference. Scale bars 50 µm, 20 µm.

Explored the optimal concentration of drugs for MIN6 cells in vitro.

(A) The cell viability of MIN6 treated with different dose STZ for 24 h using CCK-8 assay (n = 3). (B) The safe dose ranges of EUG to maintain cell viability was determined by CCK-8 assay (n = 3). (C) The dose-dependent effect of EUG on MIN6 cell viability after STZ-induced (n = 3). (D) The imaging results of MIN6 cells under an inverted microscope in bright field with different treatments. Mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001 indicate significant differences, and ns > 0.05 means no significance difference. Scale bars 100 µm.