The EUG treatment effectively alleviated symptoms associated with T1DM mice.

(A) The schematic diagram depicts the progress of animal experiments in each group of mice. Different colors indicate different treatments for mice. (B) The fasting weight levels of mice were measured weekly in each group (n = 30 mice). (C) The fasting blood glucose levels of mice were measured weekly in each group (n = 30 mice). (D) The water intake/cage in each group of mice (n = 6 cages, 5 mice/cage). (E) The food intake/cage in each group of mice (n = 6 cages, 5 mice/cage). (F) Urine ketones in each group were detected by ELISA (n = 30 mice). (G) The urine glucose levels of mice was measured by biochemical test in each group of mice (n = 30 mice). (H) The curve graph of oral glucose tolerance test (OGTT) from 0 min to 120 min at week 1, week 2, week 3, week 5, and week 10 (n = 30 mice). (I) The quantitative results of OGTT at week 1, week 2, week 3, week 5, and week 10 (n = 30 mice). Mean ± SEM. All experiments were repeated at least three times independently. Compare with the Control group *p < 0.05, compare with the Control group **p < 0.01 compare with the Control group ***p < 0.001, compare with the T1DM group #p < 0.05, compare with the T1DM group ##p < 0.01, compare with the T1DM group ##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. The quantification of western blot gel bands in different groups (n = 3 independently repeated experiments). (C) The gene levels of Ins1 in different groups (n = 5 independently repeated experiments). (D) ELISA analysis of serum fasting insulin levels at different time points (n = 5 independently repeated experiments). (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 = 5 independently repeated experiments). 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 independently repeated experiments). (C) The gene levels of Nrf2 in different groups (n = 5 independently repeated experiments). (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 independently repeated experiments). (F) The gene levels of Keap1, Nqo-1, and Ho-1 in different groups (n = 5 independently repeated experiments). (G) The levels of serum biochemical indexes (MDA, SOD, CAT, and GSH-Px) in each group of mice (n = 5 independently repeated experiments). 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 independently repeated experiments). (C) The representative immumohistochemical staining of γH2AX in pancreas islet in each group of mice. Black arrows were employed to highlight the presence of brown-stained islet β cells. Scale bars 100 µm, 25 µm. (D) The quantitative analysis of immumohistochemical staining (n = 5 independently repeated experiments). 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 independently repeated experiments). (C) The gene levels of Bax, Bcl2, and Bcl2/Bax in different groups (n = 5 independently repeated experiments). (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 = 5 independently repeated experiments). 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 schematic diagram depicts the different interventions in cell experiments in MIN6 cells. (B) The detection of Insulin expression in different groups using western blot. (C) The quantification of western blot gel bands in different groups (n = 3 independently repeated experiments). (D) The gene levels of Ins1 in different groups (n = 5 independently repeated experiments). (E) ELISA analysis of serum insulin levels of MIN6 cell in different groups (n = 5 independently repeated experiments). (F) The detection of T-NRF2, N-NRF2 expression in different groups using western blot. (G) The representative immunofluorescence staining images of NRF2 (green) in each group of MIN6 cells. Nuclei were stained with DAPI (blue). Scale bar 10 μm. (H) The quantification of western blot gel bands in different groups (n = 3 independently repeated experiments) (I) The gene levels of Nrf2 in different groups (n = 5 independently repeated experiments). (J) The quantification of immunofluorescence staining in different groups (n = 5 independently repeated experiments). 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 independently repeated experiments). The gene levels of Keap1, Nqo-1, and Ho-1 in different groups (n = 5 independently repeated experiments). (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 generation of mitochondrial ROS in each group was detected by MitoSOX (red) and DAPI (blue) staining. Scale bar 100 μm. (F) The quantification of immunofluorescence staining in different groups (n = 5 independently repeated experiments) (G) The quantitative analysis of immunofluorescence staining in different groups (n = 5 independently repeated experiments). (H) The cell ROS in each group was analyzed using flow cytometry after DCFH-DA staining. (I) The quantitative analysis of flow cytometry after DCFH-DA staining (n = 3 independently repeated experiments). 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 independently repeated experiments). (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 = 5 independently repeated experiments) 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 independently repeated experiments). (C) The gene levels of Bcl2, Bax, and Bcl2/Bax in different groups (n = 5 independently repeated experiments). (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 = 5 independently repeated experiments). (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 = 5 independently repeated experiments). 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.

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 = 5 independently repeated experiments). Scale bar 2 cm. (C) The qualitative determination of urine glucose in each group of mice (n = 5 independently repeated experiments). 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.

Statistical map of mRNA differential expression between T1DM group and EUG intervention group.

(A) The volcanic map shows the distribution of differential factors. (B) Differential expression cluster heat map, with red indicating up-regulated gene expression and blue indicating down-regulated gene expression. (C-H) GSEA enrichment curve of C: “response to glucose” pathway; D: response to carbohydrate pathway; E: “cellular glucose homeostasis” pathway; F: “positive regulation of insulin secretion” pathway; G: “hydrogen peroxide-mediated programmed cell death” pathway; H: “cellular response to hydrogen peroxide” pathway. The curve represents the cumulative enrichment score (ES), the vertical axis represents the enrichment score, each bar code on the horizontal axis represents a gene, and the color change represents the contribution of that gene to the enrichment score.

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 = 5 independently repeated experiments). (B) The safe dose ranges of EUG to maintain cell viability was determined by CCK-8 assay (n = 5 independently repeated experiments). (C) The dose-dependent effect of EUG on MIN6 cell viability after STZ-induced (n = 5 independently repeated experiments). (D) The imaging results of MIN6 cells under an inverted microscope in bright field with different treatments. Scale bars 100 µm. Mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001 indicate significant differences, and ns > 0.05 means no significance difference.