Ginseng extract (batch number, S230101; date of production, 1 January 2023) was provided by Hongjiu Biotech Co., Ltd. (Jiling, China). Ginseng extract was procured from the dried roots and rhizome of P. ginseng, sourced from five-year-old plants cultivated in Changbai Mountain, Jilin Province. The test reports of the ginseng extract are shown in Supplementary Figures S1, S2. The flow chart of the extraction process of ginseng extract is shown in Supplementary Figure S3. The extraction process involved several sequential steps: comminuting the ginseng into coarse powder, extracting ginsenosides with hot water, and adding alkali (CaO) to eliminate impurities. Subsequently, the extract’s pH was adjusted to neutral with acid solution. To further refine the extract, it was subjected to a macrocellular resin column. The pigment was removed by washing with water until colorless, followed by an additional wash with 70% ammonia alcohol until it achieved colorlessness. The ginsenosides were dissolved in ethanol and eluted, with subsequent recovery of ethanol under reduced pressure, yielding the total ginsenosides.

The quantification of total ginsenoside content was conducted through high-performance liquid chromatography (HPLC). The chromatographic conditions were provided in the Supplementary Material S1. HPLC revealed that the total ginsenoside content is 30.5%. Among the constituents, ginsenoside Rb1 accounted for 13.73%, followed by ginsenoside Rd at 4.83%, ginsenoside Rg1 at 5.51%, and ginsenoside Re at 6.43% (Supplementary Figure S4). The HPLC fingerprint of ginseng extract is shown in Supplementary Figure S5. The HPLC fingerprint of ginsenoside Rg1, Re, Rb1, and Rd is shown in Supplementary Figure S6. The quality standard and extraction protocol were referenced from the Chinese Pharmacopoeia (2020 edition; page 408–409, item: TOTAL GINSENOSIDE GINSENG ROOT) (Supplementary Figure S7).

We obtained male C57BL/6 mice aged 7 weeks from ZhuHai Bestest Bio-Tech Co., Ltd. (Zhuhai, China). During the experiment, mice were kept in cages that were specific pathogen-free. The housing environment was kept at a controlled temperature of 22°C ± 3°C with a light/dark cycle of 12 h each. Both water and food were provided ad libitum to the mice. Guangdong Pharmaceutical University’s Animal Ethics Committee evaluated and authorized the animal experiments (Approval No. gdpulac2020080).

After a week of acclimatization, the control group was given a regular diet, whereas the remaining groups received a high-fat diet (HFD). The methodology employed for establishing the T2DM mouse model drew upon the experimental approach outlined by Yang et al. (Yang et al., 2022). Briefly, following a 4-week period of HFD consumption, mice were injected intraperitoneally with 40 mg/kg streptozotocin (STZ) (dissolved in 0.1 M citric acid-sodium citrate buffer, pH 4.5) for 5 days to establish the T2DM mouse model. Concurrently, the control group was kept on a regular diet and received intraperitoneal injections of the same amount of citrate buffer. The fasting blood glucose (FBG) was assessed 2 days following the last STZ injection. FBG levels ≥11.1 mmol/L suggested that the T2DM mice model had been successfully established.

On the initial day of STZ injection, concomitant preventive drug administration was initiated. Six groups of mice were randomly assigned (n = 10 per group): control group, T2DM model group, GE-L group (T2DM mice treated with 60 mg/kg ginseng extract), GE-M group (T2DM mice treated with 120 mg/kg ginseng extract), GE-H group (T2DM mice treated with 240 mg/kg ginseng extract), and MET group (T2DM mice treated with 350 mg/kg metformin) (Inoue et al., 2021). A 0.5% solution of carboxymethylcellulose sodium (CMC-Na) was used to dissolve all drugs administered. Ginseng extract or metformin was administered daily via gavage each morning. Both the control and T2DM model mice orally received an equivalent amount of the vehicle (0.5% CMC-Na). Following the start of drug administration, the mice were split into two separate groups and euthanized after 7 and 28 days of treatment, as shown in the animal testing diagram (Supplementary Figure S8).

Following a 6-h fast, glucose (2 g/kg) was given by gavage (Luo et al., 2021; Mao et al., 2022). Blood glucose levels were assessed at designated intervals following the intake of glucose, including at 0, 30, 60, and 120 min. The measurements were carried out utilizing a glucose meter. The Area Under the Curve (AUC) was computed to measure the results of OGTT.

After anesthesia with tribromoethanol (0.01 mL/g), blood samples were obtained via the orbital vein extraction technique using capillary tubes, then euthanasia was performed by cervical dislocation after a 12 h fasting period. After being collected, these blood samples were kept at room temperature in enzyme-free tubes for 1 h. Subsequently, the samples were subjected to centrifugation for 20 min at 4°C and 3500 r/min, collecting the supernatants. Levels of insulin, glucagon, IL-1β, TNF-α, IL-10, and GLP-1 were quantified following the recommendations outlined by Meimian (Jiangsu, China).

The techniques for H&E, immunohistochemical, and immunofluorescent staining were performed in accordance with the methods described previously (Hsiao et al., 2019; Wei et al., 2020). After the mice were sacrificed, the pancreatic tissues were collected and fixed in 4% paraformaldehyde before being embedded in paraffin. Following that, 4 µm thick sections were prepared and stained. Sections were subjected to hematoxylin and eosin staining for histological analysis, and pancreatitis scores were assessed utilizing the 0–4 scoring system as previously described (Schmidt et al., 1992). To prepare for immunohistochemical staining, 5% BSA was used to block any non-specific binding sites for 1 h. Following this, the sections were left to incubate overnight with an anti-ARX antibody (diluted at a ratio of 1:200; Abcam, Cambridge, UK). Sections were rinsed thrice with PBS before being treated for 1 h with goat anti-rabbit secondary antibody. Following another round of washing, the sections were exposed to diaminobenzidine solution prior to microscopic examination. Immunofluorescence staining involved labeling sections with anti-PCNA antibody (1:1000; Abcam), then applying anti-insulin antibody (1:200, Abcam), or a combination of anti-insulin (1:200) and anti-glucagon (1:400) antibodies (Proteintech, California, United States).

A microscope (BX53; Olympus, Tokyo, Japan) was used to examine the slides. We manually counted the amount of newborn islet β-cells (INS⁺PCNA⁺). The calculation of islet cells was carried out using ImageJ software (National Institutes of Health, Bethesda, MD, United States). The ratio of α- and β-cells was counted using the Image-Pro Plus software (Media Cybernetics, Silver Spring, MD, United States).

The Western blotting procedure adhered to the methodology outlined by Chen et al., 2023b. RIPA buffer with protease and phosphatase inhibitors (Beyotime, Shanghai, China) was used to extract protein samples from pancreatic tissue. A BCA protein quantification kit (Beyotime) was used to determine protein concentration. After being separated by a 10% SDS-PAGE gel, protein samples were subsequently moved to a PVDF membrane (Millipore, Boston, United States). The membranes were obstructed using 5% nonfat milk for 1 h at room temperature, followed by an overnight incubation at 4°C with a primary antibody. This study utilized primary antibodies (diluted at a ratio of 1:1000) against GAPDH (Cell Signaling Technology, Danvers, MA, United States), FOXM1 (Abcam), cyclin D2 (Abcam), AKT (Cell Signaling Technology), P-AKT (Cell Signaling Technology), MAFA (Cell Signaling Technology), and MAFB (Santa Cruz, CA, United States). Following the primary antibody incubation, the membrane was treated with either HRP-conjugated anti-rabbit or anti-mouse secondary antibody at room temperature for 1 h (1:1000 dilution) (Cell Signaling Technology). Subsequently, the membrane was rinsed using TBST, following incubation with ECL solution (Yeasen, Shanghai, China). Protein bands were identified with a Bio-Rad chemiluminescence imager (Hercules, United States), following calculation with Image Lab 6.0 software (Bio-Rad).

All statistical analyses were performed using GraphPad Prism 8 (GraphPad Software Inc., La Jolla, United States). The statistical evaluations were performed by either one-way ANOVA or two-way ANOVA to ascertain the significance of variations among different groups. A p-value less than 0.05 was considered to have statistical significance.

Compared with the control group, mice that received STZ injections exhibited a slight reduction in body weight and a notable rise in blood glucose levels (p < 0.001), as depicted in Figures 1A–D. After 7 days of medication, in comparison to the model mice, no marked alterations were observed in terms of body weight (Figure 1A). Whereas, mice treated with high-dose ginseng extract and metformin both showed a marked decrease in FBG, as shown in Figure 1B (p < 0.01). As illustrated in Figures 1C, D, after a 28-day administration period, when contrasted with the model mice, the medium- and high-dose ginseng extract-treated groups and metformin-treated group exhibited an enhancement in body weight (p < 0.05), along with a marked reduction in FBG levels (p < 0.001). These results suggest that ginseng extract holds promise for effectively alleviating T2DM symptoms in mice.

As depicted in Figures 2A–D, the blood sugar levels of the mice reached their highest point 30 min after receiving glucose. It is noteworthy that the FBG in the control group gradually decreased to its original level after reaching a peak. Conversely, the model group of mice sustained high FBG even after the peak, resulting in a notable enhancement in the area under the glucose tolerance curve (AUC) (Figures 2A–D). These observations suggest that mice in the model group exhibited pronounced insulin resistance and severe impairment of pancreatic islet function. Although the low-dose ginseng extract-treated group exhibited no substantial improvement in OGTT outcomes following 7 days or 28 days of ginseng extract administration, the middle-dose and high-dose ginseng extract-treated groups showcased a decrease in AUC compared to the model mice (Figures 2C, D). Similar results were observed in the metformin-treated group, which displayed a notable decrease in AUC when compared with the model mice (Figures 2C, D) (p < 0.001). These results suggest that both ginseng extract and metformin possess the capacity to improve impaired glucose tolerance of T2DM mice.

In comparison to the control mice, the model mice exhibited a marked decrease in serum insulin levels as well as a substantial rise in serum glucagon levels (Figures 2E–H) (p < 0.001). When compared with the model mice, all treatment groups exhibited enhanced serum insulin levels, concomitant with decreased glucagon levels (Figures 2E–H). These outcomes collectively demonstrate that ginseng extract could promote insulin secretion, suppress glucagon secretion, improve pancreatic islet function, and ameliorate insulin resistance in T2DM mice.

Pancreatic tissue was evaluated for structural pathological changes using H and E staining. As indicated in Figures 3A, B, control mice showed normal structural characteristics, including clear boundaries and no obvious infiltration of inflammatory cells. Conversely, within the model group, there was a pronounced atrophy of pancreatic islets, severe morphological disruption, and irregular structure. Furthermore, the boundaries of these pancreatic islets were indistinct, accompanied by the emergence of numerous vacuoles and severe infiltration of inflammatory cells. Following treatment with ginseng extract, the morphology of the pancreatic islet exhibited a return to regularity, with visible boundaries. Notably, the inflammatory cell infiltration observed in the model group was significantly alleviated. These results suggest that ginseng extract confers a degree of protection to pancreatic islets and inhibits inflammatory cell infiltration. These findings align with the pancreatitis scoring results, as depicted in Figures 3C, D.

Apoptosis and β-cell dysfunction are known to be induced by cellular inflammatory cytokine stress (Ortis et al., 2010). ELISA kits were employed to measure the serum levels of specific inflammatory cytokines after 28 days of treatment. Results showed a notable reduction in serum IL-10 levels (p < 0.001) in mice from the model group, with increased IL-1β and TNF-α levels when compared with the control mice (p < 0.001) (Figures 3E–G). After receiving ginseng extract and metformin, the IL-1β and TNF-α levels were noticeably reduced when compared with the model mice (p < 0.05), while the IL-10 level exhibited a marked increase (p < 0.001) (Figures 3E–G). Furthermore, the serum levels of GLP-1 were assessed, revealing a decline in GLP-1 levels within the model group, while administration of ginseng extract or metformin for 28 days resulted in a notable enhancement of GLP-1 levels (p < 0.001) (Figure 3H). However, we are uncertain if the changes in insulin and glucagon levels are due to the shift in GLP-1 levels. This may be a concomitant phenomenon, or it might indicate a causal relationship and further research is needed. These findings collectively indicate that ginseng extract inhibits inflammatory response, promotes GLP-1 secretion, and protects β-cells against inflammatory damage in T2DM mice.

Immunofluorescence double staining with PCNA and INS, which represents the neogenesis of β-cells, was performed to assess the β-cell proliferation rate. As illustrated in Figures 4A, B, the islets from both batches of mice in the model group were all destroyed, as evidenced by reduced islet area, diminished insulin secretion, and scarcely observed neogenesis of islet β-cells. Following the administration of ginseng extract, discernible improvements were noted in the pancreatic islet morphology, indicating ginseng extract prevents β-cell destruction. Furthermore, the area size of pancreatic islets increased, and noticeable β-cell neogenesis was observed. Importantly, ginseng extract stimulated insulin secretion and the high-dose ginseng extract treatment group showed better effect. The metformin treatment produced similar results to the low-dose ginseng extract treatment. Specifically, in comparison to the model mice, the metformin-treated group exhibited a gradual recovery in pancreatic islet morphology, a significant increase in area size, and a relatively limited presence of β-cell neogenesis. Consistent with the double staining results, quantification of the INS⁺PCNA⁺ β-cells revealed a decrease in the model mice compared to the control mice, while treatment with medium or high doses of ginseng extract markedly enhanced PCNA⁺INS⁺ β-cell number (Figures 4C, D). These results suggest that ginseng extract can promote the regeneration of pancreatic β-cells in T2DM mice.

To investigate alterations in the pancreatic islet structure and changes in α- and β-cell ratio within the islets, pancreatic sections were subjected to dual immunofluorescence staining for insulin and glucagon. As depicted in Figures 5A, B, normal islets predominantly consist of a substantial population of β-cells with a smaller quantity of α-cells. Additionally, in the control mice, islet β-cells are generally found in the central region of the pancreatic islet, while α-cells are located on the outer periphery of the islet, surrounding the β-cells. By comparison, in the model group, serious damage to the pancreatic islet structure led to a significant reduction of β-cell proportion and an enhancement of α-cell proportion (Figures 5A–D). After being treated with ginseng extract, a gradual restoration of islet structure was observed, along with a rise in the number of β-cells and a reduction of α-cells (Figures 5A–D). Additionally, in mice treated with ginseng extract, we observed the emergence of a small group of yellow insulin⁺glucagon⁺ cells, commonly considered transitional cells, during the transformation from α-cells to β-cells (Zhang et al., 2019) (Figures 5A, B). Comparative analysis of the quantitative outcomes from the two batches of mice revealed that the effect between the 7-day administration and 28-day administration showed a slight difference (Figures 5C, D). This may be due to the stimulatory effects of ginseng extract on promoting β-cell proliferation and transformation occur primarily in the early stages, reaching a considerable degree when administered for 7 days. Subsequently, at later stages (administered for 28 days), the primary role of ginseng extract is to protect the newly generated and transformed β-cells and maintain their normal functions, such as insulin secretion. Overall, these studies indicate that ginseng extract may convert α-cells to β-cells as well as preserve β-cells.

Aristaless-related homeobox (ARX) is an important transcription factor that serves as a biomarker for pancreatic α-cells (Xu and Xu, 2019). Mice in the 7-day treatment group had significantly increased β-cell proportion and decreased α-cell proportion, therefore, the 7-day batch was used to conduct immunohistochemical staining for ARX. Despite the noticeable reduction in the size of pancreatic islets within the model group, the quantitative assessment of ARX-positive signal intensity did not exhibit a substantial difference compared with the control mice (Figures 6A, B). Furthermore, despite the significant enlargement of pancreatic islets after receiving ginseng extract, the ARX expression was greatly reduced when compared with the model mice, particularly in the high-dose ginseng extract-treated group (Figures 6A, B). These results indicate that ginseng extract might act by inhibiting ARX expression to facilitate the transformation of α-cells to β-cells.

MAFB is specifically expressed in maturing α-cells, whereas MAFA serves as a marker for mature β-cells (Nishimura et al., 2006). The pancreatic protein levels of MAFA and MAFB from the 7-day batch were measured. Compared with the control mice, MAFA levels were slightly elevated in the model mice, however, the observed discrepancy failed to attain statistical significance (Figures 7A, B). Additionally, compared to the model mice, all ginseng extract-treated groups, particularly the high-dose group, and the metformin-treated group exhibited increased expression levels of MAFA (Figures 7A, B). The MAFB levels exhibited a modest increase in the model mice in comparison to the control mice, but without a significant difference (Figures 7A, C). All ginseng extract-treated mice showed decreased MAFB expressions in comparison to the model mice. These findings suggest that ginseng extract might facilitate the transformation from α-cells to β-cells by regulating MAFA and MAFB expression levels.

Pancreatic AKT, FOXM1, and cyclin D2 levels in the 7-day batch were examined through Western blot analysis, which demonstrated that in the model mice, P-AKT/AKT, FOXM1, and cyclin D2 levels were slightly decreased in comparison to the control mice. Following intervention with ginseng extract, the expressions of these proteins showed an upward trend (Figures 8A–D). Upon administration of metformin, the P-AKT/AKT, and FOXM1 levels were increased compared to the model mice (Figures 8A–C). These findings suggest that ginseng extract might enhance the proliferation of islet β-cells through modulation of the AKT pathway.