Radix Astragali (lot no. 20108) from Gansu Province of China, Atractylodes Rhizoma (lot no. 20115) from Liaoning Province of China, Radix Puerariae Lobatae (lot no. 20117) from Jiangxi Province of China, and Salviae Miltiorrhizae Radix et Rhizoma (lot no. 20201) from Zhejiang Province were purchased from Zhejiang University of Traditional Chinese Pharmaceuticals Co., Ltd. (http://www.zjzyyp.com/Default.aspx); all were inspected based on the Pharmacopoeia 2015 edition and the enterprise standard TS-ZL-03. YD comprises Radix Astragali 30 g, Atractylodes Rhizoma 30 g, Radix Puerariae Lobatae 30 g, and Salviae Miltiorrhizae Radix et Rhizoma 20 g. After the medicines were crushed, 550 g of water was added for decoction for 1 h. The filtrate was adjusted to pH 7.0 using sodium citrate and sodium carbonate. The obtained YD had a crude drug content of 3 g/mL, determined by a colorimetric method. The YD liquid was added to pure water, placed in a freeze-drying box, pre-frozen at −50°C for 4 h in a medicinal vacuum freeze dryer (TA, lyo_0.5), dried, sublimated at −20°C for 12 h, and dried at 32°C for 8 h. The YD freeze-dried powder was obtained, which was refrigerated at 4°C for later use. Before use, the powder was dissolved in the corresponding medium to the required concentration, centrifuged at 4000 ×g for 30 min to remove the precipitate, and the supernatant was used.

The chemical analysis of YD was carried out by ultra-high performance liquid chromatography-Q precision hybrid quadrupole orbitrap high-resolution mass spectrometry (UHPLC-Q-Orbitrap HRMS, Thermo Fisher Scientific Inc., Grand Island, NY, USA). UHPLC (Thermo Scientific Dionex Ultimate 3000) was controlled with the Chromeleon 7.2 software. The autosampler was set to 10°C and protected from light. The column was set to 40°C. A Waters ACQUITY UPLC BEH C18 Column (2.1 × 100 mm, 1.7 μm) was used. The mobile phase consisted of A (0.1% formic acid) and B (methanol) at a flow rate of 0.3 mL/min, gradient elution: 0–4 min (4% B), 4–10 min (4%–12% B), 10–30 min (12%–70% B), 30–35 min (70% B), 35–38 min (70%–95% B), 38–42 min (95% B), 42–45 min (95% B) −4% B). The injection volume was 2 μL. The mass spectrometer Q-Orbitrap system was connected to the UHPLC system via heated electrospray ionization and controlled by the Xcalibur 4.1 software for data acquisition and analysis. The electrospray ionization source was run and optimized under positive and negative ionization models. The mass spectrometry parameters were capillary temperature: 320°C; sheath gas (N2) flow rate: 35 arbitrary units; auxiliary gas (N2) flow rate: 13 arbitrary units; purge gas flow rate: 0 arbitrary units; spray voltage: 2.8 kV (negative) and 3.2 kV (positive); S lens RF level: 50 V; auxiliary gas heater temperature, 300°C; scan mode: full MS; scan range: 80–1,200 m/z. The maximum injection time (IT) was 200 ms. The scan resolution was 70,000 FWHM (m/z/s). The automatic gain control (AGC) target was 1.0e6. The typical chromatographic fingerprint of the YD extract is shown in Figure 1.

Thirty-two specific pathogen-free (SPF) 8-week-old male ZDF (fa/fa) rats, weighing 200–220 g, and eight SPF 8-week-old male ZL (fa/+) rats, weighing 200–220 g, were purchased from Beijing Weitong Lihua Laboratory Animal Technology Co., Ltd. (Beijing Viton Lever Laboratory Animal Company, Beijing, China). The animals were housed in the SPF experimental rearing room at the Animal Experiment Center of Zhejiang University of Traditional Chinese Medicine. All animal studies were approved by the Institutional Animal Care Committee of Zhejiang University of Traditional Chinese Medicine (#2017A610167). All experiments were performed with humane care according to the 3R principles (Hubrecht and Carter, 2019) and in accordance with the Association for the Assessment and Accreditation of Laboratory Animal Care (AAALAC) (https://www.aaalac.org/) and the Institutional Animal Care and Use Committee (IACUC) guidelines.

All rats were acclimatized for 1 week. The ZDF rats were fed a high-glucose and high-fat diet (4.0% crude fiber, 7.55% total fat, 23.75% protein, and digestible energy of 14.6 kJ/kg) for 4 weeks, while the control ZL rats were fed a normal diet for 4 weeks. A fasting blood glucose ≥11.1 mmol/L was used as the success criterion for establishing T2DM rats. After successful modeling, the ZDF rats were randomly divided into four groups, with eight rats in each group. Referring to a previous study (Gong et al., 2013), the doses of YD in the high-concentration (YD-hi) and low-concentration (YD-lo) groups were 30 and 15 g/kg, respectively. The positive control group was given 10 mg/kg TAK-875 (No.A8339, APE x BIO, Houston, USA) according to previous studies (Negoro et al., 2010; Leifke et al., 2012). The model group was given the same amount of distilled water once a day for 8 weeks. The study drugs (including the distilled water) were given by gavage. The ZL rats were fed with normal chow for 8 weeks as a control group.

Before the end of the experiment, the rats in each group were fasted for 12 h, euthanized with pentobarbital, and blood was collected by cardiac puncture. The abdominal cavity was quickly cut open, and the pancreas was taken out. Part of the pancreatic tissue was taken and fixed in a 10% formalin solution to prepare sections.

After successful modeling, each rat was weighed weekly, blood samples were collected from the tail vein once a week, and the fasting blood glucose level was measured using glucose test strips.

The OGTT test and IRT were conducted every 2 weeks. For the OGTT, the rats were fasted for 12 h, and the blood was collected from the tail vein. The glucose oxidase method (Toecho Super 2, Kagawa, Japan) was used to test the plasma fasting blood glucose levels. At the same time, 0.2 mL of blood was collected, the serum was collected after centrifugation, and the fasting insulin levels were determined by ELISA. Each group was given the corresponding drugs or distilled water for 30 min to measure the blood glucose and insulin levels, then the rats were given 20% glucose by gavage at 10 mL/kg body weight, and the glucose dose was 2 g/kg. Blood glucose and insulin were measured at 30, 60, 120, and 180 min after glucose administration. The area under the curve (AUC) of blood glucose and insulin was observed at each time point after glucose administration in each group. The formula was AUC = (0 min + 30 min × 0.25 + (30 min + 60 min) × 0.25 + (60 min + 90 min) × 0.5 + (90 min + 120 min) × 0.5.

For the GSIS, the rats were fasted for 12 h and anesthetized by an intraperitoneal injection of 3% pentobarbital (45 mg/kg) (Sharma K. et al., 2018; Sharma et al., 2018c). The rats were immobilized in the supine position, the right jugular vein was cannulated, and 50% glucose 0.5 g/kg was infused. At 0, 3, 5, and 10 min, 0.5 mL of blood was collected. After centrifugation at 5,000 r/min for 15 min, the supernatant was collected, and insulin was measured by ELISA. During the experiment, normal saline was injected to maintain the blood volume of the rats.

The rats were sacrificed after fasting for 12 h. Blood was collected from the heart, and serum total cholesterol (CHOL) and triglyceride (TG) levels were detected using a U2700 automatic biochemical analyzer (Hitachi, Tokyo, Japan) using the manufacturer’s reagents and instructions.

The rat pancreatic tissues were fixed in 10% formalin for 5 days. The specimens were dehydrated with ethanol, treated with xylene for 30 min, and placed in paraffin for 5 h. The paraffin blocks were sectioned at 4 μm thickness. The paraffin sections were dewaxed with xylene, rehydrated with ethanol at 100%, 95%, 85%, and 70%, stained with hematoxylin, treated with 0.2% ammonia, and stained with 0.5% eosin. After ethanol dehydration and xylene transparency, the sections were sealed with neutral gum. The histopathological changes in the pancreas were observed under light microscopy. The nuclei were blue, the cytoplasm was pink, and the erythrocytes were orange-red. The Schmidt pathology scale was used to perform the pathological score of pancreas (Schmidt et al., 1992). The score was based on the degree of tissue edema, necrotic area, bleeding and inflammatory cell infiltration.

The paraffin sections were dewaxed and rehydrated with xylene I for 20 min, xylene II for 20 min, 100% ethanol I for 5 min, 100% ethanol II for 5 min, 95% ethanol for 5 min, 80% ethanol for 5 min, and PBS three times, 3 min/time. The sections were placed in 0.01 M citrate buffer (pH 6.0) for antigen retrieval in a microwave, cooled to room temperature naturally, and washed with PBS three times, 3 min/time. The primary anti-rabbit polyclonal GPR40 antibody (1:50; C02655F; SAB Biotherapeutics, Sioux Falls, SD, USA) was incubated overnight at 4°C in a refrigerator, transferred to room temperature to equilibrate for 30 min, and washed with PBS three times, 5 min/time. Fluorescent secondary antibody donkey anti-rabbit antibody (1:300; Alexa Fluor® 488 donkey anti-Rabbit IgG secondary antibody; Life Technologies Co., Grand Island, NY, USA) was incubated at 37°C for 60 min, rinsed with PBS three times, 5 min/time. The nuclei were stained with DAPI (Abcam, Cambridge, United Kingdom) at room temperature for 10 min. After sealing with glycerol PBS sealer (IH0271; Shanghai Ruji Biotechnology Company, Shanghai, China), the sections were observed using a laser confocal microscope (Model 800, Carl Zeiss GmbH, Oberkochen, Germany).

RIN-m5f cells were purchased from the National Collection of Typical Cell Cultures (Shanghai, China). The RINm5f cell line was inoculated in a 5.6 mmol/L glucose low-glucose DMEM medium (11885084; GIBCO, Invitrogen Inc., Carlsbad, CA, USA), 10% FBS (GIBCO, Invitrogen Inc., Carlsbad, CA, USA) with 1% penicillin/streptomycin and incubated in 5% CO₂ at 37°C with saturated humidity. The modeling was performed as previously described (Ji et al., 2017). The model group was added with 30 mmol/L glucose, and sodium oleate/sodium palmitate (Xi’an Kunchuang Technology Co., Ltd., Xi’an, China, SYSJKJ001) was added to the final concentration of 500 μM for 48 h. The control group was cultured in a low-glucose medium. The RINm5f cells were divided into five groups: YD low-dose group (YD-lo), YD high-dose group (YD-hi), positive control group (GPR40 agonist TAK-875 50 nmol/L), model group (equal amount of culture medium), and untreated RINm5f cells as control.

The effect of different concentrations of YD on cell proliferation was determined by the CCK8 assay. YD freeze-dried powder was added in a gradient of crude drug content of 0.01%, 0.1%, 1%, 5%, 10%, and 100%. The optimum concentration and time (24, 48, and 72 h) were screened for the subsequent experiments. The RIN-m5f cells in the logarithmic growth phase were adjusted to 2.5 × 10⁷/L and inoculated in a 96-well plate. After the cells adhered, the serum-free medium was replaced, and the culture was continued for 12 h. The cells were treated as above for 48 h. Then, 10 μL of CCK8 detection solution (UNOCI, China) was added to each well and incubated at 37°C for 2 h in the dark. The optical density was measured at 450 nm with a microplate reader. Cell viability (%) = OD value of experimental group/OD value of non-drug group×100%. The experiment was repeated three times independently.

The cell supernatant after drug intervention was discarded, and the cells were incubated in a 5.6 mmol/L glucose medium for 1 h. Then, the supernatant was taken, and the basal insulin secretion (BIS) levels were measured by ELISA using an insulin ELISA kit (Thermo Fisher, USA). The cells were incubated in a 30 mmol/L glucose medium for 1 h, and the supernatant was taken. GSIS levels were determined by ELISA. The total protein of each well was extracted and expressed as unit mass insulin (insulin content per well/corresponding protein content).

Total cell RNA was extracted using a TRIZOL RNA extraction kit (Invitrogen Inc., Carlsbad, CA, USA), followed by RT-PCR amplification after reverse transcription. The primers were designed with Primer 5 software and synthesized by Beijing Liuhe Huada Gene Technology Co., Ltd. (Beijing, China). The sequences are shown in Table 1. The PCR conditions were 1) pre-denaturation at 95°C for 1 min, and 2) denaturation at 95°C for 15 s, annealing at 63°C for 25 s, and extension at 72°C for 25 s, for 40 cycles. Statistical analysis was performed based on the ΔCt value, and the average relative expression (2^−ΔΔCt) represented the relative amount between groups.

Pancreatic tissue and RIN-m5f cells were lysed in NP-40 lysis buffer (FNN0021, Invitrogen Inc., Carlsbad, CA, USA) and centrifuged for 20 min (12,000 ×g, 4°C). The protein concentration was measured using a BCA kit (Sigma, St. Louis, MO, USA). The proteins were separated by SDS-PAGE and transferred to PVDF membranes (Millipore Corp., Billerica, MA, USA). The membrane was blocked with 5% nonfat milk powder for 1 h at room temperature. Primary anti-rabbit monoclonal anti-GPR40 (32 kDa, 1:1000, SAB Biotherapeutics, Sioux Falls, SD, USA), anti-IP3R (314 kDa, 1:1000, Abcam, Cambridge, United Kingdom) and anti-GAPDH (36 kDa; 1:2000, Abcam, Cambridge, United Kingdom.) were incubated overnight at 4°C. The membranes were incubated with HRP-conjugated secondary antibody goat anti-rabbit (1:5000; Alexa Fluo, A21206:1723019; Thermo Fisher Scientific, Waltham, MA, USA) for 1 h at room temperature. The bands were visualized using enhanced chemiluminescence in the VersaDoc 4000 MP system (Bio-Rad, Hercules, CA, USA). Optical density values were analyzed using the Image-Pro Plus 6.0 software (National Institutes of Health, https://imagej.nih.gov/ij/) to determine the relative expression levels of GPR40 and IP3R proteins (Sharma et al., 2020).

All data were shown as mean ± standard deviation. One-way ANOVA was used for comparison among multiple groups, with the least significant difference (LSD) post hoc test (consistent variance) or Dunnett’s-T3 post hoc test (uneven variance). p < 0.05 was considered statistically significant. SPSS 23.0 (IBM, Armonk, NY, USA) was used for statistical analysis. All graphs were drawn using Prism 6.02 (GraphPad Software Inc., San Diego, CA, USA) and Illustrator CS6 (Adobe Systems, San Jose, CA, USA).

Nine compounds were analyzed to obtain the characteristic spectra: salvianic acid A (characteristic component of Salviae Miltiorrhizae Radix Et Rhizoma), protocatechualdehyde (characteristic component of Salviae Miltiorrhizae Radix Et Rhizoma), chlorogenic acid (characteristic component of Atractylodis Rhizoma), puerarin (characteristic component of Puerariae Lobatae Radix), calycosin-7-O-β-D-glucopyranoside (characteristic component of Astragali Radix), salvianolic acid B (characteristic component of Salviae Miltiorrhizae Radix Et Rhizoma), atractylenolide I (characteristic component of Atractylodis Rhizoma), astragaloside IV (characteristic component of Astragali Radix) and astragaloside II (characteristic component of Astragali Radix) were used for quantitative and qualitative analysis.The contents of salvianolic acid A, protocatechuic aldehyde, chlorogenic acid, puerarin, verbascoside-7-O-β-D-glucopyranoside, salvianolic acid B, atractylodes I, astragaloside IV, astragaloside IV in YD were 0.32 mg/g, 0.35 mg/g, 0.10 mg/g, 0.34 mg/g, 0.14 mg/g, 0.35 mg/g, 0.11 mg/g, 0.10 mg/g and 0.11 mg/g (Table 2).

After 8 weeks of treatment, the body weight of the rats in the model group increased from 141.3% of the control group to 148.8%, and the weight of the YD-hi group was 136.8% of the control group. The body weight of the YD-hi group was significantly lower compared with the model group (p < 0.05). The YD-lo and TAK-875 groups (positive control) were similar to the model group (p > 0.05). Hence, YD appeared to prevent weight gain in the YD-hi group (Figure 2A; Table 3). Moreover, the FBG of the rats in the model group increased from 287.3% of the control group to 332.8% after 8 weeks of treatment, while the YD-hi and TAK-875 groups were 196.6% and 191.4% of the control group, respectively. The FBG of the YD-hi and TAK-875 groups were reduced compared with the model group (p < 0.05). The YD-lo group was similar to the model group. These results indicated that YD-hi and TAK-875 could reduce FBG in rats (Figure 2B; Table 4).

After 8 weeks of treatment, it was observed that the blood glucose levels (AUC value of OGTT) of the YD-lo, YD-hi, and TAK-875 groups were all decreased compared with the model group after 8 weeks of treatment (p < 0.05), while the blood glucose levels in the YD-hi and TAK-875 groups were decreased more than in YD-lo group, and these two groups were similar (p > 0.05) (Figure 2C; Table 5).

The levels of insulin release (AUC value of IRT) in the YD-lo, YD-hi, and TAK-875 groups were increased compared with the model group after 8 weeks of treatment (p < 0.05), while the insulin release levels in the YD-hi and TAK-875 groups were higher than in YD-lo group, and these two groups were similar (p > 0.05) (Figure 2D; Table 6).

The GSIS AUC values in the YD-hi and TAK-875 groups were 133.9% and 134.2% of the model group, respectively, which were significantly higher than in the model group (p < 0.05); the YD-lo group was 106.4% of the model group (Figure 2F). The insulin secretion levels after glucose stimulation in the YD-hi, YD-lo, and TAK-875 groups were higher than in the model group at 3 and 5 min (all p < 0.05), while those in the YD-lo group were lower than in the YD-hi and TAK-875 groups. The insulin secretion levels in the YD-hi and TAK-875 groups were close (p > 0.05) (Figure 2E; Table 7). Hence, compared with the control group, the model group showed higher glucose levels and lower insulin secretion (all p < 0.05), while YD decreased glucose and increased insulin levels (all p < 0.05). YD-hi had similar effects to TAK-875 (p > 0.05).

The serum TG and CHOL levels in the YD-hi group were decreased at 60.8% and 77.4% of the model group, respectively (p < 0.05). The TG levels in the YD-lo and TAK-875 groups were 88.6% and 96.7% of the model group, respectively, higher than in the YD-hi group (p < 0.05). The CHOL levels in the YD-lo and TAK-875 groups were 93.4% and 98.8% of the model group, respectively, which were also higher than in the YD-hi group (p < 0.05) (Figure 2G).

Histopathological observation of the pancreas in the control group showed that the structure of the pancreas in the control group was normal, the cell morphology was intact, and no obvious pathological changes were found. In the model group, the acinar cells in the pancreatic tissue of the model group were normal, there were flaky fat deposits between the acinar lobules, and acinar cell hyperplasia and telangiectasia were observed in the pancreatic islets, and the pathology scores were higher than those in the control group (p < 0.05). A small amount of fat deposition was observed in the YD-hi group, and no acinar cell hyperplasia or telangiectasia was found. Partial fat deposition was seen in the YD-lo and TAK-875 groups, and a small amount of acinar cell dysplasia and telangiectasia were found. The pathological scores of YD-lo, YD-hi and TAK-875 groups were lower than those of the model group (p < 0.05), while those of YD-hi group were lower than those of TAK-875 group (p < 0.05) (Figure 3).

The GPR40 protein was observed using immunofluorescence (Figure 4). The fluorescence in the control group was the strongest, and the fluorescence in the YD-hi and TAK-875 groups was slightly weaker than in the control group, accounting for 79.6% and 85.8%, respectively (p < 0.05). The YD-lo and model groups showed the weakest GPR40 expression, accounting for 53.8% and 42.6% of the control group, respectively (p < 0.05). There were no significant differences between the YD-lo and YD-hi groups. Therefore, the increased insulin levels observed in the YD-hi group might be due to a higher expression of GPR40.

The results of drug concentration screening showed that 100% YD had the strongest influence on cell proliferation, with YD 10%, 5%, and 1% also having an effect (all p < 0.05), but YD at 0.1% and 0.01% had no effect (p > 0.05) (Figure 5A). Because the YD components could interfere with optical density measurement, 10%, 5%, and 1% YD were selected for the subsequent experiments. The original decoction concentration was 3 g/mL; therefore, the 1% dose was 30 mg/mL, and the 5% dose was 150 mg/mL. Then, 1% YD was selected for drug intervention time screening, and the results showed that the cell survival rates at 48 and 72 h were better than at 24 h. Although the density of cells in the 72-h group was higher, the cell state was far inferior to that in the 48-h group (Figure 5B). Therefore, 48 h was selected.

The BIS and GSIS levels of the YD-hi group (150 mg/mL YD) were 124.5% and 178.3% of the model group, respectively (p < 0.05), and were 123.1% and 172.6% of the model group in the TAK-875 group, respectively (p < 0.05), without significant differences between the YD-hi and TAK-875 groups. The levels of BIS and GSIS in the YD-lo (30 mg/mL YD) group were not significantly different from the model group (p > 0.05) (Figures 6A, B).

The relative expression levels of the GPR40 mRNA in the YD-hi and TAK-875 groups were higher than in the model group (p < 0.05), the YD-lo group was close to the model group (p > 0.05), and the TAK-875 group was higher than the YD-hi group (p < 0.05) (Figure 7A). The protein expression levels of GPR40 were similar to that of mRNA, but there were no significant differences between the TAK-875 and YD-hi groups (p > 0.05) (Figure 7C). The relative expression of IP3R-1 mRNA in the YD-hi and TAK-875 groups was higher than in the model group (p < 0.05). There were no differences between the YD-lo and model groups. There were no differences in mRNA expression between the YD-hi and TAK-875 groups (Figure 7B). The trend of IP3R-1 protein expression was close to the mRNA expression but was higher in the TAK-875 group than in the YD-hi group (p < 0.05) (Figure 7D).