Effects of Huanglian-Renshen-Decoction, a Fixed Mixture of Traditional Chinese Medicine, on the Improvement of Glucose Metabolism by Maintenance of Pancreatic β Cell Identity in db/db Mice

Abstract

Huanglian-Renshen-Decoction (HRD) is widely used to treat type 2 diabetes mellitus (T2DM) in China. However, the underlying mechanism is unclear. We aimed to investigate the mechanism by which HRD regulates the glucose level. Forty 7-8-week-old db/db (BSK) mice were randomly assigned to the following four groups: model, low dose HRD (LHRD), high dose HRD (HHRD), and saxagliptin (SAX). Additionally, 10 db/m mice were assigned to control group. The experimental mice were administered 3.03g/kg/d and 6.06g/kg/d of HRD in the LHRD and HHRD groups, respectively, and 10mg/kg/d saxagliptin in the SAX group for 8 weeks. The control and model groups were supplied with distilled water. After the intervention, the pancreas and blood were collected and tested. Compared with that of model group, the fasting blood glucose (FBG) was significantly decreased in all intervention groups (p < 0.05 or 0.01), whereas fasting serum insulin (FINS) was increased significantly in both HHRD and SAX groups. The immunofluorescence images showed that the mass of insulin⁺ cells was increased and that of glucagon⁺ cells was reduced obviously in experimental groups compared to those of the model group. In addition, the coexpression of insulin, glucagon, and PDX1 was decreased in HHRD group, and the level of caspase 12 in islet was decreased significantly in all intervention groups. However, little difference was found in the number and morphology of islet, and the expression of ki67, bcl2, bax, caspase 3, and cleaved-caspase 3 in the pancreas among groups. Interestingly, the cleaved-Notch1 level was increased and the Ngn3 level in islet was decreased significantly in HHRD group. The HRD showed dose-dependent effects on glucose metabolism improvement through maintenance of β cell identity via a mechanism that might involve the Notch1/Ngn3 signal pathway in db/db mice.

Figure 1

HPLC fingerprint of the Huanglian-Renshen-Decoction. (a) HPLC fingerprint of the standard substances showing six different peaks. (b) HPLC fingerprint of Coptidis Rhizoma with the peaks 2, 3, and 4 and (c) HPLC fingerprint of Panax ginseng with peaks 1, 5, and 6, respectively. (d) The 3D fingerprint of the Huanglian-Renshen-Decoction. (1) Rg1 (PubChem CID: 441923); (2) coptisine (PubChem CID: 72322); (3) palmatine (PubChem CID: 19009); (4) berberine (PubChem CID: 2353); (5) Rb1 (PubChem CID: 449635); and (6) Rc (PubChem CID: 12855889).

Figure 2

Effects of HRD on glucose metabolism. (a) The FBG in each group is presented as the mean ± SEM. The difference between the control and model groups was significant throughout the experiment (p < 0.0001); p < 0.05 for model versus LHRD at the second and seventh weeks; p < 0.05 for model versus HHRD from the second to eighth weeks; no difference was detected between the model and SAX groups. (b) An IPITT was conducted. Blood glucose level is shown as mean ±SEM. The significant gap between control and model existed (p< 0.0001). p < 0.01 for model versus HHRD at 120 min; p < 0.05 for model versus SAX at 60 min, 90 min, and 120 min. (c) The data are shown as the mean ± SEM. p < 0.0001 for control versus model at all time-points; p < 0.05 for model versus LHRD at 90 min and 120 min; p < 0.05 for model versus HHRD at 0 min and 90 min; p < 0.05 for model versus SAX at 90 min and 120 min. (d) The AUC in the IPGTT is shown as the mean ± SD. (e) The FINS is shown as the mean ± SEM. (F) The HOMA-IR is shown as the mean ± SD. n=5-8 mice in each group. ∗∗ represented p < 0.01 versus control group, ∗∗∗∗ represented p < 0.0001 versus control group, ^# represented p < 0.05 versus model group, ^## represented p < 0.01 versus model group, ^### represented p < 0.001 versus model group.

Figure 3

Islet histology based on H&E staining. The islet quantity and area were analysed by one-way ANOVA. (a) Pictures of H&E-stained sections showing the islet histology. (b) The scatter diagram presents the islet area in a slide as the mean ± SD. (c) The column shows the islet number in a slide as the mean ± SEM. No difference was found between groups. n=6-10 mice in each group. ns, not significant. All images were taken with electronic microscope (×400).

Figure 4

Immunofluorescence images of insulin and glucagon in the pancreas. (a) Immunofluorescence images showing the glucagon (red) and insulin (green) expression. DAPI staining indicates the nuclei (blue). (b) The data show the ratio of glucagon⁺ cells to insulin⁺ cells as the mean± SEM. (c) The data show the total ratio of glucagon⁺ and/or insulin⁺ cell to islet cells as the mean± SD. ∗∗ represented p < 0.01 model versus control group, ∗∗∗∗ represented p < 0.0001 model versus control group, ^# represented p < 0.05 intervention versus model group, ^## represented p < 0.01 intervention versus model group, ^### represented p < 0.001 intervention versus model group. All immunofluorescence images were taken with a fluorescence microscope (×400).

Figure 5

Immunofluorescence images for NKX6.1 in the pancreas. (a) Immunofluorescence images showing glucagon (blue), insulin (green), and NKX6.1 (red) coexpression. (b) The data show the ratio of NKX6.1⁺insulin⁺ cells to insulin⁺ cells as the mean± SEM. n= 4-8 mice in each group. ∗∗ represented p < 0.01 versus control group, ^# represented p < 0.05 intervention versus model group. All immunofluorescence images were taken with a fluorescence microscope (×400).

Figure 6

Immunofluorescence images of PDX1/insulin/glucagon coexpression in pancreas. (a) The immunofluorescence images show glucagon (blue), insulin (green), and PDX1 (red) coexpression in the islets. (b) The data showed the ratio of PDX1⁺ insulin⁺ glucagon⁺ cells to the islets area (um²) as the mean ± SEM. n= 6-8 mice in each group. ∗∗∗ represented p < 0.001 versus control group, ^# represented p < 0.05 versus model group. All immunofluorescence images were taken with a fluorescence microscope (×400).

Figure 7

Immunofluorescence images of ki67/insulin/glucagon and caspase 12/insulin/glucagon coexpression and Western blotting images of cleaved-caspase 3/caspase 3, bcl2/bax expression in pancreas. (a) Immunofluorescence images showing glucagon (blue), insulin (green), and Ki67 (red) coexpression in the islets. (b) Immunofluorescence images showing glucagon (blue), insulin (green), and caspase 12 (red) coexpression in the islets. (c) Western blotting showing t cleaved-caspase 3/caspase 3, bcl2/bax expression in the pancreas. n=4-6 mice in each group. All immunofluorescence images were taken with a fluorescence microscope (×400) and Odyssey Scanning.

Figure 8

Immunohistochemistry images for cleaved Notch1 and Ngn3. (a) Representative immunohistochemistry images of cleaved Notch1 in the islets. (b) Representative immunohistochemistry images of Ngn3 in the islets. (c) and (d) show statistical analysis figures and Western blotting images. n=3-5 mice in each group. All IHC images were taken with an electronic microscope (×400).