Yunpi Heluo decoction reduces ectopic deposition of lipids by regulating the SIRT1-FoxO1 autophagy pathway in diabetic rats

Abstract

Context: Yunpi Heluo (YPHL) decoction is a Chinese herbal formula with particular advantages for treating type 2 diabetes. Yet, its exact mechanism of action is not fully understood.

Objective: To examine the therapeutic effect of YPHL on ectopic lipid deposition (EDL) in Zucker diabetic fatty (ZDF) rats and the underlying mechanism.

Materials and methods: The ZDF Rats were randomized into five groups, including model, YPHL (200 mg/kg/d for 10 weeks), SIRT1-overexpression (injected with HBAAV2/9-r-SIRT1-3'-flag-GFP), NC (injected with HBAAV2/9-CMV-GFP as blank control) and control group. Pancreatic β-cells obtained from high-lipid-high-glucose fed rats were treated with YPHL (10 mg/mL) for 48 h. Lipid deposition and autophagosomes were analyzed by transmission electron microscopy. Intracellular H₂O₂ and ROS concentrations were measured by flow cytometry. SIRT1, FOXO1, LC3 and P62 mRNA and protein levels were analyzed using qRT-PCR and Western blots.

Results: Compared with the model group, blood glucose levels in YPHL and si-SIRT1 groups were reduced by 19.3% and 27.9%, respectively. In high-lipid-high-glucose cells treated with YPHL, lipid droplets were reduced and decrease in apoptosis rate (38.6%), H₂O₂ (31.2%) and ROS (44.5%) levels were observed. After YPHL intervention or SIRT1 overexpression, LC3 and p62 expression increased. Protein expression of SIRT1 and LC3 in model, si-SIRT1, si-NC and si-SIRT1 + YPHL groups was lower than those in control group, while FoxO1 expression was increased. All of these protein level alterations were reversed in the si-NC + YPHL group.

Discussion and conclusions: YPHL reduced EDL by regulating the SIRT1-FoxO1 autophagy pathway in diabetic rats, which could lead to future perspectives for the treatment of diabetes.

Keywords: Insulin resistance; pancreas; type 2 diabetes.

Figure 1.

The effect of YPHL decoction on BW (A), fast FBG (B), AUC of blood glucose (C), HbA1C, (D) HOMA-IR index (E), and serum lipid (F). Data are shown by means ± SEM (n = 8). ^ΔΔp < 0.01 and ^Δp < 0.05 vs. control group, **p < 0.01 and *p < 0.05 vs. model group, ^##p < 0.01 and ^# p < 0.05 vs. NC group. (A) There was no statistically significant difference in BW between YPHL group and model group (p > 0.05). (B) The FBG value of model group was significantly increased than that of control group (p < 0.05), the FBG value of YPHL group was lower than model group by 30.9% (p < 0.01), the FBG value of SIRT1 group was lower than NC group by 40.8% (p < 0.01), while the difference in the FBG value between YPHL group and SIRT1 group was not statistically significant (p > 0.05). (C) Compared with control group, the AUC of blood glucose in model group was increased by 184%, while the AUC in YPHL group was reduced by 19.3% as compared with model group (p < 0.01). Compared with NC group, the AUC in SIRT1 group was reduced by 27.9% (p < 0.01), and the difference of AUC between YPHL group and SIRT1 group was not statistically significant (p > 0.05). (D) Compared with model group, the HbA1c level in YPHL group decreased by 16.1%, the HbA1c level in SIRT1 group decreased by 22.2% compared with the NC group (p < 0.05), while there was no significant difference between YPHL group and SIRT1 group (p > 0.05). (E) Compared with control group, the HOMA-IR index of model group increased significantly (p < 0.01). Compared with model group and NC group, the index of YPHL group and SIRT1 group was reduced by 65.7% and 81.7%, respectively (p < 0.01). Compared with SIRT1 group, the index of YPHL group was increased (p < 0.05). (F) The levels of serum TC in model group were increased by about 1.4 times compared with control group (p < 0.05), while there was no significant difference between model group, YPHL group and SIRT1 group (p > 0.05). Compared with control group, the levels of serum TG in model group and NC group increased by about 17 times and 16 times, respectively (p < 0.01). Compared with model group and NC group, these levels were reduced by 37% and 40% in YPHL group and SIRT1 group, respectively (p < 0.05).

Figure 2.

Morphology of the pancreas. Pancreas specimens (n = 5) were collected and stained with HE (× 200) (Figure 2(A–E)). (A) Histological examination results showed that in normal group, the structure of pancreas was normal, the cell morphology was complete, and no obvious lipid deposition was observed in the cytoplasm. (B–D) In model group and NC group, large amounts of lipid deposition were observed between the acinar lobules, the acinar cells ectopic hyperplasia and telangiectasia were seen in the pancreatic islets. (C,E) In YPHL group and SIRT1 group, lipid deposition was observed in acini, and most of the lipids were located in the lobules alone without telangiectasia (the lipid deposition was indicated by a red arrow).

Figure 3.

(A–C) Lipid deposition in the RIN-m5f cells (n = 3) was collected and stained with oil red O staining (× 400). (D–F) Transmission electron microscopical observation of autophagosomes (× 10000). (B,C) The lipid droplets in model group were significantly increased compared with the control group, while they in YPHL group were significantly decreased compared with model group (all p < 0.05). (D) TEM observation showed that no obvious autophagosomes were observed in normal organelles in control group (× 10000). (E) In model group, the organelles disappeared, and many autophagosomes were seen. (F) In YPHL group, a low number of organelles were observed, while the number of early autophagosomes was increased; the enlarged structure of the mitochondria was improved, and the degree of autophagy was lower compared with model group (the autophagosomes was indicated by a yellow arrow).

Figure 4.

Proliferation activity of RIN-m5f cells (A), model group vs. control group, ^ΔΔp < 0.01, YPHL group vs. model group; **p < 0.01. H₂O₂ concentration in RIN-m5f cells (C), model group vs. control group, ^ΔΔp < 0.01; YPHL group vs. model group, **p < 0.01. Relative mean fluorescence intensity of ROS in RIN-m5f cells (D), model group vs. control group, ^ΔΔp < 0.01; YPHL group vs. model group, **p < 0.01. Apoptosis rate of RIN-m5f cells (E), model group vs. control group, ^ΔΔp < 0.01; YPHL group vs. model group, **p < 0.01. Data are shown by mean ± SEM (n = 3). (A) The survival rate of cells performed by CCK8 assay showed that the rate was decreased in model group, while it was increased in YPHL group (all p < 0.05). (B) The effect of YPHL on the concentration of H₂O₂ in the HLHG model cells. The H₂O₂ content was increased in model group and decreased in YPHL group significantly (all p < 0.05). (C) The rate of glucose consumption in HLHG model cells were decreased compared with control group, while the rate of glucose consumption in YPHL group was increased compared with model group. (D,E) The ROS content and the rate of cell apoptosis in the RIN-m5f cells were detected by flow cytometry. The content of ROS and the rate of cell apoptosis were both increased in model group and decreased in YPHL group significantly (all p < 0.05).

Figure 5.

Photographs of each RIN-m5f cell group under the laser confocal microscope (× 400). Laser scanning confocal microscopy showed that compared with control group, the expression of SIRT1 was reduced and the expression of FoxO1 was increased significantly in model group (p < 0.05). Compared with model group, the expression of SIRT1 and FoxO1 in YPHL group had no significant difference (p > 0.05).

Figure 6.

The results of SIRT1 and FoxO1 mRNA expressions. Expressions of SIRT1 and FoxO1 mRNA in pancreas tissues (A) and RIN-m5f cells (B) were detected by qRT-PCR analysis. GAPDH was used as the internal control. n = 8, ^Δp < 0.05, ^ΔΔp < 0.01 vs. control group, *p < 0.05 vs. model group, ^# p < 0.05 vs. NC group, ^▲p < 0.05 vs.si-NC group. (A) In pancreatic tissue, the relative expression of SIRT1 mRNA in model group and NC group was decreased significantly compared with control group (p < 0.05). The relative expression of SIRT1 mRNA was increased in YPHL group and SIRT1 group compared with model and NC groups, respectively (p < 0.05). On the contrary, the relative expression of FoxO1 mRNA in model group and NC group was increased significantly compared with control group (p < 0.05). The relative expression of FoxO1 mRNA was decreased in YPHL group and SIRT1 group compared with model and NC groups, respectively (p < 0.05). (B) In RIN-m5f cells, the relative expression of SIRT1 mRNA was significantly decreased in model, si-SIRT1, si-NC, and si-SIRT1 + YPHL groups compared with control group (p < 0.05). On the contrary, compared with model, si-SIRT1, si-NC, and si-SIRT1 + YPHL groups, the relative expression of SIRT1 mRNA was increased in si-NC + YPHL group (p < 0.05). On the contrary, the relative expression of FoxO1 mRNA was significantly increased in model, si-SIRT1, si-NC, and si-SIRT1 + YPHL groups compared with control group (p < 0.05). Compared with model, si-SIRT1, si-NC, and si-SIRT1 + YPHL groups, the relative expression of FoxO1 mRNA was decreased in si-NC + YPHL group (p < 0.05).

Figure 7.

The results of protein expressions. The protein expressions of SIRT1, Foxo1, LC3, and P62 in pancreas tissues (A,B) and cells (C,D) were detected by Western blotting. The figure represents one of three experiments with similar results (A,C). In (B) and (D), ^Δp < 0.05, ^ΔΔp < 0.01 vs. control group, *p < 0.05, **p < 0.01 vs. model group, ^#p < 0.05, ^##p < 0.01 vs. NC group^{, ▲}p < 0.05 vs. si-NC group. (A–B) In pancreas tissue, compared with control group, the relative expression of protein SIRT1 and LC3 was significantly decreased in both model and NC groups (p < 0.05), while the relative expression of protein FoxO1 and p62 was significantly increased in model group and NC group (p < 0.05). On the contrary, compared with model group and NC group, the relative expression of protein SIRT1 and LC3 was significantly increased in both YPHL and SIRT1 groups (p < 0.05), while the relative expression of protein FoxO1 and p62 was significantly decreased in both YPHL and SIRT1 groups than that in model and NC groups (p < 0.05) (the relative expression of LC3 was expressed as LC3B/LC3A). (C–D) In RIN-m5f cells, compared with control group, the relative expression of protein SIRT1 and LC3 was significantly decreased in model, si-SIRT1, si-NC, and si-SIRT1 + YPHL groups (p < 0.05), while the relative expression of protein FoxO1 was significantly increased (p < 0.05). On the contrary, the relative expression of protein SIRT1 and LC3 was significantly increased in si-NC + YPHL group than that in model, si-SIRT1, si-NC, and si-SIRT1 + YPHL groups (p < 0.05), while the relative expression of protein FoxO1 was significantly decreased in the si-SIRT1 + YPHL group (p < 0.05).