Endocrinol Metab.  2021 Feb;36(1):185-195. 10.3803/EnM.2020.835.

Danshen Extracts Prevents Obesity and Activates Mitochondrial Function in Brown Adipose Tissue

• Cho YH
• Ku CR
• Choi YS
• Lee HJ
• Lee EJ

• KMID: 2513301
• DOI: http://doi.org/10.3803/EnM.2020.835

Abstract

Background
Danshen has been widely used in oriental medicine to improve body function. The purpose of this study is to investigate the effect of water-soluble Danshen extract (DE) on weight loss and on activation proteins involved in mitochondrial biogenesis in brown adipose tissue (BAT) in obese mice.
Methods
BAT was isolated from 7-week-old male Sprague-Dawley rats, and expression of proteins related to mitochondrial biogenesis was confirmed in both brown preadipocytes and mature brown adipocytes treated with DE. For the in vivo study, low-density lipoprotein receptor knock out mice were divided into three groups and treated for 17 weeks with: standard diet; high fat diet (HFD); HFD+DE. Body weight was measured every week, and oral glucose tolerance test was performed after DE treatment in streptozotocin-induced diabetic mice. To observe the changes in markers related to thermogenesis and adipogenesis in the BAT, white adipose tissue (WAT) and liver of experimental animals, tissues were removed and immediately frozen in liquid nitrogen.
Results
DE increased the expression of uncoupling protein 1 and peroxisome proliferator-activated receptor gamma coactivator 1-alpha in brown preadipocytes, and also promoted the brown adipocyte differentiation and mitochondrial function in the mature brown adipocytes. Reactive oxygen species production in brown preadipocytes was increased depending on the concentration of DE. DE activates thermogenesis in BAT and normalizes increased body weight and adipogenesis in the liver due to HFD. Browning of WAT was increased in WAT of DE treatment group.
Conclusion
DE protects against obesity and activates mitochondrial function in BAT.

Keyword

Adipose tissue, brown; Organelle biogenesis; Dan-shen root extract; Thermogenesis; Obesity

Figure

• 

  Fig. 1 Danshen extracts (DEs) increase the expression of mitochondrial biogenesis-related proteins in brown preadipocyte. (A) The effect of DE on cell proliferation and viability was assessed by using MTS assay. Brown preadipocytes were incubated for 2 days in the presence of various concentrations of DE on 96 well plate. Later, cell viability was measured by enzyme-linked immunosorbent assay. (B) Western blotting analysis of uncoupling protein 1 (UCP1), proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α), and AMP-activated protein kinase (AMPK) activity in brown preadipocyte treated with DE (50 and 100 μg/mL) or vehicle (control) for 24 hours. β-Actin serves as a loading control. (C) Number of mitochondria were visualized by staining with MitoTracker Red 580 (red fluorescence) followed by treating with and without DE (100 μg/mL, 24 hours) and tumor necrosis factor-α (TNF-α; 100 ng/mL, 1 hour) in brown preadipocyte. Bars, 20 μm. (D) The effect of DE on intracellular reactive oxygen species (ROS). Synchronized brown preadipocytes were pretreated with DE (50 and 100 μg/mL, 24 hours). CM-H2DCF-DA emission was recorded using a fluorescence cytometer. The experiments were performed in triplicate and results are expressed as the mean±standard error of mean. p-AMPK, phosphorylated AMPK. aP<0.05 and bP<0.01 statistically significant differences between vehicle and DE groups.

• 

  Fig. 2 Danshen extracts (DEs) promotes the brown adipocyte differentiation and mitochondrial functions in brown adipocytes. (A) After induction of adipogenesis for 10 days in brown preadipocytes treated with DE (10, 25, 50, 100, and 200 μg/mL) or vehicle (control) for 7 days, Oil Red O staining was performed. Images were obtained with optical microscope at ×200 magnification. (B) Western blot analysis of CCAAT/enhancer binding protein (C/EBP) α and C/EBPβ after induction of adipogenesis of brown preadipocytes for 10 days and treatment with DE (50 and 100 μg/mL) or vehicle (control) for 24 hours. (C) Western blotting analysis of uncoupling protein 1 (UCP1), proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α), and AMP-activated protein kinase (AMPK) activity after induction of adipogenesis of brown preadipocytes for 10 days and treatment with DE (50 and 100 μg/mL) or vehicle (control) for 24 hours. β-Actin serves as a loading control. Total-AMPK (t-AMPK) acts as a loading control for phosphorylated AMPK (p-AMPK). The experiments were performed in triplicate and results are expressed as the mean±standard error of mean. aP<0.05 and bP<0.005 statistically significant differences between vehicle and DE (50 and 100 μg/mL) groups.

• 

  Fig. 3 Danshen extracts (DEs) reduces weight gain, blood glucose and fatty liver. (A) DE was administrated orally (50 mg/kg on daily) during 17 weeks; Calorie composition of dietary fat (w/wo 0.2% of DE) was described in Methods (high fat [HF]-diet; 60% of calories from fat). Number of animals in each experimental group of animals was five and starting weight was average of 25 g. Weight measurements were performed once a week and indicated the average of weight gain (ΔG)±standard deviation. (B) Streptozotocin (STZ)-induced diabetic mice, DE supplementation significantly decreased blood glucose compared to the STZ+sodium citrate (pH 4.5). (C) DE ameliorate non-alcoholic fatty liver disease through upregulating AMP-activated protein kinase (AMPK) activation in HF fed mice. H&E staining and Western blotting analysis of peroxisome proliferator-activated receptor γ (PPARγ) and AMPK activity in the liver tissue of experimental animals. Images were obtained with optical microscope at ×200 magnification. p-AMPK, phosphorylated AMPK; t-AMPK, total AMPK; HF+DE, high fat diet supplemented with 0.2% DE. aP<0.05 statistically significant differences between HF group vs. HF+DE groups; bP<0.01; cP<0.005.

• 

  Fig. 4 Danshen extracts (DEs) activate brown adipose tissue (BAT) metabolism and browning of white adipose tissue (WAT) in obese mice. (A) H&E staining of BAT from each experiment groups. Scale bars, 100 μm. The changes in number of cells of BAT was also measured. (B) Protein levels in interscapular BAT of key genes involved in BAT metabolism were analysis by using the Western blotting. β-Actin serves as a loading control. All animal experiments were measured at 17 weeks in obese mice with or without 0.2% of DE supplement. (C) Analysis of mRNA expression of key genes involved in browning of WAT. UCP1, uncoupling protein 1; PGC-1α, proliferator-activated receptor gamma coactivator 1-alpha; p-AMPK, phosphorylated AMP-activated protein kinase; PPARγ, peroxisome proliferator-activated receptor γ; HF, high fat; Prdm16, PR domain containing 16. aP<0.005 vs. normal; bP<0.01 vs. HF; cP<0.01, dP<0.001 statistically significant differences between HF group vs. HF+DE groups; eP<0.05 vs. normal; fP<0.05 and gP<0.01 vs. HF.

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