Radix Stellariae extract prevents high-fat-diet-induced obesity in C57BL/6 mice by accelerating energy metabolism

Preparation of Radix Stellariae extract

RS was purchased from the Ningxia province. In a spherical extractor, 4L of 95% ethanol was added to 500 g of RS. Extraction was performed for 2 h at 85 °C, followed by cooling and filtering of the extract. Extraction was repeated using 50% ethanol. The extracted solutions were combined for rotatory evaporation at 60 °C, under reduced pressure, till the taste of alcohol was undetectable. Finally, the concentrated solution was freeze-dried and stored at −20 °C.

Liquid chromatograph-high resolution mass spectrometry

Liquid chromatography-high resolution mass spectrometry (LC-HRMS) was performed using a Waters ACQUITY UPLC system, equipped with a binary solvent delivery manager and a sample manager. This system was, coupled with a Waters Micromass Q-TOF Premier Mass Spectrometer, equipped with an electrospray interface (Waters Corporation, Milford, MA).

An Acquity BEH C18 column (100 mm × 2.1 mm; i.d., 1.7 µm; Waters, Milford, USA) was maintained at 50 °C and eluted with gradient solvent from A : B (95 :) to A : B (0 : 100) at a flow rate of 0.40 mL/min, where A is aqueous formic acid (0.1% (v/v) formic acid) and B is acetonitrile (0.1% (v/v) formic acid) with an injection volume of 5.0 µL. The following gradient was applied (0–4.00 min: 95.0% A + 5.0% B; 4.00–6.00 min: 80.0% A + 20.0% B; 6.00–8.00 min: 75.0% A + 25.0% B; 8–12.5 min: 50.0% A + 50.0% B; 12.5–13.5 min: 15.0% A + 85.0% B; 13.5–15 min: 0.0% A + 100.0% B).

The MS analyses were performed using positive and negative ions channels. The ionization conditions were optimized, and the operating parameters were as follows: Polarity: positive/ negative; Capillary voltage: 3.0 kV/2.8 kV; Sampling cone: 35 V/45 V; Collision energy: 3 eV/3 eV; Source temperature: 115 °C/115 °C; Desolvation temperature: 350 °C/350 °C; Desolvation gas: 600 L/hr/ 600 L/hr; Scan range: m/z 50–1,500/m/z 50–1,500; Scan time: 0.3 s/0.3 s; Interscan time: 0.02 s/0.02 s.

Animals and diets

Six-week-old female C57BL/6 mice were purchased from the SLAC Laboratory (Shanghai, China). The animal protocols used in this study were approved by the Shanghai University of Traditional Chinese Medicine (approval number 2014019). The mice were housed under 22 °C–23 °C with a 12 h light/dark cycle. After a 1 week adaptation period, we randomly divided the seven-week-old mice into three groups. They were fed a chow diet (Chow, 10% of calories derived from fat, Research Diets; D12450B), an HF diet (HF, 60% of calories derived from fat, Research Diets; D12492), or a diet supplemented with 1% RSE (HF + RSE). The mice had free access to food and water for 8 weeks. We weighed the food intake and body weight every 2 days.

Rectal temperature measurement

At the end of 8 weeks, the rectal temperature of the mice was recorded three times at 3 PM by using an instrument for measuring rectal temperature, at intervals of 2 days.

Intraperitoneal glucose tolerance test

All mice were fasted for 12 h overnight at the end of the preventive experiment. For the intraperitoneal glucose tolerance test (IPGTT), we collected the blood samples from the tail vein for determination of baseline glucose values (0 min). Next intraperitoneal injections of glucose (1 g/kg body weight) were administered to all the mice in 15 min, and blood glucose levels were measured at regular intervals (15, 30, 60, and 90 min) after the injection of glucose.

Intraperitoneal insulin tolerance test

The mice were not fasted for the intraperitoneal insulin tolerance test (IPITT). Similar to the IPGTT, the basal blood glucose levels (0 min) were measured from the tail vein before the injection of insulin (0.75 U/kg body weight). The insulin was diluted in physiological saline. Next, additional blood glucose levels were measured at 15, 30, 60, 90, and 120 min after the injection of insulin.

Serum chemistry analysis

The mice were fasted for 10 h overnight at the end of the animal preventive experiment; the next day, all mice were anesthetized using 20% urethane before sample and tissue collection. Blood samples were drawn from the heart using a 1 mL syringe. After clotting at room temperature for over 2 h, the serum was separated from the blood samples. After centrifugation, 120 µL of serum was drawn from every sample, and the serum total cholesterol (TC), triglyceride (TG), low-density lipoprotein cholesterol (LDL-c), high-density lipoprotein cholesterol (HDL-c), alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels were analyzed using a Hitachi 7020 Automatic Analyzer.

Morphological analysis of white adipose tissue

To examine the structure of WAT, the WAT samples were fixed in 4% paraformaldehyde. The tissue samples were sectioned at 5 µm intervals and stained with Hematoxylin and Eosin (H & E). The stained samples were examined under a light microscope.

Reporter assay

The reporter assay was performed using the Dual-Luciferase Reporter Assay System (Promega, USA) as previously described. The expression plasmids for pCMXGal-hPPAR α, β, γ and the Gal4 reporter vector MH100 × 4-TK-Luc were co-transfected with a reporter construct so that 1 µg of the relevant plasmid combined with 1µg of reporter plasmids and 0.1 µg of pREP7 (Renilla luciferase) reporter could be used to normalize transfection efficiencies. The transfection mixture, which contained 10 µg of total plasmids and 15 µl FuGENE^®HD per ml of DMEM, was added to HEK293T cells for 24 h and then removed. The PPAR α, β, γ agonists (Fenofibric acid, GW7647, Pioglitazone) and 2.5, 5, 10, 20, 50, 100, 200, 400, 600, 800, 1,000 µg /ml of RSE were added to fresh media and the cells were incubated for another 24 h to determine luciferase activity.

Quantitative real-time polymerase chain reaction (Real time qPCR)

The total RNA of WAT and BAT was extracted using the RNAiso Plus (Takara, Dalian, China). RNA is unstable, and to facilitate stable long-term preservation, we used the RevertAid First Strand cDNA Synthesis Kit (Thermo Scientific, Wilmington, Delaware, USA) for the first-strand cDNA (42 °C, 1 h; 70 °C, 5min). The gene expression levels were analyzed using quantitative real-time RT-PCR conducted using the ABI StepOnePlus real-time PCR system (Applied Biosystems, USA). The relative primers involved in the experiments are listed in Table 1. β-Actin was considered an internal control to normalize the expression levels of genes. The cDNA was denatured at 95 °C for 10 min followed by 40 cycles of PCR (95 °C for 15 s, 60 °C for 60 s).

Statistical analysis

Data were analyzed using SPSS 18.0, and the results were presented as mean ± SEM. Differences were considered significant if P < 0.05. Statistical analysis included one-way analysis of variance, the Student’s t-test, the Kruskal–Wallis H Test, and repeated measures analysis of variance.

LC–HRMS detection of the main chemical contituents

We performed the LC–HRMS assay to characterize the constituents in the extract. Ten compounds were putatively identified in the extract according to a previous report (Chen et al., 2010). We detected some β-carboline alkaloids, such as stellarine A–C, dichotomine B, H, and L, glucodichotomine B as well as vanillin, 5-hydroxymethylfurfural (Fig. 1 and Table 2).

RSE inhibited body weight gain in C57BL/6 mice induced by high-fat diet

To investigate the effect of RSE on body weight gain, we selected the most widely used inbred strain C57BL/6 mouse, which is susceptible to diet-induced obesity, type 2 diabetes and atherosclerosis (Sun et al., 2016; Xia et al., 2016). The dose of RSE was determined by using a series of complex mathematical operation according to the dose of humans (15 g natural plant medicine/60 kg body weight/day) and pharmacology of traditional Chinese medicine (Zhang, 2002). The C57BL/6 mice were fed on a chow diet, HF diet or HF diet supplemented with 1% RSE for 8 weeks. The data revealed that the average body weight in the HF group was significantly higher than that of the Chow group (Fig. 2A), whereas the body weight of the HF + RSE group was evidently lower than that in the HF group from Week 2 to Week 8 (Fig. 2A). The data indicated that RSE could inhibit body weight gain induced by HF-diet in mice. There was no significant difference of food intake between the HF and HF + RSE groups (Fig. 2B). Furthermore, we observed the weekly food intake of the mice. The weekly intake of the three groups remained in a state of equilibrium (Fig. 2C). Thus, the lower body weight in RSE-treated mice was not caused by a lower calorie intake.

Next, we measured the adipocyte size using the H & E stain. The results revealed that the size of WAT in the HF group was considerably larger than that of the Chow group, and RSE treatment reduced the size of WAT in HF + RSE group (Figs. 2D–2E). The data supported the conclusion that RSE inhibits body weight gain.

Inhibition of lipid absorption in the intestine or increase of energy expenditure may result in weight reduction. To test whether RSE affected the lipid absorption and energy expenditure, we determined the total cholesterol and triglyceride content of the feces of the mice, and measured rectal temperature. The RSE-treated mice did not exhibit an increase in the TC and TG content of feces (Figs. 2F–2G); however, the body temperature of the RSE-treated mice was notably higher than that of the mice in the other groups (Fig. 2H). Hence, an increase in energy consumption, and not the inhibition of intestinal lipid absorption, may be responsible for the reduction in body weight in HF + RSE group mice.

RSE reduced fasting blood glucose and ameliorated insulin tolerance in high-fat diet-induced obese C57BL/6 mice at 15 and 30 min

Obesity is a cause for insulin resistance and type II diabetes. Therefore, we measured the fasting blood glucose levels and glucose tolerance in the mice (Fig. 3A). RSE- treated mice exhibited lower fasting glucose levels than did the HF-fed mice (Fig. 3B). However, the blood glucose levels did not change following intraperitoneal injection of glucose. Then we tested the insulin tolerance in the mice. The results revealed that the blood glucose levels of the HF + RSE group were lowered than those of the HF group at 15, and 30 min (Fig. 3C).

RSE lowered the lipid profile in serum and liver

Obesity may be accompanied by hyperlipidemia. Therefore, we measured the lipid levels in serum and liver tissue. The fasting serum TG, TC, and LDL-c levels of the HF + RSE group were slightly lower than those of the HF group, although the levels were not significantly different between both groups (Fig. 4A). Similarly, the hepatic TG and TC levels were also lower in the RSE-treated mice (Fig. 4B). Then, we tested the serum levels of ALT and AST, the two indicators of liver damage. The levels of ALT of the HF group were evidently higher than those of the Chow diet-fed mice, indicating potential damage to liver function. RSE treatment, however, did not change the ALT and AST concentrations in HF group mice (Figs. 4C and 4D).

RSE induced the expression of metabolic gene in vivo

The genes for uncoupling proteins (UCPs), namely UCP1, UCP2, and UCP3, are closely related to energy metabolism. In view of the rise of body temperature, we tested the expression of these genes in BAT, which participates in energy consumption and heat production. The expression of the genes in the RSE-treated mice BAT was not notably different from that of the HF-fed mice (Fig. 5A). White beige fat, indicated by high UCP1 expression (Nedergaard & Cannon, 2014), which increases energy metabolism, may also be a mechanism of fat reduction. Therefore, we analyzed the expression of UCPs in the WAT. The data revealed that the mRNA of UCP1 and UCP3 increased markedly, suggesting the induction of WAT browning by RSE (Fig. 5B).

PPARs are the ligand-activated nuclear transcription factors regulating the gene expression of glucose and lipid metabolism. A reporter assay was performed to test whether the RSE alters transactivities of PPAR α, β, γ. The results showed that RSE did not change the transcription activity of PPARs, suggesting that RSE does not activate PPARs directly.

We examined the mRNA expression levels of PPARs and their target genes in the WAT. RSE clearly increased the mRNA expression of PPARs and acetyl coenzyme A carboxylase (ACC), acyl-CoA oxidase (ACO), adipose fatty acid-binding protein (aP2), cluster of differentiation 36 (CD36), peroxisome proliferator-activated receptor coactivator-1 α and −1 β (PGC-1 α and PGC-1 β), glucose transporter 4 (GLUT 4) as well as UCP1, and UCP3. Among them, PPAR β, PPAR γ, UCP1, UCP3, ACO, aP2, and CD 36 were significant (Fig. 5B). Taken together, the data suggested that RSE may regulate body weight and blood glucose levels through the enhancement of PPAR signaling.