JMP is composed of SC, which is produced in Xianqiao, Zhejiang and RCL, which is produced in Qichun, Hubei, in a 1.5:1 (w/w) ratio. SC and RCL were purchased from Tianji Chinese Herbal Medicine Co., Ltd. (Wuhan, China). All herbal drugs were soaked in eight volumes of distilled water (1:8, w/v) for 12 h at room temperature, followed by boiling three times (2 h each). The JMP water (JW) extract was filtered and concentrated to a concentration equivalent to 0.2 g raw herbs/1 ml water extract (23). The JMP ethanol (JE) extract was concentrated and precipitated with 60% ethanol for 12 h. The solution was evaporated under reduced pressure (~0.1 MPa) to produce a concentration equivalent to 0.2 g raw herbs in 1 ml ethanol extract (33). The SC (SW) extract (0.3 g/ml) and SC ethanol (SE) extract (0.3 g/ml), RCL water (RW) extract (0.4 g/ml) and RCL ethanol (RE) extract (0.4 g/ml) were prepared using the same method used for the JW extract and JE extract. The extracts were stored at 4°C until they were used.

M199 medium was from Gibco; Thermo Fisher Scientific, Inc. (Waltham, MA, USA), bovine serum albumin (BSA) and 4-(2-hydroxyethyl) piperazine-1-ethanesulfonic acid sodium salt (HEPES) were from Sigma-Aldrich; Merck Millipore (Darmstadt, Germany). The glycerol assay kit and NEFA assay kit were from Applygen Technologies Inc. (Beijing, China). ISO was from Hefeng Pharmaceutical Co., Ltd. (Shanghai, China), 100 µg/ml streptomycin and 100 U/ml penicillin were from Beyotime Institute of Biotechnology (Shanghai, China), TRIzol reagent and the reverse transcription kit were from Takara Bio, Inc. (Tokyo, Japan), primers were from Wuhan Qingke Innovative Biotechnology Co., Ltd. (Wuhan, China), NP-40 lysis buffer and the BCA protein assay kit were from Beyotime Institute of Biotechnology. Rabbit anti-P-HSL ser563 and rabbit anti-P-HSL ser660 (cat. nos. 4139 and 4126, respectively) were from Cell Signaling Technology Inc. (Danvers, MA, USA), rabbit anti-β-actin (cat. no. TDY051) was from Beijing TDY Biotech Co., Ltd. (Beijing, China).

Adult male Sprague-Dawley rats are purchased from the Experimental Animal Center of Wuhan University (Wuhan, China). All animal experiments were performed in compliance with the Principles for Care and Use of Laboratory Animals approved by the Laboratory of Tongji Medical College, Huazhong University of Science and Technology (Wuhan, China). The animals were housed in a room with temperature controlled at 20±5°C and 55±5% humidity, under a 12-h light-dark cycle. The rats are fed with standard rat chow (23). Rats with a weight of 300±30 g were used during the experiments. The rats were weighed and anesthetized, and the retroperitoneal WAT was rapidly excised in an aseptic laparotomy procedure (34). The explants of WAT were immediately placed in a culture dish containing pre-warmed (37°C) phosphate-buffered saline (PBS) solution, and washed briefly to dispel blood. The explants were transferred, discarding the tissue residue and fat droplets, into preheated PBS in another culture dish with. A surgical scalpel was used to cut the explants into uniform sections of ~100±5 mg. The sections were gently washed with PBS, resuspended in PBS, and left standing for 5 min to allow the sections rich in adipocytes to float. The floating sections were collected and transferred into a culture dish containing 25 ml of serum-free M199 with 25 mM HEPES and antibiotics (100 U/ml penicillin-G and 100 µg/ml streptomycin), and cultured at 37°C with an atmosphere of 95% air and 5% CO₂ for 24 h. Studies have shown that live adipocytes can survive in fresh adipose tissue and in adipose tissue cultivated for 24 h, with survival rates of 94% each and 87%, respectively (35). At the end of the 24-h incubation period, the dish was removed from the incubator, and pre-warmed PBS was used to clean the adipose tissue, using a sterile gauze to dry the tissues. The tissue sections were distributed into a 24-well culture plate (100 mg tissue/well). The wells are then filled with 1 ml Krebs-Ringer buffer containing 25 mM HEPES, 5.5 mM glucose, and 2% (w/v) BSA. Immediately, the cultures are treated with normal saline (vehicle), 750 nM ISO [which is known to stimulate the release of NEFA in rat WAT through activation of the β-adrenergic receptors (36)] as a positive control, or different concentrations of JW extract, JE extract, SW extract, SE extract, RW extract and RW extract. The cultures were incubated at 37°C in 5% CO₂ and 95% humidity for 90 min. At the end of the incubation period, the conditioned media were collected from each well. The NEFA and glycerol content were measured using NEFA and glycerol assay kits.

The assessment of lipolysis was measured by NEFA and glycerol release from WAT into the incubation media. The NEFA and glycerol concentrations in the conditioned media from all samples were determined according to the manufacturer's instructions of the commercial NEFA and glycerol assay kits. The results are expressed as mmol/l of NEFA and mmol/l of glycerol released per 100 mg WAT.

The expression of proteins was determined using western blot analysis. The explants of WAT were collected and washed in PBS. The total cellular protein was extracted using NP-40 lysis buffer containing phenylmethanesulphonyl fluoride. The protein concentrations were measured using a BCA protein assay kit according to the manufacturer's instructions. Equal quantities (40 µg) of protein samples were separated by 10 or 12% SDS-PAGE electrophoresis, and then transferred onto polyvinylidene fluoride membranes. The membranes were blocked with 5.0% fat-free dry milk in Tris-buffered saline and Tween-20 (TBST) at room temperature for 2 h and incubated with rabbit anti-P-HSL Ser563 (1:1,000), rabbit anti-P-HSL Ser660 (1:1,000), and rabbit anti-β-actin (1:2,000) at 4°C overnight. Following washing with TBST four times, the membranes were incubated with the corresponding HRP-conjugated secondary antibodies (cat. no. 074-1506, 1:3,000; KPL, Inc., Gaithersburg, MD, USA) for 1 h at room temperature on a rocker with gentle agitation. The bands were visualized using ECL reagent, and the band intensities were quantified by densitometry using ImageJ software version 2.0 (National Institutes of Health, Bethesda, MD, USA). The results are presented as the ratio of the optical density of the target bands to β-actin.

The explants of WAT treated with different concentrations of JCM, SC, RLC were collected, and sections of each explant were fixed in 4% paraformaldehyde for further embedding in paraffin wax. The tissues blocks were further processed using a routine procedure for hematoxylin and eosin (H&E) staining. The stained adipose tissue sections were observed and images were captured under an optical microscope (magnification, ×400).

All experiment data are presented as the mean ± standard error of the mean of three or more independent experiments. Statistical significance was determined using GraphPad Prism 6.0 software (GraphPad Software, Inc., La Jolla, CA, USA) by one-way analysis of variance. P<0.05 was considered to indicate a statistically significant difference.

To determine whether JMP and its ingredients, SC and RCL, can stimulate lipolysis in rat WAT, the present study investigated the release of NEFA from JMP-, SC- and RCL-treated rat WAT in vitro. The results revealed that 750 nmol/l ISO significantly increased the release of NEFA, compared with vehicle (Fig. 1). Furthermore, the rat WAT was treated with JW extract at various concentrations (2.5, 5.0, 10, 20 and 200 mg/ml). Compared with the vehicle, the release of NEFA was significantly increased in WAT in the rats treated with JMP concentrations of 5.0, 20 and 200 mg/ml, and the maximum effect was observed at a dose of 20 mg/ml (Fig. 1A). JE extract stimulated the release of NEFA at the concentrations of 10, 20 and 200 mg/ml in a dose-dependent manner, compared with the vehicle-treated group, with the maximum effect at 200 mg/ml (Fig. 1B). Treatment with 30 and 300 mg/ml SW extract stimulated the release of NEFA in a dose-dependent manner, with the maximum effect at 300 mg/ml, compared with the vehicle-treated group (Fig. 1C). Treatment with 15 and 300 mg/ml SE extract stimulated the release of NEFA, with the maximum effect at a dose of 300 mg/ml, however this effect was not dose-dependent manner (Fig. 1D). Treatment with 20, 40 and 400 mg/ml RW extract, and 40 and 400 mg/ml RE extract stimulated the release of NEFA in a dose-dependent manner. The maximum effects of RW extract and RE extract were observed at 400 mg/ml (Fig. 1E and F).

The present study also determined the release of glycerol from the rat WAT treated with ISO, JMP and its ingredients, SC and RCL. Consistent with the release of NEFA, the effects on lipolysis by the treatments on glycerol were similar in the medium from the cultured rat WAT. Treatment with 750 nmol/l ISO, 5.0, 20 and 200 mg/ml JW extract (maximum at 20 mg/ml), 20 and 200 mg/ml JE extract (dose-dependent with maximum at 200 mg/ml), 30 and 300 mg/ml SW extract (dose-dependent with the maximum at 300 mg/ml), 15 and 300 mg/ml SE extract (maximum at 300 mg/ml), 20, 40 and 400 mg/ml RW extract (dose-dependent, maximum at 400 mg/ml), 40 and 400 mg/ml RE extract (dose-dependent, maximum at 400 mg/ml) stimulated the release of glycerol, compared with release in the vehicle-treated group (Fig. 2A-F).

The HSL at Ser563 and Ser660 are major phosphorylation sites associated with HSL activity, and are well known markers of lipolysis (14,18–22). ISO is known to phosphorylate HSL at Ser563 and Ser660 sites, and results in increasing lipolysis, which is used as a positive control (21,22). According to the results of the release of NEFA and glycerol (Figs. 1 and 2), the present study examined the phosphorylation levels of HSL at Ser563 and Ser660 in cultured rat WAT treated with 20 mg/ml JW extract, 200 mg/ml JE extract, 300 mg/ml SW extract, 300 mg/ml SE extract, 400 mg/ml RW extract and 400 mg/ml RE extract. As shown in Fig. 3, consistent with the release of NEFA and glycerol, 750 nmol/l ISO, JW extract, JE extract, SW extract, SE extract, RW extract and RE extract significantly upregulated the phosphorylation levels of HSL at Ser563 (P-HSL Ser563) and Ser660 (P-HSL Ser660), compared with that in the vehicle-treated group. This suggested that JMP, SC and RLC significantly upregulated the expression of P-HSL Ser563 and P-HSL Ser660 in the stimulation of lipolysis in rat WAT in vitro. RCL had the most marked effect on increasing the levels of P-HSL Ser563 and P-HSL Ser660, particularly RW extract (Fig. 3).

To confirm whether JMP, SC and RCL reduce the size of adipocytes, the cultured rat WAT was stained using HE staining. Histological analysis of the adipose tissue to confirmed the sizes of the adipocytes (Fig. 4). The size of adipocytes was markedly smaller in the ISO (750 nm) group, compared with that in the vehicle group. The adipocytes were also smaller in the 20 mg/ml JW extract, 200 mg/ml JE extract, 300 mg/ml SW extract, 300 mg/ml SE extract, 400 mg/ml RW extract and 400 mg/ml RE extract groups, compared with size in the vehicle group.