Weaned (3-week-old) Sprague-Dawley male rats (Model Animal Research Center of Nanjing Medical University, China) were divided into the normal diet (ND) and HFD groups. All animals were housed under controlled environmental conditions (22°C, 40-60% relative humidity, 12-/12-h light/dark cycle) and were given free access to water and food. All animals were allowed 1 week of feeding adaptation. The ND group was given a regular diet, and the HFD group was given a diet comprising 45 kcal% fat (Research Diets, D12451). After feeding for 10 weeks, part of the HFD group served as a control group, while others received ES and drug treatment. To maintain the state of obesity, the animals were fed the high-fat diet until the end of the experiment. All experiments were performed according to the Principles of Laboratory Animal Care and the Guide for the Care and Use of Laboratory Animals published by the National Science Council, China (Animal Committee Number: 201809A015).

The animals were anesthetized with isoflurane (0.5%-1.5%) for ES. Electroacupuncture was performed at the forelimb LI11 (Quchi) acupoint by inserting a pair of stainless-steel acupuncture needles (diameter: 0.3 mm) up to 3 mm in depth at each site. LI11 is located midway between the lateral end of the transverse cubital crease and the lateral humeral epicondyle (17). The needles were connected to Han’s electroacupuncture instrument (LH402A; Beijing Huawei Technologies Co. Ltd). ES was performed with a stimulation current of 2 mA, a pulse width of 0.4 ms, an alternating frequency between 2 and 15 Hz every 3 s, and a stimulation time of 20 min. The procedure was performed six times a week and lasted for 4 weeks.

The rats from the HFD group were administered the NLRP3 inflammasome agonist (nigericin sodium salt) and were assigned to three groups: (1) solvent control (SC); (2) nigericin sodium salt treatment (Nig); (3) nigericin sodium salt with ES (Nig+ES) groups. After 10 weeks of HFD feeding, the solvent control and Nigericin sodium salt (M7029, AbMole, USA) were administered in the epididymal visceral adipose tissue (eWAT) in rats via intraperitoneal injection (i.p.) before ES. The NLRP3 agonist (Nig) was administered three times a week (Nig 1 mg/kg body weight) for 4 weeks.

The adipose tissues were fixed in 4% paraformaldehyde and embedded in paraffin, sectioned at 8-μm thickness. Hematoxylin and eosin staining (H&E staining) were performed according to the standard process. We used Image-Pro Plus software to collect image fields of each rat for adipocyte area, and more than 250 cells per rat were selected for measuring (18).

eWAT was harvested using the Adipose Protein Extraction Kit (MinuteTM) for protein extraction. Eighty milligrams of adipose tissue was homogenized with 250 ml of extraction buffer. After centrifugation at 2,000 g for 1 min, the tissue was transferred to a filter cartridge with a collection tube and incubated at -20°C for 20 min. After incubation, the tissue was immediately centrifuged at 2000 rpm for 1 min with the cap open. The flow-through contained total proteins from the adipose tissue. Then, 30 mg of total tissue lysate was separated by SDS-PAGE (10% separation gel and 5% concentration gel). Electrophoresis was performed at 70 V for 0.5 h and 110 V for 1 h. The protein bands were transferred to a PVDF membrane using e-Blot (Genscript), and 5% BSA was added for 1 h to block the membrane. The membrane was then made to react with primary antibodies against ATGL (1:1000, Abcam), HSL (1:3000, Abcam), p-HSL (1:1000, Sab), NLRP3 (1:1000, Abcam), Caspase-1 (1:1000, Abcam), IL-1β (1:1000, Abcam), MAOA (1:1000, Abcam), β-actin (1:1000, Abcam), and tyrosine hydroxylase (1:1000, Abcam) at 4°C overnight. Incubation with the corresponding secondary antibodies (diluted to 1:3000, Abcam) was performed at room temperature (18°C -25°C) for 1 h.

After tissue perfusion and separation, the samples were fixed with 4% paraformaldehyde at 4°C over 12 h and then immersed in 30% sucrose solution at 4°C overnight. Tissues were embedded in optimal cutting temperature (OCT) compounds, and sections (thickness, 50 μm) were prepared using a freezing microtome (Leica, German). The sections were blocked with 0.3% Triton X-100 Sea Block blocking buffer (Thermo Fisher Scientific, United States) for 1 h, and incubated with primary antibodies against the following proteins at 4°C overnight: tyrosine hydroxylase (TH, 1:500, Abcam) and F4/80 (1:50, Sab). After washing with PBS three times, the sections were incubated with secondary antibodies: anti-Alexa Fluor 488, Alexa Fluor 594 goat anti-chicken, and anti-rabbit (1:500, Abcam) antibodies at room temperature for 1 h. For samples were mounted using the ProLong Diamond Antifade Mountant (Thermo Fisher Scientific, United States) and cured for 24 h.

Total RNA was extracted from 300 mg of eWAT by using Trizol (TaKaRa, Japan). The total RNA was reverse-transcribed to produce cDNA by using a thermal cycler (Bio-Rad, United States) with PrimeScript RT Master Mix (TaKaRa, Japan) at 37°C for 15 min and 98°C for 15 s. The PCR reaction mixture (20 mL) contained 4 μL of a forward primer, 4 μL of a reverse primer, 2 μL of cDNA, and 10 μL of SYBR Green Mix (TaKaRa, Japan). The PCR protocol was as follows: 40 cycles of amplification for 30 s at 95°C, 5 s at 95°C, and 30 s at 60°C. Data were processed using the 2^-△△CT method. The primers used in the experiment are shown in Table 1 .

Serum level were detected according to the manufacturer’s recommendations. For free fatty acid, cholesterol and triglyceride, standards and each 2.5 μl of mice serum was added into 96 well coated plate with 250μl Working fluid, incubated at 37°C for 10 min. OD values were detected at 510 nm by using microplate reader (Bio-Tek, USA). Free fatty acid, cholesterol and triglyceride level were quantified by standard curve.

Plasma concentrations of TNFα, IL-6,IL-4, IL-1β, IL-18 were assayed by ELISA. Standard or sample solution (100 mL) was added to 96-well polystyrene microplates and incubated at 37°C for 90 min. Then, 10 mL of biotinylated antibodies (Nanjing Jiancheng Bioengineering Institute, Nanjing) was added to the wells and incubation was performed at 37°C for 60 min. The solution in each well was then aspirated, and each well was washed three times with 350 mL of 1* wash buffer. Next, 100 mL of tetramethylbenzidine (TMB) substrate was added to each well and incubation was performed for 10 min. To stop the reaction, 100 mL of stop solution was added, and the absorbance in the wells was read at 450 nm.

The caspase-1 activity in eWAT was measured by Abbkine Caspase 1 Assay Kit (Colorimetric). This assay is performed on the basis of the ability of caspase-1 to change acetyl-Tyr-Val-Ala-Asp p-nitroaniline (Ac-YVAD-pNA) into the yellow formazan product pNA. After treatment, the adipose tissues were harvested and lysed. The protocol was determined according to the manufacturer’s instructions. Absorbance was measured at 405 nm by microplate reader (Bio-Tek, USA). Standard curves for the assay system were obtained from dilutions of the standards of pNA. Caspase-1 activity was then obtained by determining the amount of pNA according to the standard curve of pNA.

GraphPad Prism6 was used for statistical analysis. Data are expressed as mean ± SEM values. To test whether obtained data were normally distributed the Shapiro-Wilk test was performed. Levene’s test was used to assess homogeneity of variance. Data between two groups were analysed by unpaired t-test, if the data were in Gaussian distribution and had equal variance, or by Mann-Whitney rank-sum test if the data were not normally distributed. Data among more than two groups were compared using one-way analysis of variance (ANOVA) followed by post hoc Student and Newman-Keuls tests. P < 0.05 was considered to indicate statistical significance.

We first compared the bodyweight of the ND and HFD groups. HFD feeding for 10 weeks induced obesity ( Figure 1A ). eWATs were weighed after 10 weeks of HFD feeding ( Figure 1C ). Since HFD consumption not only leads to obesity but also leads to low-grade inflammation (19), we measured blood cytokine levels. The proinflammatory cytokine IL-6 and TNF-α significantly accumulated in blood serum in the HFD group ( Figures 1E, F ), while the level of the anti-inflammatory cytokine IL-4 reduced in this group ( Figure 1G ). ES at ST25 reduced body weight (20), and eWAT analysis showed that ES at LI11 (2.0 mA) reduced body weight and fat weight ( Figures 1B, C ). Also, ES reduced food intake during 4 weeks ( Figure 1D ). Interestingly, ES at LI11 reduced blood serum levels of the proinflammatory cytokine IL-6 and TNF-α in the HFD group ( Figures 1E, F ) but did not promote the expression of anti-inflammatory cytokines, such as IL-4 ( Figure 1G ).

Obesity is associated with dynamic adipose tissue remodeling characterized by adipocyte hypertrophy, especially in the visceral adipose tissue (21, 22). The size of adipocytes in eWAT increased in the HFD group and reduced on ES ( Figures 2A, B ). Visceral fat is the largest lipid reservoir in the body, and its excessive expansion causes lipid metabolism disorder (23). As shown in Figures 2C–F , HFD induced dyslipidemia, including abnormal triglyceride (TG), non-esterified fatty acid (NEFA), and total cholesterol (TCH) levels. ES significantly reduced NEFA and TCH levels. As the low-grade inflammation caused by visceral adipose tissue inflammatory accumulate, we measured cytokine levels in eWAT. As shown in Figures 2G–I , the mRNA expression of the proinflammatory cytokine IL-1β and TNF-α in eWAT significantly increased in the HFD group compared to that in the ND group and decreased on ES compared to that in the HFD group. Thus, there may be a connection between lipid metabolism and inflammation in ES.

As shown in Figures 3A–C, G , ES induced lipolysis by activating p-HSL and ATGL. The SNS plays a crucial role in facilitating noradrenaline (NE) to stimulate lipolysis in white adipose tissue (WAT) (24). Next, we measured the level of NE in eWAT. The level of NE was higher in ES than that in the HFD group ( Figure 3D ). Consequently, we examined TH expression, which is related to NE generation ( Figures 3A, E ). The TH level in ES was not significantly greater than that in the HFD mice. Thus, NE generation did not increase, and we considered that ES might attenuate the degradation of NE. MAOA was an essential enzyme in the metabolism of monoamines, which can degrade NE in adipose tissue (25). As shown in Figures 3A, F , ES attenuated the activation of MAOA in eWAT. The results indicated that ES induced lipolysis by attenuating the activation of MAOA in eWAT but did not increase NE generation.

The presence of NAMs has been recently reported in adipose depots. In inguinal subcutaneous white adipose tissue (iWAT), inflammatory NAMs express machinery for the degradation (MAOA) of noradrenaline, thereby dampening β3-adrenergic receptor (β3-AR)-driven lipolysis (26). In obesity, NAMs accumulate in eWAT ( Figure 4A ). As shown in Figure 4B , ES decreased the co-localization expression of F4/80 (macrophage) and TH (sympathetic nerve), which were labeled NAMs. These findings indicate that ES may induce lipolysis through immune adjustment.

In eWAT, the increasing activity of the NLRP3 inflammasome leads to increases in MAOA oxidation, thereby dampening lipolysis and promoting obesity (11). Therefore, we hypothesized that ES might attenuate the activation of MAOA in NAMs by suppressing the activation of the NLRP3 inflammasome. As shown in Figures 5A–E , ES attenuated the relative protein level of the NLRP3 inflammasome, including NLRP3, caspase-1, and IL-1β, but not procaspase-1. In Figure 5F , ES attenuated the activity of caspase-1. Also, we measured the cytokines processed by the NLRP3 inflammasome such as IL-1β and IL-18. ES decreased the level of IL-1β and IL-18 in the serum ( Figures 5G, H ). To verify whether ES regulated NAM via suppression of the NLRP3 inflammasome, we injected the NLRP3 inflammasome agonists (Nigericin sodium salt) into the abdominal cavity of rats. As shown in Figures 6A–E , the agonist activated the NLRP3 inflammasome in the Nig and Nig+ES groups, which increased the expression of NLRP3, caspase-1, IL-1β and the activity of caspase-1 in eWAT. Also, the agonist increased the production of IL-1β and IL-18 in the serum ( Figures 6F, G ). It also increased the co-localization expression of F4/80 (macrophage) and TH (sympathetic nerve), which blocked the effect of ES-regulated NAMs ( Figures 7A, B ). These data suggest that ES regulated NAM via suppression of the NLRP3 inflammasome.

To verify whether the NLRP3 inflammasome agonist inhibited ES-induced lipolysis, we tested the enzymes related to lipolysis. As shown in Figures 8A, D , the agonist increased the expression of MAOA oxidation, suggesting that the catecholamine signal pathway was inactivated. Furthermore, it decreased the expression of p-HSL/HSL and ATGL. Thus, ES suppressed the NLRP3 inflammasome to attenuate the activation of MAOA, which induces lipolysis in eWAT ( Figures 8A–C, E ).