Experimental animals

C57BL/6 J mice (male, 7–8 weeks, 20–23 g), were purchased from Yuxiu Biotechnology Co., Ltd. (Shanghai, China). All of them were housed in a room with a temperature of 22–24 °C, humidity ranging from 50 to 60%, and a 12-h light/dark cycle, with free access to food and water. After one week of acclimatization to the environment, experiments were conducted. All animal experiments were was reviewed and approved by the Ethics Committee for Laboratory Animals, School of Basic Medical Sciences, Fudan University (20240229–050).

Partial hepatectomy model

The mice were divided into Intact, partial hepatectomy (HT), and HT + EA groups. All animals underwent 30 min of daily restraint adaptation for a period of 3 days. The surgical trauma model used in this experiment is as described earlier [17]. In brief, after intraperitoneal injection of 0.2 mL/10 g Avertin for anesthesia, a surgical incision was made in mice, approximately 3 cm long along the midline from the xiphoid process to the pubic symphysis. The abdominal cavity was fully opened, and 10% of the liver was resected from the left lobe. After removing 10% of the liver lobe, hemostasis was immediately achieved using a disinfected dry cotton ball for approximately 5 min. Finally, the abdominal cavity was sutured. Throughout the surgical procedure, environmental temperature and aseptic techniques were strictly controlled, and the surgery was conducted from 8:00 to 10:00 in the morning.

EA

Mice in each group were habituated to the self-made fixing devices (50 mL centrifuge tubes with enough holes to make sure mice could breathe normally and facilitate EA stimulation) once a day for three consecutive days before the first EA. In the EA procedure, mice were safely restrained and kept awake, while the Intact and HT groups were also restrained but received no other interventions. The acupoints chosen for EA were “Zusanli” (ST36, located on the outer side below the knee joint, about 2 mm below the head of the fibula [28]) and “Sanyinjiao” (SP6, located 5 mm below the head of the fibula and 2 mm outside the anterior tibial tubercle) on the right hind limb. The EA occurred on the morning of the day before surgery and immediately after the abdominal closure [29]. The 0.5-inch (0.22 × 13 mm) acupuncture needles (Hua Tuo, Suzhou, China) were used, inserted vertically into the ST36 to a depth of approximately 5 mm, and inserted horizontally from bottom to top into the ST6 to a depth of about 5 mm. Both acupoints were connected to a HANS Acupoint Nerve Stimulator (LH202H, Beijing, China). The EA parameters were set as follows: dense-sparse wave, frequency of 2–15 Hz, intensity ranging from 1–2 mA, with slight tremors observed in the lower limbs as the endpoint, and a duration of 30 min. At the end of the experiment (24 h post-HT), animals in all groups were decapitated following administration of Avertin. Blood was then collected via retro-orbital bleeding, and the hypothalamus was harvested on ice.

Enzyme linked immunosorbent assay

After peripheral blood collection via retro-orbital bleeding, centrifugation was conducted at 4 °C and 3000 rpm for 30 min. The upper serum layer was collected post-centrifugation. Enzyme-linked immunosorbent assay (ELISA) kits, purchased from Shanghai Lengton Biotechnology Co., Ltd. (Shanghai, China), were employed for the quantification of plasma ACTH and CORT levels.

Real-time polymerase chain reaction

Total RNA was isolated from hypothalamic tissues using TRIzol Reagent (15596026, Life Technologies, USA). Subsequently, the RNA was reverse transcribed into cDNA using the PrimeScript RT Reagent Kit (RR036A, Takara, Japan) according to the manufacturer’s instructions. PCR was performed with the SYBR Premix Ex Taq kit (RR420B, Takara, Japan) and the QuantStudio 3 Real-Time PCR System (ThermoFisher, USA). The reaction volume consisted of 10 μL SYBR Premix Ex Taq mixture, 0.8 μL primer mixture, 2 μL cDNA template, and 6.8 μL ddH2O. Transcript levels were normalized to the GAPDH within the same sample. The mRNA primers were synthesized by Shanghai Sangon Biotech Co., Ltd. (Shanghai, China).

The primer sequences were as follows: NUCB2 (Forward: 5′AAG AAG TAG GAA GAC TGC GGA TGC3′; Reverse: 5′AGG ATT CTG GTG GTT CAG GTG TTC3′); CRH (Forward: 5′CTG TCG TCC TGC CTG CCT TG3′; Reverse: 5′TTC ACC CAT GCG GAT CAG AAC C3′); GAPDH (Forward: 5′AGA AGG TGG TGA AGC AGG CAT C3′; Reverse: 5′CGA AGG TGG AAG AGT GGG AGT TG3′). The relative mRNA levels were analyzed using the 2^−ΔΔCt method, normalized to GAPDH.

Western blot

At 24 h post-surgery, hypothalamic tissues of mice were collected after the administration of Avertin. The hypothalamic tissues were lysed using RIPA lysis buffer (Biosharp, China) containing a mixture of proteinase and phosphatase inhibitors (Beyotime, China) and subjected to ultrasonic homogenization. The supernatant was centrifuged at 4 °C, 12,000 rpm for 20 min. Protein concentration was determined using a BCA assay kit (Beyotime, China). Equal amounts of protein were separated by 12% SDS-PAGE (ACE, China) and transferred onto PVDF membranes (Millipore, German). The membranes were blocked with TBST containing 5% skimmed milk powder or Rapid Protein-Free Blocking Buffer (ACE, China). Subsequently, the membranes were incubated overnight at 4 °C with primary antibodies against CRH (10944–1-AP, anti-rabbit, 1:1000, Proteintech), Nesfatin-1 (AF6895, anti-sheep, 1:1000, R&D), β-tubulin (10094–1-AP, anti-rabbit, 1:10000, Proteintech), phosphor-ERK1/2 (4370, anti-rabbit, 1:1000; Cell Signaling Technology), ERK1/2 (4695, anti-rabbit, 1:2000; Cell Signaling Technology), phosphor-CREB Ser133 (9198S, anti-rabbit, 1:1000; Cell Signaling Technology), and CREB (12208–1-AP, anti-rabbit, 1:1000; Proteintech). Following washes with TBST, the membranes were incubated with secondary antibodies, either HRP-conjugated goat anti-rabbit (L-3012, 1:10000; SAB) or rabbit anti-sheep (AS023, 1:10000; ABclonal). Signal visualization was performed using ECL (Epizyme, China), and protein bands were detected using the Amersham ImageQuant 800 Protein Blot Imaging System. ImageJ software was employed for quantifying the grayscale values of the bands, and the relative expression of the target proteins was calculated based on the grayscale values of internal reference bands.

Open field tests

The anxiety levels of mice were assessed using the open field tests (OFT) 24 h after the surgery [30]. The apparatus consisted of a square arena with opaque plastic walls (length = width = 50 cm, height = 40 cm). Each mouse was gently placed in the center of the arena, and allowed to explore the area for 10 min, during which their movement trajectory was recorded. Parameters evaluated included time spent in the central zone, central zone cross counts, the ratio of central distance to total distance, and grooming episode. After each trial, residual odors were eliminated using 75% ethanol.

Elevated plus maze

Elevated plus maze (EPM) apparatus consists of two enclosed arms (length 20 cm, width 4 cm, height 12 cm) and two similar open arms arranged in a cross. A 10-min test is employed to determine the time spent in the open arms and entries made into the open arms. The apparatus is cleaned with 75% ethanol to eliminate residual odors left by the preceding animal.

Light–dark box tests

The apparatus used for this experiment consisted of a box (35 × 25 × 30 cm) divided into two compartments: one-third of the box (dark) and two-thirds of the box (light). Mice were placed in the light compartment and allowed to freely explore the room for 10 min. A video camera was utilized to record the mouse's movements in the light area. Anxiety-like behaviors in mice were assessed based on the time spent and the distance traveled in the light area. The apparatus was cleaned with 75% ethanol to eliminate residual odors left by the preceding animal.

Immunofluorescence

Mouse coronal brain slices (thickness 40 μm) were obtained, and selected slices were subjected to immunofluorescent staining. In brief, brain samples were incubated or co-incubated with antibodies against c-Fos (226008, anti-rabbit, 1:400, SYSY), Nesfatin-1 (AF6895, anti-sheep, 1:200, R&D), CRH (C36806, anti-rabbit, 1:200, SAB), or phosphor-ERK1/2 (4370, anti-rabbit, 1:200, Cell Signaling) at 4 °C overnight. The slices were then incubated with the respective secondary antibodies, Alexa Fluor 488 (A-21206, 1:1000, ThermoFisher) or Alexa Fluor 594 (A-11016, 1:1000, ThermoFisher), at room temperature in the dark for 2 h. Visualization was conducted using the integrated fluorescence microscopy system BZ-X (KEYENCE, Japan). For quantitative analysis of immunostained cells, ImageJ software was used to count the numbers of CRH, Nesfatin-1, c-Fos, phosphor-ERK1/2 positive cells, and co-labeled cells within a 400 µm2 area adjacent to the third ventricle (3 V).

Virus injection

After intraperitoneal injection of Avertin for anesthesia, mice were placed in a stereotaxic apparatus. Following fixation, the hair on the head was shaved, and the surgical site was treated with a dilute iodine solution. An incision was made in the scalp, and the surgical area was swabbed with a surgical sponge soaked in hydrogen peroxide until the skull was exposed. The location of the PVN of the hypothalamus was (AP 0.6 mm, ML ± 0.2 mm) [29, 31]. Using a dental drill, a hole was slowly drilled into the skull, and a Hamilton 2.5 μL microsyringe, coupled with a glass electrode, was used to extract the virus. The viruses used were rAAV2/9-CMV-Nesfatin-1-EGFP-WPRE-hGH or rAAV2/9-U6-CMV-shRNA (scramble)-mCherry-SV40 obtained from BrainVT A Co., Ltd. (Wuhan, China). The virus was slowly inserted into the PVN to a depth of 4.5 mm at a rate of 40 nL/min, and a total injection volume of 200 nL. After the injection was completed, a 5-min waiting period was observed to prevent viral overflow. Subsequently, the skin on the head was sutured.

Cell culture and plasmid transfection

The mouse neuroblastoma-2a (N2a) cells were obtained from the Center for Excellence in Molecular Cell Science, Chinese Academy of Sciences, and cultured in DMEM (Biosharp, China) supplemented with 10% fetal bovine serum, 50 units/mL penicillin, and 100 µg/mL streptomycin (Biosharp, China) at 37 °C in a humidified atmosphere with 5% CO2.

Overexpression or knockdown of Nesfatin-1 in N2a cells was achieved through plasmid transfection. The plasmid backbone is consistent with the viral nucleic acid framework. For transfection, N2a cells were plated at 60% density in 6-well culture plates, and JetPrime transfection reagent (PolyPlus Transfection, France) was used. mRNA or protein was extracted 48 h post-transfection, with a transfection efficiency of approximately 75%. In interventions with inhibitors, SCH772984 (HY-50846, MCE) or 666–15 (HY-101120, MCE) was added 24 h after plasmid transfection, following the same steps as before.

Transcriptome sequencing

Sprague–Dawley (SD) rats (male, 7–8 weeks, 180–220 g) were purchased from the Slack Laboratory Animal Center (Shanghai Branch of the Chinese Academy of Sciences, Shanghai, China). Rats were in good health without any pre-existing conditions that could affect the study results. They successfully adapted to the experimental environment for one week before the experiment. Rats were randomly assigned to the three groups (Intact, HT, EA) using a random number generator. To ensure objectivity and reduce bias, all experimental assessments and subsequent analyses were conducted by researchers blinded to the group allocations. The groups were coded, and the code was not revealed until the completion of the data analysis. The samples were prepared using the Seq-RNA sequencing method provided by Novo Gene Biotech Co. Ltd. (Beijing, China). Three SD rats from each group were included. The surgical and EA procedures were identical to those performed in mice. 24 h after HT, the hypothalamic tissues were isolated from the rats, preserved in RNA storage solution, and sent to Novo Gene Biotech Company for subsequent analysis. The construction of cDNA libraries, library purification, and transcriptome sequencing were conducted following the protocols provided by Novo Gene Biotech Company. The normalized RNA count data was used for subsequent Principal Component Analysis (PCA) in R. Differential Expression analysis for RNA-Seq data was performed using R/Bioconductor package edgeR. The threshold of significance was set as a p value < 0.05 to find transcriptionally regulated genes. Heatmaps were made using R/Bioconductor package pheatmap (https://CRAN.R-project.org/package=pheatmap). Volcano plots were made using ggplot2 in R.

Stereotactic cannula implantation and PVN administration

The bilateral PVN cannulation in mice was performed under Avertin anesthesia. The drug delivery system was designed by RWD Life Science Co., Ltd. (RWD, Shenzhen, China). Modeling and drug administration were carried out two weeks after the cannulation. On the day before surgery and immediately after surgery, mice were administered ERK1/2 inhibitor SCH772984 (0.1 nmol/µL, 0.5 µL/side, HY-50846, MCE), CREB inhibitor 666–15 (0.1 nmol/µL, 0.5 µL/side, HY-101120, MCE), or normal saline (NS) at a rate of 0.1 µL/min into the PVN [29]. After injection, the mice were left undisturbed for 5 min to allow for drug diffusion. All mice were euthanized under anesthesia 24 h after the last administration. Peripheral blood and hypothalamic tissue were collected and frozen for further experiments.

Statistical analysis

All data were analyzed using GraphPad prism 9.0 software. Data were presented as mean ± SEM, and comparisons between two groups were performed using unpaired two-tailed t-tests. Multiple group comparisons were conducted using one-way ANOVA or two-way ANOVA. A p-value < 0.05 was considered statistically significant.