Animals and nobiletin administration

All animal experiments were performed according to the protocol approved by the Ethics Committee of Chengdu University of Traditional Chinese Medicine (License: 2021-05). Ninety 8-week-old male C57BL/6 mice were purchased from Spafford; 10 of these mice were fed low-fat chow (D12450J, USA) as a normal control group, and the remaining mice were fed high-fat chow (D12492, USA). The obese mice were identified according to our previous publication (Xia et al. 2022) and randomly grouped for further intervention. The nobiletin (Ruifensi Biotech Co., Ltd., Chengdu, China) (purity > 98%) concentration was determined according to previous methods (Pang et al. 2022). In the early stages of the experiment, a dose of 40 mg/kg/d was used, but the results were the same as those for 100 mg/kg/d, so the 40 mg/kg/d was omitted in the writing. Low- (NOL, 100 mg/kg/d) and high-dose nobiletin (NOH, 200 mg/kg/d) mice were treated for 2 w. Equal volumes of PBS were used in the CON and HFD groups.

Determination of the obesity index

Body weights were measured and recorded weekly. The body length of the mice was measured at the end of the experiment, and Lee’s index was calculated. An oral glucose tolerance test (OGTT) was simultaneously performed.

Serum and tissue sample collection

The mice were anesthetized with isoflurane (RWD Life Sciences, Shenzhen, China), and approximately 500 μl of serum sample was collected from each subject. Serum levels of high-density lipoprotein (HDL), low-density lipoprotein (LDL), total cholesterol (TC) and triglyceride (TG) were assayed using an automated biochemical analyzer (BS-240VET, Myriad Co., Shenzhen, China). The assay kits were purchased from Myriad Corporation (Shenzhen, China). The small intestine, liver and epididymis were anatomically removed and fixed with 4% paraformaldehyde. The remaining small intestine samples were quickly frozen in liquid nitrogen and stored at − 80 °C.

Hematoxylin–eosin (HE) staining and immunofluorescence

HE staining was performed via a conventional approach. Samples of small intestinal tissues were embedded in paraffin. After dewaxing, the 5 μm sections were stained with hematoxylin and eosin, dehydrated and sealed. Photographs were obtained by a light microscope (Leica, Italy). The images were analyzed with ImageJ.

The above tissue sections were further subjected to immunofluorescence staining. The sections were first deparaffinized with xylene and ethanol and then antigenically repaired with EDTA. Bovine serum was added for 10 min at room temperature, and the samples were incubated with anti-ZO-1/Occludin antibodies for 1 h. The samples were subsequently washed five times, incubated with AlexaFluor®488 (ab150129) and AlexaFluor®647 in a dark environment for 1 h, and incubated with DAPI for 1 h. Images were taken with a fluorescence microscope (Leica, Italy). Four fields of view were collected from the upper, lower, left and right parts of each section. The optical density was quantified and determined using ImageJ software.

RT-qPCR

Total RNA was extracted from small intestinal tissues using an RNA extraction kit (Forgene, China) and reverse transcribed with a SuperMix (Vazyme Biotechnology Co., Ltd., Nanjing, China). cDNA libraries were constructed on a PROMETHION platform (Biomarker, Inc., Beijing, China). The expression levels of the genes were determined via real-time PCR (Vazyme Biotechnology Co., Ltd., Nanjing, China). The β-actin gene was used as an internal control (Table 1). The 2−∆∆Ct method was used to calculate the fold change.

Table 1 Sequences of the primers used for RT-qPCRFull-length transcriptomic sequencing

Differences of interest (p < 0.05, |fold change|> 1.2) were considered significant. Volcano maps of the differentially expressed genes (DEGs) were generated using the DESeq package (1.18.0) (https://cloud.oebiotech.cn). Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Ontology (GO) enrichment analyses were performed (https://Metascape.org/gp/), and the results are plotted in http://www.bioinformatics.com.

Statistics

Statistical analysis and plotting were performed using GraphPad Prism (V9.1.0). Normally distributed data are expressed as the means ± SDs. Statistical analysis was performed via one-way or two-way ANOVA. All tests were considered to be statistically significant at p < 0.05.