All the animal experiments were approved by Shanghai Ninth People’s Hospital, Shanghai Jiao Tong University, School of Medicine, Shanghai, China. Female nude mice (aged 6 to 8 weeks) were housed in individual cages with a 12-hour light/dark cycle and provided with standard food and water ad libitum.
2.2 Fat Grafting Model and Treatments
The mice were randomly divided into three groups: saline, 10 μmol/L, and 50 μmol/L. Each mouse was injected subcutaneously on the back with 0.2 ml of Coleman fat using a 1 ml syringe with a blunt infiltration cannula (Supplementary material Fig. 1A). The grafts were injected into a spherical shape. The mice were locally injected with 0.2 ml of saline or salvianolic acid B (10 μmol/L, 50 μmol/L) once every two days. The mice were sacrificed after 2, 4, and 12 weeks (n = 5 per time point per group), the grafts were harvested and carefully separated from surrounding tissue, and their volumes and weights were measured. Each harvested sample was assessed histologically and immunohistochemically.
2.3 RAW264.7 Culture
RAW264.7 cells (ATCC, USA) were grown in culture medium, which was changed every 48 h. When the cells reached approximately 90% confluence, they were washed using high-sugar DMEM (Gibco, USA) with 10% foetal bovine serum (FBS; Gibco, USA). The cell suspension was further divided into three culture flasks containing growth medium.
2.4 Cell viability assays
The effects of salvianolic acid B on the viability of RAW264.7 cells were tested using Cell Counting Kit-8 (Beyotime, China) according to the manufacturer’s instructions. Briefly, 5000 cells/well were seeded in a 96-well plate. The cells were cultured in growth medium with various concentrations of salvianolic acid B (0, 10, 25, 50, 75, 100 μmol/L). At 72 h of culture, 10% CCK-8 reagent was mixed with medium and added to each well. The 96-well plate was incubated at 37 °C for 2 h. The relative number of cells was measured at an absorbance of 450 nm using a microplate reader (Thermo, USA).
2.5 2.6 5-Ethynyl-2′-deoxyuridine (EdU) proliferation assay
Cells were seeded in 24-well plates and incubated under standard conditions with various concentrations of salvianolic acid B (0, 10, 50, 100 μmol/L). Twenty-four hours after incubation, cell proliferation was detected using the EdU Cell Proliferation Assay Kit (Invitrogen, USA) according to the manufacturer’s protocol. Briefly, cells were incubated with 50 μM EdU for 2 hours before fixation, permeabilization, and EdU staining. Next, cell nuclei were stained with Hoechst 33342 (Invitrogen, USA) for 30 minutes. The proportion of cells that incorporated EdU was determined by inverted fluorescence microscopy (Nikon, Japan). The cells were counted manually in each field, three fields were counted for each of the 3 experiments, and three technical replicates were performed in each of the 3 experiments. The proportion of Edu+ cells (%)=100*Number of Edu+(Green) cells/Number of total cells (Hoechst33342+ cells, Blue). The significance of differences between the control and treated groups was set at P<0.05 and assessed by ANOVA with GraphPad Prism 8 (GraphPad Software, La Jolla, CA, USA).
2.6 Flow cytometry
After 3 days in culture with various concentrations of salvianolic acid B pretreatment, RAW264.7 cells were resuspended in PBS buffer according to the number of cells (5,000/ml). For Annexin V and propidium iodide staining, 195 μL of cell suspension was mixed well with 5 μL of Annexin V-FITC, followed by incubation at room temperature for 10 minutes. The cells were washed with PBS and resuspended in 190 μL of deliquated binding buffer, and then 10 μL of 20 µg/ml propidium iodide was added. The samples were analyzed by flow cytometry using CytoFLEX LX (Beckman Coulter, USA). The data were analyzed using CytExpert (Beckman Coulter, USA).
2.7 Histological analysis and immunofluorescence staining
Tissues were fixed in paraformaldehyde overnight, embedded in paraffin, cut at a thickness of 5 μm and then stained with haematoxylin and eosin. We used the methods of Shoshani O and Yu P to evaluate histologic parameters, such as cell integrity, tissue inflammation, the presence of cysts/vacuoles, and the extent of fibrosis. Each parameter was scored as follows: 0 = absence, 1 = minimal presence, 2 = minimal to moderate presence, 3 = moderate presence, 4 = moderate to extensive presence, and 5 = extensive presence. The scoring was performed independently by 3 authors who were unaware of the grouping.
For immunofluorescent staining, tissue sections were incubated with the following primary antibodies: Perilipin (#15294-1-AP; 1:200; Proteintech, China), F4/80 (#27044-1-AP; 1:200; Proteintech, China), CD11c (ab11029; 1:200; Abcam, UK) and CD206 (#60143-1-lg; 1:200; Proteintech, China) diluted in blocking solution overnight at 4 °C. After incubation with Alexa Fluor 488–conjugated goat anti-rabbit immunoglobulin G (#A-21206; 1:500; Invitrogen, USA), Alexa Fluor 555–conjugated goat anti-rabbit immunoglobulin G (#A-31572; 1:500; Invitrogen, USA) and Alexa Fluor 488–conjugated goat anti-mouse immunoglobulin G (#A-21202; 1:500; Invitrogen, USA), the nuclei were stained with 4′,6-diamidino-2-phenylindole (Southern Biotech, USA).
For immunocytofluorescence, RAW264.7 cells were incubated with a primary antibody against p-p65 (#3033; 1:500; Cell Signaling Technology, USA) diluted in blocking solution overnight at 4 °C. After incubation with Alexa Fluor 488–conjugated goat anti-rabbit immunoglobulin G (#A-21206; 1:500; Invitrogen, USA), the nuclei were stained with 4′,6-diamidino-2-phenylindole (Southern Biotech, USA).
ImageJ software was used for quantitative analysis. Image analysis was performed according to the website (https://imagej.net/imaging/image-intensity-processing) and the method of Keskin.
2.8 Micro-CT analysis
The fat grafts were scanned using micro-CT (PerkinElmer, USA) and analyzed by ProPlan CMF 3.0.
2.9 RNA Extraction and Real-time RT–PCR
To investigate the polarization level of RAW264.7 cells, RAW264.7 cells were incubated under standard conditions with various concentrations of salvianolic acid B (0, 10, 50, and 100 μmol/L). After 72 hours of culture, the cells were stimulated with ultrapure LPS (10 ng/ml; Sigma, USA), and then the transcriptional levels of iNOS and TNF-α in RAW264.7 cells were assessed by real-time PCR. Initially, the total RNA of RAW264.7 cells was extracted using a total RNA miniprep kit (Axygen, USA), and RT–qPCR was performed using an ABI 7900HT system and SYBR Premix (Takara, Japan) according to the manufacturer’s instructions. mRNA quantification was performed using glyceraldehyde 3-phosphate dehydrogenase (GAPDH) for normalization. The SYBR green primers for qRT–PCR are listed in Supplementary Table 1.
2.10 Western blotting
Cultured cells were lysed with RIPA buffer (Beyotime, China) supplemented with protease inhibitor (PMSF; Biosharp, China). Briefly, 20 μg of protein was resolved by 10% or 12% SDS–PAGE and electroblotted onto polyvinylidene difluoride membranes (Millipore Sigma, USA). The membranes were blocked with 5% nonfat milk at room temperature for 1 hour. The separated proteins were then immunoblotted and probed with the following primary antibodies: anti–glyceraldehyde 3-phosphate dehydrogenase (#10494-1-AP; 1:5,000; Proteintech, China), anti-NF-κB p65 (ab32536; 1:1,000; Abcam, UK), anti-NF-κB p65 (phosphor S536) (ab76302; 1:1,000; Abcam, UK), anti–JNK1+JNK2+JNK3 (ab179461; 1:1,000; Abcam, UK) and anti–JNK1+JNK2+JNK3 (phosphor T183+T183+T221) (ab124956; 1:1,000; Abcam, UK) at 4 °C overnight. The next day, the membranes were incubated with peroxidase-conjugated secondary antibody (ab205718; 1:10,000; Abcam, UK) at room temperature for 1 hour after washing with Tris-buffered saline with Tween 20 for 10 minutes three times. ImageJ software was used for quantitative analysis, which was conducted on immunoreactive bands. The number of experimental or technical replicates was three.
2.11 RNA-Seq analysis
RNA sequencing samples were acquired after the addition of LPS (10 ng/ml)+Sal-B (50 μmol/L), LPS (10 ng/ml) or solvent to RAW264.7 cells for 3 days in growth medium. The RNA quantity and quality were measured using the NanoDrop ND-1000 system. The cDNA library was constructed using the KAPA Stranded RNA-Seq Library Preparation Kit (Illumina) following the manufacturer’s protocol. The final double-stranded cDNA samples were verified using an Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA). After cluster generation (TruSeq SR Cluster Kit v3-cBot-HS; Illumina), sequencing was performed using an Illumina HiSeq 4000 sequencing platform. Image analysis, base calling, and error estimation were performed using Illumina/Solexa Pipeline (Off-Line Base Caller software, version 1.8). Quality control was checked on the raw sequence data using FastQC (https://en.wikipedia.org/wiki/FASTQ_format). Raw data were preprocessed using Solexa CHASTITY and Cutadapt to remove adaptor sequences, ribosomal RNA, and other contaminants that may interfere with clustering and assembly. The trimmed reads were mapped to the corresponding reference genome using HISAT2 (version 2.0.4) for RNA sequencing, and StringTie (version 1.2.3) was used to reconstruct the transcriptome. Ballgown software was applied to calculate the fragments per kilobase of exon per million fragments mapped in RNA sequencing data and analyze differentially expressed genes, with the fragments per kilobase of exon per million fragments mapped ≥ 0.5 (Cuffquant) considered for the analysis. The cut-off for defining which genes were differentially expressed was a fold change greater than 1.5. Gene Ontology functional and Kyoto Encyclopedia of Genes and Genomes pathway enrichment analyses were performed for differentially expressed genes using the Database for Annotation, Visualization and Integrated Discovery and Kyoto Encyclopedia of Genes and Genomes Orthology-Based Annotation System online tools (http://www.geneontology.org and http://www.genome.jp/kegg).
2.12 Molecular docking
The PDB file for the crystal structures of IKKβ was obtained using the protein data bank code 4KIK. The molecular docking procedure was performed under the C-DOCKER protocol of Accelry’s Discovery Studio 2019 software. For ligand preparation, the structure of Sal-B was constructed using ChemDraw Professional 17.0 software, saved in the SDF file format and minimized using Accelry’s Discovery Studio 2019 software. The protein structure was cleaned and inspected for errors, hydrogens were added, and the water molecules were deleted. The IKKβ proteins were defined as receptors, and the centroid of the binding site was defined based on the ligand in the cocrystal structure. Next, the original ligand was removed, and the molecule of Sal-B was placed in the sphere position to perform molecular docking. For energy minimization, the CHARMM force field was used within Accelry’s Discovery Studio 2019 software. Finally, the types of interactions between the docked proteins and Sal-B were analyzed.
2.13 Statistical analysis
In the present study, all in vitro experiments were conducted 3 times. Single blinding was used for statistical analysis. Two blinded data analysts independently analyzed the data. The final data were consistent between the two analysts. The data were expressed as means ± SD. The continuous variables between the groups were compared by the independent samples t-test. One-way ANOVA with Tukey’s post hoc test was employed for pairwise comparisons among multiple groups. The significance of differences between the control and treated groups was set at P<0.05 and assessed using GraphPad Prism 8 (GraphPad Software, La Jolla, CA, USA).