Chemicals and reagents

ZSP is composed of three herbs: Rhizoma Anemarrhenae, Cortex Phellodendri and Cinnamomum cassia. Extracts of Rhizoma Anemarrhenae, Cortex Phellodendri and Cortex Cinnamomi (extraction ratio: 10:1) were purchased from Shaanxi Zhongxin Biotechnology (Xi’an, Shaanxi, China). To produce the decoction, the three extracts were mixed at a ratio of 10:10:1, dissolved in distilled water to 0.468 g/mL and thoroughly stirred before gavage.

Liquid chromatography (LC)-mass spectrometry (MS)/MS analysis

The components of ZSP were detected by LC–MS/MS analysis. LC–MS/MS analysis was conducted using an Agilent 1290 ultra-high performance liquid chromatography system (Agilent Technologies, Santa Clara, CA, US) with a UPCL BEH C18 column (1.7 μm*2.1 mm*100 mm, Waters, Milford, MA, US). 5 μL of ZSP sample was loaded onto the system and flow rate was set at 400 μL/min. The elution program is described in Additional file 1: Table S1. The MS and MS/MS data were obtained by a Q Exactive Focus mass spectrometer (Thermo Fisher Scientific, Bremen, Germany). Mass ranged from 100 to 1500 in each cycle, and the top three of every cycle were screened for further MS/MS data acquisition. The MS/MS data was matched to the database provided by Shanghai BIOTREE biotech Co., Ltd to identify the materials.

Animals

The animal experiments in this study were approved by the Animal Care and Ethics Committee of Beijing University of Chinese Medicine (approval No. BUCM-4-2019031003-1089). A total number of 14 6 week-old male C57BL/KsJ-db/db mice and 7 C57BL/KsJ-wt/wt mice of the same age and gender were purchased from Nanjing Biomedical Research Institute of Nanjing University (Nanjing, China). Mice were kept in individually ventilated cages with free access to food (normal chow diet) and water. The cages were placed in a specific-pathogen-free environment with a standard 12/12 h artificial light/dark cycle.

After 1 week of accommodation, db/db mice were randomly divided into two groups (ZSP group, receiving 3.3 g/kg ZSP by gavage; model group, receiving distilled water by gavage) according to their blood glucose levels and body weights. Wt/wt mice were used as normal group (receiving distilled water by gavage). The dosage of ZSP treatment is calculated according to the dosage of human by the body surface area estimation method determined according to our previous study [9]. The treatment lasted for 40 days and mice received normal chow diet throughout. The body weight and food intake were measured every 3 days. Overnight fasting glucose level was measured every 7 days using blood drawn from the tail vein. At the end of treatment, the body length of mice was measured and body mass index (BMI) calculated as follows:

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Oral glucose tolerance test (OGTT)

OGTT was conducted on mice at day 35 after over-night fasting. All mice were orally administered 1 g/kg body weight of glucose (dissolved in distilled water to 10%w/v). Blood glucose levels were measured at administration and after 30, 60, and 120 min of glucose treatment. The area under curve (AUC) of OGTT was calculated as follows:

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(G0, G30, G60 and G120 represent blood glucose at 0, 30, 60, and 120 min, respectively).

Tissue harvest and histological observation

After 40 days of treatment, mice were sacrificed after anesthesia with isoflurane. Blood was drawn from abdominal aorta and collected in EP tubes, then centrifuged at 3000 rpm for 15 min at 4 °C to obtain serum. Serum was stored at − 20 °C. Liver and adipose tissue were removed and weighed. Tissue samples were snap frozen with liquid nitrogen and stored at − 80 °C.

A slice of each tissue was kept in a 4% paraformaldehyde-PBS solution (Cat.P1110, Solarbio Science & Technology, Beijing, China) for 24 h before being embedded in paraffin. Hematoxylin & eosin (H&E) staining was conducted for morphological observation, oil red O staining and periodic acid-Schiff (PAS) staining was performed to examine the lipid accumulation and glycogen content, respectively.

Biochemical examinations

Serum lipids including total cholesterol (TC), triglyceride (TG), high-density-lipoprotein (HDL) and low-density-lipoprotein (LDL), as well as hepatic function indicator alanine transaminase (ALT) and aspartate aminotransferase (AST) levels were measured by a chemistry analyzer (AU480, Beckman Coulter, Brea, CA, US). Hepatic glycogen was tested by colorimetric analysis using a commercial kit (Cat. BC0345, Solarbio Science & Technology). Fasting serum insulin levels were measured by enzyme-linked immunosorbent assay (ELISA) using Mouse Ultrasensitive Insulin ELISA kit (Cat. 80-INSMSU-E01, ALPCO Diagnostics, Salem, NH, US). Homeostatic model assessment for insulin resistance (HOMA-IR) was calculated as follows:

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Transcriptomics

Total RNA from liver were extracted from 6 random-chosen mice using an RNA isolation kit (Cat. AM1560, Ambion, Austin, TX, US) according to the manufacturer’s protocol of extracting total RNA. After quality and integrity checks, RNA was used for library construction with TruSeq Stranded mRNA Sample Prep Kit (Cat. RS-122-2101, Illumina, San Diego, CA, US). Raw reads of RNA were generated by the Illumina HiSeq™ X Ten platform. Reads containing ploy-N and low-quality reads were removed. Clean reads were mapped to NCBI database (GRCm39) using HISAT2 [18].

Bioinformatic analysis

Fragments per kb per million reads (FPKM) value [19] of gene reads was calculated. DESeq2 were applied to standardize and analyze mRNA expression levels. Threshold for differentially expressed genes (DEGs) were defined as p < 0.05 and foldchange >1.5. Principal component analysis (PCA) was used to analyze the relationship between transcriptome of samples. For functional enrichment, Gene Ontology (GO) database and Kyoto Encyclopedia of Genes and Genomes (KEGG) database were used to annotate and analyze identified DEGs.

Quantitative real-time PCR (RT-qPCR)

Total RNA was extracted using RNA Easy Fast Tissue/Cell Kit (Cat. DP451, Tiangen Biotech, Beijing, China) according to the manufacturer’s instructions. Tissue was homogenized with lysis buffer, the suspensions were filtered through a gDNA eraser column set and an RNase-free spin column. RNA solutions were obtained after column elution with RNase-free double-distilled water. Reverse transcription was performed using HiScript III RT SuperMix for qPCR (Cat. R323-01, Vazyme, Nanjing, China) with 0.5 ng RNA per reaction. RT-qPCR was performed on an Applied Biosystems 7500 Real-Time PCR instrument (Thermo Fisher Scientific) using GoTaq® qPCR Master Mix Kit (Promega Biotech, Madison, WI, US). Primers used in RT-qPCR are listed in Additional file 1: Table S2. The RNA expression levels were normalized to Gapdh and quantitated by 2−ΔΔCT method.

Immunohistochemistry (IHC) examinations

Glycogen synthase (GYS) expression levels were assessed by IHC staining. Liver slices were incubated with anti-GYS primary antibody (Cat. 3886, Cell Signaling Technology) at 4 °C overnight, then incubated with HRP-conjugated goat anti-rabbit IgG secondary antibody (Cat. PV-6000, ZSGB-Bio, Beijing, China) for 20 min and stained with 3,3 N-Diaminobenzidine tertrahydrochloride (DAB) and counterstained with hematoxylin.

Western blot analysis

Total protein was extracted from liver and adipose tissues using a Total Protein Extraction kit (Cat. KGP250, KeyGen Biotech, Nanjing, China). Protein samples were run in polyacrylamide gels and transferred onto polyvinylidene fluoride membranes (Cat. IPVH00010, Millipore, Bedford, MA, US). After 1 h of blocking at room temperature, membranes were incubated with primary antibodies: PI3K p85 (Cat. 4257, Cell Signaling Technology), p-PI3K (phosphorylation sites: p85 Tyr458/p55 Tyr199, Cat. 4228, Cell Signaling Technology), AKT (Cat. 4685, Cell Signaling Technology), p-AKT (phosphorylation site: Thr308, Cat. 13038, Cell Signaling Technology), and β-actin (Cat. 4970, Cell Signaling Technology) overnight at 4 °C. Membranes were then incubated with HRP-conjugated AffiniPure goat anti-rabbit IgG secondary antibody (Cat. SA00001-2, Proteintech Group, Rosemont, IL, US) for 1 h at room temperature. Blots were visualized using an ultra-sensitive enhanced chemiluminescence reagent (Cat. PE0010, Solarbio). The protein expression levels were analyzed by Image Lab software (Version 3.0, Bio-Rad, Hercules, CA, US) and normalized to β-actin.

Statistical analysis

All data are shown as means ± standard deviation (SD), where n represents the number of biological replicates. Statistical analysis was performed using IBM SPSS Statistics software (Version 23.0, SPSS, Chicago, IL, US). Graphs were composed by GraphPad Prism (Version 7.0, GraphPad Software, San Diego, CA, US). Normally distributed data were analyzed by one-way analysis of variation (ANOVA) with Fisher’s Least Significant Difference (LSD) tests or Dunnett’s test post hoc. Non-normal data were analyzed by nonparametric Kruskal–Wallis test. Comparisons with p value < 0.05 were considered statistically significant.