Potential role of tea drinking in preventing hyperuricaemia in rats: biochemical and molecular evidence

Chemicals and reagents

Hypoxanthine and monosodium urate (MSU) were purchased from Sigma–Aldrich (St. Louis, MO, USA). Potassium oxonate (PO) was purchased from Adamas Reagent Co.Ltd. (Shanghai, China).Assay kits for serum ALT, UA, BUN, CRE, and XOD were purchased from Nanjing Jian Cheng Bioengineering Institute (Nanjing, China). Enzyme-linked immune sorbent assay (ELISA) kits for IL-1β were purchased from DAKEWE (Beijing, China). All antibodies were obtained from Abcam (Cambridge, UK).

Turquoisepearls (green tea), golden bricks (yellow tea), golden fungi (black tea), red garment (cyan tea), old eyebrows (white tea), and red grape (red tea) were purchased from Bud-Chem Tea Co. Ltd. (Jiangkou, Guizhou, China).

Tea preparation

Green tea, cyan tea, and red tea were brewed with 80 °C, 95 °C, and 99 °C water for 2–5 min, respectively. White tea, yellow tea, and black tea were boiled for 15–20 min to make the tea. All final tea preparations had a concentration of 0.3 g/mL. The detailed protocols for the tea preparation were as described previously[37].

Animal study

Male Sprague–Dawley (SD) rats (body weight 200 ± 20 g) were purchased from Shanghai Slac Laboratory Animal Co. Ltd. (Certificate No.: 20170005059225). The rats were maintained in specific pathogen-free conditions, with a 12 h light/dark cycle at 20–22 °C and 45 ± 5%humidity. All rats were randomly assigned to ten groups: (a) control group (Con, n = 10), (b) hyperuricaemia and gout model group (Veh, n = 10), (c) benzbromarone treatment group (Ben, n = 10, 4 mg/kg/day), (d) TCM Simiao San treatment group (SMS, n = 10, 480 mg/kg/day), (e) green tea treatment group (G, n = 10,10 mL/kg/day), (f) yellow tea treatment group (Y, n = 10, 10 mL/kg/day), (g) black tea treatment group (B, n = 10, 10 mL/kg/day), (h) white tea treatment group (W, n = 10, 10 mL/kg/day), (i) red tea treatment group (R, n = 10, 10 mL/kg/day), and (j) cyan tea treatment group (C, n = 10, 10 mL/kg/day). Except for the Con group, rats in each group were intraperitoneally injected PO (300 mg/kg/day) with a: hypoxanthine (300 mg/kg/day) suspension once a day. The rats in the Con group were treated with PBS according to the same schedule. Groups (c)–(j) were gavaged with the prepared tea at a concentration of 0.3 g/mL once a day, and the Con and Veh groups were treated similarly with water. On Day 21, the rats were killed after anesthetization, and blood samples were collected by cardiac puncture. The kidney and liver were removed and fixed in 4% paraformaldehyde.

Histological analyses

For histological evaluation, the right kidneys and livers were fixed with 4% paraformaldehyde and embedded in paraffin. The kidneys and livers were cut into 3-μm sections and stained with haematoxylin and eosin for general morphological analysis and Masson staining for collagen deposition and interstitial lesion assessment. The sections were photographed under a light microscope and analysed by a pathologist blinded to the animal groups and drug treatments.

Immunohistochemistry (IHC)

The expression of NLRP3 and F4/80 in the kidney and liver was evaluated by IHC staining. For antigen retrieval, 4-μm-thick paraffin sections were placed in citrate buffer and microwaved with medium heat for 8 min and then allowed to stand for 8 min, followed by moderate-low heat for 7 min. After blocking with 3% bovine serum albumin, the sections were incubated with anti-NLRP3, and anti-F4/80 antibodies. Next, they were incubated with the appropriate secondary antibody. Finally, the sections were stained with diaminobenzidine and counterstained with haematoxylin. Positive staining was visible a brown–yellow under a microscope (Eclipse Ci-L, Nikon, Tokyo, Japan).

Enzyme-linked immunosorbent assay (ELISA)

We assayed the serum contents of IL-1β with ELISA kits. The details of the ELISA have been described previously [38].

ALT, XOD, BUN, CRE and UA activity assay

The blood samples were kept at 4 °C for 4 h to clot and then centrifuged at 2500×g for 10 min to obtain the serum. Then, the ALT, XOD, BUN, CRE and UA activities in the serum were determined using assay kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, China) according to the manufacturer's guidelines.

LC–MS-based untargeted metabolomics analysis

The prepared teas were analysed with an LCMS/MS system, a Waters 2D ultra-performance liquid chromatograph (Waters, USA) coupled with a high-resolution mass spectrometer Q Exactive HF (Thermo Fisher Scientific, USA). Chromatographic separation was performed using the Hypersil GOLDaQ column (100 × 2.1 mm, 19 μm, Thermo Fisher Scientific, USA). The column temperature and detection wavelength were set at 40 °C, and the injection volume was set to 5 μL. The mobile phase consisted of 0.1% formic acid in 100% water (A) and 0.1% formic acid in 100% acetonitrile (B) at a flow rate of 5 μL/s. The linear gradient elution was as follows: 0–2 min, 5% B; 2–22 min, 5–95%; 22–27 min, hold at 95%; 27–30 min, decrease to 5%.

The raw data for MS1 and MS2 were acquired from a Q Exactive mass spectrometer (Thermo Fisher Scientific, USA). The parameters of the electrospray source under negative ionization were as follows: spray voltage, 3.2 kV (ESI−); capillary temperature, 320 °C; aux gas heater temperature, 350 °C; and m/zrange, 150–1500 m/z. The parameters of the MS1 scan were as follows: resolution (70,000), automatic gain control (AGC) target (1.0 × 106), and maximum injection time (100 ms). For the data-dependent MS2, the following parameters were used: resolution (35,000), AGC target (2.0 × 105), and maximum injection time (50 ms). The stepped normalized collision energy (NCE) values were set at 20, 40, and 60 eV. Each sample was tested in six repetitions.

The raw data were processed by Compound Discoverer 3.1 (Thermo Fisher Scientific, USA), including peak extraction, alignment, and quantification. Compound identification was verified by authentic standards and MS/MS fragmentations. Meanwhile, PubMed and HMDB were also used to identify the compounds.

Gene set of the identified compounds in yellow tea and hyperuricaemia

The identified compounds were filtered using ADMET properties. ADME parameters were acquired from the TCMSP (https://tcmsp-e.com/), and used to evaluate the chemicals' absorption, distribution, metabolism, and excretion. The yellow tea compounds were filtered based on the criteria of oral bioavailability (OB) ≥ 30% and drug-likeness ≥ 0.18. After initial filtering, the gene set of the bioactive compounds was collected from the TCMSP, and HERB databases (http://herb.ac.cn/).

Genes related to the treatment and prevention of hyperuricaemia were collected from two databases: GeneCards (https://www.genecards.org/) and DisGeNet (https://www.disgenet.org/). Gene sets obtained from taking the intersection between yellow tea (YT) compound-related genes and antihyperuricaemia-related genes were used for further analysis.

Construction of the PPI network and compound-target network

The STRING database was used to construct the protein–protein interaction network for the overlapping targets between hyperuricaemia and yellow tea. The species “Homo sapiens” was selected. Meanwhile, the interaction network of the yellow tea compounds and the overlapping targets was established using Cytoscape 3.9.0 software. Crucial targets were identified using molecular complex detection (MCODE) (a plugin in Cytoscape).

Enrichment analysis of GO and KEGG pathways

Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses were used to unveil the potential molecular mechanism and the critical signalling pathways. The “ClusterProfile” package was used to perform enrichment analysis for GO and KEGG pathways in R software version 3.4.0.

Statistical analyses

The values are expressed as mean ± s.e.m. The unpaired t-test (GraphPad Prism 9 Software) was used for statistical analysis. Data points were not excluded. The researchers involved in this study were not blinded during sample collection or data analysis. The sample size was selected based on the preliminary results to ensure sufficient power. P values < 0.05 were considered significant.

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