Wistar rats

Thirty-two male pathogen-free Wistar rats (200–220 g) were provided by Beijing HFK Bioscience Co., Ltd. (Beijing, China). Animal care was carried out in accordance with the Guidelines for Animal Experimentation of Shenyang Pharmaceutical University (Shenyang, China) and the protocol was approved by the Animal Ethics Committee of the Institution. They were fed with a certified standard diet and tap water ad libitum. All rats were randomly divided into four groups (n = 8/group) as follows:

Petroleum ether group (PEG), rats were orally treated with PE section of EPR at a dose of 3 g/kg/day;

Acetic ether group (AEG), rats were orally treated with AE section of EPR at a dose of 3 g/kg/day;

n-Butanol group (BUTG), rats were orally treated with BUT section of EPR at a dose of 3 g/kg/day;

Healthy control group (HCG), rats were orally treated with the approximately same volume water.

Blood and kidney samples of rats in the four groups were collected after continuous oral administration of different EPR sections and water for 10 weeks. Blood samples were used in the serum biochemical parameters test and the nephrotoxic constituents’ investigation. Kidney samples (left) were used in the histopathology experiment and kidney samples (right) were used in the metabolomics study.

Wistar rats (male, 7–8 weeks old, 200–220 g) were purchased from Beijing HFK Bioscience Co., Ltd. Experiments were performed under a project license (No.: 2017055) granted by the Institutional Animal Care and Use Committee of Wuhan University, in compliance with the Center for Animal Experiment/Animal Biosafety Level-III Laboratory of Wuhan University Guidelines for the care and use of animals.
Twenty-two rats were randomly divided into two groups: the control group (n = 4) and the carbon tetrachloride (CCl₄) group (n = 18). Liver fibrosis/cirrhosis model was constructed by intraperitoneal injection of 40% CCl₄ dissolved in maize oil (1.5 mL/kg, twice a week) for at least 8 weeks. Then, five to six rats including one belonging to the control group were sacrificed every two weeks until 16 weeks. The liver tissues were immediately stored in RNAStore (TiangenBiotech, Beijing, China) or fixed in 4% paraformaldehyde for subsequent experiments. Hematoxylin and eosin (H&E) staining and Masson’s trichrome staining were performed by Servicebio Company (Wuhan, China) and observed by an inverted microscope (Olympus IX3). Liver fibrosis stages (F1–F4) were evaluated by experienced investigators according to the Metavir system [21 (link)].

Eight-week-old healthy male Wistar rats weighing 180–220 g were purchased from Beijing HFK Bioscience Co. Ltd. (Beijing, China). The rats were placed in a room with controlled temperature conditions (21–22 °C) and lighting (12 h light/dark cycle), with access to sterile food and water. After 1 week of acclimatization, the rats were divided randomly into two groups: no Vaspin injection group (n = 6, intraperitoneal injection with the same amount of normal saline) and Vaspin injection group (n = 6, Vaspin intraperitoneal injection at a dose of 1 μg/kg/day for 14 days). Rat recombinant Vaspin protein was purchased from Abbexa (Cambridge, UK). After 14 days, articular cartilage with subchondral bone in distal femur was isolated for the subsequent histological experiments. Ethical approval was received from the ethics committee of Peking University International Hospital (2018–068(BMR)). We have complied with the Guide for the Care and Use of Laboratory Animals, published by the United States National Institutes of Health (2011). Animal experiments were conducted in accordance with the internationally accepted ethical guidelines.