After the 10th day of administration, all rats were fasted but had free access to water for 12 h while urine samples were collected. The 12 h urines of all rats were collected into a 50 mL centrifuge tube containing 20 µL of 1% sodium azide (NaN
3). After centrifugation at 4,500 rpm min
−1 for 10 min in a 4°C low-temperature centrifuge, the supernatant was taken and stored at −80°C until metabonomics analysis. Afterwards, blood samples were drawn from the orbit and collected in two 2 mL centrifuge tubes, one of which contained 20 µL heparin sodium solution for preparing plasma samples, the other for preparing serum samples. Then, centrifugation was performed at 4,000 rpm min
−1 for 10 min in a 4°C low-temperature centrifuge, and the supernatant was taken to obtain plasma samples and serum samples. The plasma and serum samples were stored at −80°C until plasma metabonomics analysis and serum biochemical analysis.
Biochemical indicators included the hypothalamus-pituitary-adrenal axis in kidney-yang deficiency rats (Chen and Wang, 2015 (
link); Liu et al., 2016 (
link); Zhang et al., 2020 (
link)): CORT, ACTH and 17-OHCS; Hypothalamus-pituitary-thyroid axis related indicators: T3, T4, and TSH; Hypothalamus-pituitary-gonad axis related indicators: LH, T, and FSH.
Thereafter, all rats were euthanized in parallel. The kidney tissues were quickly excised, directly frozen in liquid nitrogen, and stored at −80°C for subsequent quantitative real-time PCR assays. Simultaneously, the hypothalamus, pituitary, thyroid, adrenal gland, testis and kidney of all rats were collected and fixed in formalin solution for histopathological analysis.
Sun E., Huang R., Ding K., Wang L., Hou J., Tan X., Wei Y., Feng L, & Jia X. (2023). Integrating strategies of metabolomics, network pharmacology, and experiment validation to investigate the processing mechanism of Epimedium fried with suet oil to warm kidney and enhance yang. Frontiers in Pharmacology, 14, 1113213.