Collection and extraction of the plant material
The C. flabellata plants were collected in September in the Xixia County, Henan province, China, and authenticated by Prof. Sui-qing Chen of the Henan University of Chinese Medicine. The plants (1 kg) were refluxed with 50% ethanol (3 × 12 L, each 1 h), and the mixture was filtered. The combined filtrates were dried by rotary evaporation using a freeze drier. Finally, the percentage yield of 50% ethanol crude extract of C. flabellata was 15.5%. The dried extract was kept in a fridge until further use.
Another separation process previously reported was used in this study to obtain the water elution fraction, 20% ethanol elution fraction, 30% ethanol elution fraction, and 40% ethanol elution fraction from C. flabellata . The 40% ethanol fraction was separated using Sephadex LH-20 and silica gel column and purified by semi-preparative high-performance liquid chromatography (HPLC) to finally obtain compounds such as SDC-0-14,16, SDC-1-8, SDC-0-60 (p-hydroxybenzyl alcohol).
Cell culture and in vitro study
Normal rat kidney epithelial cells (NRK-52E cells) purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China) were cultured to investigate the effect of C. flabellata extract on cell aging caused by D-galactose (S11050, Yuanye, Shanghai, China). The NRK-52E cells were grown in Dulbecco's modification of Eagle's medium Dulbecco (DMEM) supplied with 10% fetal bovine serum in an incubator at 37 °C and in the presence of 5% CO2. The NRK-52E cells were plated in 96-well or 6-well plates for 24 h. Then, the cells were treated with D-galactose (20 mg/mL), C. flabellata extract (75 µg/mL), and its monomeric compound (10 µM) for 48 h. Subsequently, the methyl thiazolyl tetrazolium assay was used to detect cell viability, and β-galactosidase staining was used to observe cell senescence.
Animals and administration
Six-month-old male senescence-accelerated mouse-prone 8 (SAMP8) and SAMR1 mice from the First Affiliated Hospital of Tianjin University of Traditional Chinese Medicine (Tianjin, China) were used in this study. The animals were housed under controlled light (12-h light/dark cycle), temperature (23 °C–25 °C), and humidity (45–55%) conditions and received a standard diet and water ad libitum. All animal experiments were approved by the ethics committee of Henan University of Traditional Chinese Medicine and performed under the institutional guidelines. A total of 40 SAMP8 mice were divided into four experimental groups (n = 10/group): SAMP8 model mice, low-dose CF-treated SAMP8 mice (CF-L, 387.5 mg kg− 1, intragastrically), medium-dose CF-treated SAMP8 mice (CF-M, 775 mg kg− 1, intragastrically), and high-dose CF-treated SAMP8 mice (CF-H, 1550 mg kg− 1, intragastrically). The mice in the SAMR1 control and SAMP8 model groups were treated with physiological saline (0.9%). All mice were treated orally for 1 month.
At the end of the experiment, the mice were housed individually in metabolic cages for 12-h urinary collection. All blood samples were collected by retro-orbital bleeding. The kidneys from each mouse were then surgically removed and kept at − 80 °C until the analyses.
The serum and kidney homogenate samples were thawed to room temperature, and the enzyme-linked immunosorbent assay kit method was employed according to the manufacturer’s protocols to measure serum superoxide dismutase (SOD, Wuhan Huamei Biological Technology, Wuhan, China) levels and activities of interleukin-1β (IL-1β, ABclonal Technology, Wuhan, China) and tumor necrosis factor-α (TNF-α, ABclonal Technology), and the expression levels of collagen type Ⅰ (Col-Ⅰ, Bio-Swamp Life Science Lab, Wuhan, China), α-smooth muscle actin (α-SMA, Bio-Swamp Life Science Lab), and fibronectin (FN, Bio-Swamp Life Science Lab) in kidney tissue. Serum creatinine (Cr), blood urea nitrogen, glutathione peroxidase (GSH-Px), and urinary total protein levels were measured using detection kits (Nanjing Jiancheng Institute of Biotechnology, Nanjing, China).
Senescence-associated β-galactosidase staining
Frozen kidneys from mice sliced into 10-µm-thick sections and NRK-52E cells were stained with senescence-associated β-galactosidase (SA-β-gal) (C0602; Beyotime Biotechnology, Shanghai, China) following the manufacturer’s protocols.
The kidney sections were fixed with 4% buffered paraformaldehyde, and 10-µm-thick paraffin-embedded sections were stained with Masson's trichrome (G1006; Servicebio, Wuhan, China) and observed microscopically. The blue-colored areas in Masson’s trichrome–stained sections were measured quantitatively from six randomly selected fields and analyzed by Image-Pro Plus 6.0 software.
The immunohistochemical analysis was performed using the routine method . The antibodies used included the following: Wnt4 (14371-1-AP; Proteintech, IL, USA), β-catenin (17565-1-AP; Proteintech), renin (14291-1-AP; Proteintech), type 1 angiotensin II receptors (AGTR1) (25343-1-AP; Proteintech), p-nuclear factor erythroid 2–related factor 2 (p-Nrf2) (ab76026; Abcam, Cambridge, MA, USA), p-c-Fos (ab27793; Abcam), connective tissue growth factor (CTGF) (GB11078; Servicebio), and Kelch-like Ech-associated protein-1 (Keap1) (GB11847; Servicebio). The sections were observed under a microscope (Olympus, Tokyo, Japan), and the mean of density value was measured using the Image-Pro Plus analysis software 6.0.
Western blot analysis
Western blot assay was conducted as described in a previous study . Briefly, the kidney tissues were homogenized in lysis buffer and quantified using a Bradford Protein Assay Kit (Wuhan Boster Biological Technology, Ltd., China). The homogenates were then subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transferred to polyvinylidene fluoride membrane, and blocked in blocking buffer (4% nonfat dry milk) for 90 min. They were then incubated with primary antibodies (Wnt4; renin; AGTR1; p-Nrf2; p-c-Fos; Keap1; β-actin; AC026; Abclonal, Wuhan, China; and glyceraldehyde-3-phosphate dehydrogenase, AC033, Abclonal) overnight at 4℃, followed by incubation with an appropriate fluorescence-conjugated secondary antibody for 1 h at room temperature. The proteins of interest were scanned with an Odyssey IR scanner (LI-COR Biosciences, CO, USA), and the signal intensities were quantified using Image Studio software.
UPLC-Q-TOF-MS analysis for kidney samples
The renal tissues were weighed and homogenized in ice-cold physiological saline (w/v = 1:1). Then, 1 mL of acetonitrile was added to 200 µL of tissue homogenate samples, followed by ultrasonic extraction for 30 min. The extract was centrifuged at 12,000 g and 4℃ for 10 min. The supernatant was taken into the vial for analysis. Chromatographic separation was carried out on ultraperformance liquid chromatography (UPLC) (Dionex UltiMate 3000 System, Thermo Scientific, MA, USA), with the LC system comprising an Acclaim RSLC 120 C18 column (2.2 µm, 2.1 × 100 mm; Thermo Scientific). The mobile phase consisted of solvent A (acetonitrile) and water with 0.1% formic acid (B). The separation was performed by gradient elution as follows: 10–70% A from 0 to 3 min, 70–78% A from 4 to 13 min, 78–90% A from 14 to 15 min, 90–10% A from 15 to 16 min, and 10% A from 16 to 20 min. The injection volume of the test sample was 2 µL. The optimal conditions for mass spectrometry (MS) were as follows: capillary voltage, 3.0 kV; cone voltage, 40 V; desolvation gas temperature, 500 °C; source temperature, 110 °C; desolvation gas flow, 800 L/h; and cone gas flow, 50 L/h. The scan range was from 50 to 1200 m/z. Leucine–enkephalin was used for accurate mass acquisition. Waters MassLynx v4.1 was used for the acquisition and analysis of data in both positive and negative ion modes.
The acquired raw data from ultra-performance liquid chromatography coupled to quadrupole time-of-flight mass spectrometry (UPLC-Q/TOF-MS) analysis were first preprocessed using profile analysis (version 2.1, Bruker, Germany). The “bucket table” was obtained and imported into the SIMCA-P software (version13.0 Umetrics AB, Sweden) for principal component analysis (PCA). Other results were presented as mean ± standard deviation (SD). Comparisons between groups were conducted using one-way analysis of variance. A P value less than 0.05 was considered statistically significant.