Chemical reagents and antibodies

Rhein (purity > 98%, Batch No. DD0029) was purchased from Chengdu Desite Bio-Technology Co., Ltd. (Sichuan, China). Transforming growth factor-β1 with the purity ≥ 98% was purchased from Sigma-Aldrich (St. Louis, MO, USA). Kits for quantifying serum creatinine (Scr) and blood urea nitrogen (BUN) were purchased from the Nanjing Jiancheng Bioengineering Research Institute (Nanjing, China). HPLC-grade trichloromethane, acetonitrile, and methanol were obtained from Merck (Darmstadt, Germany). Trypsin digestive juice, streptomycin mixture solution, high-glucose DMEM, phosphate buffer (PBS), and foetal bovine serum (FBS) were obtained from Gibco (USA); 4% paraformaldehyde fixative, Triton X-100, and DAPI were purchased from Solarbio, China; α-SMA (14395-1-AP), FN (15613-1-AP), Collagen I (No. AB34710), GAPDH (10494-1-AP), JNK (No. 24164-1-AP), P38 (No. 14064-1-AP), P65 (No. 14220-1-AP), IKK (No. 15649-1-AP), and AP-1 (No.22114-1-AP) antibodies were obtained from Proteintech, China; AKT (#75692) and p-AKT (#9018) antibodies were obtained from Cell Signaling Technology, USA; p-JNK (No.AF3318), p-P38 (No.AF4001), p-IKK (No.AF3013), and p-P65 (No.AF2006) antibodies were obtained from Affinity, China; horseradish peroxidase-conjugated secondary antibodies and fluorescent dye-conjugated secondary antibodies were obtained from Proteintech, China; and the BCA kit was obtained from Thermo Fisher, USA. Dimethyl sulfoxide (DMSO) was obtained from Solarbio (China) and bovine serum albumin (BSA) was obtained from Dalian Meilun Biotechnology Co., Ltd. (China).

Animals

SD rats (male, n = 18, 200–220 g) were purchased by the Laboratory Animal Centre of Wenzhou Medical University (ethical approval number: wydw2021-0720). Rats in each group were housed in the same cage at 25 °C with a humidity of approximately 50% and was adapted for 1 week in a well-ventilated environment, during which they were provided free access to food and water. They were randomly divided into three groups (6 rats each): sham operation group, ischemia–reperfusion injury group, and rhein group. Based on our previous work [22,23,24], the dose level of rhein was 120 mg/kg, prepared using 0.5% sodium carboxymethyl cellulose (CMC-Na). The sham operation and model groups were daily treated with the same volume of blank CMC-Na solution.

Effect of TGF-β on cells and determination of the concentration of rhein

Cells in the experiments were cultured in high-glucose DMEM containing 10% FBS and 1% penicillin–streptomycin mixture and placed in a constant temperature cell incubator under 5% CO2, saturated humidity and at 37 °C. Cells in the logarithmic growth phase were digested with trypsin and inoculated at 2 × 105 cells/mL into a 6-well culture plate. After 24 h of culture, high-glucose DMEM containing 1% FBS was used to starve the cells overnight. Control and 10 ng/mL TGF-β1 induction groups. The rhein concentrations in the administration groups were 1, 5, 10, and 20 ng/mL. After cells were cultured for 48 h, they were washed twice with pre-cooled PBS. The NP-40 cell lysate was added, and the cells were lysed in an ice bath at 4 °C for 30 min. Cells were subsequently collected into 2.0 mL EP tubes using a cell scraper, and the cell supernatant was taken after centrifugation at 12,500g for 8 min at 4 °C. Then, 10 μL of the supernatant was taken out and the protein content was determined using the BCA method. The remaining supernatant was added to 4 × SDS loading buffer, denatured by heating, and separated using 10% SDS-PAGE. After blocking the membrane with skim milk for 1.5 h, the membrane was washed twice and incubated with the specific primary antibodies for 12 h at 4 °C. After washing twice, the membrane was incubated with horseradish peroxidase-conjugated secondary antibodies, and the concentration of rhein was determined according to the observed band.

Cell sample preparation

Cells in the logarithmic growth phase were treated with trypsin and then inoculated into 6-well culture plates at 7.5 × 105 cells/mL. After 24 h, cells were synchronized by overnight starvation in high-glucose DMEM containing 1% FBS. After synchronization, the cells were divided into three groups (n = 6/group), namely the control group, 10 ng/mL TGF-β1 induction group, and rhein administration group, and were treated under 5% CO2 for 48 h and at 37 °C.

For the metabolomics experiments, after 48 h, the medium was quickly discarded, and cells were washed with 1 mL of cold PBS three times. Immediately after washing, the cells were quenched with liquid nitrogen to prevent continued metabolism. After the liquid nitrogen was evaporated, 1 mL of pre-cooled cell extract was added (water: methanol: chloroform = 1:9:1), and the cells were collected using a scraper. Cells were treated in liquid nitrogen for 3 min and thawed at 25 °C. This process was repeated five times to disrupt cells, dissolve cellular metabolites, and precipitate proteins. The cell supernatant was collected after centrifugation at 12,500g for 15 min at 4 °C and dried with nitrogen. Before injection, the samples were reconstituted in 300 μL of methanol solution, vortexed, and centrifuged, and 60 μL of the supernatant was injected into UPLC-QTOF-MS.

For pharmacological experiments, after treating the cells for 48 h, the cells were washed three times with 1 mL of pre-cooled PBS, 100 μL of NP-40 lysate was added, and cells were lysed in a 4 °C ice bath for 30 min and scraped off with a cell scraper. The scraped cells were centrifuged at 12,000g for 5 min at 4 °C. Then, the supernatant, which contained the total protein in the sample, was aspirated. Immunoreactive bands were measured after incubation with horseradish peroxidase-conjugated secondary antibodies using an enhanced chemiluminescence reagent (Bio-Rad). ImageJ was used for protein quantification and normalization to the respective controls. GAPDH was used to quantify cytoplasmic proteins, and total protein was used to quantify phosphorylated proteins.

Immunofluorescence

NRK-49F cells in the logarithmic phase were inoculated onto coverslips at a density of 50,000 cells/mL. After treatment for 48 h, the cells were fixed with 4% paraformaldehyde for 30 min, permeabilized with 1% Triton X-100 in an ice bath for 30 min, blocked with 3% BSA at room temperature for 2 h, and incubated with specific primary antibodies at 4 °C for 12 h. The cells were then treated with fluorescent dye-conjugated secondary antibodies at room temperature for 2 h. Finally, the cells were counterstained with DAPI. Imaging was performed using an upright fluorescence microscope.

Sample collection

Urine excreted by rats 12 h before unilateral ischemia–reperfusion injury (UIRI) modeling, after UIRI modeling, and on the 14th day of rhein administration was collected in metabolic cages. After centrifugation at 12,000g for 10 min to precipitate impurities, the supernatant was taken and stored at − 80 °C for urine metabolomics. After the samples were thawed at 4 °C, acetonitrile was added at a ratio of sample: acetonitrile = 1:1, vortexed for 3 min, and centrifuged at 12,000g for 10 min. The supernatant was used for testing.

For serum collection, 1–1.5 mL of tail vein blood was collected in EP tubes before UIRI modeling, after modeling, and on days 3, 7, and 14 after rhein administration. The upper serum layer was taken after centrifugation at 3000 rpm for 10 min, divided into three parts, and stored at − 80 °C for the determination of Scr and BUN levels and detection of endogenous metabolites in serum samples. The samples were thawed at 4 °C, and acetonitrile was added at a volume ratio of sample: acetonitrile = 1:3. The samples were vortexed for 3 min, centrifuged at 12,000g for 15 min, and the supernatants were collected for testing.

For tissue collection, rats in each group were anesthetized using 10% chloral hydrate on day 14, and cardiac perfusion was performed. The right kidney was removed and divided longitudinally into two parts. Then one part was fixed in paraformaldehyde for subsequent pathological examination and the other was stored in a − 80 °C for subsequent tissue metabolomic assays. After each sample (0.1 g) was thawed at 4 °C, 800 μL of acetonitrile was put in 1.5 mL EP tube. The samples were homogenized for 6 min, centrifuged at 12,000g for 15 min, and the supernatants were collected for testing.

Determination of biochemical indexes and pathological changes

Serum samples stored in a − 80 °C refrigerator were thawed at 4 °C. Serum creatinine and BUN levels were determined using the manufacturer’s instructions. Hematoxylin–eosin (HE) and Masson staining were performed on kidney tissues after embedding and sectioning. The pathological status of the kidney tissues after UIRI modeling was observed using an upright fluorescence microscope.

UPLC-QTOF-MS conditions

The UPLC-QTOF-MS system consisted of an Acquity ultra-performance liquid chromatograph and a Xevo G2-XS QTof quadrupole tandem time-of-flight mass spectrometer (Waters Corporation, Milford, MA, USA). All samples were separated using HSS T3 C18 column (2.1 × 100 mm, 1.8 μm) (Waters Corporation). The mobile phases consisted of 0.1% aqueous formic acid (solvent A) and acetonitrile (solvent B), and the program of gradient elution was as follows: 0–0.5 min, 5% B; 0.5–20.5 min, 5–95% B, 20.5–25 min, 5% B. The flow rate was 0.3 mL/min, the column temperature was 30 °C, the injection volume was 2 μL, and the injector temperature was 10 °C.

The mass spectrometer used MSE continuum mode, and ESI with both positive and negative ion modes were detected, capillary voltage was 2.5 kV, cone voltage was 50 V, ion source temperature was 100 °C, the gas in the cone hole was nitrogen and at a flow rate of 50 L/h, the desolvation gas was nitrogen at a flow rate of 800 L/h, the temperature of the desolvation gas was 400 °C, scan time was 0.2 s, scan interval was 0.015 s, low-channel collision energy was 6 eV, high-channel collision energy was 20–30 eV, and mass range was 50–1200 m/z.

Leucine-enkephalin (5 ng/mL) was used as the calibration solution to determine whether the axis of the mass spectrum was biased. In addition, 10 μL of each sample was mixed to prepare quality control samples (QCs) to evaluate the precision and repeatability of the method. Before sample analysis, 3–5 QCs were continuously tested, and a QC sample was inserted after every 10 samples.

Data processing method

The raw data for each sample obtained by UPLC-QTOF-MS were imported into Progenesis QI ver.2.2 (Nonlinear Dynamic) for peak alignment, deconvolution, and normalization. The molecular structures of the detected metabolites were identified and confirmed based on the consistency of their MS and MS/MS data with those of the metabolites in the HMDB, METLIN, and LIPD MAPS databases. The errors of MS and MS/MS values were set at < 0.1 and 0.5 Da, respectively. To verify the reliability of the obtained data, using the Noreva 2.0 website (http://47.99.36.124/noreva/), the data were pre-processed according to the QC samples as follows. Metabolites with more than 80% missing values in the samples were deleted, the missing values were supplied using the k-nearest neighbour algorithm, the RSD of the QC was < 30%, and a local polynomial fit was performed.

The reliability and precision of UPLC-QTOF-MS method were verified through duplicate analyses of 6 injections of the same QCs and 6 parallel samples. The RSD of the peak retention times and areas were < 5.0%. The precision and reliability of the proposed cell, serum, serum, and tissue methods were satisfactory for metabolomics.

Multivariate statistical methods, including a principal component analysis (PCA), partial least squares discriminant analysis (PLS-DA), and orthogonal partial least squares discriminant analysis (OPLS-DA), were performed on the pre-processed data using SIMICA (version 14.0, Umetrics AB, Sweden). Differential metabolites were screened based on the variable importance in projection (VIP) in the loading map (≥ 1.0). One-way ANOVA (P = 0.05) was evaluated using IBM SPSS (version 22.0, IBM Corporation, Armonk, NY, USA), and differential metabolites were screened using fold change (FC) > 2 or < 0.5 as thresholds. Pathway enrichment was evaluated using the MetaboAnalyst 5.0 platform. The obtained data are expressed as mean ± standard deviation (x ± s).

Network pharmacology and molecular docking of rhein

The STITCH database was employed to identify potential targets of rhein with a high confidence level of 0.7. Rhein was docked to potential targets using AutoDock Vina. The crystal structures were obtained from the Protein Data Bank. The mean values and standard deviations were obtained through the calculations of three molecular docking. PYMOL was performed to visualize the processing of the molecular docking results. String database (Version:12.0, https://cn.string-db.org/) with a high confidence of 0.7 was used to search for interactions between the potential targets of rhein and proteins P65, IKK, AKT, P38, JNK, and AP-1.