Chemical and reagent

Human NQO1 recombinant protein was obtained from Synpeptide Co., Ltd. (Nanjing, China). Aristolochic acid I (AAI, A5512), mercaptopropyltrimethoxysilane (MPTS), N,N-Dimethylformamide (DMF), and N-(4-maleimide butyryloxide) succinimide (GMBS) were obtained from Sigma (St. Louis, MO, USA). Silica gel was obtained from Qingdao Meigao Chemical Co., Ltd. (Qingdao, China). Skullcapflavone II (SFII) was bought from Shanghai Huizhou Bio-Technology Co., Ltd. (HZ-HQXI98-50, Shanghai, China). Dicoumarol was obtained from Selleck Chemicals (S4299, Houston, TX, USA). Oroxylin A (B20958), wogonin (B20489), and tectochrysin (B21459) were purchased from Shanghai Yuanye Bio-Technology Co., Ltd. (Shanghai, China). Dimethyl sulfoxide (DMSO) was purchased from WAK-Chemie (WAK-DMSO-70, Berlin, Germany). CCl4, olive oil, and NaHCO3 were bought from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China).

Preparation of NQO1 biochromatographic stationary phase

The synthesis of the NQO1 biochromatographic stationary phase was done according to the published research method [21] and was shown in the second step of Fig. 1. Synthesis process: 1 g silica gel (5 μm, 200 Å) and 1 mL MPTS were added in 100 mL DMF, and the solution system was stirred under the conditions of nitrogen protection and 60 °C for 5 h. Then, the suspension obtained from the reaction was centrifuged at 5000× g and washed with DMF 3 times. Subsequently, the obtained precipitate was stirred in 5% GMBS (dissolved in 500 mL DMSO) at room temperature for 2 h. DMSO was used to wash the MPTS/GMBS-modified silica gel for 3 times and the gel was dried in a vacuum for 48 h. Under the condition of 4 °C, 20 μg of human NQO1 recombinant protein and 40 mg MPTS/GMBS-modified silica gel were stirred in phosphate buffer solution (PBS) for 12 h, and the NQO1 recombinant protein could be immobilized in the stationary phase. The NQO1 Activity Assay Kit (ab184867, Abcam, Cambridge, MA, USA) was used to determine the biological activity of the chromatographic stationary phase, and the color development and rate (mOD/min) per sample well were recorded.

Fig. 1

Synthetic route of NQO1 biochromatographic stationary phase and screen workflow of NQO1 binding candidate components from the extract of Scutellaria baicalensis.

Comprehensive 2D NQO1 biochromatography system

The comprehensive 2D NQO1 biochromatography system was aboard on an Agilent 1200 series high-performance liquid chromatography system combined with time-of-flight mass spectrometer (TOFMS), controlled by Agilent MassHunter Workstation (Agilent Technologies, Palo Alto, CA, USA). With ammonia acetate as the mobile phase, the NQO1 column was used as the first-dimensional column. Besides, an Agilent Poroshell 120 EC-C18 column was used as the second dimension, with acetonitrile and 0.1% formic acid as the mobile phase. The relevant operating details of the comprehensive 2D NQO1 column/C18 column/TOFMS system could be referred to in the literature [22, 23]. The system was equilibrated for 30 min, and 3 µL of the Scutellaria baicalensis extract was injected into the system. In order to obtain the comprehensive 2D NQO1 biochromatographic analysis results, system collects at least two fractions across a retention peak. Therefore, the collection period of each round was set to 2.5 min. Subsequently, the machine collected the information on the stable retention behavior and molecular structure of components in extract of Scutellaria baicalensis. Relevant data were analyzed for baseline correction and 2D mapping of retention behavior.

NQO1 enzyme activity test

The inhibition activities of candidate compounds on human NQO1 recombinant protein, cell and mouse samples were detected by the NQO1 Activity Assay Kit (ab184867, Abcam, Cambridge, MA, USA). The sample and reaction buffer were added to 96 well plates, and the absorbance at 440 nm was recorded by a microplate reader. GraphPad Prism 8.0.1 (LaJolla, CA, USA) was used to calculate the 50% inhibiting concentration (IC50) and fit the inhibition curve.

Surface plasmon resonance (SPR) analysis

The Biacore T200 system (GE Healthcare Life Sciences, Marlborough, MA, USA) was used for SPR assays. The acetic acid buffer (pH = 4.0) was used to dilute NQO1 recombinant protein (His tag), and the protein was covalently immobilized in Series S Sensor Chip CM5 (BR100530, GE Healthcare Life Sciences, Marlborough, MA, USA). The candidate compounds were subjected to gradient dilution with PBS buffer containing 5% DMSO and then injected into the NQO1 recombinant protein channel and the control channel, and the flow rate of the running buffer was set to 30 mL/min. The coupling time was 60 s and the dissociation time was 300 s. The Biacore T200 was used to analyze the experimental data, calculate the equilibrium dissociation constant (KD), and fit the affinity curve.

Molecular docking

The structure of NQO1 protein was obtained from the Protein Data Bank (PDB ID: 1D4A). The molecular structures of candidate compounds were downloaded from PubChem. Schrö-dinger v2019. 03 (Schrödinger Inc., Plainview, NY, USA) software was used to simulate the candidate molecular docking to NQO1 protein, and to predict the binding sites of the candidate molecule and NQO1 protein.

Animal experiment

C57BL/6 mice were purchased from the Model Animal Resource Information Platform (Nanjing, China). All animal experiments were performed following the NIH guidelines and approved by the Ethics Committee of the Shanghai Cancer Center, Fudan University. Ethical Approval Number: FUSCC-IACUC-S2022-0114.

For the C57BL/6 adult mice (male) acute renal injury model, animals were given indicated doses of AAI or SFII at the age of 8 weeks. Mice were divided into four groups: NaHCO3 group, AAI (5 mg/kg) group, and SFII (30 or 60 mg/kg) + AAI (5 mg/kg) group. Mice were sacrificed 3 days after the first dose of AAI administration.

For the adult mice (male) renal fibrosis model, animals were divided into four groups: NaHCO3 group, AAI (10 mg/kg) group, and SFII (30 or 60 mg/kg) + AAI (10 mg/kg) group. The AAI group was pretreated with olive oil 12 h by gavage in advance, followed by a single intraperitoneally (i.p.) injection of AAI and subsequent (olive oil) for 3 days. SFII and AAI combined treatment groups were pretreated with SFII (30 or 60 mg/kg) by gavage 12 h in advance, followed by a single i.p. injection of AAI and subsequent SFII by gavage for 3 days. Mice were sacrificed 14 days after the first dose of AAI.

For the infant mice acute injury model, male mice were given indicated doses of AAI or SFII at the age of 14 days. Infant mice were divided into the following groups: NaHCO3 group, AAI group, and SFII (10 mg/kg) + AAI group. Mice were sacrificed 24 h after AAI administration. For the AAI-induced liver cancer model, a single injection of AAI or NaHCO3 was given to male infant mice on the 14th day after birth. Mice (6 mice/group) were divided into five groups: NaHCO3 group, AAI (20 mg/kg) group, and the SFII (10, 30, or 60 mg/kg) + AAI (20 mg/kg) group. For each group, mice were sacrificed 16 weeks after a single dose of NaHCO3 or AAI.

Additional details of the animal models are available in Supplementary Materials.

Blood coagulation assay

Mice (4 mice/group) were administered with dicoumarol, oroxylin A and SFII orally at dose of 6 mg/kg respectively and then sacrificed after 24 h. Blood samples of mice were taken to detect the activated partial thromboplastin time (APTT), prothrombin time (PT), and thrombin time (TT). The coagulation test reagents of APTT, PT, and TT were purchased from Siemens (Berlin, Germany). The Sysmex CS-5100 (Kobe, Japan) was used for coagulation assays of blood samples.

Cell culture

Human normal liver cell line (L02) and human renal proximal tubular epithelial cell line (HK-2) were obtained from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). L02 was cultured with DMEM medium (Gibco, Grand Island, NY, USA), which was supplemented with 1% antibiotic-antimycotic solution and 10% fetal bovine serum (FBS, Biological Industries, Kibbutz Beit Haemek, Israel). HK-2 was cultured with Dulbecco’s modified Eagle’s medium/Nutrient Mixture F-12 (DMEM/F12, Gibco, Grand Island, NY, USA), which contained 1% antibiotic-antimycotic solution and 10% FBS (Gibco, Grand Island, NY, USA). All cells were cultured in a humidified incubator containing 5% carbon dioxide at 37 °C.

Construction of NQO1 knockdown cell line

Recombinant lentivirus vectors containing the interference NQO1 plasmid (shNQO1) and negative control (shNC) were purchased from Shanghai Genechem (Shanghai, China). L02 with 50% density in a 6-well plate was infected by a concentrated virus (200 μL) with polybrene for 10 h. Lentivirus infected L02 cells were treated with puromycin (2 μg/mL) for 48 h. The knockdown effect of NQO1 in infected cells was verified by quantitative real-time PCR (qRT-PCR) and Western blot.

Cell proliferation assay

The cells were inoculated into 96 well plates and cultured overnight in a 5% CO2 incubator at 37 °C. AAI (dissolved in 1% NaHCO3) and SFII (dissolved in DMSO) were added to the culture medium. At the time points of 0 h, 24 h, and 48 h, the culture medium in the well was replaced with a CCK-8 reagent (CK04, Dojindo, Kumamoto, Japan), and the plates were placed in the 37 °C incubators for 2 h. Subsequently, the absorbance per well was measured at 450 nm by a microplate reader.

Western blot

Cell lysis buffer for Western blot (P0013, Beyotime, Shanghai, China) was added to the cells and tissues of mice to extract the whole protein. The protein concentration was determined by BCA Protein Assay Kit (23227, Invitrogen, Carlsbad, CA, USA). The SDS-PAGE gel (PG113, Epizyme, Shanghai, China) was used for electrophoresis, and then the proteins were transferred to the nitrocellulose (NC) blotting membrane. The NC membranes were incubated with primary antibodies which included P53 (SC-126, Santa Cruz, CA, USA), NQO1 (3187S, CST, Danvers, MA, USA), β-Actin (AC004, Abclonal, Wuhan, China), GAPDH (AC033, Abclonal, Wuhan, China), γ-H2AX (ab11174, Abcam, Cambridge, MA, USA), cleaved caspase-3 (9664T, CST, Danvers, MA, USA) and PARP-1 (9532T, CST, Danvers, MA, USA). The membranes were scanned by an Odyssey CLx image scanner system (Li-COR, Lincoln, NE, USA).

Flow cytometry analysis

The cells were inoculated in a 6-well plate and cultured overnight in a 5% CO2 incubator at 37 °C. AAI and SFII were added to the culture medium for 24 h. The cells were digested with trypsin, centrifuged at 1000 rpm for 3 min, and washed with PBS. Then, 5 μL Annexin V-FITC reagent (C1062L, Beyotime, Shanghai, China) or Annexin V-APC reagent (550474, BD Pharmingen, San Diego, CA, USA), 10 μL PI reagent, and 195 μL binding solution was added to per sample tube. Cells and reagents were incubated at room temperature in the dark for 20 min, and then BD FACSCelesta (BD, Franklin Lakes, NJ, USA) was used for flow cytometry analysis.

The quantitative real-time PCR (qRT-PCR) assay

Total RNA was extracted from cells using Trizol (Invitrogen, Carlsbad, CA, USA). The cDNA was synthesized using a reverse transcription system which included M-MLV reverse transcriptase (M1701, Promega, Madison, USA), Ribonuclease Inhibitor (N2515, Promega, Madison, USA), dNTP, and N6 randomized primer. Subsequently, SYBR® Green Master (4913914001, Roche, Basel, Switzerland) based, the indicated gene was amplified with cDNA as the template by specific primers through LightCycler (Roche, Basel, Switzerland). The primer sequences were listed in Supplementary Table S1.

Sirius red staining

The sections of tissues were dewaxed in xylene and rehydrated in gradient alcohol. Sirius red dye (9046, Chondrex, Redmond, WA, USA) was added to the sections and incubated in 37 °C incubator for 30 min. The sections were washed in ddH2O, and then dehydrated in 100% ethanol for 5 min. Subsequently, the sections were soaked in xylene for 5 min, and mounted in a resinous medium.

TUNEL staining

We detected the apoptosis of mouse tissues with the Biotin TUNEL Assay Apoptosis Detection Kit (T6068, US EVERBRIGHT INC, Suzhou, China). The sections of tissues were dewaxed in xylene and rehydrated in gradient alcohol. Protein K was added to the sections and incubated at room temperature for 20 min. The sections were exposed to 100 µL of 0.3% H2O2 solution and incubated at room temperature for 30 min to inactivate the endogenous peroxidases. The TUNEL reaction solution (50 µL) was added to sections and incubated at 37 °C incubators in the dark for 2 h. Subsequently, the sections were exposed to 50 μL Streptavidin-HRP solution and incubated at 37 °C incubators in the dark for 30 min. Finally, DAB (K3468, Dako, Glostrup, Denmark) was used for the chromogenic reaction of sections.

H&E (hematoxylin-eosin) staining

The tissues were fixed in 10% formaldehyde solution for 24 h, and dehydrated by Leica ASP300S (Leica, Wetzlar, Germany). The tissues were embedded in paraffin, and cut into 3 μm section. H&E staining was performed on sections using the Leica Autostainer XL (Leica, Wetzlar, Germany Germany), according to the Leica staining protocol.

Immunohistochemistry (IHC)

For IHC, the sections of tissues were incubated with the primary antibody at 4 °C overnight, such as the anti-alpha 1 fetoprotein antibody (AFP, 1:500, 009P, Celplor, NC, USA) and anti-Ki-67 antibody (1:300, ab15580, Abcam, Cambridge, MA, USA). Subsequently, the sections were incubated with SupervisionTM anti-Rabbit-HRP (D-3002, Longislandbio, Shanghai, China) at 37 °C for 30 min. The DAB (K3468, Dako, Glostrup, Denmark) was used for the chromogenic reaction of section.

Biochemical parameter

The culture medium of L02 was collected to detect the cell damage by Alanine Aminotransferase Test Kit (ALT, Mindray Biomedical, Shenzhen, China) and Aspartate Aminotransferase Test Kit (AST, Mindray Biomedical, Shenzhen, China). The renal function of mice was detected by Creatinine Test Kit (CRE, Mindray Biomedical, Shenzhen, China) and Urea Test Kit (UREA, Mindray Biomedical, Shenzhen, China). The mice were sacrificed at the defined time point, and the serum was separated at the speed of 3000 rpm for 10 min. All biochemical tests were carried out with the full-automatic biochemical analyzer (BS-200, Mindray Biomedical, Shenzhen, China).

Statistical analysis

The GraphPad Prism 8.0.1 (LaJolla, CA, USA) was used for data analysis. Values were expressed as mean ± Standard Deviation (SD). When the data satisfied the normality and homogeneity of variance, the data were statistically analyzed by T-test. The P-value tests were two sides in which less than 0.05 were represented as statistically significant.