Rat renal tubular epithelial cells (NRK-52E cells) were purchased from the National Laboratory Cell Resource Sharing Platform (Beijing, China). Burdock roots were obtained from Yishunkang (Jiangsu, China). The kits for superoxide dismutase (SOD) and catalase (CAT) were acquired from Jiancheng Bioengineering Institute (Nanjing, Jiangsu, China). BCA protein assay kit, ROS assay kit and mitochondrial membrane potential detection kit were acquired from Beyotime Institute of Biotechnology (Haimen, Jiangsu, China). Antibodies specific for Nrf2, HO-1, Bcl-2, Bax, and β-actin were obtained from ABclonal (Wuhan, Hubei, China).

BFO was isolated and fractionated following our previously reported method (Hao et al., 2005; Yuan et al., 2021). Briefly, burdock roots were submerged in hot water and 95% ethanol for alcohol precipitation. The precipitate was dissolved in distilled water and deproteinized using the Sevag method (Sevag, 1938). The aqueous phase was collected and decolorized using D101 macroporous resin (Solarbio, Beijing, China), followed by loading onto a DEAE-cellulose-52 chromatographic column (Solarbio). The collected fractions were filtered using a 0.22-μm filter membrane, inserted in a 1 kDa regenerated cellulose dialysis bag (Solarbio), and dialyzed at 4°C for 72 h. Then, BFO was further purified by gel filtration chromatography on a Sephadex G75 column (Solarbio) and eluted with distilled water at a flow rate of 0.5 ml/min. The homogeneous fractions from the eluted single peak were gathered, concentrated, and lyophilized to powder (BFO). The homogeneity and molecular weight of BFO were determined by high performance gel permeation chromatography (HPGPC) on a Shimadzu Lc-l0Avp instrument (Shimadzu, Japan) equipped with an Ultrahydrogel™ liner column. Elution was monitored using a Shimadzu RID-10A refractive index detector. A series of standard dextran solutions was run under the same conditions and a standard curve linear over a wide range (1–10 kDa) was obtained by correlation analysis between the dextran standard molar mass and retention time (Liu et al., 2014). The total sugar content of BFO was measured as D-fructose equivalents using the phenol-sulfuric acid method (Dubois et al., 1956). The presence of starch-type polysaccharides was detected using the triiodination reaction (Liu et al., 2018). The Bradford method was adapted to determine the total protein content using bovine serum albumin (BSA) as the standard (Bradford, 1976).

NRK-52E cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) (Gibco, CA, United States) supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin in a humidified atmosphere of 5% CO₂ at 37°C. The cells were digested and passaged every 1–2 days and seeded into 6- or 96-well plates for experiments. BFO was dissolved in phosphate-buffered saline to prepare the stock solution.

NRK-52E cells were cultured in normal glucose (NG, 5.5 mM glucose), HG (30 mM glucose), and HG + different BFO concentrations (62.5, 125, and 250 μg/ml) for 48 h using 96-well plates. Then, CCK-8 kit reagent was added to wells and then the plates were incubated at 37°C for 1–2 h. A microplate reader (Biotek, Winooski, VT, United States) was used to measure the absorbance at 450 nm.

NRK-52E cells were subjected to the various culture conditions described above. Thereafter, 5 µL of propidium iodide and 10 µL of Annexin V-fluorescein isothiocyanate (FITC) were added to each sample for 15 min at room temperature (RT) in the dark, followed by the addition of 500 µL binding buffer and filtration through a 300 µm mesh cell filter. Finally, flow cytometry was performed immediately.

NRK-52E cells were cultured with different substances, as described previously. Samples were then incubated with DCFH-DA for 30 min at 37°C. The percentage of fluorescently positive cells was measured by flow cytometry at 488 nm excitation wavelength and 525 nm emission wavelength.

NRK-52E cells were cultured with different substances, as described previously. Thereafter, JC-1 dyeing working solution was added to the samples, mixed well, and incubated at 37°C for 20 min. At the end of the treatment, JC-1 fluorescence was measured by flow cytometry at 490 nm excitation wavelength and 530 nm emission wavelength.

NRK-52E cells were exposed to different substances, as described previously. The levels of SOD and CAT were determined using relevant detection kits according to the manufacturer’s instructions. At the end of the reaction, a microplate reader was used to measure the absorbance of the samples.

NRK-52E cells were incubated with different substances, as described previously. The protein concentration was determined using a BCA protein assay kit, and the denatured protein was separated on SDS-PAGE gel. Protein was transferred to a nitrocellulose membrane under constant current (300 mA) conditions, and then the membrane was blocked with 5% skim milk at RT for 2 h. The membranes were incubated with Nrf2 (1:2000), HO-1 (1:500), Bax (1:1000), and Bcl-2 (1:1000) antibodies at 4°C overnight. Next, the membranes were washed with TBST (3 × 10 min) and then incubated with the secondary antibodies (1:10,000) for 1 h. ECL detection reagent and a chemiluminescence imaging system were used to examine the membranes. The results were analyzed using the ImageJ software.

Total RNA was isolated from NRK-52E cells using TRIzol reagent (Beyotime), and cDNA was synthesized from total RNA using Prime Script RT kit (Thermo Fisher Scientific, Waltham, MA, United States) according to the manufacturer’s instructions. GAPDH was used as an internal standard. The primer pairs used for real-time PCR are shown in Table 1. The cycle threshold (Ct) value was determined, and the level of the housekeeping gene GAPDH was used for normalization. The relative mRNA level of each target gene was calculated with the 2^−△△Ct method.

Statistical analysis was conducted with SPSS 22.0. Data are shown as the mean ± standard deviation. Differences among groups were compared by one-way analysis of variance, followed by Dunnett’s post hoc test. p < 0.05 was considered statistically significant.

The elution curve of gel filtration chromatography on a Sephadex G75 column presented BFO as a single component (Figure 1A). The total sugar content of BFO was determined to be 99.7%. After concentration and lyophilization, BFO presented as a white powder and tested negative for the triiodide reaction, indicating the absence of starch-type polysaccharides. The Bradford test result was negative with no absorption at 280 or 260 nm, suggesting the absence of proteins and nucleic acids in BFO. The homogeneity and molecular weight of BFO were determined by high-performance gel permeation chromatography (HPGPC). The retention time and purity of BFO were 12.366 min and 99.753%, respectively. BFO presented a single and symmetrically sharp peak (Figure 1B), indicating a homogenous fraction with a molecular weight of 3,194 Da.

The CCK-8 assay was conducted to investigate the effect of BFO on the viability of NRK-52E cells under HG condition. The cell viability of the HG group was significantly reduced compared to that of the NG group. Conversely, when NRK-52E cells were incubated with HG + different BFO concentrations (62.5, 125, and 250 μg/ml), the cell viability increased (63.16, 72.97, and 77.98% of the control value, respectively) (Figure 2).

To study the effect of BFO on the apoptosis rate of NRK-52E cells under HG condition, the cell apoptosis rate was measured by flow cytometry. The apoptosis rate of the cells cultured with NG, HG, and HG + different BFO concentrations (62.5, 125, and 250 μg/ml) were 7.183 ± 1.230; 30.210 ± 1.741; and 20.719 ± 1.280, 16.510 ± 1.697, and 10.84 ± 0.172%, respectively. The apoptosis rate of the HG group was significantly higher than that of the NG group. Conversely, compare with HG, the intervention with HG + different BFO concentrations significantly reduced the cell apoptosis rate (Figure 3).

The DCFH-DA method was used to measure ROS level in NRK-52E cells exposed to HG and HG + different BFO concentrations. The results indicated that the ROS level of the HG group was higher than that of the NG group. Compare with HG, the intervention with HG + different BFO concentrations significantly reduced the level of ROS in a dose-dependent manner (Figure 4).

To elucidate the effect of BFO on the mitochondrial membrane potential in NRK-52E cells under HG condition, the mitochondrial membrane potential was determined by flow cytometry. The results demonstrated that in the HG group, the mitochondrial membrane potential was significantly decreased (Figure 5). The mitochondrial membrane potential of cells treated with HG + 62.5 μg/ml BFO did not increase significantly, and BFO at 125 and 250 μg/ml effectively inhibited the decrease in cell mitochondrial membrane potential induced by HG.

To elucidate whether BFO could protect NRK-52E cells against HG-induced oxidative stress damage, the activities of SOD and CAT were detected. The activities of SOD and CAT in the HG group were significantly lower than those in the NG group; both 125 μg/ml and 250 μg/ml BFO effectively inhibited the HG-induced reduction in SOD and CAT levels (Figures 6A,B).

To observe the molecular mechanisms underlying the protective effects of BFO on NRK-52E cells, the protein and mRNA expression of Nrf2, HO-1, Bax, and Bcl-2 in NRK-52E cells induced by HG was determined by western blotting and real-time PCR, respectively. The protein levels of Bax in the HG group were significantly increased compared to those in the NG group, and the protein levels of Nrf2, HO-1, and Bcl-2 in the HG group were significantly decreased. In addition, the protein levels of Bax in the HG + BFO group were significantly decreased in a dose-dependent manner, and the protein levels of Nrf2, HO-1, and Bcl-2 in the HG + BFO group were significantly increased (Figures 7A–E). Furthermore, the mRNA expression of Nrf2, HO-1, Bax, and Bcl-2 in these groups was in accordance with the protein expression (Figures 7F–I).