Animal models

Six C57BL/6 mice (female, 60–80 g, specific-pathogen-free (SPF) grade, 11 weeks old) and 18 lupus erythematosus MRL/lpr mice (female, 61–82 g, SPF grade, 11 weeks old) were purchased from Huachuang sino Pharmaceutical Technology Co., LTD. (Taizhou, Jiangsu, China).

Experimental design

This study was approved by the Experimental Animal Welfare and Ethics Committee of ZVAST BIO Co., LTD. (No.2021100701). All six C57BL/6 mice and the 18 MRL/lpr mice were randomly divided into each group. The experiment was carried out in two parts:

1)

Experimental protocol A: for HE staining and flow cytometry. It was divided into the following four groups: control group, MRL/lpr group (hereafter referred to as the model group), MRL/lpr mice with Nrf2 gene adenovirus overexpression interference empty-load group (hereafter referred to as the Nrf2 empty-load group), and MRL/lpr mice with Nrf2 gene adenovirus overexpression interference vector group (hereafter referred to as the Nrf2 vector group). Three mice were randomly assigned to each group(n = 3), with C57BL/6 mice in the control group and MRL/lpr mice in another three groups.

2)

Experimental protocol B: for genetic testing in mice, including qPCR, Western Blotting, ELISA detection. It was also divided into four groups: control group, model group, Nrf2 empty-load group, Nrf2 vector group. Three mice were randomly assigned to each group (n = 3), with C57BL/6 mice in the control group and MRL/lpr mice in another three groups.

All animals entered the experiment after 1 week of adaptive feeding. Throughout the experiment, animals were fed a standard diet and had ad libitum access to food and water. Animals were maintained in a clean room at a temperature of 20–26 °C and humidity of 40–70%. The levels of circulating immune complexes in MRL/lpr mice increased significantly at approximately 12 weeks after birth. Therefore, 12-week-old mice were chosen as the starting point for this study. The experiments lasted for four weeks, starting at the beginning of the 12th week of age and ending by the 16th.

Adenoviral plasmids containing the Nrf2 gene (pAdEasy-EF1-MCS-CMV-EGFP-Nrf2, the same sequence as NM_010902.4) and an empty vector plasmid (pAdEasy-EF1-MCS-CMV-EGFP) were purchased from Hanbio BIO Co., Ltd. (Shanghai, China). Mice in the Nrf2 empty-load and Nrf2 vector groups were anesthetized, the abdominal skin was pre-prepared, and the renal pelvis was bilaterally exposed. Each mouse was injected bilaterally into the renal pelvis with 50 μl of virus, and the skin was subsequently sutured. Overexpression of Nrf2 was verified by immunofluorescence before proceeding with additional experiments.

On the one week after orthotopic virus injection, the kidneys of mice in each group were collected, embedded in paraffin, sectioned, and stained for renal histology by hematoxylin–eosin (HE) staining. Enzyme-linked immunosorbent assay (ELISA) was performed in mouse serum to detect the levels of IL-1β, TNF-α, and NF-κB. Quantitative polymerase chain reaction (qPCR) was performed to detect Nrf2 (Nfe2l2, NM_010902.4), HMGB1 (NM_ 010439.4), and TLR4 (NM_021297.3) expression in kidney tissues. Western blotting (WB) was conducted to evaluate Nrf2, HMGB1, and TLR4 protein levels in kidney tissues. Flow cytometry was employed to detect the TLR4+CXCR4+ PC ratio in the blood and kidney.

HE staining

The mouse kidney tissue samples were dehydrated, soaked, embedded in wax, and sectioned using a microtome. Samples were then stained with hematoxylin and eosin (HE) according to the manufacturer’s guidelines, dehydrated recursively with a series of ethanol dilutions, and sealed with xylene. Images were acquired using a microscope.

Flow cytometry

A single-cell suspension of approximately 107 cells/ml was prepared, and 100 μl was added to a flow tube. Five µL of each reagent (CD38 FITC, CD138 PE, CD27 APC)/(CXCR4 APC, TLR4 PE) were added to the cell suspension, incubated in the dark for 30 min, and centrifuged at 400 × g for 5 min at room temperature. The supernatant was discarded, 2 mL of PBS was added, and the sample was centrifuged again at 400 × g for 5 min at room temperature, with a subsequent repetition of these processes. After centrifugation, 500 µL of PBS and a flow cytometer probe (NovoCyte 2060R, ACEA BIO Co., Hangzhou, China) were added for detection. CD38 FITC, CD138 PE, CD27 APC, CXCR4 APC, and TLR4 PE were purchased from BioLegend (San Diego, CA, USA).

ELISA detection

Blood samples were collected and centrifuged for serum retrieval. All samples and reagents from the IL-1β (MM-0040M1, MEIMIAN, Jiangsu, China), TNF-α (MM-0132M1, MEIMIAN, Jiangsu, China), and NF-κB kits (ML063331-2, MEILIAN, Shanghai, China) were equilibrated at room temperature for 120 min before starting the protocol. The ELISAs were conducted according to the manufacturer’s instructions. Briefly, 100 μL of horseradish peroxidase (HRP)-labeled detection antibody were added to the samples in the well and incubated for 60 min in a closed thermostat. After plate washing five times, 50 μL each of substrate A and B were added to each well and incubated in the dark at 37 °C for 15 min. Then, 50 μL of stop solution were added to each well, and the optical density value was measured at a wavelength of 450 nm using a microplate reader (WD-2012B, LIUYI BIO Co., Ltd., Beijing, China).

qPCR

Kidney tissue samples were processed using the TRIzol Reagent kit (CW0580S, CWBIO, Beijing, China), total RNA was extracted using an Ultrapure RNA Extraction Kit (CW0581M, CWBIO, Beijing, China), and RNA concentration and purity were determined using an ultraviolet spectrophotometer (NP80, Implen NanoPhotometer, Munich, Germany). RNA was converted into cDNA using HiScript II Q RT SuperMix for qPCR (+ gDNA wiper) (R223-01, Vazyme, Nanjing, China) and used for fluorescent quantitative PCR, with the following reaction parameters: 10 μl of 2 × SYBR Green PCR Master Mix (Q711-02, Vazyme, Nanjing, China), 1 μl of cDNA, 0.4 μl of forward primers, 0.4 μl of reverse primers, and 8.2 μl of RNase-free ddH2O. The qPCR program consisted of 40 cycles of amplification. β-actin was used as the housekeeping gene, and the relative expression of Nrf2, HMGB1, TLR4, and NF-κB was calculated according to the 2−△△Ct method. (Primer sequences are shown in Table 1).

Table 1 Primers used for qPCR experimentsWestern blotting

Mouse kidney samples were treated with radioimmunoprecipitation assay (RIPA) buffer for protein extraction. Protein concentration was subsequently determined using a BCA kit. The samples were denatured for Western blotting. Briefly, protein samples were loaded into the gel, and sodium dodecyl benzene sulfonate gel electrophoresis was performed for 1–2 h. Proteins were then transferred to the PVDF membrane by the wet method, blocked for 1 h in a 3% TBS-T skim milk solution, and incubated overnight in the primary antibody solution at 4 °C. The next day, the membranes were washed three times with standard TBS-T buffer, incubated for 2 h at room temperature in a secondary antibody solution, washed three times, and incubated with ECL prior to detection in an imaging system. Images were analyzed in the "ImageJ" software. The mouse anti-GAPDH antibody was purchased from TransGen Biotech Co., Ltd. (Beijing, China). HRP-conjugated Goat Anti-Mouse IgG (H + L) and HRP-conjugated Goat Anti-Rabbit IgG (H + L) were purchased from Servicebio Technology Co., Ltd. (Wuhan, China). Mouse Anti-Nrf2 was acquired from Proteintech Group Inc. (Chicago, IL, USA), and Rabbit Anti-HMGB1 and Rabbit Anti-TLR4 antibodies were purchased from Affinity Biosciences Ltd. (Melbourne, Australia).

Statistical analysis

SPSS19.0 software was used for statistical analysis. Data are presented as mean ± standard deviation (SD). Comparisons between three or more groups were conducted using one-way analysis of variance, and then comparisons between the two groups were conducted using the LSD method. Graphpad 8.0 was used for graph design, and the inspection level was set at α = 0.05. Gray values were analyzed using Image Pro J software.