2.1. Reagents
The reagents used in the present experiments include: TCE and Freund’s complete adjuvant (FCA) (Sigma, St. Louis, MO, USA), olive oil (Sinopharm Chemical Regent Company, Shanghai, China), acetone (Xilong Scientific, Guangzhou, China), poly I:C (InvivoGen, MA, USA), DAPI, TRIzol and Revert Aid First Strand cDNA Synthesis Kit (ThermoFisher Scientific, MA, USA), LightCycler 480 SYBR Green Ⅰ Master kit (Roche, Basel, Switzerland), hematoxylin and eosin staining kit, periodic acid-schiff staining kit, SP-9100 immunohistochemistry detection kit and DAB kit (ZSGB-BIO, Beijing, China), and sarcosine oxidase and urease method kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, China).The details of the antibodies used in this study include anti-HMGB1, GAPDH, VCAM-1, ICAM-1, and E-selectin (Abcam, Cambridge, United Kingdom); TLR3 (ZENBIO, Chengdu, China); Syndecan 1, Glypican 1, Histone H3, CD31(Santa Cruz, Texas, USA);Acetyl-HMGB1-K2(ABclonal, Wuhan, China).
2.2. Mice and treatments
Female BALB/c mice (6 ~ 8 wks, n = 63) were obtained from the Experimental Animal Center of Anhui Province and kept in a specific pathogen-free environment with food and water ad libitum. The housing conditions were maintained at 22.5 ± 0.5°C, 50 ± 5% humidity, and a 12 h light/dark cycle. After a week of adaptation, the BALB/c mouse model of TCE sensitization was established according to our previousstudies (Wang et al., 2015; Zhang et al., 2018). To mimic the status of viral infection, mice were pretreated with a single dose of 100 µl 0.5 mg/ml poly I:C via intraperitoneal injection 3 hrs before the challenge on Day 19 (Fig. 1A). Depending on whether presented with erythema and/or blisters, mice were grouped into the TCE sensitization-positive group (TCE positive), TCE sensitization-negative group (TCE negative), poly I:C pretreatment + TCE sensitization-positive group (TCE + poly I:C positive), and poly I:C pretreatment + TCE sensitization-negative group (TCE + poly I:C negative). All experimental protocols were approved by the Experimental Animal Ethics Committee of Anhui Medical University (No. LLSC 20180310) and conducted under the roles of care and use of laboratory animals by the National Institute of China.
2.3. Histopathologic analysis
Mouse kidney samples were isolated and fixed in 4% paraformaldehyde for 48 hrs. Then, 5 µm thickness FFPE sections were cut via standard manufacture protocols. For hematoxylin & eosin (H&E) staining, the sections were stained with hematoxylin and eosin in order. Thereafter, the histological changes in mouse kidneys were evaluated under an optical microscope (Olympus BX53, Japan).
2.4. Renal function assessment
Mouse serum samples were obtained to detect blood urea nitrogen (BUN) and creatinine (Cre) according to the kit instructions. The standard series were manipulated and analyzed in parallel. The optical density (OD) value was read in a microplate reader (Bio-Tek µ Quant, USA) at 640 nm wavelength for BUN and 546 nm wavelength for Cre, respectively.
2.5. Immunohistochemistry (IHC) test
After dewaxing and hydration, mouse kidney sections were gently washed using PBS solution and then treated with 3% H2O2 to eliminate the activity of endogenous peroxidase. Next, the sections were heated in 0.01 M sodium citrate buffer for antigen retrieval, followed by incubation with 10% normal goat serum in PBS for 30 min at 37°C to block non-specific binding. For the IHC test, antibodies against VCAM-1 (Abcam, ab134047, UK), E-selectin (Abcam, ab18981, UK), or ICAM-1 (Abcam, ab171123, UK) were incubated with the sections at 4°C overnight, followed by incubation with biotin-labeled secondary antibody and horseradish enzyme-labeled oval chain enzyme (ZSJQ-BIO, China). Finally, a DAB kit was used to detect the target protein under an optical microscope (Olympus BX53, Japan).
2.6. Immunofluorescence (IF) test
Frozen OCT-embedded kidney samples were sectioned at 4 µm thickness in a freezing microtome (Leica, CM1950, Germany). The sections were fixed in pre-cold acetone for 5 min, and then gently washed with PBS 3 times for 5 min each time. After blocking with goat serum for 2 h at 37°C, antibodies against HMGB1 (Abcam, ab79823, UK) and CD31 (Santa Cruz, sc-376764, USA) were added to the sections. Next, fluorescein-labeled secondary antibody was added to the sections for 2 hrs. Then 4′,6-diamidino-2-phenylindole (DAPI, Merck, USA) was used for nuclear staining. Finally, the sections were analyzed under a fluorescence microscope (Olympus, IX73, Japan).
2.7. Quantitative real-time PCR (qRT-PCR)
A total of 25 mg of mouse kidney tissue was used for extraction of total RNA by TRIzol reagent (ThermoFisher, USA). The concentration and purity of RNA was checked by a NanoDrop One (Thermo Fisher, USA), and the final concentration of total RNA was diluted to 0.5 mg/mL with RNase-free water. The cDNA was obtained by RevertAid First Strand cDNA Synthesis kit (Thermo Scientific, Waltham, MA) according to the manufacturer's protocols. Then, the PCR amplification reaction was carried out in a Light Cycler 480 system (Roche, Switzerland) as follows: 95°C for 10 min for initial denaturation; 95°C for 15 s for denaturation, 45 cycles; 60°C for 15 s to annealing; 72°C for 20 s for extension. The primer sequences for qRT-PCR were listed as follows: TLR3: Forward, 5’-GTGAGATACAACGTAGCTGACTG-3’, Reverse, 5’-TCCTGCATCCAAGATAGCAAGT-3’; HMGB1: Forward, 5’-CGGAGAAACTTCAGACCGGA-3’, Reverse, 5’-CCCATGTTTAGTTGATTTTCCAGC-3; GAPDH: Forward, 5’-TGACCTCAACTACATGGTCTACA-3’; Reverse, 5’-CTTCCCATTCTCGGCCTTG-3’.
2.8. Western blot
Total proteins were extracted from mouse kidneys by radioimmunoprecipitation lysis buffer (RIPA) containing protease and phosphatase inhibitors. The concentration of total protein was measured by BCA assay, and the final protein concentration was adjusted to 10 mg/ml. A total of 10 µl sample was loaded in sodium dodecyl sulfate polyacrylamide gels (SDS‒PAGE) for electrophoresis. Next, the proteins were transferred to PVDF membranes (0.45 µm, Millipore, USA) and blocked in BSA working solution for 2 hrs. Afterwards, antibodies against HMGB1 (Abcam, ab79823, UK, dilution 1:1000), ac-HMGB1 (ABclonal, A16002, China, dilution 1:1000), TLR3 (ZEN-BIOSCIENCE, 120101, China, dilution 1:1000), histone H3 (Santa Cruz, sc-517576, USA, dilution 1:1000), syndecan-1 (Santa Cruz, sc-390791, USA, dilution 1:1000), glypican-1 (Santa Cruz, sc-365000, USA, dilution 1:1000), or GAPDH (Abcam, ab18160, UK, dilution 1:1000) were incubated with the PVDF membrane at 4°C overnight. After gently washing in TBST 3 times for 10 min each time, the PVDF membrane was placed in TBST solution containing goat anti-rabbit IgG antibody (dilution 1:20,000). Finally, the membrane protein was detected in a chemiluminescence system (Tanon, Fine-do X6, China).
2.9. Statistical analysis
Data are presented as means ± SD and analyzed by the SPSS13.0 software package (Chicago, USA). One-way ANOVA and Bonferroni’s test were performed to check the differences between multiple groups. A p-value < 0.05 was considered as statistically significant difference in the present study.