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Protein was extracted from renal tissue using RIPA reagent, and the total protein content was determined by a BCA protein quantitative kit (Solarbio Biotechnology, Beijing, China). SDS–PAGE (TransGen Biotech, Beijing, China) was followed by film transfer and closure and incubation with primary and secondary antibodies; rabbit pAb anti-GAPDH (1:1000, Wanleibio, Shenyang, China), rabbit pAb anti-P65 (1:500, Wanleibio, Shenyang, China), rabbit pAb anti-IFN-γ (1:1000, Wanleibio, Shenyang, China), rabbit pAb anti-IL-6 (1:1000, Wanleibio, Shenyang, China), and rabbit pAb anti-IL-8 (1:1000, Wanleibio), followed by the corresponding HRP-conjugated secondary antibodies (1:5000, Bioss, Beijing, China). Then, the signal was detected with a Bio–Rad Chemidoc Touch imager (Bio–Rad Chemidoc Touch, CA, USA). Finally, the gray value of the corresponding protein was analyzed by ImageJ software (National Institute of Health, Bethesda, Maryland, USA).

Total protein lysates were extracted using RIPA lysis buffer (Beyotime) supplemented with protease inhibitors (Roche, Penzberg, Germany), and the concentrations of the samples were determined using a BCA Protein Assay Kit (Beyotime). Equal amounts of total proteins were separated by SDS-PAGE (Transgen, Beijing, China), transferred to the PVDF membrane (Millipore, Billerica, MA, USA) and probed with a rabbit anti-MMP2 polyclonal antibody (1:2000, Proteintech, Wuhan, China) and mouse anti-GAPDH antibody (1:10,000, Proteintech). After overnight incubation with HRP-conjugated secondary antibodies (1:10,000, Proteintech), protein bands were visualized using Immobilon Western Chemiluminescent HRP Substrate (Millipore) on a Tanon-5200 chemiluminescence detection system (Tanon, Shanghai, China). The bands were analyzed by using Image J software (NIH, Bethesda, MD, USA).