| Part Ⅰ: The role of Nrf2 in reducing renal calcium oxalate crystal deposition and renal tubular epithelial cell injury by inhibiting M1 macrophage polarization Objective: Macrophages(Mφs)are crucial innate immune cells that affect the occurrence and development of calcium oxalate kidney stones,among which M1Mφ and M2Mφ can promote and inhibit calcium oxalate crystal deposition,respectively.This study aimed to explore whether the pharmacological activator of Nrf2,sulforaphane(SFN),can reduce renal calcium oxalate crystal deposition and the inflammatory injury of renal tubular epithelial cells by activating Nrf2 and inhibiting M1Mφ polarization in vitro and in vivo.Methods: In animal experiments,C57BL/6J mice were pre-administered intraperitoneal injection of different doses of SFN,and the calcium oxalate crystal deposition mouse model was established by intraperitoneal injection of glyoxylate.Renal calcium oxalate crystal deposition of mice in each group was detected by polarized light microscopy and Pizzolato staining.The degree of renal tubular damage and necrosis of renal cells in each group of mice were assessed by PAS staining and TUNEL staining.Differentially expressed genes in the genome-wide gene expression profile of Nrf2 knockout mouse bone marrow-derived macrophages in the Gene Expression Omnibus(GEO)database was analyzed,and validated by immunohistochemistry and qRT-PCR.The polarization status of Mφs in mouse kidney was assessed by immunofluorescence and immunohistochemistry.In cell experiments,mouse bone marrow-derived macrophages co-cultured with calcium oxalate monohydrate-stimulated renal tubular epithelial cells were treated with different concentrations of SFN,Nrf2 overexpression plasmid or interfering RNA,respectively.The expression of Mφs polarization markers and related proteins in each group were analyzed by qRT-PCR and Western Blot.Immunofluorescence staining and flow cytometry were used to detect the polarization subtypes of Mφs.Results: Polarized light microscopy and Pizzolato staining showed that with the increase of SFN dose,renal calcium oxalate crystal deposition of mice was significantly reduced.PAS staining and TUNEL staining showed that the degree of renal tubular damage and necrotic cells in the SFN intervention group were significantly reduced,and there was a positive correlation between the degree of reduction and the dose of SFN.Analysis of the dataset of the GEO(GSE71695)suggested that after Nrf2 knockout,M1Mφ polarization factors TLR4 and IRF1,Mφs marker gene NOS2 and cytokine-related gene IL-1b was significantly up-regulated.Immunohistochemistry and qRT-PCR showed that the expression of Nrf2 was negatively correlated with the expression of TLR4 and IRF1.Immunofluorescence and immunohistochemistry showed that the expression of M1Mφmarker iNOS in SFN intervention group mice was gradually decreased,and the expression of M2Mφ marker ARG-1 was gradually increased.qRT-PCR and Western Blot showed that after SFN treatment or overexpression of Nrf2,the expression of Nrf2 was up-regulated,and the expressions of TLR4 and IRF1 were down-regulated.At the same time,iNOS was also down-regulated,while the expression of ARG-1 was up-regulated,while Nrf2 knockdown with si RNA had the opposite effect.Immunofluorescence staining showed that after SFN treatment or overexpression of Nrf2,Mφs expressed more ARG-1and less iNOS,while Nrf2 knockdown with si RNA showed the opposite effect.Flow cytometry showed that after SFN treatment or overexpression of Nrf2,Mφs expressed more M2Mφ marker CD206 and less M1Mφ marker CD11c,while Nrf2 knockdown with si RNA showed the opposite effect.Conclusions: SFN inhibits M1Mφ polarization and promotes M2Mφ polarization by up-regulating the expression and function of Nrf2,thereby reducing renal calcium oxalate crystal deposition and the inflammatory injury of renal tubular epithelial cells,and finally inhibiting the the occurrence and development of calcium oxalate kidney stone.Part Ⅱ: The mechanism of Nrf2 inhibiting M1 macrophage polarization to reduce renal calcium oxalate crystal deposition and renal tubular epithelial cell injury via Nrf2-miR-93-TLR4/IRF1 axisObjective: The results of Part I confirmed that SFN inhibited M1Mφ polarization by activating Nrf2,thereby reducing calcium oxalate crystal deposition and the inflammatory injury of renal tubular epithelial cells.This study aimed to further explore the potential molecular mechanism of the inhibition of M1Mφ polarization after Nrf2 activation.Methods: We searched for miRNAs that can both be transcriptionally activated by Nrf2 and simultaneously target TLR4 and IRF1 by analyzing the miRNA expression profiles of Mφs.Chromatin immunoprecipitation assay was used to detect whether Nrf2 could bind to the promoter region of miR-93-5p.Luciferase reporter assay,qRT-PCR and Western Blot were used to confirm the regulation of miR-93-5p on TLR4 and IRF1 and the effect of miR-93-5p on Mφs polarization.Mouse bone marrow-derived macrophages co-cultured with calcium oxalate monohydrate-stimulated tubular epithelial cells were treated with SFN and/or miR-93-5p inhibitor,and the polarization status of Mφs was detected by immunofluorescence and flow cytometry.The crystal phagocytic ability of Mφs was detected by fluorescence microscopy.The necrosis of renal tubular epithelial cells was detected by flow cytometry.The levels of inflammatory cytokines in the culture medium were detected by ELISA.Based on the calcium oxalate crystal deposition mouse model,mice were given intraperitoneal injection of SFN and/or tail vein injection of antagomiR-93,a long-acting inhibitor of miR-93-5p.Polarized light microscopy and Pizzolato staining were used to detect renal calcium oxalate crystal deposition of mice in each group.PAS staining and TUNEL staining were used to assess the degree of renal tubular damage and necrosis of renal cells in each group of mice.Micro PET-CT and ELISA were used to evaluate renal inflammatory response of mice.Immunofluorescence and immunohistochemistry were applied to assess the polarization status of Mφs in mouse kidney.Results: miR-93-5p can both be transcriptionally activated by Nrf2 and simultaneously target TLR4 and IRF1.Chromatin immunoprecipitation showed that Nrf2 could bind to the promoter region of miR-93-5p.Luciferase reporter assay found that miR-93-5p could directly target the 3’UTR of TLR4 and IRF1.qRT-PCR and Western Blot found that SFN up-regulated the expression of Nrf2,down-regulated the expression of TLR4 and IRF1,decreased M1Mφ,and increased M2Mφ,while miR-93-5p inhibitor had the opposite effect.Immunofluorescence and flow cytometry showed that SFN reduced M1Mφ and increased M2Mφ,while miR-93-5p inhibitor had the opposite effect.Fluorescence microscopy found that SFN enhanced phagocytic ability of Mφs,while miR-93-5p inhibitor had the opposite effect.Flow cytometry showed that SFN reduced the ratio of renal tubular epithelial cell necrosis,while miR-93-5p inhibitor had the opposite effect.ELISA showed that SFN reduced pro-inflammatory cytokines and increased anti-inflammatory cytokines,while miR-93-5p inhibitor had the opposite effect.Polarized light microscopy and Pizzolato staining indicated that SFN could reduce glyoxylate-induced calcium oxalate crystal deposition in mouse kidneys,while antagomiR-93 had the opposite effect.PAS staining and TUNEL staining showed that SFN could reduce the degree of renal tubular damage,while antagomiR-93 had the opposite effect.Micro PET-CT and ELISA show that SFN can reduce calcium oxalate crystal-induced renal inflammation,while antagomiR-93 has the opposite effect.Immunofluorescence and immunohistochemistry show that SFN can reduce M1Mφ and increase M2Mφ in mouse kidneys,while antagomiR-93 had the opposite effect.Conclusions: SFN activates Nrf2 and inhibits M1Mφ polarization via the Nrf2-miR-93-TLR4/IRF1 axis,thereby reducing the inflammatory injury of renal tubular epithelial cells and promoting the phagocytosis of calcium oxalate crystals by Mφs,reducing renal calcium oxalate crystal deposition,and finally inhibiting the the occurrence and development of calcium oxalate kidney stone. |
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