Abstract：
BACKGROUND: Myoglobin (Mb) induced death of renal tubular epithelial cells (RTECs) is a major pathological factor in crush syndrome-related acute kidney injury (CS-AKI). It is unclear whether ferroptosis is involved and could be a target for treatment.
OBJECTIVE: This study aimed to evaluate the potential therapeutic effects of combining the natural small molecule rosemarinic acid (RA) and the iron chelator deferasirox (Dfe) on CS-AKI through inhibition of ferroptosis.
METHODS: Sequencing data were downloaded from the GEO database, and differential expression analysis was performed using the R software limma package. The CS-AKI mouse model was constructed by squeezing the bilateral thighs of mice for 16 h with 1.5 kg weight. TCMK1 and NRK-52E cells were induced with 200 μM Mb and then treated with RA combined with Dfe (Dfe + RA, both were 10 μM). Functional and pathological changes in mouse kidney were evaluated by glomerular filtration rate (GFR) and HE pathology. Immunofluorescence assay was used to detect Mb levels in kidney tissues. The expression levels of ACSL4, GPX4, Keap1, and Nrf2 were analyzed by WB.
RESULTS: We found that AKI mice in the GSE44925 cohort highly expressed the ferroptosis markers ACSL4 and PTGS2. CS-AKI mice showed a rapid decrease in GFR, up-regulation of ACSL4 expression in kidney tissue, and down-regulation of GPX4 expression, indicating activation of the ferroptosis pathway. Mb was found to deposit in renal tubules, and it has been proven to cause ferroptosis in TCMK1 and NRK-52E cells in vitro. We found that Dfe had a strong iron ion scavenging effect and inhibited ACSL4 expression. RA could disrupt the interaction between Keap1 andNrf2, stabilize Nrf2, and promote its nuclear translocation, thereby exerting antioxidant effects. The combination of Dfe and RA effectively reversed Mb induced ferroptosis in RTECs.
CONCLUSIONS: In conclusion, we found that RA combined with Dfe attenuated CS-AKI by inhibiting Mb-induced ferroptosis in RTECs via activating the Nrf2/Keap1 pathway.
OBJECTIVE: This study aimed to evaluate the potential therapeutic effects of combining the natural small molecule rosemarinic acid (RA) and the iron chelator deferasirox (Dfe) on CS-AKI through inhibition of ferroptosis.
METHODS: Sequencing data were downloaded from the GEO database, and differential expression analysis was performed using the R software limma package. The CS-AKI mouse model was constructed by squeezing the bilateral thighs of mice for 16 h with 1.5 kg weight. TCMK1 and NRK-52E cells were induced with 200 μM Mb and then treated with RA combined with Dfe (Dfe + RA, both were 10 μM). Functional and pathological changes in mouse kidney were evaluated by glomerular filtration rate (GFR) and HE pathology. Immunofluorescence assay was used to detect Mb levels in kidney tissues. The expression levels of ACSL4, GPX4, Keap1, and Nrf2 were analyzed by WB.
RESULTS: We found that AKI mice in the GSE44925 cohort highly expressed the ferroptosis markers ACSL4 and PTGS2. CS-AKI mice showed a rapid decrease in GFR, up-regulation of ACSL4 expression in kidney tissue, and down-regulation of GPX4 expression, indicating activation of the ferroptosis pathway. Mb was found to deposit in renal tubules, and it has been proven to cause ferroptosis in TCMK1 and NRK-52E cells in vitro. We found that Dfe had a strong iron ion scavenging effect and inhibited ACSL4 expression. RA could disrupt the interaction between Keap1 andNrf2, stabilize Nrf2, and promote its nuclear translocation, thereby exerting antioxidant effects. The combination of Dfe and RA effectively reversed Mb induced ferroptosis in RTECs.
CONCLUSIONS: In conclusion, we found that RA combined with Dfe attenuated CS-AKI by inhibiting Mb-induced ferroptosis in RTECs via activating the Nrf2/Keap1 pathway.
摘要：
背景：肌红蛋白（Mb）诱导的肾小管上皮细胞（RTEC）死亡是挤压综合征相关急性肾损伤（CS-AKI）的主要病理因素。目前尚不清楚铁性凋亡是否涉及并可能成为治疗目标。
目的：本研究旨在评估天然小分子迷迭香酸（RA）和铁螯合剂地拉罗司（Dfe）联合抑制铁凋亡对CS-AKI的潜在治疗作用。
方法：测序数据从GEO数据库下载，并使用R软件limma软件包进行差异表达分析。以1.5kg体重挤压小鼠双侧大腿16h构建CS-AKI小鼠模型。用200μMMb诱导TCMK1和NRK-52E细胞，然后用RA联合Dfe（DfeRA，两者均为10μM)。通过肾小球滤过率（GFR）和HE病理学评估小鼠肾脏的功能和病理变化。免疫荧光法检测肾组织中的Mb水平。通过WB分析ACSL4、GPX4、Keap1和Nrf2的表达水平。
结果：我们发现GSE44925组群中的AKI小鼠高表达铁凋亡标志物ACSL4和PTGS2。CS-AKI小鼠GFR快速下降,肾组织ACSL4表达上调,和下调GPX4的表达，表明铁凋亡途径的激活。发现Mb沉积在肾小管中，并已被证明在体外引起TCMK1和NRK-52E细胞的铁凋亡。我们发现Dfe具有较强的铁离子清除作用并抑制ACSL4的表达。RA可以破坏Keap1和Nrf2之间的相互作用，稳定Nrf2，并促进其核易位，从而发挥抗氧化作用。Dfe和RA的组合有效地逆转了RTEC中Mb诱导的铁凋亡。
结论：结论：我们发现RA联合Dfe通过激活Nrf2/Keap1通路抑制Mb诱导的RTEC铁凋亡,从而减弱CS-AKI.
目的：本研究旨在评估天然小分子迷迭香酸（RA）和铁螯合剂地拉罗司（Dfe）联合抑制铁凋亡对CS-AKI的潜在治疗作用。
方法：测序数据从GEO数据库下载，并使用R软件limma软件包进行差异表达分析。以1.5kg体重挤压小鼠双侧大腿16h构建CS-AKI小鼠模型。用200μMMb诱导TCMK1和NRK-52E细胞，然后用RA联合Dfe（DfeRA，两者均为10μM)。通过肾小球滤过率（GFR）和HE病理学评估小鼠肾脏的功能和病理变化。免疫荧光法检测肾组织中的Mb水平。通过WB分析ACSL4、GPX4、Keap1和Nrf2的表达水平。
结果：我们发现GSE44925组群中的AKI小鼠高表达铁凋亡标志物ACSL4和PTGS2。CS-AKI小鼠GFR快速下降,肾组织ACSL4表达上调,和下调GPX4的表达，表明铁凋亡途径的激活。发现Mb沉积在肾小管中，并已被证明在体外引起TCMK1和NRK-52E细胞的铁凋亡。我们发现Dfe具有较强的铁离子清除作用并抑制ACSL4的表达。RA可以破坏Keap1和Nrf2之间的相互作用，稳定Nrf2，并促进其核易位，从而发挥抗氧化作用。Dfe和RA的组合有效地逆转了RTEC中Mb诱导的铁凋亡。
结论：结论：我们发现RA联合Dfe通过激活Nrf2/Keap1通路抑制Mb诱导的RTEC铁凋亡,从而减弱CS-AKI.
