Structure of RSL3
CAS No.: 1219810-16-8
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RSL3 ((1S,3R)-RSL3) is an inhibitor of glutathione peroxidase 4 (GPX4), which reduces GPX4 expression and induces ferroptosis in head and neck cancer cells. In HN3 resistant cells, it increases the protein levels of p62 and Nrf2, leading to Keap1 inactivation.
Synonyms: (1S,3R)-RSL3
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Zeng, Xi ; Zhang, Liefeng ; Lu, Hong ; Xue, Xinru ; Yang, Ling ; Yang, Xiuling , et al.
Abstract: Background and Purpose: Radiation enteritis (RE) is a severe complication after radiotherapy with no specific therapeutic agents. Here, we have attempted to identify the key therapeutic targets for RE, to advance drug development. Experimental Approach: Therapeutic targets were screened and identified using RE patients' intestinal samples, bioinformatics, and mouse models. RNA sequencing, electron microscopy, metabolomics, glycolytic flux, co-immunoprecipitation, molecular docking, point mutation were used to identify mechanisms. Key Results: Analysis of gene changes in response to ionising radiation showed extensive regulation of several differentially expressed genes by PPARγ, as well as its deficiency in activation and expression in RE. Both activation and overexpression of PPARγ significantly antagonised RE in vivo. Mechanistically, PPARγ specifically limited ferroptosis in intestinal epithelial cells exposed to ionising radiation, and its selective activation was more effective than full activation because of the reduced effect on the ferroptosis-driving genes PTEN and SAT1. Furthermore, ionising radiation caused the greatest changes in glucose metabolism. PPARγ targeted GAPDH at Lys107 to shift glycolysis to the hexosamine biosynthesis pathway, thereby enhancing glycosylation. In ionising radiation-induced ferroptosis, O/N-GlcNAcylation initially played antagonistic roles and later mediated the process, and they assisted PPARγ in restraining lysosomal degradation of heavy-chain ferritin (FTH1) and the transferrin receptor TFRC, thus controlling storage and transport of iron, and consequently alleviated ferroptosis. Conclusion and Implications: PPARγ is a potential therapeutic target for RE, as it elicits GAPDH-mediated glucose metabolic reprogramming and alleviates ionising radiation-induced ferroptosis, in a glycosylation-dependent manner.
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Keywords: ferroptosis ; GAPDH ; glucose metabolic reprogramming ; peroxisome proliferator-activated receptor γ ; radiation enteritis
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Shi, Shengbo ; Zhang, Zijing ; Shi, Pengpeng ; Gong, Changsheng ; Zhao, Zetian ; Wang, Tienan , et al.
Abstract: Spinal cord injury (SCI) is a severe traumatic disorder of the central nervous system, often resulting in partial or complete loss of sensory and motor functions. Ferroptosis, a lipid peroxidation-driven apoptotic process triggered by iron overload, has emerged as a novel form of programmed cell death and a focal point in post-SCI cell death research. Exosomes (Exo), as delivery vehicles, exhibit multiple advantages, including superior encapsulation capacity, high targeting efficiency, and enhanced blood–brain barrier penetration to reach the central nervous system. Previous studies have identified exosomes as key carriers of bioactive molecules, including miRNAs. Our prior investigations demonstrated that miR-494 attenuates SCI progression. To investigate the therapeutic mechanism of Exo-miR-494 in SCI, this study conducted in vitro and in vivo experiments using PC12 cell models and rat SCI models. In vitro, qRT-PCR and Western blot were employed to detect the expression levels of miR-494, SIRT1, HO-1, GPX4, and 4HNE, while intracellular oxidative stress markers were measured to clarify the regulatory effects of Exo-miR-494 on cellular oxidative stress and ferroptosis. In vivo, rats with successful SCI modeling were intravenously injected with Exo-miR-494. Fluorescence quantitative PCR, Western blot, and immunofluorescence were comprehensively used to analyze the changes in SIRT1/HO-1 signaling pathway molecules and ferroptosis-related indicators in injured spinal cord tissues. HE staining and Nissl staining were applied to evaluate spinal cord tissue damage and neuronal morphology. Transmission electron microscopy was utilized to observe cellular ultrastructural changes, and Prussian blue staining was combined to detect iron deposition, thereby deeply exploring the ferroptosis mechanism. Finally, Basso, Beattie, Bresnahan (BBB) locomotor scores and inclined plane tests were used to systematically assess the motor function recovery of rats. Results revealed a significant downregulation of miR-494 post-SCI. Exo-miR-494 effectively restored miR-494 levels in injured spinal tissues, suppressed ferroptosis through SIRT1/HO-1 pathway activation, mitigated SCI progression, and enhanced functional recovery in rats. Exosomes derived from rat bone marrow mesenchymal stem cells (BMSCs) delivering miR-494 represent a potential therapeutic strategy for SCI. miR-494 targets injury sites, reduces neuronal ferroptosis after spinal cord injury by regulating the SIRT1/HO-1 signaling pathway, alleviates damage caused by spinal cord injury, and promotes motor function recovery in rats.
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Keywords: Spinal cord injury ; Exosome ; MiR-494 ; SIRT1/HO-1 ; Ferroptosis ; Neural repair
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Han, Linfen ; Gao, Chengchang ; Jin, Xiaorui ; Li, Yingping ; Chen, Liangjie ; Li, Donglin , et al.
Abstract: Introduction: Ferroptosis is a form of regulated cell death driven by the accumulation of iron–dependent lipid peroxides, and ferroptosis–mediated cancer therapy has gained considerable attention. Despite emerging evidence that ferroptosis induction effectively suppresses hepatocellular carcinoma (HCC) progression and enhances chemosensitivity, the development of resistance to ferroptosis-targeting therapies remains a critical challenge. Natural active compounds have great potential in cancer treatment. Methods: The impact of 6-ME on the cell viability of HCC cells was assessed using the Cell Counting Kit-8 (CCK-8) assay and colony formation assay. Furthermore, cellular morphology of HCC cells was visualized under inverted fluorescence microscopy. Intracellular reactive oxygen species (ROS) and lipid peroxidation levels were quantified using fluorescence probes and determined by flow cytometry analysis. The expression of ferroptosis-related proteins and genes was determined via Western blot and quantitative real-time PCR analyses. Results: Here, we demonstrate that 6–Methoxydihydrosanguinarine (6–ME), an alkaloid from Macleaya cordata, exerts anti–tumor functions in HCC cells via ferroptosis. Stimulation with 6–ME induces intracellular ROS production, cell growth inhibition, and cell death in HCC cells, and these effects can be weakened by the ROS scavenger GSH or NAC and ferroptosis inhibitors deferoxamine mesylate (DFO) or ferrostatin–1 (Fer–1). Mechanistically, 6–ME downregulates the expression of the key ferroptosis defense enzyme GPX4 at the transcriptional level, leading to excessive lipid peroxidation and ferroptosis in HCC cells. Importantly, low concentrations of 6–ME also enhanced the ferroptosis sensitivity induced by RSL3 and IKE in HCC cells. Conclusion: These findings reveal that the natural product 6–ME exerts anti–tumor functions in HCC cells via ferroptosis and underscore the potential of 6–ME administered alone or in combination with canonical ferroptosis inducers for the treatment of HCC patients.
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Keywords: 6-methoxydihydrosanguinarine ; HCC ; ROS ; ferroptosis ; GPX4
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Shi, Shengbo ; Zhang, Zijing ; Huang, Xingxing ; Gong, Changsheng ; Zhao, Zetian ; Wang, Tienan , et al.
Abstract: Background: Spinal cord injury (SCI) is a debilitating neurological condition that often results in long-term disability and functional impairment. Recent studies have identified ferroptosis as a significant pathological mechanism in SCI. SIRT2, a deacetylase enzyme, is closely associated with inflammatory responses and apoptosis, playing a crucial role in the pathogenesis of various neurological disorders. This research aims to elucidate the specific mechanisms by which SIRT2 overexpression inhibits ferroptosis and promotes SCI repair through the deacetylation of nuclear factor-κB (NF-κB) p65. Results: Utilizing cell models and a rat SCI model, we discovered that SIRT2 overexpression promotes NF-κB p65 deacetylation, subsequently inhibiting ferroptosis and oxidative stress. Conversely, the use of AK-7 elevated NF-κB p65 acetylation levels, exacerbating ferroptosis and oxidative stress. In SCI rats, intrathecal injection of SIRT2-overexpressing recombinant adenovirus successfully inhibited NF-κB p65 acetylation and ferroptosis in the dorsal root ganglia, thereby reducing neuronal apoptosis and enhancing motor function recovery. Conclusion: In summary, these findings indicate that SIRT2 overexpression can suppress ferroptosis through NF-κB p65 deacetylation, facilitating SCI repair. Therefore, a deeper understanding of the interaction between SIRT2 and NF-κB p65 and their roles in the regulation of ferroptosis is of paramount importance for developing novel therapeutic approaches for spinal cord injury.
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Keywords: Spinal cord injury ; SIRT2 ; NF-κB/P65 ; Acetylation ; Oxidative stress ; Ferroptosis ; Neural injury and repair
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Li, Donglin ; Li, Yingping ; Chen, Liangjie ; Gao, Chengchang ; Dai, Bolei ; Yu, Wenjia , et al.
Abstract: The natural product auraptene can influence tumor cell proliferation and invasion, but its effect on hepatocellular carcinoma (HCC) cells is unknown. Here, we report that auraptene can exert anti-tumor effects in HCC cells via inhibition of cell proliferation and ferroptosis induction. Auraptene treatment induces total ROS and lipid ROS production in HCC cells to initiate ferroptosis. The cell death or cell growth inhibition of HCC cells induced by auraptene can be eliminated by the ROS scavenger NAC or GSH and ferroptosis inhibitor ferrostatin-1 or Deferoxamine Mesylate (DFO). Mechanistically, the key ferroptosis defense protein SLC7A11 is targeted for ubiquitin–proteasomal degradation by auraptene, resulting in ferroptosis of HCC cells. Importantly, low doses of auraptene can sensitize HCC cells to ferroptosis induced by RSL3 and cystine deprivation. These findings demonstrate a critical mechanism by which auraptene exhibits anti-HCC effects via ferroptosis induction and provides a possible therapeutic strategy for HCC by using auraptene or in combination with other ferroptosis inducers.
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Keywords: auraptene ; HCC ; ferroptosis ; SLC7A11
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MDH2 promotes hepatocellular carcinoma growth through ferroptosis evasion via stabilizing GPX4
Yu, Wenjia ; Li, Yingping ; Gao, Chengchang ; Li, Donglin ; Chen, Liangjie ; Dai, Bolei , et al.
Abstract: The crosstalk between tumor progression and ferroptosis is largely unknown. Here, we identify malate dehydrogenase 2 (MDH2) as a key regulator of ferroptosis. MDH2 deficiency inhibits the growth of hepatocellular carcinoma (HCC) cells and enhances their sensitivity to ferroptosis induced by RAS-selective lethal 3 (RSL3), a compound known to cause ferroptosis. MDH2 knock-down enhances RSL3-induced intracellular reactive oxygen species, free iron ions and lipid per-oxides levels, leading to HCC ferroptotic cell death which is rescued by ferrostatin-1 and iron chelator deferiprone. Importantly, the inhibition of HCC cell growth caused by MDH2 deficiency is partially rescued by ferroptosis blockade. Mechanistically, MDH2 resists RSL3-induced ferroptosis sensitivity dependent on glutathione peroxidase 4 (GPX4), an enzyme responsible for scavenging lipid peroxides, which is stabilized by MDH2 in HCC. The protein expressions of MDH2 and GPX4 are positively correlated with each other in HCC cell lines. Furthermore, through our UALCAN website analysis, we found that MDH2 and GPX4 are highly expressed in HCC samples. These findings reveal a critical mechanism by which HCC evades ferroptosis via MDH2-mediated stabilization of GPX4 to promote tumor progression and underscore the potential of MDH2 inhibition in combi-nation with ferroptosis inducers for the treatment of HCC.
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Keywords: tumor progression ; ferroptosis ; MDH2 ; HCC
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Yang, Haoying ; Dai, Bolei ; Chen, Liangjie ; Li, Yingping ; Jin, Xiaorui ; Gao, Chengchang , et al.
Abstract: Ferroptosis, a recently elucidated style of regulated cell death, has emerged as a significant area of investigation in cancer biology. Natural active compounds that have anti-cancer effects are promising candidates for cancer prevention. Iberverin, a natural compound derived from Brassica oleracea var. capitata, has been shown to exert anti-tumor activities in some cancers. However, its role in hepatocellular carcinoma (HCC) cells and the molecular mechanisms are still poorly understood. In this study, we proved that iberverin can induce intracellular reactive oxygen species (ROS) generation to inhibit cell proliferation and initiate ferroptotic cell death in HCC cells, which can be eradicated by the ferroptosis inhibitor ferrostatin-1 (Fer-1) or deferoxamine mesylate (DFO) and ROS scavenger (GSH or NAC). Mechanistically, iberverin treatment can simultaneously downregulate SLC7A11 mRNA level and degrade GPX4 through the ubiquitination pathway, leading to lipid peroxidation and ferroptotic cell death in HCC cells. Significantly, a low dose of iberverin can remarkably increase the sensitivity of HCC cells to ferroptosis induced by canonical ferroptosis inducers RSL3 and imidazole ketone erastin (IKE). This study uncovers a critical function of iberverin in preventing HCC through ferroptosis and provides a promising strategy for HCC treatment either via iberverin alone or in combination with canonical ferroptosis inducers in the future.
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Keywords: iberverin ; HCC ; ferroptosis ; GPX4 ; SLC7A11
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ALOX5 deficiency contributes to bladder cancer progression by mediating ferroptosis escape
Liu, Tianyao ; Xu, Xinyan ; Li, Jiazheng ; Bai, Ming ; Zhu, Wenjie ; Liu, Yanqing , et al.
Abstract: Ferroptosis is an iron-dependent form of regulated cell death driven by the lethal lipid peroxides. Previous studies have demonstrated that inducing ferroptosis holds great potential in cancer therapy, especially for patients with traditional therapy failure. However, cancer cells can acquire ferroptosis evasion during progression. To date, the therapeutic potential of inducing ferroptosis in bladder cancer (BCa) remains unclear, and whether a ferroptosis escape mechanism exists in BCa needs further investigation. This study verified that low pathological stage BCa cells were highly sensitive to RSL3-induced ferroptosis, whereas high pathological stage BCa cells exhibited obviously ferroptosis resistance. RNA-seq, RNAi-mediated loss-of-function, and CRISPR/Cas9 experiments demonstrated that ALOX5 deficiency was the crucial factor of BCa resistance to ferroptosis in vitro and in vivo. Mechanistically, we found that ALOX5 deficiency was regulated by EGR1 at the transcriptional level. Clinically, ALOX5 expression was decreased in BCa tissues, and its low expression was associated with poor survival. Collectively, this study uncovers a novel mechanism for BCa ferroptosis escape and proposes that ALOX5 may be a valuable therapeutic target and prognostic biomarker in BCa treatment.
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| CAS No. : | 1219810-16-8 |
| Formula : | C23H21ClN2O5 |
| M.W : | 440.88 |
| SMILES Code : | O=C([C@H]1CC2=C([C@H](C3=CC=C(C(OC)=O)C=C3)N1C(CCl)=O)NC4=C2C=CC=C4)OC |
| Synonyms : |
(1S,3R)-RSL3
|
| English Name : | (1S,3R)-Methyl 2-(2-chloroacetyl)-1-(4-(methoxycarbonyl)phenyl)-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indole-3-carboxylate |
| MDL No. : | MFCD30187526 |
| InChI Key : | TXJZRSRTYPUYRW-NQIIRXRSSA-N |
| Pubchem ID : | 1750826 |
In Vitro:
| Concentration | Treated Time | Description | References |
| HT22 mouse hippocampal neuronal cells | 80 nM | 24 hours | To evaluate the protective effect of catechol estrogens against erastin/RSL3-induced ferroptosis. Results showed that four catechol estrogens (2-OH-E1, 2-OH-E2, 4-OH-E1, and 4-OH-E2) significantly inhibited erastin/RSL3-induced cell death and reduced the accumulation of NO, ROS, and lipid-ROS. | Sci Rep. 2024 Oct 14;14(1):23988. |
| Pfa1 Gpx4-KO cells | 300 nM | 24 hours | To assess the sensitivity of FSP1 mutants to ferroptosis | Nat Struct Mol Biol. 2023 Nov;30(11):1806-1815. |
| Pfa1 cells | 500 nM | 2 days | To identify possible mutations affecting FSP1's ferroptosis-suppressive function | Nat Struct Mol Biol. 2023 Nov;30(11):1806-1815. |
| synovial fibroblasts | 0.125 μM | 12 hours | Evaluate the effect of RSL3 on ferroptosis in synovial fibroblasts, results showed RSL3 induced cell death and lipid peroxidation | Nat Commun. 2022 Feb 3;13(1):676 |
| C4-2 | 0.5 μM | 9 days | To evaluate the effect of RSL3 on prostate cancer cell colony formation, results showed that RSL3 significantly inhibited colony formation of prostate cancer cells. | Cancer Res. 2021 Mar 15;81(6):1583-1594. |
| PC3 | 0.125, 0.25, 0.5 μM | 72 h | To evaluate the effect of RSL3 on prostate cancer cell growth, results showed that all prostate cancer cell lines were sensitive to RSL3-induced ferroptosis. | Cancer Res. 2021 Mar 15;81(6):1583-1594. |
| DU145 | 0.125, 0.25, 0.5, 1, 2, and 4 μM | 72 h | To evaluate the effect of RSL3 on prostate cancer cell growth, results showed that all prostate cancer cell lines were sensitive to RSL3-induced ferroptosis. | Cancer Res. 2021 Mar 15;81(6):1583-1594. |
| G-361 | 0.5 µM | 24 h | RSL3 induced ferroptosis in G-361 cells, and overexpression of miR-137 suppressed RSL3-induced ferroptosis | Cell Death Differ. 2018 Aug;25(8):1457-1472. |
| A375 | 0.1 µM | 24 h | RSL3 induced ferroptosis in A375 cells, and overexpression of miR-137 suppressed RSL3-induced ferroptosis | Cell Death Differ. 2018 Aug;25(8):1457-1472. |
| HT29 cells | 3 µM | 24 h | RSL3 induced ferroptosis by increasing ROS levels and the cellular labile iron pool, leading to cell death. | Front Pharmacol. 2018 Nov 22;9:1371. |
| LoVo cells | 3 µM | 24 h | RSL3 induced ferroptosis by increasing ROS levels and the cellular labile iron pool, leading to cell death. | Front Pharmacol. 2018 Nov 22;9:1371. |
| HCT116 cells | 3 µM | 24 h | RSL3 induced ferroptosis by increasing ROS levels and the cellular labile iron pool, leading to cell death. | Front Pharmacol. 2018 Nov 22;9:1371. |
| PANC1 cells | 0.5 µM | 6 h | RSL3 induced cell death and lipid peroxidation in PANC1 cells, and CQ inhibited RSL3-induced cell death and lipid peroxidation. | Autophagy. 2021 Nov;17(11):3361-3374. |
| HT1080 cells | 0.5 µM | 6 h | RSL3 induced cell death and lipid peroxidation in HT1080 cells, and CQ inhibited RSL3-induced cell death and lipid peroxidation. | Autophagy. 2021 Nov;17(11):3361-3374. |
| 60 human cancer cell lines | 0.5 µM | 3 and 6 h | RSL3 exhibits wider and stronger activity in the upregulation of MAP1LC3B-II or downregulation of SQSTM1 in 80% (48/60) or 63% (38/60) of cell lines, respectively. Both RSL3 and erastin failed to affect SLC7A11 expression, but they led to GPX4 downregulation in 12% (7/60) and 3% (2/60) of cell lines, respectively. | Autophagy. 2021 Nov;17(11):3361-3374. |
| SH-SY5Y cells | 20 μM | To evaluate the inhibitory effect of RSL3 and erastin-induced ferroptosis in SH-SY5Y cells, it was found that ThA increased cell viability in a dose-dependent manner at concentrations of 2-16 μM. | Theranostics. 2024 Sep 23;14(16):6161-6184. | |
| PC-12 cells | 0.5 μM | 4, 8, 12, 24 h | To evaluate the inhibitory effect of RSL3-induced ferroptosis in PC-12 cells, it was found that ThA significantly increased cell survival and effectively prevented ferroptosis at all observed time points. | Theranostics. 2024 Sep 23;14(16):6161-6184. |
| Huh7 | 5 μM | RSL3 significantly suppressed the viability of Huh7 cells and induced ferroptosis. | Drug Des Devel Ther. 2021 Sep 18;15:3965-3978. | |
| HepG2 | 5 μM | RSL3 significantly suppressed the viability of HepG2 cells and induced ferroptosis. | Drug Des Devel Ther. 2021 Sep 18;15:3965-3978. | |
| human epidermal keratinocytes (HEKa) | 10 μM | 6 h | To assess the effect of RSL3 on cell viability, results showed that RSL3 treatment significantly reduced cell viability. | J Clin Invest. 2024 Nov 21;135(2):e183219. |
| EOC 20 microglial cells | 500 nM | 5 h | To test the sensitivity to RSL3-induced ferroptosis, M1-activated cells showed high resistance to RSL3, while M0 and M2-activated cells were susceptible. | Nat Chem Biol. 2020 Mar;16(3):278-290. |
| Bone marrow-derived macrophages (BMDM) | 500 nM | 5 h | To test the sensitivity to RSL3-induced ferroptosis, M1-activated cells showed high resistance to RSL3, while M0 and M2-activated cells were susceptible. | Nat Chem Biol. 2020 Mar;16(3):278-290. |
| RAW 264.7 macrophages | 500 nM | 5 h | To test the sensitivity to RSL3-induced ferroptosis, M1-activated cells showed high resistance to RSL3, while M0 and M2-activated cells were susceptible. | Nat Chem Biol. 2020 Mar;16(3):278-290. |
| HK2 cells | 1 μM | 24 h | RSL3 induces ferroptosis in HK2 cells, leading to increased lipid peroxidation and cell death. | Redox Biol. 2023 Dec;68:102939. |
In Vivo:
| Administration | Dosage | Frequency | Description | References |
| NSG (NOD-SCID-IL2R γ) male mice | Prostate cancer xenograft model | Intraperitoneal injection | 100 mg/kg | Twice per week, until the end of the experiment | To evaluate the effect of RSL3 on tumor growth in prostate cancer xenograft models, results showed that RSL3 significantly delayed tumor growth of DU145 and PC3 xenografts. | Cancer Res. 2021 Mar 15;81(6):1583-1594. |
| Nude mice | Subcutaneous tumor model | Intraperitoneal injection | 15 mg/kg | Twice every other day for 20 days | Knockdown of miR-137 enhanced erastin-induced ferroptosis and inhibited tumor growth | Cell Death Differ. 2018 Aug;25(8):1457-1472. |
| Mice | C57BL/6 mice | Topical application | 10 μM (120 μL) | Twice daily for 2 weeks | To evaluate the effect of RSL3 on mouse skin, results showed that RSL3 increased ferroptosis-specific oxPE species in the epidermis but did not induce psoriasis-like epidermal changes or immune cell infiltration. | J Clin Invest. 2024 Nov 21;135(2):e183219. |
| Mice | Zymosan-induced peritonitis model | Intraperitoneal injection | 40 mg/kg | Single injection, lasting 5 hours | To test the effect of RSL3 on M2 macrophages in vivo, RSL3 significantly reduced the number of M2 macrophages, while the NO donor DETA NONOate suppressed this reduction. | Nat Chem Biol. 2020 Mar;16(3):278-290. |
| Mice | AKI-CKD model | Intraperitoneal injection | 5 mg/kg | Daily for 14 days | RSL3 pretreatment significantly increased renal fibrosis and ferroptosis in AKI-CKD model mice and abrogated the anti-ferroptosis and renoprotective effects of RGFP966. | Redox Biol. 2023 Dec;68:102939. |
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