Curcumin alleviates septic lung injury in mice by inhibiting TXNIP/TRX-1/GPX4-mediated ferroptosis

doi:10.12122/j.issn.1673-4254.2024.09.21

Abstract

Abstract:

Objective To investigate whether curcumin alleviates septic lung injury by inhibiting ferroptosis through modulating the TXNIP/TRX-1/GPX4 pathway. Methods Male C57BL/6 mice were randomly divided into Sham group, cecal ligation puncture (CLP)-induced sepsis group, CLP with curcumin treatment (50, 100, and 200 mg/kg) groups, and CLP with both curcumin (200 mg/kg) and TRX-1 inhibitor PX-12 (25 mg/kg) treatment group. Inflammatory factors, MDA, MPO, and GSH levels in the lung tissue of the mice were detected. Beas-2B cells stimulated with lipopolysaccharide (LPS; 1 μg/mL) were treated with 2.5, 5, or 10 μmol/L curcumin or with 10 μmol/L curcumin combined with 5 μmol/L PX-12, and the changes in MDA, Fe²⁺ and ROS levels were assessed. Western blotting was performed to detect the protein expressions of TXNIP, TRX-1, GPX4 and X-CT in both the mouse lung tissues and Beas-2B cells. Results The mice with CLP-induced sepsis showed severe lung injury with elevated expressions of IL-6, IL-1β, TNF-α, MDA and MPO and decreased GSH expression. In Beas-2B cells, LPS stimulation significantly increased MDA and Fe²⁺ levels and ROS release, increased TXNIP protein expression, and lowered the protein expression levels of TRX-1, GPX4 and X-CT, and these changes were also observed in the septic mice. Curcumin treatments at different concentrations obviously alleviated lung injury in the septic mice and reduced LPS-induced injury in Beas-2B cells. Curcumin significantly decreased the release of inflammatory factors, MDA and MPO, increased GSH level, lowered Fe²⁺, MDA and ROS levels, increased TXNIP protein expression, and lowered the protein expressions of TRX-1, GPX4 and X-CT in both septic mouse lung tissues and LPS-stimulated Beas-2B cells. The protective effect of curcumin was effectively blocked by PX-12 treatment. Conclusion Curcumin inhibits ferroptosis and alleviates septic lung injury in mice by elevating TRX-1 and GPX4 and decreasing TXNIP in the lung tissue.

Key words: sepsis lung injury, curcumin, ferroptosis, TXNIP/TRX-1/GPX4 pathway

Kai CHEN, Zhaofei MENG, Jingting MIN, Jiahui WANG, Zhenghong LI, Qin GAO, Junfeng HU. Curcumin alleviates septic lung injury in mice by inhibiting TXNIP/TRX-1/GPX4-mediated ferroptosis[J]. Journal of Southern Medical University, 2024, 44(9): 1805-1813.

Figures/Tables 10

Fig.1 HE staining of lung tissue of the mice and lung injury scores in each group. A: HE staining of paraffin sections of the lung tissues. B: Comparison of lung injury scores among the groups. **P<0.01 vs Sham group; #P<0.05, ##P<0.01 vs CLP group; &&P<0.01 vs CLP+Cur-H group (Mean±SD, n=6).

Fig.2 Expressions of IL-1β (A), IL-6 (B) and TNF-α (C) in the lung tissues of the mice in each group. **P<0.01 vs Sham group; #P< 0.05, ##P<0.01 vs CLP group; &P<0.05, &&P<0.01 vs CLP+Cur-H group (Mean±SD, n=6).

Fig.3 Determination of MDA （A）, MPO （B） and GSH （C） levels in the lung tissues of the mice in each group. **P<0.01 vs Sham group; #P<0.05, ##P<0.01 vs CLP group; &&P<0.01 vs CLP+Cur-H group （Mean±SD, n=6）.

Fig.4 Results of Western blotting in each group. A： Western blots of TXNIP, TRX-1, GPX4 and X-CT proteins in the lung tissues of the mice in each group. B-E： Quantitative analysis of the protein levels. **P<0.01 vs Sham group; ##P<0.01 vs CLP group; &&P<0.01 vs CLP+Cur-H group （Mean±SD, n=3）.

Fig.5 CCK8 assay for determining the optimal concentration and time of LPS for cell treatment. A： Cell viability assay after LPS treatment at 0, 0.1, 1, 5 and 10 μg/mL for 24 h. B： Cell viability assay after LPS treatment at 0, 0.1, 1, 5 and 10 μg/mL concentrations for 6, 12 and 24 h. *P<0.05, **P<0.01 vs 0 μg/mL group （Mean±SD, n=4）.

Fig.6 Effects of curcumin and PX-12 on cell viability. A: Effect of curcumin on cell viability; B: Effect of PX-12 on cell viability. *P<0.05, **P<0.01 vs 0 μg/mL group (Mean±SD, n=4)

Fig.7 CCK8 assay of cell viability in each group of cells. **P<0.01 vs CON;##P<0.01 vs LPS; &&P<0.01 vs LPS+Cur-H (Mean±SD, n=4).

Fig.8 Changes in MDA （A） and Fe2+ （B） levels in the cells in each group. **P<0.01 vs control; #P<0.05, ##P<0.01 vs LPS; &&P<0.01 vs LPS+Cur-H （Mean±SD, n=4）.

Fig.9 ROS immunofluorescence and fluorescence intensity in each group of cells. A: Immunofluorescence images of Beas-2B cells in each group. B: Comparison of ROS fluorescence intensity of the cells. **P<0.05 vs control group; ##P<0.01 vs LPS group; &&P<0.01 vs LPS+Cur-H group (Mean±SD, n=4).

Fig.10 Western blotting for the proteins in the TXNIP/TRX-1/GPX4 pathways in each group of cells. A: Western blots of the proteins. B-E: Quantitative analysis of the protein expression levels. **P<0.01 vs control group; ##P<0.01 vs LPS group; &&P<0.01 vs LPS+Cur-H group （Mean±SD, n=3）.

References 31

1	Sun BS, Lei MX, Zhang JQ, et al. Acute lung injury caused by sepsis: how does it happen[J]? Front Med, 2023, 10: 1289194.
2	Li WL, Li D, Chen YS, et al. Classic signaling pathways in alveolar injury and repair involved in sepsis-induced ALI/ARDS: new research progress and prospect[J]. Dis Markers, 2022, 2022: 6362344.
3	Wang YF, Zhao ZJ, Xiao ZY. The emerging roles of ferroptosis in pathophysiology and treatment of acute lung injury[J]. J Inflamm Res, 2023, 16: 4073-85.
4	Chen Y, Fang ZM, Yi X, et al. The interaction between ferroptosis and inflammatory signaling pathways[J]. Cell Death Dis, 2023, 14(3): 205.
5	Huo L, Liu CF, Yuan YJ, et al. Pharmacological inhibition of ferroptosis as a therapeutic target for sepsis-associated organ damage[J]. Eur J Med Chem, 2023, 257: 115438.
6	Zhang J, Zheng YP, Wang Y, et al. YAP1 alleviates sepsis-induced acute lung injury via inhibiting ferritinophagy-mediated ferroptosis[J]. Front Immunol, 2022, 13: 884362.
7	Zhang XY, Liu CM, Ma YH, et al. The TXNIP/Trx-1/GPX4 pathway promotes ferroptosis in hippocampal neurons after hypoxia-ischemia in neonatal rats[J]. Chin J Contemp Pediatr, 2022, 24(9): 1053-60.
8	Sadeghi M, Dehnavi S, Asadirad A, et al. Curcumin and chemokines: mechanism of action and therapeutic potential in inflammatory diseases[J]. Inflammopharmacology, 2023, 31(3): 1069-93.
9	Wang Y, Wang YJ, Cai N, et al. Anti-inflammatory effects of curcumin in acute lung injury: in vivo and in vitro experimental model studies[J]. Int Immunopharmacol, 2021, 96: 107600.
10	Yuan R, Li YQ, Han S, et al. Fe-curcumin nanozyme-mediated reactive oxygen species scavenging and anti-inflammation for acute lung injury[J]. ACS Cent Sci, 2022, 8(1): 10-21.
11	Zheng YX, Wang JP, Ling ZY, et al. A diagnostic model for sepsis-induced acute lung injury using a consensus machine learning approach and its therapeutic implications[J]. J Transl Med, 2023, 21(1): 620.
12	Tao HP, Shen LH. Research progress of curcumin in the treatment of sepsis[J]. Shock, 2024, 61(6): 805-16.
13	Liu RF, Fang XH, Meng C, et al. Lung inflation with hydrogen during the cold ischemia phase decreases lung graft injury in rats[J]. Exp Biol Med, 2015, 240(9): 1214-22.
14	Zhou X, Liao YX. Gut-lung crosstalk in sepsis-induced acute lung injury[J]. Front Microbiol, 2021, 12: 779620.
15	Zhang H, Liu JL, Zhou YL, et al. Neutrophil extracellular traps mediate m⁶A modification and regulates sepsis-associated acute lung injury by activating ferroptosis in alveolar epithelial cells[J]. Int J Biol Sci, 2022, 18(8): 3337-57.
16	Wang W, Xu RL, He P, et al. CircEXOC5 aggravates sepsis-induced acute lung injury by promoting ferroptosis through the IGF₂BP₂/ATF3 axis[J]. J Infect Dis, 2024, 229(2): 522-34.
17	Ahmad S, Zaki A, Manda K, et al. Vitamin-D ameliorates sepsis-induced acute lung injury via augmenting miR-149-5p and downregulating ER stress[J]. J Nutr Biochem, 2022, 110: 109130.
18	Jiang X, Stockwell BR, Conrad M. Ferroptosis: mechanisms, biology and role in disease[J]. Nat Rev Mol Cell Biol, 2021, 22(4):266-82.
19	Shi X, Han B, Zhang B, et al. Schisandra chinensis polysaccharides prevent cardiac hypertrophy by dissociating thioredoxin-interacting protein/thioredoxin-1 complex and inhibiting oxidative stress[J]. Biomed Pharmacother, 2021, 139:111688.
20	Lv HM, Zhu C, Wei W, et al. Enhanced Keap1-Nrf2/Trx-1 axis by daphnetin protects against oxidative stress-driven hepatotoxicity via inhibiting ASK1/JNK and Txnip/NLRP3 inflammasome activation[J]. Phytomedicine, 2020, 71: 153241.
21	Choi EH, Park SJ. TXNIP: A key protein in the cellular stress response pathway and a potential therapeutic target[J]. Exp Mol Med, 2023, 55(7): 1348-56.
22	Chen YF, Yin HW, Sun J, et al. TrxR/Trx inhibitor butaselen ameliorates pulmonary fibrosis by suppressing NF‑κB/TGF‑β1/Smads signaling[J]. Biomedecine Pharmacother, 2023, 169: 115822.
23	周滟, 李晓茜, 汪思琴. 血清OSM、Trx-1及HBP与脓毒症严重程度相关性及对临床转归的预测研究[J].临床和实验医学杂志, 2024, 23(5): 473-7.
24	Shen K, Wang XJ, Wang YW, et al. MiR-125b-5p in adipose derived stem cells exosome alleviates pulmonary microvascular endothelial cells ferroptosis via Keap1/Nrf2/GPX4 in sepsis lung injury[J]. Redox Biol, 2023, 62: 102655.
25	Kumar A, Harsha C, Parama D, et al. Current clinical developments in curcumin-based therapeutics for cancer and chronic diseases[J]. Phytother Res, 2021, 35(12): 6768-801.
26	Shi Y, Wu Q, Lu Y, et al. Arginine-Glycine-aspartic acid-anchored curcumin-based nanotherapeutics inhibit pyroptosis-induced cytokine release syndrome for in vivo and in vitro sepsis applications[J]. Curr Pharm Des, 2023, 29(4): 283-94.
27	Ahmadabady S, Beheshti F, Shahidpour F, et al. A protective effect of curcumin on cardiovascular oxidative stress indicators in systemic inflammation induced by lipopolysaccharide in rats[J]. Biochem Biophys Rep, 2021, 25: 100908.
28	Zhou Y, Gao LP, Xia P, et al. Glycyrrhetinic acid protects renal tubular cells against oxidative injury via reciprocal regulation of JNK-connexin 43-thioredoxin 1 signaling[J]. Front Pharmacol, 2021, 12: 619567.
29	Li WC, Xu XT, Dong DD, et al. Up-regulation of thioredoxin system by puerarin inhibits lipid uptake in macrophages[J]. Free Radic Biol Med, 2021, 162: 542-54.
30	林全德, 闫艳, 刘晴, 等. PX-12促进硼替佐米诱导多发性骨髓瘤细胞H929凋亡的实验研究[J]. 中国实验血液学杂志, 2021, 29(2): 515-9.
31	Lin Y, Chen XW, Yu CC, et al. Radiotherapy-mediated redox homeostasis-controllable nanomedicine for enhanced ferroptosis sensitivity in tumor therapy[J]. Acta Biomater, 2023, 159: 300-11.

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