姜黄素通过抑制TXNIP/TRX-1/GPX4通路介导的铁死亡减轻脓毒症小鼠肺损伤

doi:10.12122/j.issn.1673-4254.2024.09.21

摘要/Abstract

摘要：

目的探究姜黄素（Cur）是否通过调控TXNIP/TRX-1/GPX4通路抑制铁死亡减轻脓毒症肺损伤。方法雄性C57BL/6小鼠随机分为Sham组、脓毒症（盲肠结扎穿孔术，CLP）组、CLP+各浓度Cur（50、100和200 mg/kg）组、CLP+Cur（200 mg/kg）+TRX-1抑制剂PX-12（25 mg/kg）组，10只/组，检测各组小鼠肺组织炎症因子及铁死亡关键因子MDA、MPO、GSH水平。Beas-2B细胞分为Con组、脂多糖组（LPS，1 μg/mL）、LPS+各浓度Cur（2.5、5、10 μmol/L）及LPS+Cur （10 μmol/L）+PX-12 （5 μmol/L）组，检测MDA和Fe²⁺水平，DHE染色评估ROS变化。Western blotting检测肺组织和Beas-2B细胞TXNIP、TRX-1、GPX4和X-CT蛋白表达水平。结果与Sham组相比，CLP组小鼠肺损伤严重，IL-6、IL-1β、TNF-α、MDA、MPO含量增加，GSH含量下降（P<0.01）；与CON组相比，LPS组MDA、Fe²⁺、ROS水平升高；CLP组肺组织和LPS组Beas-2B细胞TXNIP蛋白表达增加，TRX-1、GPX4及X-CT蛋白表达减低（P<0.01）；与CLP组相比，CLP+Cur各浓度组肺损伤减轻，炎症因子和脂质过氧化因子含量降低，GSH水平升高；与LPS组相比，LPS+Cur各浓度组Fe^2＋、MDA和ROS水平下降（P<0.05）；TXNIP蛋白表达降低，TRX-1、GPX4及X-CT蛋白表达增加（P<0.01）。PX-12干预后，取消了Cur对脓毒症肺组织和Beas-2B细胞的保护作用（P<0.05）。结论姜黄素通过升高TRX-1、GPX4，降低TXNIP抑制铁死亡，减轻脓毒症肺损伤。

关键词: 脓毒血症肺损伤, 姜黄素, 铁死亡, TXNIP/TRX-1/GPX4通路

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

陈凯, 孟兆菲, 闵静婷, 王佳慧, 李正红, 高琴, 胡俊锋. 姜黄素通过抑制TXNIP/TRX-1/GPX4通路介导的铁死亡减轻脓毒症小鼠肺损伤[J]. 南方医科大学学报, 2024, 44(9): 1805-1813.

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.

图/表 10

图1 各组小鼠肺组织HE染色和肺损伤评分

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).

图2 各组小鼠肺组织IL-1β、IL-6和TNF-α含量表达结果

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).

图3 各组小鼠MDA、MPO和GSH水平的结果

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）.

图4 各组小鼠Western blotting的结果

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）.

图5 CCK8法检测LPS最适造模浓度和时间结果

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）.

图6 姜黄素和PX-12对细胞活力的影响

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)

图7 各组细胞CCK8活性检测结果

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).

图8 各组细胞MDA和Fe2+含量表达

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）.

图9 各组细胞ROS免疫荧光和荧光强度的结果

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).

图10 各组细胞Western blotting的结果

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）.

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