橙皮素通过调控AMPK/NLRP3通路减轻阿霉素诱导的小鼠心肌毒性

doi:10.12122/j.issn.1673-4254.2025.09.05

摘要/Abstract

摘要：

目的探讨橙皮素（Hes）通过调控AMP依赖的蛋白激酶（AMPK）/NOD热蛋白结构域相关蛋白3（NLRP3）信号减轻炎症反应改善阿霉素（DOX）诱导的心肌损伤。方法采用DOX处理C57/bl6小鼠和H9c2细胞，并随机分为：对照组/假手术组、DOX处理组、Hes干预DOX组（DOX+Hes）以及Hes联合AMPK抑制剂Compound C干预DOX组（DOX+Hes+CC）。观察细胞形态变化，采用CCK-8法检测细胞活力，超声检测心脏功能，ELISA法检测培养基和心肌组织中LDH活性，TUNEL染色法检测细胞凋亡，RT-PCR检测TNF-α、IL-6和IL-1β的mRNA水平，Western blotting法检测cleaved caspase-3、Bcl2、Bax、IL-1β、IL-18、p-AMPK、AMPK、p-mTOR、mTOR、NLRP3、ASC和caspase-1的表达。结果与对照组相比，DOX组细胞肿胀，活力降低，培养基中LDH活性增加（P<0.01）；cleaved caspase-3表达和TUNEL染色阳性细胞数增加，Bcl2/Bax比值降低（P<0.01）。与假手术组相比，DOX组心肌纤维肿胀并出现炎性浸润，心脏功能降低，LDH活性增加；同时TNF-α、IL-6和IL-1β的mRNA水平以及IL-1β和IL-18的蛋白表达增加（P<0.01），p-AMPK和p-mTOR蛋白表达降低，NLRP3、ASC和caspase-1的表达增加（P<0.01）。与DOX组相比，DOX+Hes组细胞肿胀减轻，活力增加，培养基中LDH活性降低（P<0.01）；cleaved caspase-3表达和TUNEL染色阳性细胞数减少，Bcl2/Bax比值增加（P<0.01）；TNF-α、IL-6和IL-1β的mRNA水平以及IL-1β和IL-18的蛋白表达降低（P<0.01）；p-AMPK和p-mTOR蛋白表达增加，NLRP3、ASC和caspase-1的表达降低（P<0.01）。与DOX+Hes组相比，Compound C可以阻断Hes对DOX诱导的细胞损伤、心脏功能、炎症反应和AMPK/NLRP3通路的作用。结论 Hes通过调控AMPK/NLRP3通路抑制炎症反应减轻DOX诱导的心脏毒性。

关键词: 橙皮素, 阿霉素, AMPK/NLRP3, 炎症反应, H9c2细胞

Abstract:

Objective To verify whether hesperetin (Hes) alleviates doxorubicin (DOX)-induced cardiotoxicity by reducing inflammation via regulating the AMPK/NLRP3 pathway. Methods C57/bl6 mice and H9c2 cells treated with DOX to mimic cardiotoxicity were randomly divided into Sham (or control) group, DOX group, DOX+Hes group, DOX+Hes+compound C (CC, an AMPK inhibitor) group. Cardiac function and myocardial pathologies of the mice were evaluated, and the changes in H9c2 cell morphology and viability were assessed. Lactate dehydrogenase (LDH) activity in mouse myocardial tissues and H9c2 cells was measured using ELISA, and H9c2 cell apoptosis was detected with TUNEL staining. In both H9c2 cells and the myocardial tissues of the mice, cellular expression levels of TNF-α, IL-6 and IL-1β mRNAs and cleaved caspase-3, Bcl2, Bax, IL-1β, IL-18, p-AMPK, AMPK, p-mTOR, mTOR, NLRP3, ASC and caspase-1 proteins were detected using RT-PCR and Western blotting. Results DOX treatment caused cell swelling, decreased cell viability and increased LDH activity in H9c2 cells, resulting also in significantly increased cell apoptosis and cleaved caspase-3 expression and decreased Bcl2/Bax ratio. The DOX-treated mice showed obvious myocardial fiber swelling and inflammatory infiltration, decreased cardiac function and significantly increased myocardial LDH activity. In H9c2 cells, DOX treatment significantly increased the mRNA expressions of TNF-α, IL-6 and IL-1β and protein expressions of IL-1β and IL-18, lowered the expressions of p-AMPK and p-mTOR, and increased the expressions of NLRP3, ASC and caspase-1. Hes treatment obviously reduced these toxic effects of DOX in H9c2 cells, but its protective effects were blocked by application of compound C. Conclusion Hes reduces DOX-induced cardiotoxicity by inhibiting inflammation via regulating the AMPK/NLRP3 pathway.

Key words: hesperetin, doxorubicin, AMPK/NLRP3, inflammatory response, H9c2 cells

闫爱丽, 罗梦瑶, 常晋瑞, 李新华, 朱娟霞. 橙皮素通过调控AMPK/NLRP3通路减轻阿霉素诱导的小鼠心肌毒性[J]. 南方医科大学学报, 2025, 45(9): 1850-1858.

Aili YAN, Mengyao LUO, Jinrui CHANG, Xinhua LI, Juanxia ZHU. Hesperetin alleviates doxorubicin-induced cardiotoxicity by regulating the AMPK/NLRP3 pathway[J]. Journal of Southern Medical University, 2025, 45(9): 1850-1858.

图/表 7

图1 Compound C阻断Hes对DOX引起的细胞损伤的改善作用

Fig.1 Compound C blocks the protective effects of Hes against DOX-induced cell injury in H9c2 cells. A: Cell morphology in each group (Original magnification: ×100). B: Comparison of cell viability among the 4 groups. C: LDH activity in the cells in the 4 groups. **P<0.01 vs Control group; ##P<0.01 vs DOX group; &&P<0.01 vs DOX+Hes group (n=6).

图2 Compound C阻断Hes对DOX引起的细胞凋亡的改善作用

Fig.2 Compound C reduces the protective effect of Hes against DOX-induced cell apoptosis.A: Representative Western blots of Bcl2, Bax, cleaved caspase-3 and GAPDH in each group. B: Bcl2/Bax ratios in each group. C: Cleaved caspase-3 expression levels in each group. D: TUNEL staining of the cells in each group (Scale bar=100 μm). **P<0.01 vs Control group; ##P<0.01 vs DOX group; &&P<0.01 vs DOX+Hes group (n=6).

图3 Compound C阻断Hes对DOX引起的细胞炎症的改善作用

Fig.3 Compound C attenuates the protective effect of Hes against DOX-induced inflammation. A-C: Relative mRNA expressions of TNF‑α, IL-6 and IL-1β in each group. D: Representative Western blots of TNF‑α, IL-6, IL-1β and GAPDH in each group. E, F: Relative expression levels of IL-1β and IL-18 in the cells in each group. **P<0.01 vs Control group; ##P<0.01 vs DOX group; &&P<0.01 vs DOX+Hes group (n=6).

图4 Compound C阻断Hes对DOX引起的AMPK/NLRP3表达的变化

Fig.4 Compound C blocks the effect of Hes against DOX-induced changes in protein expressions of AMPK/NLRP3 signaling. A: Representative Western blots of p-AMPK, AMPK, p-mTOR, mTOR, NLRP3, ASC, caspase-1 and GAPDH in each group. B-F: Relative expression levels of p-AMPK, p-mTOR, NLRP3, ASC, and caspase-1 in each group. **P<0.01 vs Control group; ##P<0.01 vs DOX group; &&P<0.01 vs DOX+Hes group (n=6).

图5 Compound C阻断Hes对DOX引起的心肌损伤的改善作用

Fig.5 Compound C blocks the protective effect of Hes against DOX-induced myocardial injury in mice. A: HE staining of the myocardial tissues in each group (×200). B: Myocardial LDH activity in each group. C: LVEF in each group. D: LVFS in each group. **P<0.01 vs Sham group; ##P<0.01 vs DOX group; &&P<0.01 vs DOX+Hes group (n=6).

图6 Compound C阻断Hes对DOX引起炎症的改善作用

Fig.6 Compound C attenuates the protective effect of Hes against DOX-induced inflammation in the myocardial tissues of the mice. A-C: Relative mRNA expressions of TNF-α, IL-6 and IL-1β in each group. D: Representative Western blots of IL-1β, IL-18 and GAPDH. E, F: Relative expression levels of IL-1β and IL-18 in each group. **P<0.01 vs Sham group; ##P<0.01 vs DOX group; &&P<0.01 vs DOX+Hes group (n=6).

图7 Compound C阻断Hes对DOX引起的小鼠心肌AMPK/NLRP3表达的变化

Fig.7 Compound C blocks the effect of Hes against DOX-induced changes in expression of AMPK/NLRP3 signaling in the mice. A: Representative Western blots of p-AMPK, AMPK, p-mTOR, mTOR, NLRP3, ASC, caspase-1 and GAPDH. B-F: Relative expression levels of p-AMPK, p-mTOR, NLRP3, ASC, and caspase-1 in the myocardial tissues of the mice in each group. **P<0.01 vs Sham group; ##P<0.01 vs DOX group; &&P<0.01 vs DOX+Hes group (n=6).

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