【摘要】 目的 探讨ZDE-1与自噬接头蛋白SQSTM1/p62的靶向调控关系，明确其通过调控SQSTM1/p62介导的自噬流缓解心肌纤维化的分子机制。方法 采用CCK-8法检测ZDE-1对心肌细胞活力的影响；免疫荧光技术检测ZDE-1干预后心肌细胞内p62蛋白成点数量的变化。为明确ZDE-1对细胞自噬流的调控效应，以p62和LC3-Ⅱ为自噬流核心检测指标，结合自噬通路特异性抑制剂(溶酶体降解抑制剂BafA1、自噬起始抑制剂3-MA)进行干预，采用蛋白免疫印迹法检测各指标表达变化。采用蛋白免疫印迹技术检测ZDE-1处理后细胞中p62、LC3-II以及p-p70S6K、p-AMPK的表达水平；实验分为以下各组：空白对照组、2.5μmol/L ZDE-1单独处理组、2.5μmol/L ZDE-1+50 nmol/L BafA1联合处理组、2.5μmol/L ZDE-1+100nmol/L Rapa联合处理组、2.5μmol/L ZDE-1+10 mmol/L 3-MA联合处理组。在抗纤维化实验中，采用蛋白免疫印迹法检测不同处理组细胞中CollagenⅠ和α-SMA的表达水平，实验分为：空白对照组、10 ng/mL TGF-β单独处理组，以及2.5、5、10μmol/L ZDE-1分别联合10 ng/mL TGF-β处理组。结果 以2.5、5、10、20μmol/L ZDE-1干预4 h为诱导心肌细胞自噬的最优方案。与对照组相比，ZDE-1处理后p62小体成点数量明显增加(P<0.05),p62蛋白表达水平下调(P<0.05),LC3-Ⅱ表达水平上调(P<0.001)。相较于空白对照组，2.5μmol/L ZDE-1与50 nmol/L BafA1联合处理组的LC3-Ⅱ表达上调(P<0.01),2.5μmol/L ZDE-1与10 mmol/L 3-MA联合处理组的LC3-Ⅱ表达量下调(P<0.01)。p62蛋白表达方面，2.5μmol/L ZDE-1与BafA1联合处理组p62表达上调(P<0.05),2.5μmol/L ZDE-1与3-MA联合处理组p62表达下调(P<0.01)。相较于空白对照组，ZDE-1单独处理组(P<0.01)、ZDE-1与BafA1联合处理组(P<0.001)、ZDE-1与Rapa联合处理组(P<0.001)以及ZDE-1与3-MA联合处理组(P<0.05)中蛋白p-p70S6K表达量均降低。相较于空白对照组，在ZDE-1单独处理组(P<0.01)、ZDE-1与BafA1联合处理组(P<0.001)、ZDE-1与Rapa联合处理组(P<0.01)以及ZDE-1与3-MA联合处理组(P<0.01)中蛋白p-AMPK表达量均升高。在TGF-β诱导的心肌成纤维细胞中，2.5μmol/L ZDE-1能够明显降低α-SMA和CollagenⅠ的蛋白表达，使其恢复到正常水平(P<0.05)。结论 2.5μmol/L浓度的ZDE-1干预心肌细胞无细胞毒性，其可通过下调p62表达水平、增加p62小体成点数量，经AMPK通路激活SQSTM1/p62介导的自噬并维持自噬流通畅，进而显著抑制TGF-β诱导的心肌成纤维细胞纤维化，缓解心肌纤维化进程。更多还原

【Abstract】 Objective To investigate the targeted regulatory relationship between ZDE-1 and the autophagy adaptor protein SQSTM1/p62, and to clarify the cellular and molecular mechanism by which it alleviates myocardial fibrosis by regulating the autophagic flux mediated by SQSTM1/p62. Methods The effect of ZDE-1 on cardiomyocyte proliferation was detected using the CCK-8 assay; in the number of p62 punctain cardiomyocytes after ZDE-1 intervention were detected by immunofluorescence. To clarify the regulatory effect of ZDE-1 on cellular autophagic flux, p62 and LC3-II were used as core indicators for autophagic flux detection. Interventions were combined with autophagy pathway-specific inhibitors(the lysosomal degradation inhibitor BafA1 and autophagy initiation inhibitor 3-MA), and changes in the expression of each indicator were detected by western blotting. Western blotting was used to detect the expression levels of p62, LC3-II, p-p70S6K, and p-AMPK in cells treated with ZDE-1. The cells were grouped into blank control group, 2.5 μmol/L ZDE-1 alone, 2.5 μmol/L ZDE-1+50 nmol/L BafA1 co-treatment group, 2.5 μmol/L ZDE-1+100 nmol/L Rapaco-treatment group, and 2.5 μmol/L ZDE-1+10 mmol/L 3-MA co-treatment group, respectively. In the anti-fibrotic experiment, Western blotting was used to detect the expression levels of Collagen I and α-SMA in cells from different treatment groups. The experiment was divided into: control group, 10 ng/mLTGF-β alone treatment group, and groups treated with 2.5, 5, and 10 μmol/L ZDE-1 combined with 10 ng/mL TGF-β, respectively. Results Intervention with 2.5, 5, 10, and 20 μmol/L ZDE-1 for 4 h was identified as the optimal protocol for inducing autophagy in cardiomyocytes. Compared to the blank control group, the number of p62 puncta increased significantly(P<0.05), p62 protein expression levels were downregulated(P<0.05), and the expression level of LC3-II increased(P<0.001) after ZDE-1 treatment. Compared with the control group, LC3-II expression was significantly up-regulated in the 2.5 μmol/L ZDE-1 and 50 nmol/L BafA1 co-treatment group(P<0.01), while it was significantly reduced in the 2.5 μmol/L ZDE-1+10 mmol/L 3-MA co-treatment group(P<0.01). Regarding p62 protein expression, the 2.5 μmol/L ZDE-1 and BafA1 group showed a significant increase(P<0.05), while the 2.5 μmol/L ZDE-1 and 3-MA group showed a significant downregulation(P< 0.01). Compared to the blank control group, p-p70S6K protein expression levels were reduced in the ZDE-1 monotherapy group(P<0.01), the ZDE-1 and BafA1 group(P<0.001), the ZDE-1 and Rapa group(P<0.001), and the ZDE-1 and 3-MA group(P<0.05). Compared with the blank control group, p-AMPK protein expression levels were increased in the ZDE-1 monotherapy group(P<0.01), the ZDE-1 and BafA1 group(P<0.001), the ZDE-1 and Rapa group(P<0.01), and the ZDE-1 and 3-MA group(P<0.01). In TGF-β-induced cardiac fibroblasts, 2.5 μmol/L ZDE-1 significantly reduced the protein expression levels of α-SMA and Collagen I, restoring them to normal levels(P<0.05). Conclusion ZDE-1 at a concentration of 2.5 μmol/L exhibited no cytotoxicity in cardiomyocytes. By downregulating p62 expression levels and increasing the number of p62 puncta, it activated SQSTM1/p62-mediated autophagy via the AMPK pathway and maintained unobstructed autophagic flux, thereby significantly inhibiting TGF-β-induced fibrosis in cardiac fibroblasts and alleviating the progression of myocardial fibrosis.更多还原