Molecular Mechanism of Celastrol Intervening in TFEB-Mediated Lysosomal Biogenesis Dysfunction Inhibiting Cataract Formation
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摘要:
目的 探讨雷公藤红素（celastrol，Cel）是否通过激活转录因子EB（transcription factor EB，TFEB）介导的溶酶体生物生成途径，从而抑制白内障的形成。 方法 采用H2O2诱导的人晶状体上皮细胞氧化应激模型和亚硒酸盐诱导的大鼠白内障模型，将细胞分为对照组、模型组、Cel干预组、Cel+TFEB siRNA组或Cel+MHY1485组（n = 5）；45只仔鼠分为对照组、模型组、Cel干预组（n = 9）。采用CCK-8法检测细胞活力；免疫荧光染色观察TFEB核转位；Western blot检测LAMP1、CTSB蛋白表达；LysoTracker Red染色评估溶酶体数量；裂隙灯显微镜观察并分级（Bahmani标准）晶状体混浊程度；HE染色观察晶状体组织形态。同时利用TFEB siRNA和mTOR激活剂（MHY1485）验证Cel的作用机制。 结果 与模型组相比，Cel干预组TFEB核转位效率显著提高了约2.1倍（P < 0.0001 ），显著上调了溶酶体功能相关蛋白的表达（P < 0.001）。LysoTracker Red染色显示，Cel干预使H2O2诱导的溶酶体数量减少得以逆转（P <0.0001 ）。在动物模型中，Cel干预显著降低了晶状体混浊程度（P < 0.05）。上述Cel的积极作用均可被TFEB siRNA或mTOR激活剂MHY1485显著拮抗（P < 0.001）。结论 Cel通过抑制mTOR信号促进TFEB核转位，增强溶酶体生物发生，改善晶状体蛋白稳态，从而抑制白内障形成。 Abstract:Objective This study aims to investigate whether celastrol （Cel） inhibits cataract formation by activating the transcription factor EB （TFEB）-mediated lysosomal biosynthesis pathway. Methods An oxidative stress model in human lens epithelial cells induced by H2O2 and a rat cataract model induced by selenite were employed. Cells were divided into control, model, Cel intervention, Cel + TFEB siRNA, or Cel + MHY1485 groups （n = 5）. 45 neonatal mice were divided into control, model, and Cel intervention groups （n = 9）. Cell viability was assessed by the CCK-8 assay, TFEB nuclear translocation was observed by immunofluorescence staining. Protein expression of LAMP1 and CTSB was detected by Western blot, lysosome quantity was assessed by LysoTracker Red staining. Lens opacity was observed and graded using the Bahmani standard under slit lamp microscopy, and lens morphology was observed by hematoxylin and eosin staining. TFEB siRNA and an mTOR activator （MHY1485） were also used to validate the mechanism of action of Cel. Results Compared with the model group, the Cel intervention group significantly increased TFEB nuclear translocation efficiency by approximately 2.1-fold （P < 0.0001 ） and significantly upregulated the expression of lysosomal function-related proteins （all P < 0.001）. LysoTracker Red staining showed that Cel intervention group reversed the H2O2-induced decrease in lysosome quantity （P <0.0001 ）. In the animal model, Cel intervention group significantly reduced lens opacity （P < 0.05）. These positive effects of Cel were significantly antagonized by TFEB siRNA or the mTOR activator MHY1485 （P < 0.001）.Conclusion Cel promotes TFEB nuclear translocation by inhibiting the mTOR signaling pathway, enhances lysosomal biogenesis, improves lens protein homeostasis, and thereby suppresses cataract formation. This study provides a theoretical basis for non-surgical cataract treatment targeting TFEB. -
Key words:
- Celastrol /
- Transcription factor EB /
- Lysosome /
- Cataract /
- mTOR
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图 1 Cel对正常HLEB3细胞活力的影响（$\bar x \pm s $， n = 5）
Figure 1. Effect of Cel on the viability of normal HLEB3 cells （$\bar x \pm s $，n = 5）
图 2 Cel对H2O2诱导的HLEB3细胞氧化应激模型活力的影响（n = 5）
Figure 2. Effect of Cel on the viability of H2O2-induced oxidative stress in HLEB3 cells （n = 5）
图 3 HLEB3细胞内ROS水平（$\bar x \pm s $，n = 5）
A：HLEB3细胞DCFH-DA荧光染色图；B：HLEB3细胞内ROS水平定量分析；****P < 0.0001。
Figure 3. Intracellular ROS levels in HLEB3 cells （$\bar x \pm s $，n = 5）
图 4 免疫荧光检测HLEB3细胞中TFEB的核转位情况（$\bar x \pm s $，n = 5）
A：TFEB免疫荧光染色HLEB3细胞图；B：HLEB3细胞内TFEB核/质荧光强度比值；****P < 0.0001。
Figure 4. Immunofluorescence detection of TFEB nuclear translocation in HLEB3 cells （$\bar x \pm s $，n = 5）
图 5 HLEB3细胞中LAMP1和CTSB蛋白的表达情况（$\bar x \pm s $，n = 5）
A：Cel给药干预HLEB3细胞后Western blot条带图；B：Cel给药干预HLEB3细胞后LAMP1相对表达水平；C：Cel给药干预HLEB3细胞后CTSB相对表达水平；***P < 0.001，****P < 0.0001。
Figure 5. Expression of LAMP1 and CTSB proteins in HLEB3 cells （$\bar x \pm s $，n = 5）
图 6 HLEB3细胞中溶酶体数量的影响（$\bar x \pm s $，n = 5）
A：HLEB3细胞LysoTracker Red荧光图；B：HLEB3细胞内LysoTracker Red荧光探针相对荧光强度；***P < 0.001；****P < 0.0001。
Figure 6. Effect on lysosome quantity in HLEB3 cells （$\bar x \pm s $，n = 5）
图 7 大鼠给药干预后体重变化和肝肾病理学变化（$\bar x \pm s $，n = 9）
A：大鼠给药干预后体重变化；B：大鼠给药干预后肝肾病理学变化；ns：P > 0.05。
Figure 7. Changes in body weight and histopathological alterations in the liver and kidneys of rats after drug intervention （$\bar x \pm s $，n = 9）
图 8 大鼠晶状体浑浊程度（$\bar x \pm s $，n = 9）
A：大鼠晶状体照片；B：大鼠晶状体浑浊程度；***P < 0.001。
Figure 8. Lens opacity in rats （$\bar x \pm s $，n = 9）
图 9 大鼠晶状体组织结构变化
A：Con组大鼠晶状体组织结构；B：Model组大鼠晶状体组织结构；C：Cel组大鼠晶状体组织结构。
Figure 9. Structural changes in rat lens tissue
图 10 大鼠晶状体组织中LAMP1和CTSB蛋白的表达情况（$\bar x \pm s $，n = 5）
A：Cel给药干预大鼠后Western blot条带图；B：Cel给药干预大鼠后晶状体中LAMP1相对表达水平；C：Cel给药干预大鼠后晶状体中CTSB相对表达水平；****P < 0.0001。
Figure 10. Expression of LAMP1 and CTSB proteins in rat lens tissue （$\bar x \pm s $，n = 5）
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