Abstract：
BACKGROUND: Diabetic cataract (DC) is a common ocular complication of diabetes. Mitofusin 2 (MFN2), a mitochondrial fusion protein, is involved in the pathogenesis of cataract and diabetic complications. However, its role and molecular mechanisms in DC remain unclear.
METHODS: DC models in rats were induced by intraperitoneal injection of streptozocin (STZ) for 12 weeks. We measured the body weight of rats, blood glucose concentrations, sorbitol dehydrogenase (SDH) activity and advanced glycation end products (AGE) content in the lenses of rats. MFN2 mRNA and protein expression levels in the lenses were detected by RT-qPCR and western blot assays. In vitro, human lens epithelial (HLE) B3 cells were treated for 48 h with 25 mM glucose (high glucose, HG) to induce cell damage. To determine the role of MFN2 in HG-induced cell damage, HLE-B3 cells were transfected with lentivirus loaded with MFN2 overexpression plasmid or short hairpin RNA (shRNA) to overexpress or knock down MFN2 expression, followed by HG exposure. Cell viability was assessed by CCK-8 assay. Flow cytometry was used to detect cell apoptosis and reactive oxygen species (ROS) level. JC-1 staining showed the changes in mitochondrial membrane potential (Δψm). The mediators related to apoptosis, mitochondrial damage, and autophagy were determined.
RESULTS: STZ-administrated rats showed reduced body weight, increased blood glucose levels, elevated SDH activity and AGE content, suggesting successful establishment of the DC rat model. Interestingly, MFN2 expression was significantly downregulated in DC rat lens and HG-induced HLE-B3 cells. Further analysis showed that under HG conditions, MFN2 overexpression enhanced cell viability and inhibited apoptosis accompanied by decreased Bax, cleaved caspase-9 and increased Bcl-2 expression in HLE-B3 cells. MFN2 overexpression also suppressed the mitochondrial damage elicited by HG as manifested by reduced ROS production, recovered Δψm and increased mitochondrial cytochrome c (Cyto c) level. Moreover, MFN2 overexpression increased LC3BⅡ/LC3BⅠ ratio and Beclin-1 expression, but decreased p62 level, and blocked the phosphorylation of mTOR in HG-treated HLE-B3 cells. In contrast, MFN2 silencing exerted opposite effects.
CONCLUSIONS: Presented findings indicate that MFN2 expression may be essential for preventing lens epithelial cell apoptosis during development of diabetic cataract.
METHODS: DC models in rats were induced by intraperitoneal injection of streptozocin (STZ) for 12 weeks. We measured the body weight of rats, blood glucose concentrations, sorbitol dehydrogenase (SDH) activity and advanced glycation end products (AGE) content in the lenses of rats. MFN2 mRNA and protein expression levels in the lenses were detected by RT-qPCR and western blot assays. In vitro, human lens epithelial (HLE) B3 cells were treated for 48 h with 25 mM glucose (high glucose, HG) to induce cell damage. To determine the role of MFN2 in HG-induced cell damage, HLE-B3 cells were transfected with lentivirus loaded with MFN2 overexpression plasmid or short hairpin RNA (shRNA) to overexpress or knock down MFN2 expression, followed by HG exposure. Cell viability was assessed by CCK-8 assay. Flow cytometry was used to detect cell apoptosis and reactive oxygen species (ROS) level. JC-1 staining showed the changes in mitochondrial membrane potential (Δψm). The mediators related to apoptosis, mitochondrial damage, and autophagy were determined.
RESULTS: STZ-administrated rats showed reduced body weight, increased blood glucose levels, elevated SDH activity and AGE content, suggesting successful establishment of the DC rat model. Interestingly, MFN2 expression was significantly downregulated in DC rat lens and HG-induced HLE-B3 cells. Further analysis showed that under HG conditions, MFN2 overexpression enhanced cell viability and inhibited apoptosis accompanied by decreased Bax, cleaved caspase-9 and increased Bcl-2 expression in HLE-B3 cells. MFN2 overexpression also suppressed the mitochondrial damage elicited by HG as manifested by reduced ROS production, recovered Δψm and increased mitochondrial cytochrome c (Cyto c) level. Moreover, MFN2 overexpression increased LC3BⅡ/LC3BⅠ ratio and Beclin-1 expression, but decreased p62 level, and blocked the phosphorylation of mTOR in HG-treated HLE-B3 cells. In contrast, MFN2 silencing exerted opposite effects.
CONCLUSIONS: Presented findings indicate that MFN2 expression may be essential for preventing lens epithelial cell apoptosis during development of diabetic cataract.
摘要：
背景：糖尿病性白内障（DC）是糖尿病常见的眼部并发症。Mitofusin2(MFN2),线粒体融合蛋白，参与了白内障和糖尿病并发症的发病机制。然而,其在DC中的作用和分子机制尚不清楚。
方法：腹腔注射链脲佐菌素(STZ)12周建立大鼠DC模型。我们测量了大鼠的体重,血糖浓度，大鼠晶状体中山梨醇脱氢酶（SDH）活性和糖基化终产物（AGE）含量。通过RT-qPCR和蛋白质印迹测定检测晶状体中的MFN2mRNA和蛋白质表达水平。体外，人晶状体上皮（HLE）B3细胞用25mM葡萄糖（高葡萄糖，HG)诱导细胞损伤。为了确定MFN2在HG诱导的细胞损伤中的作用，用装载有MFN2过表达质粒或短发夹RNA(shRNA)的慢病毒转染HLE-B3细胞以过表达或敲低MFN2表达,其次是HG暴露。通过CCK-8测定评估细胞活力。流式细胞术检测细胞凋亡和活性氧（ROS）水平。JC-1染色显示线粒体膜电位的变化（ΔΦm）。与细胞凋亡相关的介质，线粒体损伤，并测定了自噬。
结果：给予STZ的大鼠显示体重下降，血糖水平升高，SDH活性和AGE含量升高，提示DC大鼠模型的成功建立。有趣的是,MFN2表达在DC大鼠晶状体和HG诱导的HLE-B3细胞中显著下调。进一步分析表明，在HG条件下，MFN2过表达增强细胞活力并抑制细胞凋亡，同时Bax降低，HLE-B3细胞中caspase-9裂解并增加Bcl-2表达。MFN2过表达还抑制了由HG引起的线粒体损伤，表现为ROS产生减少。恢复的Δφm和增加的线粒体细胞色素c（Cytoc）水平。此外，MFN2过表达增加LC3BⅡ/LC3BⅠ比值和Beclin-1表达,但降低了p62水平，并阻断HG处理的HLE-B3细胞中mTOR的磷酸化。相比之下,MFN2沉默产生相反的效果。
结论：我们的研究结果表明，MFN2的表达可能是预防糖尿病性白内障发展过程中晶状体上皮细胞凋亡所必需的。
方法：腹腔注射链脲佐菌素(STZ)12周建立大鼠DC模型。我们测量了大鼠的体重,血糖浓度，大鼠晶状体中山梨醇脱氢酶（SDH）活性和糖基化终产物（AGE）含量。通过RT-qPCR和蛋白质印迹测定检测晶状体中的MFN2mRNA和蛋白质表达水平。体外，人晶状体上皮（HLE）B3细胞用25mM葡萄糖（高葡萄糖，HG)诱导细胞损伤。为了确定MFN2在HG诱导的细胞损伤中的作用，用装载有MFN2过表达质粒或短发夹RNA(shRNA)的慢病毒转染HLE-B3细胞以过表达或敲低MFN2表达,其次是HG暴露。通过CCK-8测定评估细胞活力。流式细胞术检测细胞凋亡和活性氧（ROS）水平。JC-1染色显示线粒体膜电位的变化（ΔΦm）。与细胞凋亡相关的介质，线粒体损伤，并测定了自噬。
结果：给予STZ的大鼠显示体重下降，血糖水平升高，SDH活性和AGE含量升高，提示DC大鼠模型的成功建立。有趣的是,MFN2表达在DC大鼠晶状体和HG诱导的HLE-B3细胞中显著下调。进一步分析表明，在HG条件下，MFN2过表达增强细胞活力并抑制细胞凋亡，同时Bax降低，HLE-B3细胞中caspase-9裂解并增加Bcl-2表达。MFN2过表达还抑制了由HG引起的线粒体损伤，表现为ROS产生减少。恢复的Δφm和增加的线粒体细胞色素c（Cytoc）水平。此外，MFN2过表达增加LC3BⅡ/LC3BⅠ比值和Beclin-1表达,但降低了p62水平，并阻断HG处理的HLE-B3细胞中mTOR的磷酸化。相比之下,MFN2沉默产生相反的效果。
结论：我们的研究结果表明，MFN2的表达可能是预防糖尿病性白内障发展过程中晶状体上皮细胞凋亡所必需的。
