School of Public Health, The Key Laboratory of Environmental Pollution Monitoring and Disease Control, Ministry of Education, Guizhou Medical University, Guiyang 550025, China.
Simonyi Károly Faculty of Engineering, University of Sopron, 9400 Sopron, Hungary.
Molecules. 2022 Dec 16;27(24):8985. doi: 10.3390/molecules27248985.
Cataracts are an ailment representing the leading cause of blindness in the world. The pathogenesis of cataracts is not clear, and there is no effective treatment. An increasing amount of evidence shows that oxidative stress and autophagy in lens epithelial cells play a key role in the occurrence and development of cataracts. Buddleja officinalis Maxim flavonoids (BMF) are natural antioxidants and regulators that present anti-inflammatory and anti-tumor effects, among others. In this study, we optimized the extraction method of BMFs and detected three of their main active monomers (luteolin, apigenin, and acacetin). In addition, a model of oxidative damage model using rabbit lens epithelial cells induced by hydrogen peroxide (H2O2). By detecting the levels of superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), malondialdehyde (MDA), and OH (OH), the expression of autophagosomes and autolysosomes were observed after MRFP-GFP-LC3 adenovirus was introduced into the cells. Western blotting was used to detect the expression of Beclin-1 and P62. Our research results showed that the optimal extraction parameters to obtain the highest yield of total flavonoids were a liquid−solid ratio of 1:31 g/mL, an ethanol volume fraction of 67%, an extraction time of 2.6 h, and an extraction temperature of 58 °C. Moreover, the content of luteolin was 690.85 ppb, that of apigenin was 114.91 ppb, and the content of acacetin was 5.617 ppb. After oxidative damage was induced by H2O2, the cell survival rate decreased significantly. BMFs could increase the levels of superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) and decrease the levels of malondialdehyde (MDA) and OH (OH). After the MRFP-GFP-LC3 virus was introduced into rabbit lens epithelial cells and detecting the expression of P62 and Beclin-1, we found that the intervention of BMF could promote the binding of autophagosomes to lysosomes. Compared with the model group, the level of P62 in the low-, middle-, and high-dose groups of BMF was significantly down-regulated, the level of Beclin-1 was significantly increased, and the difference was statistically significant (p < 0.05). In other words, the optimized extraction method was better than others, and the purified BMF contained three main active monomers (luteolin, apigenin, and acacetin). In addition, BMFs could ameliorate the H2O2-induced oxidative damage to rabbit lens cells by promoting autophagy and regulating the level of antioxidation.
白内障是全球致盲的主要原因。白内障的发病机制尚不清楚，也没有有效的治疗方法。越来越多的证据表明，晶状体上皮细胞中的氧化应激和自噬在白内障的发生和发展中起着关键作用。马钱子素（BMF）是一种天然抗氧化剂和调节剂，具有抗炎和抗肿瘤等作用。在这项研究中，我们优化了 BMF 的提取方法，并检测了其三种主要的活性单体（木犀草素、芹菜素和乙酰化）。此外，还建立了过氧化氢（H2O2）诱导兔晶状体上皮细胞氧化损伤模型。通过检测超氧化物歧化酶（SOD）、谷胱甘肽过氧化物酶（GSH-Px）、丙二醛（MDA）和 OH（OH）的水平，观察到 MRFP-GFP-LC3 腺病毒转染细胞后自噬体和自噬溶酶体的表达情况。Western blot 检测 Beclin-1 和 P62 的表达。我们的研究结果表明，获得总黄酮产量最高的最佳提取参数是液固比为 1:31 g/mL，乙醇体积分数为 67%，提取时间为 2.6 h，提取温度为 58°C。此外，木犀草素含量为 690.85 ppb，芹菜素含量为 114.91 ppb，乙酰化含量为 5.617 ppb。H2O2 诱导氧化损伤后，细胞存活率明显下降。BMF 可提高超氧化物歧化酶（SOD）和谷胱甘肽过氧化物酶（GSH-Px）水平，降低丙二醛（MDA）和 OH（OH）水平。将 MRFP-GFP-LC3 病毒导入兔晶状体上皮细胞并检测 P62 和 Beclin-1 的表达后，我们发现 BMF 的干预可以促进自噬体与溶酶体的结合。与模型组相比，BMF 低、中、高剂量组的 P62 水平明显下调，Beclin-1 水平明显升高，差异有统计学意义（p<0.05）。换句话说，优化后的提取方法优于其他方法，纯化后的 BMF 含有三种主要的活性单体（木犀草素、芹菜素和乙酰化）。此外，BMF 可通过促进自噬和调节抗氧化水平来改善 H2O2 诱导的兔晶状体细胞氧化损伤。
