Zhang Xinzhuang, Geng Aopan, Cao Di, Dugarjaviin Manglai
lnner Mongolia Key Laboratory of Equine Science Research and Technology Innovation, College of Animal Science and Technology, Inner Mongolia Agricultural University, Hohhot, China.
Front Mol Biosci. 2024 Apr 8;11:1353387. doi: 10.3389/fmolb.2024.1353387. eCollection 2024.
Horses are susceptible to oxidative stress during strenuous endurance exercise, leading to muscle fatigue and damage. Mulberry leaf flavonoids (MLFs) possess significant antioxidant properties. However, the antioxidant efficacy of MLFs can be influenced by the extraction process, and their impact on HO-induced oxidative stress in equine skeletal muscle satellite cells (ESMCs) remains unexplored. Our study employed three extraction methods to obtain MLFs: ultrasound-assisted extraction (CEP), purification with AB-8 macroporous resin (RP), and n-butanol extraction (NB-EP). We assessed the protective effects of these MLFs on HO-induced oxidative stress in ESMCs and analyzed the MLF components using metabolomics. The results revealed that pre-treatment with MLFs dose-dependently protected ESMCs against HO-induced oxidative stress. The most effective concentrations were 0.8 mg/mL of CEP, 0.6 mg/mL of RP, and 0.6 mg/mL of NB-EP, significantly enhancing EMSC viability ( < 0.05). These optimized MLF concentrations promoted the GSH-Px, SOD and T-AOC activities ( < 0.05), while reducing MDA production ( < 0.05) in HO-induced ESMCs. Furthermore, these MLFs enhanced the gene expression, including and its downstream regulatory genes (, , , , and ) ( < 0.05). In terms of mitochondrial function, ESMCs pre-treated with MLFs exhibited higher basal respiration, spare respiratory capacity, maximal respiration, ATP-linked respiration compared to HO-induced ESMCs ( < 0.05). Additionally, MLFs enhanced cellular basal glycolysis, glycolytic reserve, and maximal glycolytic capacity ( < 0.05). Metabolomics analysis results revealed significant differences in mulberrin, kaempferol 3-O-glucoside [X-Mal], neohesperidin, dihydrokaempferol, and isobavachalcone among the three extraction processes ( < 0.05). Our study revealed that MLFs enhance antioxidant enzyme activity, alleviate oxidative damage in ESMCs through the activation of the Nrf2 pathway, and improve mitochondrial respiration and cell energy metabolism. Additionally, we identified five potential antioxidant flavonoid compounds, suggesting their potential incorporation into the equine diet as a strategy to alleviate exercise-induced oxidative stress.
马匹在剧烈的耐力运动中易受氧化应激影响，导致肌肉疲劳和损伤。桑叶黄酮（MLFs）具有显著的抗氧化特性。然而，MLFs的抗氧化功效会受到提取过程的影响，其对马骨骼肌卫星细胞（ESMCs）中血红素加氧酶（HO）诱导的氧化应激的影响仍未得到探索。我们的研究采用了三种提取方法来获得MLFs：超声辅助提取（CEP）、AB - 8大孔树脂纯化（RP）和正丁醇提取（NB - EP）。我们评估了这些MLFs对HO诱导的ESMCs氧化应激的保护作用，并使用代谢组学分析了MLF的成分。结果表明，MLFs预处理可剂量依赖性地保护ESMCs免受HO诱导的氧化应激。最有效的浓度分别为：CEP的0.8mg/mL、RP的0.6mg/mL和NB - EP的0.6mg/mL，显著提高了ESMC的活力（P < 0.05）。这些优化的MLF浓度促进了谷胱甘肽过氧化物酶（GSH - Px）、超氧化物歧化酶（SOD）和总抗氧化能力（T - AOC）的活性（P < 0.05），同时降低了HO诱导的ESMCs中丙二醛（MDA）的产生（P < 0.05）。此外，这些MLFs增强了基因表达，包括Nrf2及其下游调控基因（Keap1、Bax、Bcl - 2、HO - 1和GCLC）（P < 0.05）。在线粒体功能方面，与HO诱导的ESMCs相比，用MLFs预处理的ESMCs表现出更高的基础呼吸、备用呼吸能力、最大呼吸、ATP相关呼吸（P < 0.05）。此外，MLFs增强了细胞基础糖酵解、糖酵解储备和最大糖酵解能力（P < 0.05）。代谢组学分析结果显示，三种提取过程中，桑色素、山奈酚3 - O - 葡萄糖苷[X - Mal]、新橙皮苷、二氢山奈酚和异补骨脂查尔酮存在显著差异（P < 0.05）。我们的研究表明，MLFs增强抗氧化酶活性，通过激活Nrf2途径减轻ESMCs中的氧化损伤，并改善线粒体呼吸和细胞能量代谢。此外，我们鉴定出五种潜在的抗氧化黄酮类化合物，表明它们有可能作为减轻运动诱导的氧化应激的策略纳入马的饮食中。
