竹叶黄酮对奶牛乳腺上皮细胞氧化损伤的缓解作用及对乳脂合成的影响

Alleviating Effect of Bamboo Leaf Flavonoids on Oxidative Damage in Bovine Mammary Epithelial Cells and Its Effect on Milk Fat Synthesis

本试验旨在探究竹叶黄酮(BLF)对奶牛乳腺上皮细胞(BMECs)氧化损伤的缓解作用及对乳脂合成的影响。以BMECs为研究对象,进行如下操作:对照组1是将细胞接种于不添加任何物质的维持培养基中培养24 h;损伤组是将细胞接种于含800μmol/L过氧化氢(H_(2)O_(2))的维持培养基中处理8 h;对照组2是将细胞接种于含80μg/mL BLF的维持培养基中培养24 h;保护组是将细胞接种于含80μg/mL BLF的维持培养基中培养24 h后,更换含800μmol/L H_(2)O_(2)的维持培养基中继续处理8 h。应用试剂盒检测细胞内抗氧化指标,实时荧光定量PCR方法检测细胞内抗氧化、凋亡和乳脂合成相关基因的mRNA相对表达量,免疫印迹法检测乳脂合成相关蛋白的表达量。结果显示:1)与损伤组相比,保护组细胞内超氧化物歧化酶(SOD)、过氧化氢酶(CAT)和谷胱甘肽过氧化物酶(GSH-Px)活性显著升高(P<0.05),丙二醛(MDA)含量显著降低(P<0.05)。2)与对照组1相比,损伤组细胞内活性氧(ROS)含量显著提升(P<0.05),BLF预处理可以显著抑制受氧化损伤的细胞内ROS产生(P<0.05)。3)与损伤组相比,保护组细胞内凋亡基因B细胞淋巴瘤/白血病-2相关X蛋白(Bax)和半胱氨酸天冬氨酸蛋白酶3(Caspase 3)的mRNA相对表达量显著降低(P<0.05),抗凋亡基因B淋巴细胞瘤2(Bcl2)的mRNA相对表达量显著升高(P<0.05),抗氧化基因血红素加氧酶-1(HO-1)、醌氧化还原酶1(NQO1)和核转录因子E2相关因子2(Nrf2)的mRNA相对表达量显著升高(P<0.05)。4)与对照组1相比,损伤组细胞内脂滴(LDs)含量减少,保护组细胞内LDs含量有所增加;保护组细胞内甘油三酯(TGs)含量虽未恢复到正常水平,但与损伤组相比差异显著(P<0.05)。5)与损伤组相比,保护组细胞内乳脂合成相关基因乙酰辅酶A羧化酶α(ACACA)、乙酰辅酶A合成酶2(ACSS2)、脂肪酸结合蛋白3(FABP3)、脂肪酸合成酶(FASN)、过氧化物酶体增殖物激活受体γ(PPARγ)、胆固醇调节元件结合蛋白1(SREBP1)和硬脂酰辅酶A去饱和酶(SCD)的mRNA相对表达量均显著升高(P<0.05)。6)与对照组1相比,保护组细胞内乳脂合成相关蛋白ACSS2、FABP3、PPARγ和SCD的相对表达量均显著上升(P<0.05)。综上所述,BLF不仅能通过提升Nrf2及其下游抗氧化基因的表达,从而提升氧化损伤的BMECs的抗氧化能力,降低氧化应激带来的氧化损伤,而且BLF还能够有效提升乳脂相关基因和蛋白的表达,从而促进乳脂合成,有效减少氧化损伤对机体乳脂合成的影响。

The purpose of this study was to explore the alleviating effect of bamboo leaf flavonoids(BLF)on oxidative damage in bovine mammary epithelial cells(BMECs)and its effect on milk fat synthesis.With BMECs as study subjects,the following operations were carried out:cells were seeded in maintenance medium without any substance for 24 h to set the control group 1;cells were seeded in maintenance medium containing 800μmol/L hydrogen peroxide(H_(2)O_(2))for 8 h to set the injury group;cells were seeded in maintenance medium containing 80μg/mL BLF for 24 h to set the control group 2;cells were seeded in maintenance medium containing 80μg/mL BLF for 24 h,then replaced in maintenance medium containing 800μmol/L H_(2)O_(2) for 8 h to set the protection group.The antioxidant indexes in cells were examined using the kit method,the mRNA relative expression levels of antioxidative,apoptosis and milk fat-related genes were measured by real-time fluorescence quantification PCR,and the expression levels of milk fat-related proteins were measured by Western blotting.The results showed as follows:1)compared with the injury group,the activities of superoxide dismutase(SOD),catalase(CAT)and glutathione peroxidase(GSH-Px)in cells of the protection group were significantly increased(P<0.05),and the malondialdehyde(MDA)content was significantly decreased(P<0.05).2)Compared with the control group 1,the content of reactive oxygen species(ROS)in cells of the injured group was significantly increased(P<0.05),and BLF pretreatment could significantly inhibit ROS production in oxidative damaged cells(P<0.05).3)Compared with the injured group,the mRNA relative expression levels of apoptotic genes B cell lymphoma/leukemia-2-related X protein(Bax)and cysteine aspartate protease 3(Caspase3)in cells of the protection group were significantly decreased(P<0.05),the mRNA relative expression levels of anti-apoptotic gene B lymphocytoma 2(Bcl2)was significantly increased(P<0.05),and the mRNA relative expression levels of antioxidant genes heme oxygenase-1(HO-1),quinone oxidoreductase 1(NQO1)and nuclear transcription factor E2-associated factor 2(Nrf2)were significantly increased(P<0.05).4)Compared with the control group 1,the lipid droplets(LDs)content decreased in the injured group,the LDs content increased in the protection group,and the triglycerides(TGs)content did not return to normal level but was significantly different compared with the injured group(P<0.05).5)Compared with the injured group,the mRNA relative expression levels of acetyl-CoA carboxylaseα(ACACA),acetyl-CoA synthetase 2(ACSS2),fatty acid-binding protein 3(FABP3),fatty acid synthetase(FASN),peroxisome proliferator-activated receptorγ(PPARγ),cholesterol regulatory element binding protein 1(SREBP1)and stearoyl-CoA desaturase(SCD)in cells of the protection group were significantly increased(P<0.05).The relative expression levels of milk fat synthesis related proteins ACSS2,FABP3,PPARγand SCD in cells were all significantly increased of the protection group compared with the control group 1(P<0.05).To sum up,BLF can not only by promoting the expression of Nrf2 and its downstream antioxidant genes,thus enhance the antioxidant capacity of BMECs,reduce the oxidative damage caused by oxidative stress,and BLF can also effectively improve the expression of milk fat related genes and proteins,thus promote the synthesis of milk fat,effectively reduce the effects of oxidative damage on the body milk fat synthesis.