竹叶黄酮通过调节抗氧化通路和自噬缓解奶牛乳腺上皮细胞氧化应激的作用机制

Mechanism of Bamboo Leaf Flavonoids Regulating Antioxidant Pathway and Autophagy to Alleviate Oxidative Stress in Dairy Cow Mammary Epithelial Cells

本试验旨在探究竹叶黄酮(BLF)对氧化应激的奶牛乳腺上皮细胞自噬和抗氧化通路的影响,并通过添加自噬抑制剂Mdivi-1探究自噬与Kelch样环氧氯丙烷相关蛋白1(Keap1)-核因子E2相关因子2(Nrf2)信号通路的关联性。以奶牛乳腺上皮细胞MAC-T细胞系为研究对象,将维持培养基中处理的MAC-T设为对照组,经含BLF(80μg/mL)的维持培养基处理的MAC-T设为BLF组,经含过氧化氢(H_(2)O_(2),800μmol/L)的维持培养基处理的MAC-T设为H_(2)O_(2)组(诱导氧化应激),经含BLF(80μg/mL)的维持培养基和含H_(2)O_(2)(800μmol/L)的维持培养基处理的MAC-T设为BLF+H_(2)O_(2)组,经含BLF(80μg/mL)的维持培养基、含Mdivi-1(1μmol/L)的维持培养基以及含H_(2)O_(2)(800μmol/L)的维持培养基处理的MAC-T设为BLF+H_(2)O_(2)+Midiv组,经含Mdivi-1(1μmol/L)的维持培养基处理的MAC-T设为Midiv组。培养结束后,采用实时荧光定量PCR检测自噬和Keap1-Nrf2信号通路相关基因的相对表达量,免疫印迹法检测自噬及Keap1-Nrf2、胞外信号调节激酶(MAPK)信号通路相关蛋白的表达量,免疫荧光技术检测转录因子EB(TFEB)、Nrf2蛋白表达及核移位情况。结果显示:1)BLF预处理能显著增加氧化损伤细胞中自噬标志物Beclin1、Parkin蛋白的表达量(P<0.05),显著增加溶酶体功能相关蛋白组织蛋白酶D(CTSD)和溶酶体关联膜蛋白1(LAMP1)蛋白的表达量(P<0.05),显著增加腺苷酸活化蛋白激酶(AMPK)蛋白的磷酸化程度和TFEB蛋白的表达量(P<0.05),并促进TFEB核转位,显著升高溶酶体相关基因囊泡型ATP酶H亚基(ATP6V1H)、组织蛋白酶B(CTSB)、三肽基肽酶1(TPP1)和自噬相关基因微管相关蛋白1轻链3(LC3)的相对表达量(P<0.05)。2)BLF预处理能显著减少氧化损伤细胞中Keap1蛋白的表达量(P<0.05),导致Nrf2蛋白在细胞质内稳定并大量转移到细胞核内,激活下游Nrf2靶基因血红素加氧酶-1(HO-1)和NAD(P)H:醌氧化还原酶1(NQO1)的转录与翻译。但是,通过Mdivi-1抑制自噬将抑制BLF对Keap1-Nrf2信号通路的促进作用。综上所述,本研究发现BLF通过促进细胞自噬和溶酶体功能,激活Keap1-Nrf2信号通路来缓解奶牛乳腺上皮细胞的氧化应激,并且自噬与抗氧化信号通路之间存在相互串扰。

The purpose of this experiment was to explore the effect of bamboo leaf flavonoids(BLF)on the autophagy and antioxidant pathway of dairy cow mammary epithelial cells under oxidative stress,and to explore the correlation between autophagy and Kelch-like ECH-associated protein 1(Keap1)-nuclear factor erythroid-2-related factor 2(Nrf2)signaling pathway by adding autophagy inhibitor Mdivi-1.The MAC-T cell line of dairy cow mammary epithelial cells was used as the research object.The MAC-T treated in the maintenance medium was set as the control group.The MAC-T treated with the maintenance medium containing(80μg/mL)BLF was set as the BLF group.The MAC-T treated with the maintenance medium containing(800μmol/L)hydrogen peroxide(H_(2)O_(2))was set as the H_(2)O_(2) group(induced oxidative stress).The MAC-T treated with the maintenance medium containing(80μg/mL)BLF and the maintenance medium containing(800μmol/L)H_(2)O_(2) were set as the BLF+H_(2)O_(2) group.The MAC-T treated with the maintenance medium containing(80μg/mL)BLF,(1μmol/L)Mdivi-1 and(800μmol/L)H_(2)O_(2) was set as BLF+H_(2)O_(2)+Midiv group.The MAC-T treated with the maintenance medium containing(1μmol/L)Mdivi-1 was set as Midiv group.After the culturing,real-time fluorescence quantitative PCR was used to detect the expression of autophagy and Keap1-Nrf2 signaling pathway-related genes,Western blot was used to detect the expression of autophagy-related proteins,Keap1-Nrf2 and extracellular signal-regulated kinase(MAPK)signaling pathway-related proteins,and immunofluorescence technique was used to detect transcription factor EB(TFEB)protein expression,Nrf2 protein expression and nuclear translocation.The results showed as follows:1)BLF pretreatment could significantly increase the expression levels of autophagy markers Beclin1 and Parkin proteins in oxidative damaged cells(P<0.05),significantly increase the expression levels of lysosomal function-related proteins cathepsin D(CTSD)and lysosomal associated membrane protein 1(LAMP1)(P<0.05),significantly increase the phosphorylation degree of adenosine monophosphate activated protein kinase(AMPK)protein and the expression level of TFEB protein(P<0.05),promote TFEB nuclear translocation,and significantly increase the relative expression levels of lysosomal-related genes such as ATPase H+transporting V1 subunit H(ATP6V1H),cathepsin B(CTSB)and tripeptidyl peptidase 1(TPP1)and autophagy-related gene microtubule-associated protein 1 light chain 3(LC3)(P<0.05).2)BLF pretreatment significantly reduced the expression level of Keap1 protein in oxidative damaged cells(P<0.05),resulting in the stabilization of Nrf2 protein in the cytoplasm and the transfer of Nrf2 protein to the nucleus,which activated the transcription and translation of downstream Nrf2 target genes heme oxygenase-1(HO-1)and NAD(P)H:quinone oxidoreductase 1(NQO1).However,inhibition of autophagy by Mdivi-1 inhibited the promotion of BLF on the Keap1-Nrf2 signaling pathway.In summary,this study found that BLF alleviates the oxidative stress of dairy cow mammary epithelial cells by promoting autophagy and lysosomal function and activating Keap1-Nrf2 signaling pathway,and there is a crosstalk between autophagy and antioxidant signaling pathways.