Abstract：
Subacute rumen acidosis can lead to mastitis in dairy cows. Mitochondrial dysfunction is closely related to the inflammatory response. This experiment was conducted to investigate the effects of a high-concentrate diet on mammary gland inflammation and mitochondrial damage in dairy cows. Twelve Holstein dairy cows in mid-lactation were randomly divided into 2 groups and fed a 40% concentrate (low concentrate, LC) diet or a 60% concentrate (high concentrate, HC) diet. Cows were fed individually, and the experiment lasted for 3 wk. After the experiment, mammary gland tissue, blood, and rumen fluid were collected. Compared with the LC diet, the HC diet significantly decreased rumen pH; the pH was <5.6 for more than 3 h. The HC diet also increased the concentration of LPS in the blood (7.17 ± 1.25 µg/mL vs. 12.12 ± 1.26 µg/mL), which indicated that feeding the HC diet successfully induced subacute rumen acidosis. The HC diet also increased the concentration of Ca2+ (34.80 ± 4.23 µg/g vs. 46.87 ± 7.24 µg/g) in the mammary gland and upregulated the expression of inflammatory factors IL-6 (1,128.31 ± 147.53 pg/g vs. 1,538.42 ± 241.38 pg/g), IL-1β (69.67 ± 5.86 pg/g vs. 90.13 ± 4.78 pg/g), and tumor necrosis factor-α (91.99 ± 10.43 pg/g vs. 131.75 ± 17.89 pg/g) in mammary venous blood. The HC diet also increased the activity of myeloperoxidase (0.41 ± 0.05 U/g vs. 0.71 ± 0.11 U/g) and decreased the content of ATP (0.47 ± 0.10 µg/mL vs. 0.32 ± 0.11 µg/mL) in the mammary gland. In addition, phosphorylation of JNK (1.00 ± 0.21 vs. 2.84 ± 0.75), ERK (1.00 ± 0.20 vs. 1.53 ± 0.31), and p38 (1.00 ± 0.13 vs. 1.47 ± 0.41) and protein expression of IL-6 (1.00 ± 0.22 vs. 2.21 ± 0.27) and IL-8 (1.00 ± 0.17 vs. 1.96 ± 0.26) were enhanced in cows of the HC group, indicating that the mitogen-activated protein kinase (MAPK) signaling pathway was activated. Compared with the LC diet, the HC diet reduced the protein expression of mitochondrial biogenesis-related proteins PGC-1α (1.00 ± 0.17 vs. 0.55 ± 0.12), NRF1 (1.00 ± 0.17 vs. 0.60 ± 0.10), TFAM (1.00 ± 0.10 vs. 0.73 ± 0.09), and SIRTI (1.00 ± 0.44 vs. 0.40 ± 0.10). The HC diet promoted mitochondrial fission and inhibited mitochondrial fusion by reducing protein expression of MFN1 (1.00 ± 0.31 vs. 0.49 ± 0.09), MFN2 (1.00 ± 0.19 vs. 0.69 ± 0.13), and OPA1 (1.00 ± 0.08 vs. 0.72 ± 0.07), and by increasing that of DRP1 (1.00 ± 0.09 vs. 1.39 ± 0.10), MFF (1.00 ± 0.15 vs. 1.89 ± 0.12), and TTC1/FIS1 (1.00 ± 0.08 vs. 1.76 ± 0.14), leading to mitochondrial dysfunction. The HC diet increased mitochondrial permeability by upregulating the protein expression of VDAC1 (1.00 ± 0.42 vs. 1.90 ± 0.44), ANT (1.00 ± 0.22 vs. 1.27 ± 0.17), and CYPD (1.00 ± 0.41 vs. 1.82 ± 0.43). Taken together, these results indicated that feeding the HC diet induced mitochondrial damage via the MAPK signaling pathway in the mammary gland of dairy cows.
摘要：
亚急性瘤胃酸中毒可导致奶牛乳腺炎。线粒体功能障碍与炎症反应密切相关。本试验旨在研究高浓缩日粮对奶牛乳腺炎症和线粒体损伤的影响。12头处于泌乳中期的荷斯坦奶牛随机分为2组，饲喂40％的浓缩物（低浓缩物，LC)饮食或60%浓缩物(高浓缩物，HC）饮食。奶牛被单独喂养，实验持续了3周。实验之后,乳腺组织,血,并收集瘤胃液。与LC饮食相比，HC饮食显着降低瘤胃pH；pH<5.6超过3小时。HC饮食还增加了血液中LPS的浓度（7.17±1.25µg/mLvs.12.12±1.26µg/mL)，表明饲喂HC饮食成功诱发亚急性瘤胃酸中毒。HC饮食也增加了Ca2+的浓度(34.80±4.23µg/gvs.乳腺中46.87±7.24µg/g），并上调炎症因子IL-6的表达（1,128.31±147.53pg/gvs.1,538.42±241.38pg/g），IL-1β（69.67±5.86pg/gvs.90.13±4.78pg/g)，和肿瘤坏死因子-α（91.99±10.43pg/gvs.131.75±17.89pg/g）在乳腺静脉血中。HC饮食还增加了髓过氧化物酶的活性（0.41±0.05U/gvs.0.71±0.11U/g），并降低了ATP的含量（0.47±0.10µg/mL与0.32±0.11µg/mL）在乳腺中。此外,JNK的磷酸化(1.00±0.21vs.2.84±0.75),ERK(1.00±0.20vs.1.53±0.31),和p38(1.00±0.13vs.1.47±0.41）和IL-6的蛋白表达（1.00±0.22vs.2.21±0.27）和IL-8（1.00±0.17vs.在HC组的奶牛中增强了1.96±0.26），表明丝裂原活化蛋白激酶（MAPK）信号通路被激活。与LC饮食相比，HC饮食降低线粒体生物发生相关蛋白PGC-1α的蛋白表达(1.00±0.17vs.0.55±0.12)，NRF1(1.00±0.17vs.0.60±0.10),TFAM(1.00±0.10vs.0.73±0.09),和SIRTI(1.00±0.44vs.0.40±0.10）。HC饮食通过降低MFN1蛋白表达促进线粒体裂变并抑制线粒体融合（1.00±0.31vs.0.49±0.09),MFN2(1.00±0.19vs.0.69±0.13），和OPA1(1.00±0.08vs.0.72±0.07),并通过增加DRP1的值(1.00±0.09vs.1.39±0.10),MFF(1.00±0.15vs.1.89±0.12)，和TTC1/FIS1(1.00±0.08与1.76±0.14)，导致线粒体功能障碍.HC饮食通过上调VDAC1的蛋白表达增加线粒体通透性（1.00±0.42vs.1.90±0.44)，ANT(1.00±0.22vs.1.27±0.17)，和CYPD（1.00±0.41vs.1.82±0.43）。一起来看,这些结果表明，饲喂HC日粮可通过MAPK信号通路引起奶牛乳腺线粒体损伤。
