College of Food Science and Engineering, Gansu Agricultural University, Lanzhou 730070, China; Key Laboratory of Functional Dairy, College of Food Science and Nutritional Engineering, China Agricultural University, Beijing, 100083, China.
Key Laboratory of Functional Dairy, College of Food Science and Nutritional Engineering, China Agricultural University, Beijing, 100083, China; Beijing Advanced Innovation Center for Food Nutrition and Human Health, College of Food Science and Nutritional Engineering, China Agricultural University, Beijing, 100094, China.
J Dairy Sci. 2019 Feb;102(2):985-996. doi: 10.3168/jds.2018-14946. Epub 2018 Dec 20.
Intestinal epithelial cells (IEC) are an important part of the intestinal barrier. Barrier function was disrupted under hypoxia, but milk-derived exosomes can regulate the intestinal barrier function. However, the mechanisms underlying the association between yak milk exosomes and hypoxia in IEC remain poorly understood. In this follow-up study, we proposed an effective optimization method for purifying yak-milk-derived exosomes. The Western blot analyses indicated that the expression of the proteins of the endosomal sorting complexes required for transport (TSG101), proteins of the tetraspanin family (CD63), and heat shock protein 70 (Hsp-70) proteins from yak-milk-derived exosomes were significantly higher than those in cow-milk-derived exosomes. Flow cytometry analysis showed that yak milk had 3.7 times the number of exosomes compared with cow milk. Moreover, we explored whether yak milk exosomes could facilitate intestinal cell survival under hypoxic conditions in vitro. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide results showed that yak-milk-derived exosomes significantly increased survival of IEC-6 cells with rates of up to 29% for cells incubated in hypoxic conditions for 12 h, compared with those of cow-milk-derived exosomes posttreatment (rates of up to 22% for cells incubated in hypoxic conditions for 12 h). Confocal microscopy revealed that the IEC-6 cells uptake more yak-milk-derived exosomes than cow milk in hypoxic conditions. Furthermore, the Western blot analyses indicated that yak-milk-derived exosomes significantly promote oxygen-sensitive prolyl hydroxylase (PHD)-1 expression and decrease the expression of hypoxia-inducible factor-α and its downstream target vascular endothelial growth factor (VEGF) in the IEC-6 cells. Further, yak-milk-derived exosomes significantly inhibited p53 levels. In conclusion, our findings demonstrate that yak-milk-derived exosomes more effectively activate the hypoxia-inducible factor signaling pathway, thus promoting IEC-6 cell survival, which may result in higher hypoxia tolerance than cow-milk-derived exosomes.
肠上皮细胞 (IEC) 是肠道屏障的重要组成部分。在缺氧下，屏障功能被破坏，但乳源性外泌体可以调节肠道屏障功能。然而，牦牛乳外泌体与 IEC 缺氧之间的关联的机制仍知之甚少。在这项后续研究中，我们提出了一种有效优化牦牛乳衍生外泌体纯化的方法。Western blot 分析表明，从牦牛乳衍生的外泌体中内体分选复合物所需的蛋白质 (TSG101)、四跨膜蛋白家族 (CD63) 和热休克蛋白 70 (Hsp-70) 蛋白的表达明显高于牛乳衍生的外泌体。流式细胞术分析表明，与牛乳相比，牦牛乳的外泌体数量多 3.7 倍。此外，我们还探讨了牦牛乳外泌体是否可以促进体外缺氧条件下肠细胞的存活。3-(4,5-二甲基噻唑-2-基)-2,5-二苯基四氮唑溴盐结果表明，与牛乳衍生的外泌体处理相比，牦牛乳衍生的外泌体可显著提高 IEC-6 细胞在缺氧条件下培养 12 小时后的存活率，高达 29%，而牛乳衍生的外泌体处理后，在缺氧条件下培养 12 小时后的存活率高达 22%。共聚焦显微镜显示，在缺氧条件下，IEC-6 细胞摄取的牦牛乳衍生外泌体多于牛乳。此外，Western blot 分析表明，牦牛乳衍生的外泌体可显著促进氧敏感脯氨酰羟化酶 (PHD)-1 的表达，并降低 IEC-6 细胞中缺氧诱导因子-α及其下游靶血管内皮生长因子 (VEGF) 的表达。此外，牦牛乳衍生的外泌体可显著抑制 p53 水平。综上所述，我们的研究结果表明，牦牛乳衍生的外泌体更有效地激活缺氧诱导因子信号通路，从而促进 IEC-6 细胞的存活，这可能导致比牛乳衍生的外泌体更高的缺氧耐受性。
