Swim bladder is an ideal source of collagen production in fish for improved human health. Proline (Pro) is the main proteinogenic amino acid needed for collagen production. However, the effects of Pro supplementation on the swim bladder collagen synthesis have rarely been evaluated in fish. We determined the effects of dietary Pro supplementation on swim bladder collagen synthesis and its possible signalling pathway in spotted drum, Nibea diacanthus. A total of 450 N. diacanthus (100 ± 3.05 g) were randomly assigned into six treatments and fed with diets supplemented with different levels of Pro (0, 2.5, 5, 7.5, 10 and 12.5 g/kg of dry diet, hereafter P0, P1, P2, P3, P4 and P5, respectively) for 8 weeks. At the end, we evaluated collagen synthesis in swim bladder and the expression of genes related to TGF‐β/Smad pathway in the fish. Dietary Pro levels increased significantly the contents of crude protein, total collagen (TC) and the levels of some amino acids in swim bladder than the control diet (p < .05). The optimum amount of dietary Pro inclusion in diets for swim bladder collagen synthesis in N. diacanthus was 7.6 and 7.5 g/kg based on crude protein and TC in swim bladder, respectively. Dietary Pro levels increased significantly the proline 4‐hydroxylase (P4H) content in fish serum, swim bladder, muscle and liver tissues than control (p < .05). The relative expression of collagen type I alpha 1 (COL1A1), alpha 2 (COL1A2) and mothers against decapentaplegic homolog 2 (Smad2) genes in liver and swim bladder initially increased significantly as the concentration of Pro and later decreased (p < .05). Similarly, the relative expression of transforming growth factor beta (TGF‐β), P4Ha2 and P4Ha3 genes in the swim bladder increased significantly as dietary Pro levels increased (p < .05). Using K‐means clustering analysis, dietary proline partly promoted collagen accumulation in swim bladder through upregulation of Smad2 and TGF‐βRT genes. Taken together, Pro affected the collagen metabolism in swim bladder, probably by regulating the TGF‐β/Smad pathway, most likely via transient overexpression of Smad2 gene.

中文翻译：

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日粮脯氨酸对斑纹鼓形念珠虫游泳膀胱胶原合成的影响及其可能通过TGF-β/ Smad途径调控

游泳膀胱是鱼类中产生胶原蛋白以改善人体健康的理想来源。脯氨酸（Pro）是胶原蛋白生产所需的主要蛋白质氨基酸。然而，很少在鱼类中评估补充Pro对游泳膀胱胶原蛋白合成的影响。我们确定了饮食中Pro补充剂对斑鼓鱼diacanthus中游泳膀胱胶原蛋白合成及其可能的信号传导途径的影响。总计450 diacanthus（100±3.05 g）被随机分配到六个处理中，并饲喂补充不同水平的Pro（0、2.5、5、7.5、10和12.5 g / kg干粮，以下为P0，P1，P2，P3， P4和P5），共8周。最后，我们评估了鱼体内胶原蛋白的合成以及鱼中TGF-β/ Smad通路相关基因的表达。与对照饮食相比，饮食Pro的水平显着提高了游泳膀胱中粗蛋白，总胶原（TC）的含量和某些氨基酸的含量（p  <.05）。饮食中膳食中Pro的最佳添加量，可用于结实猪笼草的游泳膀胱胶原蛋白合成分别基于泳囊中的粗蛋白和TC分别为7.6 g / kg和7.5 g / kg。饮食中Pro的水平显着提高了鱼血清，游泳膀胱，肌肉和肝组织中脯氨酸的4-羟化酶（P4H）含量（p  <.05）。I型胶原蛋白α1（COL1A1），α2（COL1A2）和母亲针对肝功能异常的scapentaplegic homolog 2（Smad2）基因的相对表达最初随着Pro的浓度而显着增加，随后又下降（p  <.05） 。同样，转化生长因子β（TGF-β），P4Ha2和P4Ha3的相对表达随着饮食中Pro水平的增加，游泳膀胱中的这些基因显着增加（p  <.05）。使用K均值聚类分析，膳食脯氨酸通过Smad2和TGF-βRT基因的上调部分促进了游泳膀胱中胶原蛋白的积累。综上所述，Pro可能通过调节TGF-β/ Smad途径影响了游泳膀胱中的胶原代谢，最有可能是通过Smad2基因的瞬时过表达来实现的。