Abstract:Proline (Pro) is a conditionally essential amino acid in animals, with important nutritional, metabolic, and immune functions. To investigate the effect and mechanism of proline on oxidative stress in vitro in fibroblastic cells from the embryonic calipash of Chinese soft-shelled turtle Pelodiscus sinensis, a lipopolysaccharide induced oxidative stress model was established in the experiment. The cells in the cell culture system were divided into control, LPS stressed, and Pro treated groups. The cell viability, reactive oxygen species (ROS) content, and mRNA expression levels of collagen metabolism related genes were determined by MTT, HE staining, DCFH-DA, and qRT-PCR. The results showed that there were significant decrease in the number and survival rate of cells, significant increase in ROS levels, and a remarkable reduction in the mRNA expression abundance of collagen synthesis related genes Col1α1, Col1α2, and Col3α1 (P<0.05) in the LPS stressed group compared with the control, with significant escalation in the mRNA expression of collagen degradation related genesMMP1 and MMP18 (P<0.05). Correspondingly, the mRNA levels of MMPs specific inhibitors TIMP2, TIMP3, and TIMP4, key enzymes for collagen degradation were found to be significantly declined (P<0.05). The supplementation of Pro led to significantly elevate the number and survival rate of stressed cells (P<0.05), and significantly reduce the content of ROS (P<0.05) compared with the LPS group. Significantly up-regulated relative mRNA expression levels of Col3α1, TIMP2 and TIMP3, and significantly down-regulated mRNA expression levels of MMP18 (P<0.05, P<0.01) were shown to be in the fibroblastic cells from the embryonic calipash of Chinese soft-shelled turtle treated with Pro, indicating that Pro can alleviate LPS induced oxidative stress, in fibroblastic cells of Chinese soft-shelled turtle, possibly by altering the ROS signaling pathway, regulating the expression of collagen metabolism related enzymes MMPs and their inhibitory factors TIMPs. The findings revealed the effects of Pro on the growth and collagen metabolism of LPS induced damage in the fibroblastic cells from the embryonic calipash of Chinese soft-shelled turtle, which can provide theoretical basis for the application of similar functional amino acids such as Pro to enhance the stress resistance of Chinese soft-shelled turtle.
朱乐妍, 周贝宁, 宋伟, 罗嘉翔, 李彩燕. 脯氨酸对中华鳖受损裙边细胞生长及胶原代谢的影响[J]. 水产科学, 2025, 44(4): 642-650.
ZHU Leyan, ZHOU Beining, SONG Wei, LUO Jiaxiang, LI Caiyan. Effect of Proline on Growth and Collagen Metabolism in the Injured Calipash Cells of Chinese Soft-Shelled Turtle Pelodiscus sinensis. Fisheries Science, 2025, 44(4): 642-650.
[1]农业农村部渔业渔政管理局,全国水产技术推广总站,中国水产学会.2024中国渔业统计年鉴[M].北京:中国农业出版社,2024:24. [2]张丹.中华鳖营养特征分析及评价[D].上海:上海海洋大学,2015. [3]杨桠楠,李彩燕,钱国英.鳖源胶原蛋白提取纯化及在生物材料中的应用[J].生物工程学报,2016,32(6):819-830. [4]TANG Y J, FENG L, JIANG W D, et al.Enhancements in flavor substances, mouthfeel characteristics and collagen synthesis in the muscle of sub-adult grass carp (Ctenopharyngodon idella):application of a dietary lysine nutrition strategy[J]. Aquaculture, 2023,565:739115. [5]CHENG J H, SUN D W, HAN Z, et al. Texture and structure measurements and analyses for evaluation of fish and fillet freshness quality:a review[J]. Comprehensive Reviews in Food Science and Food Safety,2014,13(1):52-61. [6]ZHOU B N, SONG W, LI C Y, et al. Texture quality, histological and extracellular matrix-related molecular changes of the calipash of Chinese soft-shelled turtle Pelodiscus sinensis in response to Aeromonas hydrophila challenge[J]. Aquaculture Research,2022,53(10):3706-3717. [7]唐丽花,王广军,余德光,等.几种氨基酸对日本鳗鲡摄食、消化和生长的影响[J].大连水产学院学报,2009,24(6):574-577. [8]XIE S W, TIAN L X, LI Y M, et al. Effect of proline supplementation on anti-oxidative capacity, immune response and stress tolerance of juvenile Pacific white shrimp,Litopenaeus vannamei[J]. Aquaculture,2015,448:105-111. [9]荣华,夏优,王晓雯,等.饲料中添加脯氨酸对浅色黄姑鱼生长、体组成及抗氧化能力的影响[J].上海海洋大学学报,2023,32(1):89-97. [10]邓紫连,宋伟,芮千龙,等.脯氨酸对中华鳖生长性能、抗氧化能力和抗嗜水气单胞菌感染能力的影响[J].动物营养学报,2023,35(5):3213-3227. [11]ZHOU B N, SONG W, ZHU L Y, et al. The anti-lipopolysaccharide effect of glycine via fibroblastic cell culture from the embryonic calipash tissue of Chinese soft-shelled turtle Pelodiscus sinensis[J]. Aquaculture Reports,2023,30:101575. [12]SLOMINSKI A, PISARCHIK A, ZBYTEK B, et al. Functional activity of serotoninergic and melatoninergic systems expressed in the skin[J]. Journal of Cellular Physiology,2003,196(1):144-153. [13]WU G Y, BAZER F W, BURGHARDT R C, et al. Proline and hydroxyproline metabolism:implications for animal and human nutrition[J]. Amino Acids,2011,40(4):1053-1063. [14]ZHANG K K, MAI K S, XU W, et al. Proline with or without hydroxyproline influences collagen concentration and regulates prolyl 4-hydroxylase α (I) gene expression in juvenile turbot (Scophthalmus maximus L. )[J]. Journal of Ocean University of China,2015,14(3):541-548. [15]KANG P, ZHANG L L, HOU Y Q, et al. Effects of L-proline on the growth performance, and blood parameters in weaned lipopolysaccharide (LPS)-challenged pigs[J]. Asian-Australasian Journal of Animal Sciences,2014,27(8):1150-1156. [16]KRISHNAN N, DICKMAN M B, BECKER D F. Proline modulates the intracellular redox environment and protects mammalian cells against oxidative stress[J]. Free Radical Biology and Medicine, 2008,44(4):671-681. [17]WANG W, LEE J, JIN Q X, et al. Effects of osmolytes on Pelodiscus sinensis creatine kinase:a study on thermal denaturation and aggregation[J]. International Journal of Biological Macromolecules,2013,60:277-287. [18]KAUL S, SHARMA S S, MEHTA I K. Free radical scavenging potential of L-proline:evidence from in vitro assays[J]. Amino Acids,2008,34(2):315-320. [19]WANG J C, XUE Z M, LIN J, et al. Proline improves cardiac remodeling following myocardial infarction and attenuates cardiomyocyte apoptosis via redox regulation[J]. Biochemical Pharmacology,2020,178:114065. [20]李明,张思,张建清,等.温度、盐度和 pH 对仿刺参体腔细胞活性氧产生的影响[J].水产科学,2016,35(4):346-351. [21]李文静,徐文婷,谭颖,等.活性氧类与mTOR信号通路[J].生命的化学,2010,30(6):866-870. [22]PHANG J M, LIU W, ZABIRNYK O. Proline metabolism and microenvironmental stress[J]. Annual Review of Nutrition,2010,30:441-463. [23]KARNA E, SZOKA L, HUYNH T Y L, et al. Proline-dependent regulation of collagen metabolism[J]. Cellular and Molecular Life Sciences,2020,77(10):1911-1918. [24]LI X, SUN X D, KAN C X, et al. COL1A1 A novel oncogenic gene and therapeutic target in malignancies[J]. Pathology-Research and Practice,2022,236:154013. [25]ZHOU J F, YANG Y S, ZHANG H L, et al. Overexpressed COL3A1 has prognostic value in human esophageal squamous cell carcinoma and promotes the aggressiveness of esophageal squamous cell carcinoma by activating the NF-κB pathway[J]. Biochemical and Biophysical Research Communications,2022,613:193-200. [26]RAPPU P, SALO A M, MYLLYHARJU J, et al. Role of prolyl hydroxylation in the molecular interactions of collagens[J]. Essays in Biochemistry,2019,63(3):325-335. [27]GUO H J, ZHANG Q H, LIU X, et al. Dietary hydroxyproline promotes collagen deposition in swim bladder through regulating biosynthesis of amino acid:in-vitro and in-vivo investigations in Nibea coibor[J]. Aquaculture,2023,573:739614. [28]荣华,杨钰香,王晓雯,等.脯氨酸对浅色黄姑鱼鱼鳔胶原蛋白沉积及相关基因表达的影响[J].动物营养学报,2022,34(5):3184-3193. [29]CHOW W Y, FORMAN C J, BIHAN D, et al. Proline provides site-specific flexibility for in vivo collagen[J]. Scientific Reports,2018,8(1):13809. [30]BARBUL A. Proline precursors to sustain mammalian collagen synthesis[J]. The Journal of Nutrition,2008,138(10):2021S-2024S. [31]LI P, WU G Y. Roles of dietary glycine, proline, and hydroxyproline in collagen synthesis and animal growth[J]. Amino Acids,2018,50(1):29-38. [32]GORRES K L, RAINES R T. Prolyl 4-hydroxylase[J]. Critical Reviews in Biochemistry and Molecular Biology,2010,45(2):106-124. [33]HU B Q, XIAO J, YI P P, et al. Cloning and characteristic of MMP1 gene from Hyriopsis cumingii and collagen hydrolytic activity of its recombinant protein[J]. Gene,2019,693:92-100. [34]JIANG Y, LIU L, DENG Y X, et al. MMP13 promotes the osteogenic potential of BMP9 by enhancing Wnt/β-catenin signaling via HIF-1α upregulation in mouse embryonic fibroblasts[J]. The International Journal of Biochemistry & Cell Biology,2023,164:106476. [35]刘明明,李爱玲,修瑞娟.基质金属蛋白酶的研究进展[J].中国病理生理杂志,2018,34(10):1914-1920. [36]NAGASE H, VISSE R, MURPHY G. Structure and function of matrix metalloproteinases and TIMPs[J]. Cardiovascular Research,2006,69(3):562-573. [37]LEONARDI A, CORTIVO R, FREGONA I, et al. Effects of Th2 cytokines on expression of collagen, MMP-1, and TIMP-1 in conjunctival fibroblasts[J]. Investigative Ophthalmology & Visual Science,2003,44(1):183-189. [38]YAN S J, ZHAN M, LIU Z, et al. Insight into the transcriptional regulation of key genes involved in proline metabolism in plants under osmotic stress[J]. Biochimie,2025,228:8-14. [39]HANIF S, FAROOQ S, KIANI M Z, et al. Surface modified ZnO NPs by betaine and proline build up tomato plants against drought stress and increase fruit nutritional quality[J]. Chemosphere,2024,362:142671. [40]MEENA M, DIVYANSHU K, KUMAR S, et al. Regulation of L-proline biosynthesis, signal transduction, transport, accumulation and its vital role in plants during variable environmental conditions[J]. Heliyon,2019,5(12):e02952. [41]彭捷,崔翠菊,张立楠,等.高温胁迫对高温筛选后海带配子体存活率和生理的影响[J].水产科学,2016,35(1):32-36. [42]PANDHARE J, DONALD S P, COOPER S K, et al. Regulation and function of proline oxidase under nutrient stress[J]. Journal of Cellular Biochemistry,2009,107(4):759-768. [43]季昀.羟脯氨酸对葡聚糖硫酸钠诱导的小鼠肠炎的缓解作用及机制[D].北京:中国农业大学,2018. [44]WEI Z H, ZHOU H H, ZHANG Y J, et al. Integrative analysis of transcriptomics and metabolomics profiling on flesh quality of large yellow croaker Larimichthys crocea fed a diet with hydroxyproline supplementation[J]. The British Journal of Nutrition,2018,119(4):359-367.