Anti-aging Effects and Mechanisms of Recombinant Human-derived Collagen on Aging Mouse Induced by D-Galactose
-
摘要: 为探究重组人源胶原蛋白（recombinant human-derived collagen，RHC）的抗衰老作用及机制，通过300 mg/kg D-半乳糖腹腔注射构建衰老小鼠模型，并采用不同剂量RHC进行干预，研究衰老模型小鼠皮肤透明质酸、胶原蛋白、脂褐素、羟脯氨酸含量、抗氧化酶活性、与表皮细胞增殖分化和胶原蛋白合成相关的关键基因、蛋白的表达量等变化。结果表明，与衰老模型组（SLM）相比，小鼠经过RHC干预后，皮肤的褶皱程度明显减轻，胶原蛋白含量和含水量显著增加（P<0.05），超氧化物歧化酶（Superoxide dismutase，SOD）含量增加，丙二醛（Malondialdehyde，MDA）含量显著减少（P<0.0001）。与SLM组相比，RHC处理组的抗氧化指标，如谷胱甘肽过氧化物酶（GSH-Px）含量显著增加，而褐质素含量、病理评分均有所降低，且不同剂量处理组之间也有一定的差异性。此外，RHC处理组小鼠皮肤中的关键基因（EGFR、TIMP-1、COL1A2、COL3A1、TGF-βⅠ、TGF-βⅡ）的表达水平呈上升趋势， MMP-1、MMP-3 基因和蛋白的表达降低。综上所述，RHC具有良好的抗皮肤衰老作用，其作用机制可能通过抗氧化、以及改善 EGFR、TGF 信号通路，调节 MMPs和TIMPs 动态平衡，促进表皮细胞的增殖分化和胶原蛋白合成从而起到延缓皮肤衰老的作用。Abstract: In order to explore the anti-aging effect and mechanism of recombinant recombinant human-derived collagen (RHC), the aging mouse model was constructed through 300 mg/kg D-galactose intraperitoneal injection, and different doses of RHC were used to study the changes of hyaluronic acid, collagen, lipofuscin, hydroxyproline content, antioxidant enzyme activity, key genes and protein expression related to epidermal cell proliferation and differentiation and collagen synthesis. The results showed that compared with the aging model group (SLM), the intervention groups that was gavaged RHC had significantly less skin folds, higher collagen content (P<0.05) and superoxide dismutase (SOD), while lower malondialdehyde (MDA) content in the skins (P<0.0001). Compared with the SLM group, antioxidant indicators such as glutathione peroxidase (GSH-Px) content were increased in the RHC intervention groups, while lipofuscin content and pathological scores were reduced, and significant effect were also observed. In addition, the expression levels of key genes and protein (EGFR, TIMP-1, COL1A2, COL3A1, TGF-βⅠ, TGF-βⅡ) were increased, while those of MMP-1, MMP-3 in the skin of mouse were decreased in the RHC intervention groups. In conclusion, RHC has a good anti-aging effect on the skin, and its mechanism may be related to increase antioxidant effect, promote the EGFR, TGF pathway, and modulate the expression balance of MMPs and TIMPs, promote the proliferation and differentiation of epidermal cells, and the synthesis of collagen.
-
Key words:
- recombinant human-derived collagen /
- D-galactose /
- aging model /
- anti-aging /
- lipofuscin
-
图 3 RHC对小鼠皮肤组织抗氧化指标的测定
Figure 3. Determination of antioxidant indexes of mouse skin tissue by RHC
图 6 组织切片病理及评分
注：A. NC组；B. SLM组；C. LRHC组；D. MRHC组；E. HRHC组；F. 对照组和各剂量处理组的病理评分。
Figure 6. Pathology and integral of tissue sections
表 1 实验分组与设计
Table 1. Experimental group and design
编号 组别 受试物 剂量
（mg/kg）给药
方式给药
周期小鼠
数量NC 正常对照组 − − ig 6 周 12只 SLM 衰老模型组 − − ig 6 周 12只 LRHC RHC低剂量 RHC低剂量 50 ig 6 周 12只 MRHC RHC中剂量 RHC中剂量 500 ig 6 周 12只 HRHC RHC高剂量 RHC高剂量 1000 ig 6 周 12只 DC 胶原蛋白干预组 胶原蛋白 1000 ig 6 周 12只 注：ig表示经口灌胃。 
下载: 导出CSV
表 2 皮肤损伤评分标准
Table 2. Score standard of skin injury
皮肤病理学改变 积分 最高分 A.棘层肥厚（a） 轻度（表皮为正常厚度1.5～3倍） 1 3 中度（表皮为正常厚度3～4倍） 2 重度（表皮为正常厚度4倍以上） 3 B.过度角化 （b）颗粒层增厚 1 1 （c）角层增厚 1 1 C.其他表皮改变 （d）颗粒层缺乏 1 1 （e）角化不全 1 1 （f）表皮细胞空泡化或细胞内水肿或基底细胞液化变性 1 1 （g）海绵形成 棘细胞间水肿 1 2 水疱形成 2 D.表皮坏死 （h）表皮坏死 轻度（占表皮切面的1/3以下） 8 15 中度（占表皮切面的1/3～2/3） 10 重度（占表皮切面的2/3以上） 15 E.真皮变化 （i）真皮结缔组织血管扩张充血或水肿 1 1 （j）胶原纤维变性或解离 1 1 （k）真皮炎细胞浸润 轻度 1 3 中度 2 重度 3
下载: 导出CSV
-
[1] TURNHEIM K. When drug therapy gets old: Pharmacokinetics and pharmacodynamics in the elderly[J]. Experimental Gerontology,2003,38(8):843−853. doi: 10.1016/S0531-5565(03)00133-5 [2] 魏姝玥, 王海英, 李爱兰, 等. 脂肪干细胞对D-半乳糖致裸鼠皮肤老化的拮抗作用及对皮肤恢复功能的影响[J]. 中国美容医学,2020,29(1):62−67. [WEI Z Y, WANG H Y, LI A L, et al. Antagonistic effect of adipose-derived stem cells on skin aging induced by D-galactose in nude mice and its effect on skin recovery[J]. Chinese Journal of Aesthetic Medicine,2020,29(1):62−67. doi: 10.15909/j.cnki.cn61-1347/r.003476 [3] CHATTOPADHYAY S, RAINES R T. Review collagen-based biomaterials for wound healing[J]. Biopolymers,2014,101(8):21−33. [4] 蔡东联. 不可忽视老年斑的危害[J]. 求医问药,2012(10):41. [CAI D L. Don't ignore the dangers of age spots[J]. Seek Medical,2012(10):41. [5] OGRODNIK M, SALMONOWICZ H, GLADYSHEV V N. Integrating cellular senescence with the concept of damage accumulation in aging: Relevance for clearance of senescent cells[J]. Aging Cell,2019,18(1):e12841. doi: 10.1111/acel.12841 [6] KUDRYAVTSEVA A V, KRASNOV G S, DMITRIEV A A, et al. Mitochondrial dysfunction and oxidative stress in aging and cancer[J]. Oncotarget,2016,7(29):44879−44905. doi: 10.18632/oncotarget.9821 [7] TERMAN A, KURZ T, NAVRATIL M, et al. Mitochondrial turnover and aging of long-lived postmitotic cells: The mitochondrial-lysosomal axis theory of aging[J]. Antioxidants & Redox Signaling,2010,12(4):503−535. [8] SARETZKI G. Telomeres, telomerase and ageing[J]. Sub-Cellular Biochemistry,2018,9:221−308. [9] DAVALLI P, MITIC T, CAPORALI A, et al. ROS, cell senescence, and novel molecular mechanisms in aging and age-related diseases[J]. Oxidative Medicine and Cellular Longevity,2016,2016:3565127. [10] AUNG-HTUT M T, AYER A, BREITENBACH M, et al. Oxidative stresses and ageing[J]. Subcell Biochem,2012,57:13−54. [11] GOMEZ-LINTON D R, ALAVEZ S, ALARCON-AGUILA A, et al. Some naturally occurring compounds that increase longevity and stress resistance in model organisms of aging[J]. Biogerontology,2019,20(5):583−603. doi: 10.1007/s10522-019-09817-2 [12] 米钰, 惠俊峰, 范代娣, 等. 类人胶原蛋白生物相容性实验研究[J]. 西北大学学报( 自然科学版),2004,34(1):66−72. [MI Y, HUI J F, FAN D D, et al. The biocompatibility of human-like collagen[J]. Journal of Northwest University (Natural Science Edition),2004,34(1):66−72. [13] YOON D, YOON D, CHA H J, et al. Enhancement of wound healing efficiency mediated by artificial dermis functionalized with EGF or NRG1[J]. Biomedical Materials (Bristol, England),2018,13(4):45007. doi: 10.1088/1748-605X/aaac37 [14] 余建军, 赵铧. 重组类人胶原蛋白研究应用进展[J]. 日用化学品科学,2022,45(2):48−51. [YU J J, ZHAO H. Progress in the research and application of recombinant human collagen[J]. Daily Chemicals Science,2022,45(2):48−51. doi: 10.3969/j.issn.1006-7264.2022.02.011 [15] YUAN T, ZHANG L, LI K F, et al. Collagen hydrogel as an immunomodulatory scaffold in cartilage tissue engineering[J]. Journal of Biomedical Materials Research. Part B, Applied Biomaterials,2014,102(2):337−344. doi: 10.1002/jbm.b.33011 [16] AN B, LIN Y S, BRODSKY B, et al. Collagen interactions: Drug design and delivery[J]. Advanced Drug Delivery Reviews,2016,97:69−84. doi: 10.1016/j.addr.2015.11.013 [17] JIA Q, LUO Y. The selective roles of chaperone systems on overexpression of human-like collagen in recombinant Escherichia coli[J]. J Ind Microbiol Biotechnol,2014,41(11):1667−1675. doi: 10.1007/s10295-014-1500-x [18] TANG Y, YANG X, HANG B, et al. Efficient production of hydroxylated human-like collagen via the co-expression of three key genes in Escherichia coli origami (DE3)[J]. Applied Biochemistry and Biotechnology,2016,178(7):1458−1470. doi: 10.1007/s12010-015-1959-6 [19] 齐志峰, 郑双进, 张晓瑜, 等. 射频联合重组人源胶原蛋白凝胶修复面部皮肤屏障功能的研究[J]. 中国医疗美容,2020,10(3):48−52. [QI Z F, ZHENG S J, ZHANG X Y, et al. Radiofrequency combined with recombinant human collagen-gel to repair the function of facial skin barrier[J]. China Medical Cosmetology,2020,10(3):48−52. doi: 10.19593/j.issn.2095-0721.2020.03.013 [20] 郭亚媛. 重组人胶原蛋白水凝胶复合成纤维细胞修复皮肤缺损的实验研究[D]. 西安: 西北大学, 2019.GUO Y Y. The study of recombinant human collagen hydrogel containing fibroblastsin repairing skin defect[D]. Xi'an: Northwest University, 2019. [21] 牛佳玮, 莫玲, 王子锃, 等. 恭城油茶减轻D-半乳糖致小鼠衰老的氧化应激[J]. 现代食品科技,2021,37(4):1−6. [NIU J W, MO L, WANG Z C, et al. Gongcheng oil tea attenuates oxidative stress in d-galactose-induced aging mice[J]. Modern Food Science & Technology,2021,37(4):1−6. doi: 10.13982/j.mfst.1673-9078.2021.4.0913 [22] XIAO LIAN F, XIAO FANG Z, SHI QI L, et al. Preparation of aminated fish scale collagen and oxidized sodium alginate hybrid hydrogel for enhanced full-thickness wound healing[J]. International Journal of Biological Macromolecules,2020,164:626−637. doi: 10.1016/j.ijbiomac.2020.07.058 [23] JING C, PAN W, YAN NAN L, et al. Double crosslinked HLC-CCS hydrogel tissue engineering scaffold for skin wound healing[J]. International Journal of Biological Macromolecules,2020,155:625−635. doi: 10.1016/j.ijbiomac.2020.03.236 [24] LEI H, CHEN HUI ZHU, DAI DI F. Optimization of human-like collagen composite polysaccharide hydrogel dressing preparation using response surface for burn repair[J]. Carbohydrate Polymers, 2020, 239. [25] HUI Z, YU XIAO L, CAN WEN C, et al. Responsive drug-delivery microcarriers based on the silk fibroin inverse opal scaffolds for controllable drug release[J]. Applied Materials Today, 2020, 19(C): 100540. [26] FAROKHI M, MOTTAGHITALAB F, REIS R L, et al. Functionalized silk fibroin nanofibers as drug carriers: Advantages and challenges[J]. Journal of Controlled Release, 2020, 321(C): 324-347. [27] 宋晓娟. 类人胶原蛋白治疗颜面再发性皮炎疗效观察及研究[J]. 吉林医学,2016,37(9):2259−2260. [SONG X J. Study on the efficacy of human-like collagen in the treatment of facial reoccurring dermatitis[J]. Jilin Medical Journal,2016,37(9):2259−2260. doi: 10.3969/j.issn.1004-0412.2016.09.088 [28] KONG S Z, LI JC, LI S D, et al. Anti-aging effect of chitosan oligosaccharide on d-galactose-induced subacute aging in mice[J]. Mar Drugs,2018,16(6):181. doi: 10.3390/md16060181 [29] 薛文, 秦菊, 努尔买买提·阿穆提, 等. 不同ELISA和AGID试剂盒检测马传染性贫血病毒抗体的比较[J]. 中国动物检疫,2022,39(7):103−107. [XUE W, QIN J, NURMAIMAITI·A, et al. Comparison of different ELISA and AGID kits for equine infectious anemia virus antibodies[J]. Chinese Animal Quarantine,2022,39(7):103−107. [30] 钟召兵. 澳州进口与中国本地荷斯坦牛血清抗氧化指标和微量元素含量比较研究[J]. 中国奶牛,2022(7):16−19. [ZHONG Z B. Comparative study on antioxidant index and trace element content of Australian imported and local Holstein cattle in China[J]. Chinese Dairy Cows,2022(7):16−19. doi: 10.19305/j.cnki.11-3009/s.2022.07.004 [31] 林才云, 王联珠, 许加超, 等. 培养条件对副溶血性弧菌AI-2活性及其合成关键基因表达水平的影响[J]. 食 品安全质量检测学报,2020,11(9):2924−2929. [LIN C Y, WANG L Z, XU J C, et al. Effects of culture conditions on AI-2 activity of V. parahaemolyticus and the expression levels of key genes for synthesis[J]. Food Safety and Quality Testing Journal,2020,11(9):2924−2929. [32] 康俞莉, 赵颖, 章强强. 荧光原位杂交法观察小鼠皮肤组织切片中型无绿藻感染的研究[J]. 中华皮肤科杂志,2014,47(9):646−649. [KANG Y L, ZHAO Y, ZHANG Q Q. Observation of medium-free green algae infection in mouse skin tissue sections by fluorescence in situ hybridization[J]. The Chinese Journal of Dermatology,2014,47(9):646−649. [33] 熊慧, 戢丹菊, 杨家林, 等. 九龙虫对衰老小鼠皮肤的影响[J]. 鄂州大学学报,2021,28(5):108−110. [XIONG H, YANG J L, LIU D H, et al. Effects of Kowloon worm on skin of aged mice[J]. The Journal of Ezhou University,2021,28(5):108−110. doi: 10.16732/j.cnki.jeu.2021.05.037 [34] 颜泽清, 娄红军, 王元. 透明质酸对犬皮肤水分的改善效果研究[J]. 中国工作犬业,2019(9):14−15. [YAN Z Q, LOU H J, WANG Y. Study on the effect of hyaluronic acid on the improvement of skin moisture in dogs[J]. China Working Dog,2019(9):14−15. [35] TOMOKO, ASAI T, OIKAWA F, et al. Food-derived collagen peptides, Prolyl-Hydroxyproline (Pro-Hyp), and Hydroxyprolyl-Glycine (Hyp-Gly) enhance growth of primary cultured mouse skin fibroblast using fetal bovine serum free from hydroxyprolyl peptide[J]. International Journal of Molecular Sciences,2019,21(1):229. [36] GILCHREST B A, GARMYS M, YEAR M. Aging and photoaging effect gene expression in cultured human keratinocyte[J]. Arch Dermatol,1994,130(1):82−86. doi: 10.1001/archderm.1994.01690010086013 [37] 李卫, 元发芝. 皮肤衰老及抗衰老研究进展[J]. 中华医学美学美容杂志,2004,10(1):60−61. [LI W, YUAN F Z. Advances in skin ageing and anti-ageing research[J]. Chinese Journal of Medical Aesthetics and Cosmetology,2004,10(1):60−61. [38] 伍春. 党参多糖对D-半乳糖致衰老小鼠皮肤抗氧化作用及基因表达谱的影响[D]. 兰州: 甘肃中医学院, 2014WU C. Effect of Dangshen polysaccharide on skin antioxidant effect and gene expression profile of D-galactose-induced aging mice[D]. Lanzhou: College of Traditional Chinese Medicine, 2014. [39] 王振富. 党参对大强度训练大鼠心肌线粒体抗氧化能力的的影响[J]. 江苏医药,2011,37(13):1515−1518. [WANG Z F. Effect of Dangshen on the antioxidant capacity of myocardial mitochondria in intensively trained rats[J]. Jiangsu Medicine,2011,37(13):1515−1518. [40] 王岚, 张帆, 王艳, 等. 甘肃党参水提物对 D-半乳糖所致衰老小鼠脑组织 SOD、MDA 影响的实验研究[J]. 中国中医药科技,2010,17(2):148−149. [WANG L, ZHANG F, WANG Y, et al. Experimental study on the effect of Gansu Dangshen water extract on brain tissue SOD and MDA in aging mice caused by D-galactose[J]. Chinese TCM Science and Technology,2010,17(2):148−149. [41] WILSON J, BALKWILL F. The role of cytokines in the epithelial cancer microenvironment[J]. Seminars in Cancer Biology,2002,12(2):113−120. [42] SHEN B C, LIEN H C, H N, et al. Increased expression and activation of gelatinolytic matrix metalloproteinases is associated with the progression and recurrence of human cervical cancer[J]. Cancer Research,2003,63(10):6537−6542. [43] NIETO N. 335 Kupffer cells induce a fibrogenic response by transactivating the COL1A1 and COL1A2 promoters and by decreasing collagen I degradation[J]. Journal of Hepatology,2006,44(S2):S129−S129. [44] QUAN T, HE T, KANG S, et al. Ultraviolet Irradiation alters transforming growth factor β/smad pathway in human skin in vivo[J]. Journal of Investigative Dermatology,2002,119(2):499−506. doi: 10.1046/j.1523-1747.2002.01834.x [45] 马作霖. 乳清粉调节D-半乳糖诱导衰老小鼠肠道菌群结构及抗衰老作用研究[D]. 兰州: 甘肃农业大学, 2021.MA Z L. Study on intestinal flora structure and anti-aging effect of D-galactose [D]. Lanzhou: Gansu Agricultural University, 2021. -
下载:
点击查看大图
图(6) / 表(2)
计量
- 文章访问数: 15
- HTML全文浏览量: 31
- PDF下载量: 0
- 被引次数: 0
京公网安备 11010502036328号 京ICP备17033152号