基于制备液相色谱与非靶向代谢组学筛选鹿茸促进软骨细胞增殖的关键因子

doi:10.16431/j.cnki.1671-7236.2026.01.046

摘要/Abstract

摘要：

目的鹿茸具有显著促进软骨损伤修复的作用，本研究旨在探究鹿茸中可促进软骨细胞增殖的关键因子。方法采用超声和冷水提取法分别对鹿茸顶端生长中心组织进行提取，并通过高效液相色谱法鉴定最优提取方法；采用制备液相色谱分离鹿茸提取物中不同峰组分；体外培养大鼠关节软骨细胞，通过CCK-8法比较鹿茸提取物各峰组分对细胞增殖的影响；采用非靶向代谢组学分析鹿茸提取物不同峰组分的组成差异；进一步通过CCK-8法验证鹿茸提取物关键物质对大鼠软骨细胞增殖的影响。结果高效液相色谱分析显示，超声提取法为鹿茸提取物的高效提取方法，所获峰型良好且操作便捷。制备液相色谱成功分离鹿茸提取物4个单独峰组分，各峰均与鹿茸提取物色谱峰一一对应，基线平稳、峰型良好，杂峰信号极少或无。进一步试验证明，与其他浓度的鹿茸提取物相比，0.1 mg/mL鹿茸提取物可极显著促进大鼠关节软骨细胞增殖（P<0.01）。与其他峰组分相比，峰1组分对大鼠关节软骨细胞增殖有极显著促进作用（P<0.01）。非靶向代谢组学分析表明，鹿茸提取物峰组分以脂质类为主（43.74%）；主成分分析（PCA）可清晰区分峰1~4组分；其中峰1组分的精胺相对含量极显著高于其他组分（P<0.01）。与其他浓度精胺相比，0.25 mg/mL精胺极显著促进大鼠关节软骨细胞增殖（P<0.01）。结论鹿茸提取物可显著促进大鼠关节软骨细胞增殖，制备液相色谱成功分离鹿茸提取物4个单独峰组分，峰1 组分中的精胺是鹿茸提取物显著促进软骨细胞增殖的关键因子。

关键词: 鹿茸; 制备液相色谱; 非靶向代谢组学; 软骨细胞增殖; 精胺

Abstract:

Objective Deer antler exhibit significant efficacy in promoting cartilage injury repair. This study aimed to identify the underlying key factor responsible for the cartilage-repairing effects of deer antler. Method Tissue from the apical growth center of fresh antlers was extracted using ultrasonic and cold-water extraction methods， respectively， with the optimal method identified by high-performance liquid chromatography （HPLC）. Preparative liquid chromatography was employed to isolate different peak fractions from the extract. Rat articular chondrocytes were isolated and cultured in vitro. The proliferative effects of the different peak fractions on these chondrocytes were compared using the CCK-8 assay. The compositional differences among the peak fractions were analyzed by untargeted metabolomics. Further validation of the effects of key substances in the extract on chondrocyte proliferation was performed using the CCK-8 assay. Result Ultrasonic extraction was identified via HPLC as an effective method for deer antler extract preparation， producing well-resolved peaks with convenient operation. Using preparative liquid chromatography， this study successfully isolated four distinct peak fractions from the extract， all exhibiting stable baselines， well-defined peaks， and minimal or no extraneous signals. Compared with other concentrations extract，0.1 mg/mL extract significantly promoted proliferation in rat articular chondrocytes （P<0.01）. Compared with other peak components， peak 1 exhibited an extremely significant promoting effect on chondrocyte proliferation （P<0.01）. Untargeted metabolomics analysis demonstrated that lipid-like metabolites comprised the majority of the extract （43.74%）， and principal component analysis （PCA） clearly separated peaks 1-4. The relative spermine content in peak 1 was extremely significantly higher than in other fractions （P<0.01）. Compared to other concentrations of spermine， 0.25 mg/mL of spermine extremely markedly enhanced chondrocyte proliferation （P<0.01）. Conclusion Antler extract significantly promotes the proliferation of rat articular chondrocytes. Preparative liquid chromatography successfully isolated four distinct peak components of the extract， and spermine in peak 1 was identified as the key factor responsible for significantly enhancing chondrocyte proliferation of deer antler.

Key words: deer antler; preparative liquid chromatography; untargeted metabolomics; chondrocyte proliferation; spermine

中图分类号:

S825

赵琛, 朱爽, 李吉萍, 胡鹏飞, 李春义. 基于制备液相色谱与非靶向代谢组学筛选鹿茸促进软骨细胞增殖的关键因子[J]. 中国畜牧兽医, 2026, 53(1): 509-519.

ZHAO Chen, ZHU Shuang, LI Jiping, HU Pengfei, LI Chunyi. Screening Key Factor of Deer Antler in Promoting Chondrocyte Proliferation Through Preparative Liquid Chromatography and Untargeted Metabolomics[J]. China Animal Husbandry & Veterinary Medicine, 2026, 53(1): 509-519.

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参考文献 27

[1]	王玉俗，褚文辉，李久波，等.干细胞ECM修复软骨缺损与鹿茸干细胞ECM的研究进展［J］.特产研究，2020，42（1）：64-68.
	WANG Y S， CHU W H， LI J B，et al. Research progress of stem cell extracellular matrix in repairing cartilage defects and antler stem cell extracellular matrix［J］.Special Wild Economic Animal and Plant Research，2020，42（1）：64-68.（in Chinese）
[2]	李勋胜，赵海平，丛培强，等.鹿茸软骨内血管生成研究进展［J］.黑龙江畜牧兽医，2021，23：46-50.
	LI X S， ZHAO H P， CONG P Q，et al. Research progress on agiogenesis in deer antler cartilage［J］.Heilongjiang Animal Science and Veterinary Medicine，2021，23：46-50. （in Chinese）
[3]	HU P F， WANG Z， LI J P，et al. IGF1R and LOX modules are related to antler growth rate revealed by integrated analyses of genomics and transcriptomics［J］.Animals，2022，12（12）：1522.
[4]	付晶，褚文辉，孙红梅，等.血管化鹿茸软骨组织与骨组织修复［J］.中国组织工程研究，2016，20（46）：6970-6977.
	FU J， CHU W H， SUN H M，et al. Vascularized antler cartilage and bone tissue repair［J］.Chinese Journal of Tissue Engineering Research，2016，20（46）：6970-6977.（in Chinese）
[5]	ZHOU J， ZHAO J， WANG Y，et al. Repair of mechanical cartilage damage using exosomes derived from deer antler stem cells［J］.Frontiers in Bioscience-Landmark，2024，29（8）：309.
[6]	ZHANG Z X， HE C X， BAO C H，et al. miRNA profiling and its potential roles in rapid growth of velvet antler in Gansu Red deer （Cervus elaphus kansuensis）［J］.Genes，2023，14（2）：424.
[7]	WANG Y S， CHU W H， ZHAI J J，et al. High quality repair of osteochondral defects in rats using the extracellular matrix of antler stem cells［J］.World Journal of Stem Cells，2024，16（2）：176-190.
[8]	ZHONG J H， ZHANG J C， ZHOU Z W，et al. Novel insights into the effect of deer IGF-1 on chondrocyte viability and IL-1β-induced inflammation response［J］.Journal of Biochemical and Molecular Toxicology，2023，37（1）：e23227.
[9]	高宏伟.鹿茸提取物调控软骨细胞的生物学机制研究［D］.长春：长春中医药大学，2020.
	GAO H Y. Study on the biological mechanism of pilose antler extract regulating chondrocytes［D］. Changchun：Changchun University of Chinese Medicine， 2020.（in Chinese）
[10]	ZHANG J， FENG C R， GE P，et al. High purity separation of hypericin from Hypericum perforatum L. extract with macroporous resin column coupling preparative liquid chromatography［J］. Process Biochemistry，2021，103：107-113.
[11]	高红，张炜，靳明坤.优化法结合制备型高效液相色谱分离纯化虾青素［J］.食品研究与开发，2024，45（14）：119-126.
	GAO H， ZHANG W， JIN M K. Separation and purification of astaxanthin by optimization methodology combined with preparative high performance liquid chromatography［J］.Food Research and Development，2024，45（14）：119-126.（in Chinese）
[12]	王洪宇，张慧君，黄超，等.制备液相色谱法在辅酶Q10分离纯化中的应用研究［J］.化工设计通讯，2024，50（3）：96-105.
	WANG H Y， ZHANG H J， HUANG C，et al. Application study of preparative liquid chromatography in the purification and quality enhancement of coenzyme Q10［J］.Chemical Engineering Design Communications，2024，50（3）：96-105.（in Chinese）
[13]	MUSSAP M， LODDO C， FANNI C，et al. Metabolomics in pharmacology—A delve into the novel field of pharmacometabolomics［J］.Expert Review of Clinical Pharmacology，2020，13（2）：115-134.
[14]	黄亚成，任东立，何斌，等.转录组学和代谢组学在植物非生物胁迫中的研究进展［J］.江苏农业科学，2023，51（22）：1-7.
	HUANG Y C， REN D L， HE B，et al. Research progress in transcriptomics and metabolomics in plant abiotic stress［J］.Jiangsu Agricultural Sciences，2023，51（22）：1-7.（in Chinese）
[15]	杨霞，吴柳燕，赵玉雪，等.代谢组学在植物与病原真菌相互作用研究中的应用［J］.贵州林业科技，2024，52（4）：77-81.
	YANG X， WU L Y， ZHAO Y X，et al. Applying metabolomics for plant-pathogenenic fungi interactions［J］.Guizhou Forestry Science and Technology，2024，52（4）：77-81.（in Chinese）
[16]	丛心缘.南极磷虾中虾青素的分离、鉴定及抗氧化活性研究［D］.青岛：青岛大学，2019.
	CONG X Y. Isolation，identification and antioxidant activity of astaxanthin from Antarctic Krill［D］.Qingdao：Qingdao University，2019.（in Chinese）
[17]	廖晴，曾静，陈峻，等.艾灸调节circPan3/miR-667-5p/Ghrelin信号通路减轻膝骨关节炎大鼠的软骨病变［J］.中国组织工程研究，2025，29（12）：2475-2483.
	LIAO Q， ZENG J， CHEN J，et al. Moxibustion alleviates cartilage lesions in rats with knee osteoarthritis through regulating the circPan3/miR-667-5p/Ghrelin signaling pathway［J］.Chinese Journal of Tissue Engineering Research，2025，29（12）：2475-2483.（in Chinese）
[18]	LI T， CHEN S， PEI M. Contribution of neural crest-derived stem cells and nasal chondrocytes to articular cartilage regeneration［J］. Cellular and Molecular Life Sciences， 2020，77（23）：4847-4859.
[19]	PUEYO M A， ITO K， ZAUCKE F， et al. Restoring articular cartilage： Insights from structure， composition and development［J］. Nature Reviews Rheumatology，2025，21（5）：291-308.
[20]	LI C. Residual antler periosteum holds the potential to partially regenerate lost antler tissue［J］. Journal of Experimental Zoology Part A Ecological & Integrative Physiology，2021，335（4）：386-395.
[21]	FELEKE M， BENNETT S， CHEN J， et al. New physiological insights into the phenomena of deer antler： A unique model for skeletal tissue regeneration［J］. Journal of Orthopaedic Translation，2020，27：57-66.
[22]	TAN Y Q， LOH C K， MOHD S S， et al. Improved HPLC method with automated pre-column sample derivatisation for serum pegylated L-asparaginase activity measurement in paediatric acute lymphoblastic leukaemia patients［J］. Journal of Pharmaceutical and Biomedical Analysis，2024，247：116243.
[23]	CHANG L， LI Z， GUO H， et al. Function of polyamines in regulating cell cycle progression of cultured silkworm cells［J］. Insects，2021，12（7）：624.
[24]	RAMOT Y， TIEDE S， BÍRÓ T， et al. Spermidine promotes human hair growth and is a novel modulator of human epithelial stem cell functions［J］. PLoS One，2011，6（7）：e22564.
[25]	MONTI M G， PERNECCO L， MANFREDINI R， et al. Inhibition of cell growth by accumulated spermine is associated with a transient alteration of cell cycle progression［J］. Life Sciences，1996，58（23）：2065-2072.
[26]	CARNEVALE R， NOCELLA C， SCHIAVON S， et al. Beneficial effects of a combination of natural product activators of autophagy on endothelial cells and platelets［J］. British Journal of Pharmacology，2021，178（10）：2146-2159.
[27]	LIU Y， ZHANG X， YAO Y， et al. The effect of epigallocatechin gallate on laying performance， egg quality， immune status， antioxidant capacity， and hepatic metabolome of laying ducks reared in high temperature condition［J］. Veterinary Quarterly，2023，43（1）：1-11.