乳源外泌体包载连翘酯苷A和山奈酚的制备、表征及体外抗炎效果评价

doi:10.11843/j.issn.0366-6964.2025.05.043

摘要/Abstract

摘要：

本试验旨在制备负载连翘酯苷A(forsythoside A，FTA)和山奈酚(kaempferol，KPF)的乳源外泌体(milk-derived EVs，miEVs)递药系统，并体外评价其对脂多糖(lipopolysaccharides，LPS)诱导牛乳腺上皮细胞(mammary alveolar cells-large T，MAC-T)炎症模型的细胞迁移效果及相关炎性因子的影响。利用前期已建立并优化的乙酸法提取乳源外泌体，低温超声共孵育的方法制备负载FTA乳源外泌体(FTA-miEVs)和负载KPF乳源外泌体(KPF-miEVs)，以及建立炎症模型，并通过透射电子显微镜(transmission electron microscope，TEM)和免疫印迹(Western blot)方法评估制备效果，液质联用方法进行载药量测定，免疫荧光和划痕试验观察炎症模型对负载抗炎药物的乳源外泌体摄取，及炎症模型的细胞迁移效果的影响，酶联免疫吸附测定(enzyme linked immunosorbent assay，ELISA)和实时荧光定量PCR(real-time quantitative PCR，RT-qPCR)方法验证负载抗炎药物的乳源外泌体对炎症模型中相关炎性因子分泌及基因表达水平的影响。结果表明：FTA-miEVs和KPF-miEVs均具有外泌体典型结构，外形呈圆形，有部分凹陷，似“茶托”形结构；均可表达表面及内部特异性标记蛋白白细胞分化抗原81(cluster of differentiation 81，CD-81)和热休克蛋白70(heat shock protein 70，HSP70)；载药量分别为0.78%和9.79%；在8 h内，LPS诱导的MAC-T炎症模型对其摄取效果良好；且在24 h时，FTA-miEVs和KPF-miEVs对炎症模型细胞迁移的抑制率分别为608.48%和522.88%，显著高于单独使用miEVs、FTA和KPF的效果；其对炎症模型中白细胞介素-6(interleukin-6，IL-6)、白细胞介素-8(interleukin-8，IL-8)和肿瘤坏死因子α(tumor necrosis factor α，TNF-α)的分泌及基因表达水平均优于单独使用miEVs、FTA和KPF。综上所述，本试验成功制备FTA-miEVs和KPF-miEVs递药系统，综合结果发现在同等药量下可提高FTA和KPF的抗炎效果，为外泌体在药物递送系统的制备及临床应用提供新的思路。

关键词: 乳源外泌体, 连翘酯苷A, 山奈酚, 牛乳腺上皮细胞, 炎性因子

Abstract:

The purpose of this study was to prepare a milk-derived exosome (miEVs) delivery system loaded with forsythiaside A (FTA) and kaempferol (KPF) and evaluate its effect on lipopolysaccharide (LPS)-induced bovine mammary epithelial cells (MAC-T) in vitro Effects of cell migration and related inflammatory factors in inflammation models. FTA-loaded milk-derived exosomes (FTA-miEVs) and KPF-loaded milk-derived exosomes (KPF-miEVs) were prepared by using the acetic acid method established and optimized in the early stage, and the low-temperature ultrasonic co-incubation method was usedAs well as the establishment of inflammation models, and the evaluation of the preparation effect by transmission electron microscopy (TEM) and immunoblotting (Western blot) methods, the liquid chromatography-mass spectrometry method for drug loading determination, immunofluorescence and scratch experiments to observe the effect of inflammation models on the uptake of milk-derived exosomes loaded with anti-inflammatory drugs, and the effect of cell migration in the inflammation model, enzyme-linked immunosorbent assay (ELISA) and real-time quantitative PCR (RT-qPCR) were used to verify the effects of milk-derived exosomes loaded with anti-inflammatory drugs on the secretion of related inflammatory factors and gene expression levels in inflammation models. The results showed that FTA-miEVs and KPF-miEVs both had typical exosome structures, with circular shapes and partial depressions, similar to the "saucer" structure. Both surface and internal specific marker proteins leukocyte differentiation antigen 81 (CD-81) and heat shock protein 70 (HSP70) can be expressed. The drug loading was 0.78% and 9.79%, respectively. Within 8 hours, the LPS-induced MAC-T inflammation model had a good uptake effect; At 24 h, the inhibition rates of FTA-miEVs and KPF-miEVs on cell migration in inflammation models were 608.48% and 522.88%, respectively, which were significantly higher than those of miEVs, FTA and KPF alone. It has a positive effect on interleukin-6 (IL-6), interleukin-8 (IL-8), and tumor necrosis factor α (TNF-α) in models of inflammation. The secretion and gene expression levels were better than those of miEVs, FTA and KPF alone. In summary, the FTA-miEVs and KPF-miEVs drug delivery systems were successfully prepared in this experiment, and the comprehensive results showed that the anti-inflammatory effects of FTA and KPF could be improved at the same dosage, which provided a new idea for the preparation and clinical application of exosomes in drug delivery systems.

Key words: milk-derived exosomes, forsythiaside A, kaempferol, bovine mammary epithelial cells, inflammatory factors

中图分类号:

S859.5

赵莹, 王靖雷, 王萌, 王立斌, 张倩, 李志杰, 马鑫, 余四九, 潘阳阳. 乳源外泌体包载连翘酯苷A和山奈酚的制备、表征及体外抗炎效果评价[J]. 畜牧兽医学报, 2025, 56(5): 2481-2495.

ZHAO Ying, WANG Jinglei, WANG Meng, WANG Libin, ZHANG Qian, LI Zhijie, MA Xin, YU Sijiu, PAN Yangyang. Preparation and Characterization of Forsythiaside A and Kaempferol Encapsulated in Milk-derived Exosomes and Evaluation of Anti-inflammatory Effects in vitro[J]. Acta Veterinaria et Zootechnica Sinica, 2025, 56(5): 2481-2495.

图/表 13

表 1

图 1

图 2

图 3

图 4

图 5

图 6

图 7

表 2

图 8

表 3

图 9

图 10

参考文献 43

1	刘玉峰, 朱丽君, 孙珊珊, 等. 连翘酯苷A的体内外代谢及药动学研究进展[J]. 辽宁大学学报: 自然科学版, 2019, 46 (1): 51- 59.
	LIU Y F , ZHU L J , SUN S S , et al. Research progress on metabolism and pharmacokinetics of forsythoside A[J]. Journal of Liaoning University: Natural Sciences Edition, 2019, 46 (1): 51- 59.
2	QUAN X H , LIU H H , YE D M , et al. Forsythoside A alleviates high glucose-induced oxidative stress and inflammation in podocytes by inactivating MAPK signaling via MMP12 inhibition[J]. Diabetes Metab Syndr Obes, 2021, 14, 1885- 1895. doi: 10.2147/DMSO.S305092
3	WANG F , CAO G S , LI Y , et al. Characterization of forsythoside A metabolites in rats by a combination of UHPLC-LTQ-Orbitrap mass spectrometer with multiple data processing techniques[J]. Biomed Chromatogr, 2018, 32 (5): e4164. doi: 10.1002/bmc.4164
4	SHEN P X , YU J C , LONG X C , et al. Effect of forsythoside A on the transcriptional profile of bovine mammary epithelial cells challenged with lipoteichoic acid[J]. Reprod Domest Anim, 2023, 58 (1): 89- 96. doi: 10.1111/rda.14265
5	XU J , YIN P , LIU X W , et al. Forsythoside A inhibits apoptosis and autophagy induced by infectious bronchitis virus through regulation of the PI3K/Akt/NF-κB pathway[J]. Microbiol Spectr, 2023, 11 (6): e01921- e01923.
6	ZHANG X T , DING Y , KANG P , et al. Forsythoside A modulates zymosan-induced peritonitis in mice[J]. Molecules, 2018, 23 (3): 593. doi: 10.3390/molecules23030593
7	WANG C Y , CHEN S S , GUO H Y , et al. Forsythoside A mitigates Alzheimer's-like pathology by inhibiting ferroptosis-mediated neuroinflammation via Nrf2/GPX4 axis activation[J]. Int J Biol Sci, 2022, 18 (5): 2075- 2090. doi: 10.7150/ijbs.69714
8	ZHANG X , ZHANG H Q , GAO Y K , et al. Forsythoside A regulates autophagy and apoptosis through the AMPK/mTOR/ULK1 pathway and alleviates inflammatory damage in MAC-T cells[J]. Int Immunopharmacol, 2023, 118, 110053. doi: 10.1016/j.intimp.2023.110053
9	PERIFERAKIS A , PERIFERAKIS K , BADARAU I A , et al. Kaempferol: antimicrobial properties, sources, clinical, and traditional applications[J]. Int J Mol Sci, 2022, 23 (23): 15054. doi: 10.3390/ijms232315054
10	ALMATROUDI A , ALLEMAILEM K S , ALWANIAN W M , et al. Effects and mechanisms of kaempferol in the management of cancers through modulation of inflammation and signal transduction pathways[J]. Int J Mol Sci, 2023, 24 (10): 8630. doi: 10.3390/ijms24108630
11	YANG L , GAO Y C , BAJPAI V K , et al. Advance toward isolation, extraction, metabolism and health benefits of kaempferol, a major dietary flavonoid with future perspectives[J]. Crit Rev Food Sci Nutr, 2023, 63 (16): 2773- 2789. doi: 10.1080/10408398.2021.1980762
12	ALAM W , KHAN H , SHAH M A , et al. Kaempferol as a dietary anti-inflammatory agent: current therapeutic standing[J]. Molecules, 2020, 25 (18): 4073. doi: 10.3390/molecules25184073
13	ULLAH A , MUNIR S , BADSHAH S L , et al. Important flavonoids and their role as a therapeutic agent[J]. Molecules, 2020, 25 (22): 5243. doi: 10.3390/molecules25225243
14	ZHAO L L , YUAN X Y , WANG J B , et al. A review on flavones targeting serine/threonine protein kinases for potential anticancer drugs[J]. Bioorg Med Chem, 2019, 27 (5): 677- 685. doi: 10.1016/j.bmc.2019.01.027
15	IMRAN M , RAUF A , SHAH Z A , et al. Chemo-preventive and therapeutic effect of the dietary flavonoid kaempferol: a comprehensive review[J]. Phytother Res, 2019, 33 (2): 263- 275. doi: 10.1002/ptr.6227
16	YUAN P , SUN X F , LIU X , et al. Kaempferol alleviates calcium oxalate crystal-induced renal injury and crystal deposition via regulation of the AR/NOX2 signaling pathway[J]. Phytomedicine, 2021, 86, 153555. doi: 10.1016/j.phymed.2021.153555
17	MARSH S R , PRIDHAM K J , JOURDAN J , et al. Novel protocols for scalable production of high quality purified small extracellular vesicles from bovine milk[J]. Nanotheranostics, 2021, 5 (4): 488- 498. doi: 10.7150/ntno.62213
18	RASHIDI M , BIJARI S , KHAZAEI A H , et al. The role of milk-derived exosomes in the treatment of diseases[J]. Front Genet, 2022, 13, 1009338. doi: 10.3389/fgene.2022.1009338
19	AARTS J , BOLEIJ A , PIETERS B C H , et al. Flood control: how milk-derived extracellular vesicles can help to improve the intestinal barrier function and break the gut-joint axis in rheumatoid arthritis[J]. Front Immunol, 2021, 12, 703277. doi: 10.3389/fimmu.2021.703277
20	AHADIAN S , FINBLOOM J A , MOFIDFAR M , et al. Micro and nanoscale technologies in oral drug delivery[J]. Adv Drug Deliv Rev, 2020, 157, 37- 62. doi: 10.1016/j.addr.2020.07.012
21	WU L , WANG L L , LIU X , et al. Milk-derived exosomes exhibit versatile effects for improved oral drug delivery[J]. Acta Pharm Sin B, 2022, 12 (4): 2029- 2042. doi: 10.1016/j.apsb.2021.12.015
22	SALEHI M , NEGAHDARI B , MEHRYAB F , et al. Milk-derived extracellular vesicles: biomedical applications, current challenges, and future perspectives[J]. J Agric Food Chem, 2024, 72 (15): 8304- 8331. doi: 10.1021/acs.jafc.3c07899
23	KIM H , JANG H , CHO H , et al. Recent advances in exosome-based drug delivery for cancer therapy[J]. Cancers (Basel), 2021, 13 (17): 4435. doi: 10.3390/cancers13174435
24	JANG H , KIM H , KIM E H , et al. Post-insertion technique to introduce targeting moieties in milk exosomes for targeted drug delivery[J]. Biomater Res, 2023, 27 (1): 124. doi: 10.1186/s40824-023-00456-w
25	LI Y T , XING L Y , WANG L L , et al. Milk-derived exosomes as a promising vehicle for oral delivery of hydrophilic biomacromolecule drugs[J]. Asian J Pharm Sci, 2023, 18 (2): 100797.
26	赵莹, 王靖雷, 王萌, 等. 奶牛乳源外泌体提取方法的优化及效果评价[J]. 动物营养学报, 2024, 36 (2): 1265- 1276.
	ZHAO Y , WANG J L , WANG M , et al. Optimization and evaluation of extraction methods of milk exosomes from dairy cows[J]. Chinese Journal of Animal Nutrition, 2024, 36 (2): 1265- 1276.
27	WANG M Q , ZHOU C H , CONG S , et al. Lipopolysaccharide inhibits triglyceride synthesis in dairy cow mammary epithelial cells by upregulating miR-27a-3p, which targets the PPARG gene[J]. J Dairy Sci, 2021, 104 (1): 989- 1001. doi: 10.3168/jds.2020-18270
28	TONG C , CHEN T , CHEN Z W , et al. Forsythiaside a plays an anti-inflammatory role in LPS-induced mastitis in a mouse model by modulating the MAPK and NF-κB signaling pathways[J]. Res Vet Sci, 2021, 136, 390- 395. doi: 10.1016/j.rvsc.2021.03.020
29	ZHANG J L , ZHANG Y , HUANG H L , et al. Forsythoside A inhibited S. aureus stimulated inflammatory response in primary bovine mammary epithelial cells[J]. Microb Pathog, 2018, 116, 158- 163. doi: 10.1016/j.micpath.2018.01.002
30	LIU J J , GAO Y K , ZHANG H Q , et al. Forsythiaside A attenuates mastitis via PINK1/Parkin-mediated mitophagy[J]. Phytomedicine, 2024, 125, 155358. doi: 10.1016/j.phymed.2024.155358
31	LEE C , YOON S , MOON J O . Kaempferol suppresses carbon tetrachloride-induced liver damage in rats via the MAPKs/NF-κB and AMPK/Nrf2 signaling pathways[J]. Int J Mol Sci, 2023, 24 (8): 6900. doi: 10.3390/ijms24086900
32	ZHU X , WANG X L , YING T H , et al. Kaempferol alleviates the inflammatory response and stabilizes the pulmonary vascular endothelial barrier in LPS-induced sepsis through regulating the SphK1/S1P signaling pathway[J]. Chem Biol Interact, 2022, 368, 110221. doi: 10.1016/j.cbi.2022.110221
33	DONOSO-MENESES D , FIGUEROA-VALDÉS A I , KHOURY M , et al. Oral administration as a potential alternative for the delivery of small extracellular vesicles[J]. Pharmaceutics, 2023, 15 (3): 716. doi: 10.3390/pharmaceutics15030716
34	DEL POZO-ACEBO L , DE LAS HAZAS M C L , TOMÉ-CARNEIRO J , et al. Bovine milk-derived exosomes as a drug delivery vehicle for miRNA-based therapy[J]. Int J Mol Sci, 2021, 22 (3): 1105. doi: 10.3390/ijms22031105
35	MUNIR J , NGU A , WANG H C , et al. Review: milk small extracellular vesicles for use in the delivery of therapeutics[J]. Pharm Res, 2023, 40 (4): 909- 915. doi: 10.1007/s11095-022-03404-w
36	MEHRYAB F , RABBANI S , SHAHHOSSEINI S , et al. Exosomes as a next-generation drug delivery system: an update on drug loading approaches, characterization, and clinical application challenges[J]. Acta Biomater, 2020, 113, 42- 62. doi: 10.1016/j.actbio.2020.06.036
37	吕田田, 余海艳, 薛丙权, 等. 负载连翘苷的外泌体递药系统制备及体外评价[J]. 中国新药杂志, 2022, 31 (1): 89- 94. doi: 10.3969/j.issn.1003-3734.2022.01.013
	LÜ T T , YU Y H , XUE B Q , et al. Preparation and in vitro evaluation of phillyrin-loaded exosomal drug delivery system[J]. Chinese Journal of New Drugs, 2022, 31 (1): 89- 94. doi: 10.3969/j.issn.1003-3734.2022.01.013
38	WANG Y T , HUO Y M , ZHAO C Y , et al. Engineered exosomes with enhanced stability and delivery efficiency for glioblastoma therapy[J]. J Control Release, 2024, 368, 170- 183. doi: 10.1016/j.jconrel.2024.02.015
39	GUAN P F , FAN L , ZHU Z B , et al. M2 microglia-derived exosome-loaded electroconductive hydrogel for enhancing neurological recovery after spinal cord injury[J]. J Nanobiotechnology, 2024, 22 (1): 8. doi: 10.1186/s12951-023-02255-w
40	ALBALADEJO-GARCÍA V , MORÁN L , SANTOS-COQUILLAT A , et al. Curcumin encapsulated in milk small extracellular vesicles as a nanotherapeutic alternative in experimental chronic liver disease[J]. Biomed Pharmacother, 2024, 173, 116381. doi: 10.1016/j.biopha.2024.116381
41	WANG C , LI M , XIA X H , et al. Construction of exosome-loaded LL-37 and its protection against zika virus infection[J]. Antiviral Res, 2024, 225, 105855. doi: 10.1016/j.antiviral.2024.105855
42	WEI H X , CHEN J Y , WANG S L , et al. A nanodrug consisting of doxorubicin and exosome derived from mesenchymal stem cells for osteosarcoma treatment in vitro[J]. Int J Nanomedicine, 2019, 14, 8603- 8610. doi: 10.2147/IJN.S218988
43	KANDIMALLA R , AQIL F , ALHAKEEM S S , et al. Targeted oral delivery of paclitaxel using colostrum-derived exosomes[J]. Cancers(Basel), 2021, 13 (15): 3700.

引物 Primer	引物序列(5′→3′) Primer sequence
IL-6-F	TGGGGCTGCTCCTGGT
IL-6-R	TCTCACATATCTCCTTTCTCATTG
IL-8-F	ATTCCACACCTTTCCACCC
IL-8-R	TGGGGTTTAAGCAGACCTCG
TNFα-F	GATGTGGAGCTGGCGGAG
TNFα-R	AGGGCTGTTGATGGAGGG
GAPDH-F	TATGACCACCGTCCACGCCATC
GAPDH-R	CGCCTGCTTCACCACCTTCTTG

组别 Constituencies	划痕值 Scratch value	划痕变化值 Scratch change value	迁移抑制率/% Migration inhibition rate
对照组0 h Control group 0 h	68 027
miEVs组0 h miEVs group 0 h	44 880
FTA组0 h FTA group 0 h	60 903
FTA-miEVs组0 h FTA-miEVs group 0 h	69 474
对照组24 h Control group 24 h	40 811
miEVs组24 h miEVs group 24 h	91 309
FTA组24 h FTA group 24 h	132 700
FTA-miEVs组24 h FTA-miEVs group 24 h	262 288
对照组 Control group		27 216
miEVs组 miEVs group		40 726	49.64
FTA组 FTA group		71 797	163.58
FTA-miEVs组 FTA-miEVs group		192 814	608.48

组别 Group	划痕值 Scratch value	划痕变化值 Scratch change value	迁移抑制率/% Migration inhibition rate
对照组0 h Control group 0 h	70 066
miEVs组0 h miEVs group 0 h	40 998
KPF组0 h KPF group 0 h	62 304
KPF-miEVs组0 h KPF-miEVs group 0 h	75 478
对照组24 h Control group 24 h	38 090
miEVs组24 h miEVs group 24 h	88 719
KPF组24 h KPF group 24 h	134 567
KPF-miEVs组24 h KPF-miEVs group 24 h	274 649
对照组 Control group		31 976
miEVs组 miEVs group		46 429	45.20
KPF组 KPF group		72 263	125.99
KPF-miEVs组 KPF-miEVs group		199 171	522.88

[1]	王靖, 关淑文, 赵小博, 王琳玮, 郭刚, 蒋林树. 竹叶黄酮对H₂O₂诱导奶牛乳腺上皮细胞焦亡的保护作用[J]. 畜牧兽医学报, 2025, 56(1): 281-294.
[2]	李相辰, 王林楠, 于正青, 张莉, 杨晨晨, 宋亮丽. 槲皮素抑制自噬恢复LTA诱导的奶牛乳腺上皮细胞紧密连接功能[J]. 畜牧兽医学报, 2024, 55(9): 3887-3896.
[3]	付艺乾, 梁东阁, 王铭洋, 潘佳佳, 杨彦宾, 曾磊, 康相涛. 干扰素调节因子敲减细胞系的构建及其对猪伪狂犬病病毒增殖的影响[J]. 畜牧兽医学报, 2024, 55(9): 4100-4109.
[4]	康方圆, 刘镇滔, 吴奎显, 倪晗, 钟凯, 李和平, 杨国宇, 韩立强. 脂噬对奶牛乳腺上皮细胞脂滴大小的调控研究[J]. 畜牧兽医学报, 2024, 55(3): 1095-1101.
[5]	庄翠翠, 韩博. 大肠杆菌感染奶牛乳腺上皮细胞和小鼠乳腺组织致其线粒体损伤的机制研究[J]. 畜牧兽医学报, 2024, 55(2): 822-833.
[6]	张梓璇, 张颖, 李志军, 杨婧玲, 蒋子豪, 黄华敏, 齐雪峰. 牛病毒性腹泻病毒诱导铁死亡对其复制水平的影响[J]. 畜牧兽医学报, 2024, 55(12): 5716-5724.
[7]	刘义钢, 马玉辉, 冯琦, 马雪连, 李娜, 孙亚伟, 于维浩, 姚刚. 肢体成角畸形马驹及其母马的血液生理生化、炎性因子和激素变化研究[J]. 畜牧兽医学报, 2024, 55(12): 5854-5865.
[8]	丁修虎, 林志平, 赵芳, 陈坤琳, 仲跻峰, 张燕, 高运东, 李惠侠, 王慧利, 张建丽, 丁强. 利用CRISPR/Cas9技术制备BLG基因敲除牛乳腺上皮细胞系[J]. 畜牧兽医学报, 2024, 55(10): 4475-4488.
[9]	蔡明玉, 张海龙, 海珍珍, 乔亚蕊, 杜军, 周学章. 重组克柔念珠菌14-3-3蛋白诱导奶牛乳腺上皮细胞炎症反应的分子机制[J]. 畜牧兽医学报, 2023, 54(4): 1679-1689.
[10]	苏丹, 文晓宾, 马腾, 钟儒清, 王阳, 陈亮. 羟基酪醇对肉仔鸡生长性能、抗氧化能力和肠道炎性因子的影响[J]. 畜牧兽医学报, 2023, 54(11): 4851-4859.
[11]	孟梅娟, 汪艳, 霍然, 李雪睿, 常广军, 沈向真. 抑制PERK对LPS诱导的奶牛乳腺上皮细胞自噬的影响[J]. 畜牧兽医学报, 2023, 54(1): 351-360.
[12]	李林, 曹萌, 宫彬彬, 赵梅, 王婕, 张晓辉. 丁酸钠通过AMPK通路调控LPS造成牛乳腺上皮细胞脂代谢紊乱的作用机制[J]. 畜牧兽医学报, 2022, 53(9): 3221-3230.
[13]	刘丽华, 钟震宇, 郑玉杰, 王昕. lncRNA EPB41L4A-AS通过调控ErbB3抑制奶牛乳腺上皮细胞增殖的研究[J]. 畜牧兽医学报, 2022, 53(7): 2152-2159.
[14]	郭大伟, 侯思鲁, 池宇佳, 于非可, 尉啸涵, 邓倩, 肖传明, 刘晓晔, 董虹. 芪英汤和子甘汤中药复方促进母猪繁殖性能和断奶仔猪生长性能的临床研究[J]. 畜牧兽医学报, 2022, 53(6): 1994-2004.
[15]	丁军, 付子琳, 和俊豪, 段晓微, 马露, 卜登攀. 乳源外泌体研究进展[J]. 畜牧兽医学报, 2022, 53(4): 1019-1029.