忍冬藤水提物冻干粉调控共培养体系中大鼠T淋巴细胞迁移机制研究

孙嵩; 柴立民; 冯桂宇; 李东阳; 张萍心; 刘蔚; 张英凯

doi:10.3969/j.issn.1006-2157.2023.02.014

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忍冬藤水提物冻干粉调控共培养体系中大鼠T淋巴细胞迁移机制研究

实验研究 | 更新时间：2023-03-07

- 忍冬藤水提物冻干粉调控共培养体系中大鼠T淋巴细胞迁移机制研究
- Study on the mechanism underlying the regulatory effects of freeze-dried powder of aqueous extract of Lonicera japonica Thunb. on the migration of rat T lymphocytes in a coculture system
- 北京中医药大学学报 2023年46卷第2期页码：232-241
- 作者机构：
  
  北京中医药大学东直门医院　北京　100700
- 作者简介：
  
  孙嵩，男，在读硕士生
  #柴立民，男，博士，教授，博士生导师，主要研究方向：中医药防治自身免疫性疾病，E-mail：liminchai@hotmail.com
- 基金信息：
  
  国家中医药管理局国家中医临床研究基地业务建设第二批科研专项(JDZX2015191)
- DOI：10.3969/j.issn.1006-2157.2023.02.014
  中图分类号： R285.5
- 收稿日期：2022-10-17，
  
  网络出版日期：2022-12-14，
  
  纸质出版日期：2023-02-28
- 稿件说明：
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孙嵩, 柴立民, 冯桂宇, 等. 忍冬藤水提物冻干粉调控共培养体系中大鼠T淋巴细胞迁移机制研究[J]. 北京中医药大学学报, 2023,46(2):232-241.

SUN Song, CHAI Limin, FENG Guiyu, et al. Study on the mechanism underlying the regulatory effects of freeze-dried powder of aqueous extract of Lonicera japonica Thunb. on the migration of rat T lymphocytes in a coculture system[J]. Journal of beijing university of traditional chinese medicine, 2023, 46(2): 232-241.
孙嵩, 柴立民, 冯桂宇, 等. 忍冬藤水提物冻干粉调控共培养体系中大鼠T淋巴细胞迁移机制研究[J]. 北京中医药大学学报, 2023,46(2):232-241. DOI： 10.3969/j.issn.1006-2157.2023.02.014.

SUN Song, CHAI Limin, FENG Guiyu, et al. Study on the mechanism underlying the regulatory effects of freeze-dried powder of aqueous extract of Lonicera japonica Thunb. on the migration of rat T lymphocytes in a coculture system[J]. Journal of beijing university of traditional chinese medicine, 2023, 46(2): 232-241. DOI： 10.3969/j.issn.1006-2157.2023.02.014.

摘要

目的

探讨忍冬藤水提物冻干粉调控共培养体系中大鼠T淋巴细胞迁移的作用机制。

方法

Transwell建立大鼠外周血T淋巴细胞与大鼠滑膜细胞株RSC－364细胞共培养体系，白细胞介素－1β(25 μg/L)诱导细胞成炎性损伤模型，以忍冬藤水提物冻干粉(100、250 mg/L)作为干预措施，白细胞介素－1β抑制剂(25 μg/L)作为阳性对照。采用高效液相色谱－电喷雾质谱(HPLC-ESI/MS)检测忍冬藤水提物组分，HE染色观察滑膜细胞形态改变，实时动态细胞分析技术(RTCA)检测淋巴细胞迁移指数变化，流式细胞仪检测CD3

＋

CD4

＋

T细胞及CD3

＋

CD8

＋

T细胞百分比变化，酶联免疫吸附测定(ELISA)检测共培养体系中可溶性血管内皮黏附分子－1(sVCAM－1)、可溶性细胞间黏附分子－1(sICAM－1)浓度。

结果

HPLC-ESI/MS结果显示，忍冬藤水提物冻干粉主要成分有咖啡酸、芦丁等。忍冬藤水提物冻干粉干预后，HE染色显示滑膜细胞数目减少，细胞形态发生不同程度改变，细胞呈圆形，胞质颜色深染，核区分相对明显。与模型组相比，RTCA检测忍冬藤水提物低剂量组48 h和忍冬藤水提物高剂量组24、48 h淋巴细胞迁移指数降低(

0.05，

0.01)；与模型组相比，流式细胞仪检测忍冬藤水提物低、高剂量组24、48 h CD3

＋

CD4

＋

T细胞百分比降低(

0.01)，忍冬藤水提物高剂量组48 h CD3

＋

CD8

＋

T细胞百分比降低(

0.01)。与模型组相比，ELISA检测共培养体系中忍冬藤水提物低、高剂量组24、48 h细胞因子sVCAM－1、sICAM－1浓度均降低(

0.01)。

结论

忍冬藤水提冻物干粉能够降低sICAM－1、sVCAM－1含量，抑制淋巴细胞迁移至滑膜细胞，抑制CD4

＋

T、CD8

＋

T细胞的迁移浸润以减轻炎症反应，缓解滑膜细胞的炎性增殖，减缓和阻滞类风湿性关节炎的疾病发展过程，从而达到缓解炎症的目的。

Abstract

Objective

To investigate the mechanism underlying the regulatory effects of freeze-dried powder of aqueous extract of

Lonicera japonica

Thunb. on the migration of rat T lymphocytes in a coculture system.

Methods

Transwell was used to establish a coculture system of rat peripheral blood T lymphocytes and rat synovial cell line RSC-364 cells

and interleukin-1β (25 μg/L) was used to induce the inflammatory injury model of cells. The freeze-dried powder of aqueous extract of

Lonicera japonica

Thunb. (100 and 250 mg/L) was used as the intervention measure

and interleukin-1β inhibitor (25 μg/L) was used as the positive control. The components of aqueous extract of

Lonicera japonica

Thunb. were analyzed by high-performance liquid chromatography-electro spray ionization/mass spectrometry (HPLC-ESI/MS). HE staining was used to observe the morphological changes of synovial cells. The change of lymphocyte migration index was determined by real-time cellular analysis(RTCA). The percentage changes of CD3

CD4

T cells and CD3

CD8

T cells were detected by flow cytometry

and the concentrations of sVCAM-1 and sICAM-1 in the coculture system were detected by ELISA.

Results

The result of HPLC-ESI/MS analysis showed that the main components of freeze-dried powder of aqueous extract of

Lonicera japonica

Thunb. were caffeic acid and rutin

etc. After the intervention of freeze-dried powder of aqueous extract of

Lonicera japonica

Thunb.

HE staining showed that the number of synovial cells was decreased and the cell morphology had changed to varying degrees; the cells were round

the cytoplasm was deeply stained

and the nucleus was relatively distinct. Compared with the model group

the migration index of T lymphocytes in the aqueous extract of

Lonicera japonica

Thunb. low-dose group at 48 h and the aqueous extract of

Lonicera japonica

Thunb. high-dose group at 24 and 48 h measured by RTCA decreased (

0.05

0.01). Compared with the model group

the percentage of CD3

CD4

T cells in the aqueous extract of

Lonicera japonica

Thunb. low-dose and high-dose groups decreased at 24 and 48 h (

0.01)

and the percentage of CD3

CD8

T cells in the aqueous extract of

Lonicera japonica

Thunb. high-dose group decreased at 48 h (

0.01). Compared with the model group

ELISA result showed that the concentrations of cytokines sVCAM-1 and sICAM-1 in the aqueous extract of

Lonicera japonica

Thunb. low-dose and high-dose groups in the coculture system decreased at 24 and 48 h (

0.01).

Conclusion

The freeze-dried powder of aqueous extract of

Lonicera japonica

Thunb. can reduce the contents of sICAM-1 and sVCAM-1

inhibit the migration of lymphocytes to synovial cells

inhibit the migration and infiltration of CD4

T and CD8

T cells to reduce inflammation

alleviate the inflammatory proliferation of synovial cells

slow down and block the disease development process of rheumatoid arthritis

so as to alleviate inflammation.

关键词

Keywords

references

SMOLEN JS , ALETAHA D , BARTON A , et al . Rheumatoid arthritis [J/OL]. Nat Rev Dis Primers , 2018 , 4 : 18001 [ 2022－09－13 ]. https：//www.nature.com/articles/nrdp20181 https：//www.nature.com/articles/nrdp20181 .

CHEMIN K , GERSTNER C , MALMSTRÖM V . Effector functions of CD4＋ T cells at the site of local autoimmune inflammation-lessons from rheumatoid arthritis [J/OL]. Front Immunol , 2019 , 10 : 353 [ 2022－09－13 ]. https：//www.frontiersin.org/articles/10.3389/fimmu.2019.00353/full https：//www.frontiersin.org/articles/10.3389/fimmu.2019.00353/full .

KIM M , YOO SJ , KANG SW , et al . TNFα and IL-1β in the synovial fluid facilitate mucosal-associated invariant T (MAIT) cell migration [J]. Cytokine , 2017 , 99 : 91 － 98 .

KAY J , CALABRESE L . The role of interleukin－1 in the pathogenesis of rheumatoid arthritis [J]. Rheumatology (Oxford) , 2004 , 43 ( Suppl 3 ): iii2 － iii9 .

BEN-SASSON SZ , HU-LI J , QUIEL J , et al . IL－1 acts directly on CD4 T cells to enhance their antigen-driven expansion and differentiation [J]. Proc Natl Acad Sci U S A , 2009 , 106 ( 17 ): 7119 － 7124 .

BEN-SASSON SZ , HOGG A , HU-LI J , et al . IL－1 enhances expansion, effector function, tissue localization, and memory response of antigen-specific CD8 T cells [J]. J Exp Med , 2013 , 210 ( 3 ): 491 － 502 .

WEHRENS EJ , PRAKKEN BJ , VAN WIJK F . T cells out of control: impaired immune regulation in the inflamed joint [J]. Nat Rev Rheumatol , 2013 , 9 ( 1 ): 34 － 42 .

KOMATSU N , TAKAYANAGI H . Autoimmune arthritis: the interface between the immune system and joints [J]. Adv Immunol , 2012 , 115 : 45 － 71 .

KANG I , HUNDHAUSEN C , EVANKO SP , et al . Crosstalk between CD4 T cells and synovial fibroblasts from human arthritic joints promotes hyaluronan-dependent leukocyte adhesion and inflammatory cytokine expression in vitro [J/OL]. Matrix Biol Plus , 2022 , 14 : 100110 [ 2022－09－13 ]. https：//linkinghub.elsevier.com/retrieve/pii/S2590028522000102 https：//linkinghub.elsevier.com/retrieve/pii/S2590028522000102 .

张红敏，谢春光，陈世伟 . 含药血清体外药理试验的评价 [J]. 中国中西医结合杂志， 2004 , 24 ( 8 ): 741 － 745 .

WANG C , HUANDIKE M , YANG Y , et al . Glycosides of Caulis Lonicerae inhibits the inflammatory proliferation of IL-1β-mediated fibroblast-like synovial cells cocultured with lymphocytes [J]. Phytother Res , 2021 , 35 ( 5 ): 2807 － 2823 .

LOU L , ZHOU J , LIU Y , et al . Chlorogenic acid induces apoptosis to inhibit inflammatory proliferation of IL-6-induced fibroblast-like synoviocytes through modulating the activation of JAK/STAT and NF-κB signaling pathways [J]. Exp Ther Med , 2016 , 11 ( 5 ): 2054 － 2060 .

LOU L , LIU Y , ZHOU J , et al . Chlorogenic acid and luteolin synergistically inhibit the proliferation of interleukin-1β-induced fibroblast-like synoviocytes through regulating the activation of NF-κB and JAK/STAT-signaling pathways [J]. Immunopharmacol Immunotoxicol , 2015 , 37 ( 6 ): 499 － 507 .

DAMERAU A , GABER T . Modeling rheumatoid arthritis in vitro: From experimental feasibility to physiological proximity [J/OL]. Int J Mol Sci , 2020 , 21 ( 21 ): 7916 [ 2022－09－13 ]. https：//www.mdpi.com/1422－0067/21/21/7916 https：//www.mdpi.com/1422－0067/21/21/7916 .

XING XW , SHI HY , LIU S , et al . miR－496/MMP10 is involved in the proliferation of IL-1β-induced fibroblast-like synoviocytes via mediating the NF-κB signaling pathway [J]. Inflammation , 2021 , 44 ( 4 ): 1359 － 1369 .

GUO Q , ZHANG S , HUANG J , et al . Alogliptin inhibits IL-1β-induced inflammatory response in fibroblast-like synoviocytes [J/OL]. Int Immunopharmacol , 2020 , 83 : 106372 [ 2022－09－13 ]. https：//linkinghub.elsevier.com/retrieve/pii/S1567576919328747 https：//linkinghub.elsevier.com/retrieve/pii/S1567576919328747 .

李时珍．本草纲目 [M]. 北京：人民卫生出版社， 1977 ： 1335 .

崔启东，王丽娟 . 周平安教授诊治类风湿关节炎经验探析 [J]. 光明中医， 2022 , 37 ( 12 ): 2129 － 2131 .

童惠云，熊源胤，李建武，等 . 五藤汤辨治类风湿性关节炎 [J]. 中医药临床杂志， 2007 , 19 ( 4 ): 321 － 322 .

李海，刘玉欢，赵文海 . 国医大师刘柏龄治疗类风湿性关节炎经验 [J]. 中华中医药杂志， 2021 , 36 ( 7 )： 4002 － 4004 .

WANG W , SUN W , JIN L . Caffeic acid alleviates inflammatory response in rheumatoid arthritis fibroblast-like synoviocytes by inhibiting phosphorylation of IκB kinase α/β and IκBα [J]. Int Immunopharmacol , 2017 , 48 : 61 － 66 .

SUN CL , WEI J , BI LQ . Rutin attenuates oxidative stress and proinflammatory cytokine level in adjuvant induced rheumatoid arthritis via inhibition of NF－κB [J]. Pharmacology , 2017 , 100 ( 1－2 ): 40 － 49 .

KLIMIUK PA , SIERAKOWSKI S , LATOSIEWICZ R , et al . Soluble adhesion molecules (ICAM－1, VCAM－1, and E-selectin) and vascular endothelial growth factor (VEGF) in patients with distinct variants of rheumatoid synovitis [J]. Ann Rheum Dis , 2002 , 61 ( 9 ): 804 － 809 .

MELLADO M , MARTÍNEZ-MUÑOZ L , CASCIO G , et al . T cell migration in rheumatoid arthritis [J/OL]. Front Immunol , 2015 , 6 : 384 [ 2022－09－13 ]. https：//www.frontiersin.org/articles/10.3389/fimmu.2015.00384/full https：//www.frontiersin.org/articles/10.3389/fimmu.2015.00384/full .

林翊萍，徐沪济 . 细胞因子网络与滑膜炎症 [J]. 细胞与分子免疫学杂志， 2010 , 26 ( 12 ): 1313 － 1314, 1316 .

GUDERUD K , SUNDE LH , FLÅM ST , et al . Rheumatoid arthritis patients, both newly diagnosed and methotrexate treated, show more DNA methylation differences in CD4 ＋ memory than in CD4 ＋ naïve T cells [J/OL]. Front Immunol , 2020 , 11 : 194 [ 2022－09－13 ]. https：//www.frontiersin.org/articles/10.3389/fimmu.2020.00194/full https：//www.frontiersin.org/articles/10.3389/fimmu.2020.00194/full .

WASÉN C , OSPELT C , CAMPONESCHI A , et al . Nicotine changes the microRNA profile to regulate the FOXO memory program of CD8 ＋ T cells in rheumatoid arthritis [J/OL]. Front Immunol , 2020 , 11 : 1474 [ 2022－09－13 ]. https：//www.frontiersin.org/articles/10.3389/fimmu.2020.01474/full https：//www.frontiersin.org/articles/10.3389/fimmu.2020.01474/full .

NISHIGORI R , WARABI Y , SHISHIDO-HARA Y , et al . Inflammatory cerebellar PML with a CD4/CD8 ratio of 2.9 showed a favorable prognosis in a patient with rheumatoid arthritis [J]. Intern Med , 2019 , 58 ( 22 ): 3323 － 3329 .

CHO SH , RAYBUCK AL , BLAGIH J , et al . Hypoxia-inducible factors in CD4 ＋ T cells promote metabolism, switch cytokine secretion, and T cell help in humoral immunity [J]. Proc Natl Acad Sci U S A , 2019 , 116 ( 18 ): 8975 － 8984 .

KALIA A , AGRAWAL M , GUPTA N . CD8 ＋ T cells are crucial for humoral immunity establishment by SA14－14－2 live attenuated Japanese encephalitis vaccine in mice [J]. Eur J Immunol , 2021 , 51 ( 2 ): 368 － 379 .

CARVALHEIRO H , DA SILVA JA , SOUTO-CARNEIRO MM . Potential roles for CD8 ＋ T cells in rheumatoid arthritis [J]. Autoimmun Rev , 2013 , 12 ( 3 ): 401 － 409 .

HUSSEIN MR , FATHI NA , EL-DIN AM , et al . Alterations of the CD4 ＋，CD8 ＋ T cell subsets, interleukins－1β，IL-10, IL－17, tumor necrosis factor-α and soluble intercellular adhesion molecule－1 in rheumatoid arthritis and osteoarthritis: preliminary observations [J]. Pathol Oncol Res , 2008 , 14 ( 3 ): 321 － 328 .

BOTTINI N , FIRESTEIN GS . Duality of fibroblast-like synoviocytes in RA: passive responders and imprinted aggressors [J]. Nat Rev Rheumatol , 2013 , 9 ( 1 ): 24 － 33 .

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