Mechanism of Astragaloside Ⅳ regulates extracellular vesicles⁃mediated JMJD3/H3K27me3/OPN pathway against colorectal cancer liver metastasis

LI Ling; JI Qing; ZHOU Jing

doi:10.16305/j.1007-1334.2023.2308044

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Mechanism of Astragaloside Ⅳ regulates extracellular vesicles⁃mediated JMJD3/H3K27me3/OPN pathway against colorectal cancer liver metastasis

更新时间：2023-11-24

- Mechanism of Astragaloside Ⅳ regulates extracellular vesicles⁃mediated JMJD3/H3K27me3/OPN pathway against colorectal cancer liver metastasis
- Shanghai Journal of Traditional Chinese Medicine Vol. 57, Issue 11, Pages: 42-49(2023)
- 作者机构：
  
  1.上海中医药大学附属曙光医院肿瘤科/肿瘤研究室（上海 201203）
- 作者简介：
- 基金信息：
- DOI：10.16305/j.1007-1334.2023.2308044
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李玲,季青,周晶.黄芪甲苷调控外泌体介导的JMJD3/H3K27me3/OPN通路抗结直肠癌肝转移机制研究[J].上海中医药杂志,2023,57(11):42-49.

LI Ling,JI Qing,ZHOU Jing.Mechanism of Astragaloside Ⅳ regulates extracellular vesicles⁃mediated JMJD3/H3K27me3/OPN pathway against colorectal cancer liver metastasis[J].Shanghai Journal of Traditional Chinese Medicine,2023,57(11):42-49.
李玲,季青,周晶.黄芪甲苷调控外泌体介导的JMJD3/H3K27me3/OPN通路抗结直肠癌肝转移机制研究[J].上海中医药杂志,2023,57(11):42-49. DOI： 10.16305/j.1007-1334.2023.2308044.

LI Ling,JI Qing,ZHOU Jing.Mechanism of Astragaloside Ⅳ regulates extracellular vesicles⁃mediated JMJD3/H3K27me3/OPN pathway against colorectal cancer liver metastasis[J].Shanghai Journal of Traditional Chinese Medicine,2023,57(11):42-49. DOI： 10.16305/j.1007-1334.2023.2308044.

摘要

目的,2,研究黄芪甲苷（ASⅣ）抑制结直肠癌肝转移的作用机制，探索ASⅣ对外泌体介导的Jumonji结构域包含蛋白3（JMJD3）/第27位的赖氨酸三甲基化的组蛋白H3（H3K27me3）/骨桥蛋白（OPN）信号通路的影响。,方法,2,取对数生长期的MC38细胞，超速离心法提取MC38细胞外泌体（MC38-EVs）及ASⅣ干预后MC38细胞外泌体（ASⅣ/MC38-EVs），粒径鉴定后用于动物实验。选取C57BL/6J小鼠15只，建立结直肠癌原位肝转移模型后，按照随机数字表法分为模型组、MC38-EVs组、ASⅣ/MC38-EVs组，每组5只。模型组造模前1周尾静脉注射磷酸盐缓冲液（PBS），100 μL/次，隔日1次，连续3周；MC38-EVs组和ASⅣ/MC38-EVs组造模前1周尾静脉分别注射0.1 g/L的MC38-EVs、ASⅣ/MC38-EVs，100 μL/次，隔日1次，连续3周。4周后处死小鼠，观察结直肠癌肝转移情况；苏木精-伊红（HE）染色法观察肝转移灶病理学改变；蛋白质免疫印迹（Western blot）法检测肝转移灶中JMJD3、H3K27me3、OPN蛋白表达情况；免疫荧光法检测肝转移灶中F4/80,+,、CD206,+,巨噬细胞的浸润和JMJD3、H3K27me3、OPN蛋白表达；实时荧光定量逆转录聚合酶链式反应（RT-qPCR）检测瘤体中,JMJD3,、,OPN, mRNA表达。,结果,2,与模型组比较，MC38-EVs组肝转移灶数目和肝脏质量明显增加（,P,<,0.05）；与MC38-EVs组比较，ASⅣ/MC38-EVs组肝转移灶数目和肝脏质量明显减少（,P,<,0.05）；免疫荧光结果显示，与模型组比较，MC38-EVs组中肝组织F4/80,+,、CD206,+,巨噬细胞浸润程度明显升高（,P,<,0.05）；与MC38-EVs组比较，ASⅣ/MC38-EVs组中肝组织巨噬细胞浸润程度明显降低（,P,<,0.05）；Western blot、RT-qPCR和免疫荧光结果显示，与模型组比较，MC38-EVs组JMJD3、OPN蛋白和mRNA表达水平明显升高（,P,<,0.05），H3K27me3蛋白表达水平明显降低（,P,<,0.05）；与MC38-EVs组相比，ASⅣ/MC38-EVs组JMJD3、OPN蛋白和mRNA表达水平明显降低（,P,<,0.05），H3K27me3蛋白表达水平明显升高（,P,<,0.05）。,结论,2,ASⅣ能够有效抑制结直肠癌肝转移，其机制与调控外泌体介导的JMJD3/H3K27me3/OPN通路激活相关。

Abstract

Objective,2,To explore the mechanism of Astragaloside Ⅳ （ASⅣ） in inhibiting colorectal cancer liver metastasis and to investigate the effect of ASⅣ on extracellular vesicle-mediated Jumonji domain-containing protein-3 （JMJD3） /trimethylated histone H3 at lysine 27 （H3K27me3）/osteopontin （OPN） signaling pathway.,Methods,2,MC38 cells at the logarithmic growth stage were taken， and MC38 cell extracellular vesicles （MC38-EVs） and MC38 cell extracellular vesicles after ASⅣ intervention （ASⅣ/MC38-EVs） were extracted by ultracentrifugation， and the particle size was identified and used for animal experiments. After establishing the orthotopic liver metastasis model of colorectal cancer， fifteen C57BL/6J mice were randomly divided into model group， MC38-EVs group and ASⅣ/MC38-EVs group， with 5 animals in each group. The model group was injected with PBS in the tail vein 1 week before modeling， 100 μL per time， every other day for 3 weeks； the MC38-EVs group and the ASⅣ/MC38-EVs group were injected with 0.1 g/L，100 μL per time MC38-EVs and ASⅣ/MC38-EVs in the tail vein 1 week before modeling respectively， every other day for 3 weeks.The mice were executed after 4 weeks to observe the liver metastases of colorectal cancer； HE staining was performed to observe the pathological changes of liver metastases； Western blot was performed to detect the expression of JMJD3， H3K27me3 and OPN in liver metastases； immunofluorescence was performed to detect the infiltration of F4/80,+,，CD206,+ ,macrophages and the expression of JMJD3， H3K27me3， and OPN in liver metastases； RT-qPCR detected ,JMJD3,， ,OPN, mRNA expression in the tumor.,Results,2,The number of metastases and weight of liver were significantly higher in the MC38-EVs group compared with the model group （,P,<,0.05）， while those were significantly lower in the ASⅣ/MC38-EVs group compared with the MC38-EVs group （,P,<,0.05）； the immunofluorescence results showed that the extent of F4/80,+,，CD206,+, macrophage infiltration in liver tissue was significantly higher in the MC38-EVs group compared with the model group （,P,<,0.05）， but that was significantly lower in the ASⅣ/MC38-EVs group compared with the MC38-EVs group （,P,<,0.05）； Western blot， RT-qPCR and immunofluorescence results showed that the expression levels of JMJD3 and OPN proteins and mRNA were significantly higher （,P,<,0.05） and H3K27me3 protein expression level was significantly lower （,P,<,0.05） in the MC38-EVs group compared with the model group. Compared with the MC38-EVs group， JMJD3 and OPN protein and the mRNA expression levels were significantly lower （,P,<,0.05） and H3K27me3 protein expression level was significantly higher （,P,<,0.05） in the ASⅣ/MC38-EVs group.,Conclusion,2,ASⅣ can effectively inhibit liver metastasis of colorectal cancer by a mechanism related to the regulation of extracellular vesicle-mediated activation of the JMJD3/H3K27me3/OPN pathway.

关键词

结直肠癌肿瘤转移黄芪甲苷外泌体肿瘤相关巨噬细胞中药药理作用

Keywords

colorectal cancercancer metastasisAstragaloside Ⅳextracellular vesiclestumor associated macrophagestraditional Chinese herbal medicinepharmacological action

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