健脾养正方调控肿瘤相关巨噬细胞外泌体诱导胃癌细胞失巢凋亡的机制研究

郑姗姗; 吴坚; 张瑞娟; 张翔; 郑晓霞; 卢雨佳; 黄磊; 孙庆敏

doi:10.14148/j.issn.1672-0482.2024.0906

健脾养正方调控肿瘤相关巨噬细胞外泌体诱导胃癌细胞失巢凋亡的机制研究

Study on the Mechanism of Jianpi Yangzheng Formula Regulating Tumor-Associated Macrophage Exosomes to Induce Anoikis in Gastric Cancer Cells

摘要

摘要:
目的探讨健脾养正方调控肿瘤相关巨噬细胞(TAM)外泌体对胃癌失巢凋亡的影响及其机制。
方法体外诱导人单核细胞THP-1建立TAM模型；超速离心法提取M0、TAM及TAM+健脾养正方外泌体，与胃癌细胞共孵育。流式细胞术检测各组外泌体对胃癌细胞失巢凋亡的影响。构建BALB/c移植瘤小鼠模型，Western blot检测移植瘤中凋亡蛋白的表达水平。Label-free质谱蛋白组学及生物信息学分析干预前后胃癌细胞中的差异蛋白；Western blot及qPCR实验检测差异蛋白异柠檬酸脱氢酶1(IDH1)表达水平，泛素化实验检测IDH1的泛素化水平；试剂盒检测α-酮戊二酸(α-KG)、NADPH/NADP⁺、谷胱甘肽(GSH/GSSG)水平及活性氧(ROS)含量。
结果 TAM外泌体干预胃癌细胞后其失巢凋亡率降低(P < 0.05, P < 0.01)；而TAM+健脾养正外泌体干预后则显著升高(P < 0.05, P < 0.01)。小鼠体内实验中，TAM外泌体组瘤体内Cleaved Caspase-3/Caspase-3的比值降低(P < 0.05)，而TAM+健脾养正方外泌体干预后比值明显增高(P < 0.05)。蛋白组学分析显示IDH1在干预前后差异明显，且与三羧酸循环代谢相关。相较于M0组，TAM组外泌体干预的胃癌细胞IDH1泛素化水平升高，α-KG、NADPH/NADP⁺及GSH/GSSG水平明显增加，ROS含量降低(P < 0.05)，而TAM+健脾养正外泌体则可逆转上述现象。
结论健脾养正方通过调控TAM外泌体使胃癌细胞IDH1发生泛素降解，降低胃癌细胞三羧酸循环代谢水平，促进ROS积累，诱发胃癌失巢凋亡，进而抑制胃癌发展。

Abstract:
OBJECTIVE To investigate the effect of Jianpi Yangzheng (JPYZ) formula regulating tumor-associated macrophage (TAM) exosomes on anoikis in gastric cancer and its mechanism.
METHODS TAM model was established by inducing human mononuclear THP-1 cells in vitro; M0, TAM and TAM+JPYZ formula exosomes were extracted by ultracentrifugation and co-incubated with gastric cancer cells. Flow cytometry was used to detect the effect of exosomes in each group on anoikis in gastric cancer cells. A BALB/c transplanted tumor mouse model was constructed, and the expression level of apoptotic proteins in transplanted tumors was detected by Western blot. Label-free mass spectrometry proteomics and bioinformatics were used to analyze the differential proteins in gastric cancer cells before and after intervention; Western blot and qPCR experiments were used to detect the expression level of differential protein isocitrate dehydrogenase 1 (IDH1), and ubiquitination experiments were used to detect the ubiquitination level of IDH1; kits were used to detect the levels of α-ketoglutarate (α-KG), NADPH/NADP⁺, glutathione (GSH/GSSG) and reactive oxygen species (ROS) content.
RESULTS The anoikis rate of gastric cancer cells was reduced after TAM exosomes intervention (P < 0.05, P < 0.01), while it was significantly increased after TAM+JPYZ exosomes intervention (P < 0.05, P < 0.01). In the in vivo mouse experiment, the ratio of Cleaved Caspase-3/Caspase-3 in the tumor of the TAM exosome group was reduced (P < 0.05), while the ratio was significantly increased after TAM+JPYZ exosome intervention (P < 0.05). Proteomic analysis showed that IDH1 was significantly different after intervention, and was related to tricarboxylic acid cycle metabolism. Compared with the M0 group, the IDH1 ubiquitination level of gastric cancer cells in the TAM group with exosome intervention was increased, the levels of α-KG, NADPH/NADP⁺ and GSH/GSSG were significantly increased, and the ROS content was reduced (P < 0.05), while TAM+JPYZ exosomes could reverse the above phenomenon.
CONCLUSION JPYZ Formula can regulate TAM exosomes to cause ubiquitin degradation of IDH1 in gastric cancer cells, reduce the level of tricarboxylic acid cycle metabolism in gastric cancer cells, promote ROS accumulation, induce anoikis in gastric cancer, and thus inhibit the development of gastric cancer.

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