二甲双胍通过COX-2/PGE2/STAT3途径抑制结直肠癌的机制研究

Metformin inhibits the development of colorectal cancer through the COX-2/PGE2/STAT3 pathway

摘要:

目的 探讨二甲双胍对结直肠癌的抑制作用及相关机制。方法 在体外培养小鼠结肠癌细胞系MC38和人结肠癌细胞系SW480，同时在体内使用氧化偶氮甲烷（AOM）与葡聚糖硫酸钠（DSS）诱导的结直肠癌小鼠模型，使用二甲双胍进行干预，通过CCK-8、细胞凋亡试验、Western blotting、苏木精-伊红（HE）染色、免疫组织化学检测、酶联免疫吸附试验（ELISA）、粪便16S rDNA测序来分析二甲双胍是否可抑制结直肠癌的发生、发展，并阐明其机制。结果 不同浓度的二甲双胍分别处理MC38细胞和SW480细胞24 h可在镜下看到，随着二甲双胍浓度的增加，肿瘤细胞数量明显减少。CCK-8法检测结果表明，随着二甲双胍浓度的增加，两种结直肠癌细胞细胞活性率受到抑制。两种结直肠癌细胞加入0、20 mmol/L二甲双胍的细胞凋亡率比较，差异有统计学意义（P <0.05）。Western blotting结果可见，二甲双胍干预可抑制COX-2蛋白的表达，且随着浓度的增加，COX-2蛋白表达呈药物剂量依赖性减低。两种结直肠癌细胞添加不同浓度二甲双胍后PGE2蛋白水平比较，差异有统计学意义（P <0.05）。AOM+DSS组较NaCl组肿瘤数量增多、体积增大、结肠长度缩短（P <0.05），AOM + DSS + Met组较Nacl组结肠肿瘤数量增多、体积增大、结肠长度缩短（P <0.05），AOM + DSS + Met组较AOM + DSS组结肠肿瘤数量减少、肿瘤体积减小（P <0.05）。AOM+DSS+Met组PGE2蛋白水平较AOM + DSS组低，但高于NaCl组和Met组（P <0.05）。AOM + DSS + Met组Sobs指数、Shannon指数较AOM + DSS组高（P <0.05）。各组放线菌门、变形菌门、髌骨细菌门、内脏臭气杆菌属、普雷沃菌属、双歧杆菌属、粪厌氧棒杆菌属、埃希氏-志贺氏菌属群落丰度比较，差异有统计学意义（P <0.05）。结论 在体内外二甲双胍均可以通过下调COX-2/PGE2/STAT3途径抑制结直肠癌的发生、发展，且二甲双胍可以促使结直肠癌模型小鼠的肠道菌群向对照组小鼠肠道菌群改变。

Abstract:

Objective To study the potential inhibitory effect of metformin (Met) on colorectal cancer (CRC) and its underlying mechanisms.Methods Mouse CRC cell line MC38 and human CRC cell line SW480 were cultured in vitro. In addition, the mouse CRC model, induced by azomethane (AOM) and dextran sulfate sodium (DSS), was subjected to treatment with Met in vivo. Using CCK-8 assay, apoptosis assay, Western blotting (WB), hematoxylin-eosin (HE) staining, immunohistochemical (IHC) analysis, enzyme linked immunosorbent assay (ELISA) and fecal 16S rDNA sequencing, we explored whether Met could inhibit the carcinogenesis and development of CRC, and further elucidated the underlying mechanisms.Results The MC38 and SW480 cells were treated with different concentrations of Met for 24 hours, which demonstrated that the number of tumor cells was obviously decreased with the increase in the concentration of Met. The CCK-8 assay showed the cell viability of the two cell lines were inhibited with the increase in the concentration of Met. There was a difference in the apoptosis rate between the cells treated with 20 mmol/L of Met and those untreated (P < 0.05). Western blotting demonstrated that Met suppressed the expression of cyclooxygenase-2 (COX-2) in a dose-dependent manner. The relative expression of prostaglandin E2 (PGE2) in the two cell lines was different when treated with distinct concentrations of Met (P < 0.05). Compared with the NaCl group, the number of tumors was higher, the volume of tumors was larger, and the length of colons was shorter in the AOM + DSS group (P < 0.05). The number of tumors was higher, the volume of tumors was larger, and the length of colons was shorter in the AOM + DSS + Met group than in the NaCl group (P < 0.05), whereas the number of tumors was lower and the volume of tumors was smaller in the AOM + DSS + Met group than in the AOM + DSS group (P < 0.05). The relative expression of PGE2 in the AOM + DSS + Met group was lower than that in the AOM + DSS group, but was higher than that in the NaCl group and the Met group (P < 0.05). The Sobs index and Shannon index in the AOM + DSS + Met group were higher than those in the AOM + DSS group (P < 0.05). There were differences in the abundance of Actinobacteria, Proteobacteria, Patescibacteria, Odoribacter splanchnicus, Prevotella, Bifidobacterium, Anaerostipes caccae, and Escherichia-Shigella among the groups (P < 0.05).Conclusions Met inhibits the occurrence and development of CRC through suppressing the COX-2/PGE2/STAT3 pathway both in vivo and in vitro. In addition, Met may promote the conversion of the intestinal microbiota of CRC mice to those of control mice.