四君子汤对DFMO作用下的Caco-2细胞微绒毛结构及多胺含量的影响

Effects of Sijunzi Decoction on Microvilli Structure and Polyamine Content of Caco-2 Cells with DFMO

目的探讨四君子汤(SJZT)对多胺合成特异性抑制剂二氟甲基鸟氨酸(α-difluoromethyornithine,DFMO)作用下的Caco-2人结直肠腺癌细胞微绒毛及腐胺(PUT)、精眯(SPD)、精胺(SPM)等多胺含量的影响,阐明SJZT的肠黏膜修复机制。方法大鼠灌服SJZT 17 g·kg^(-1),空白对照组给予等量生理盐水,每日2次,连续3 d,末次给药1 h后腹主动脉取血,制备SJZT含药血清。以Caco-2细胞为研究对象,实验分为对照组、DFMO组、SPD+DFMO组、SJZT(10%,5%,2.5%含药血清)+DFMO组。采用MTT法检测细胞增殖;透射电镜观察细胞微绒毛结构;柱前衍生-高效液相色谱(HPLC)法检测细胞内多胺含量。结果 DFMO组与对照组比较,DFMO对Caco-2细胞增殖、微绒毛的生长及多胺含量均有明显抑制作用,差异有统计学意义(P<0.05,P<0.01);而SPD及SJZT各剂量组能明显促进Caco-2细胞生长,改善微绒毛的生长抑制,提高细胞内多胺的含量。结论SJZT对肠黏膜损伤的修复作用可能与提高细胞多胺含量,促进Caco-2细胞增殖,改善微绒毛结构有关。

Objective To investigate the effect of Sijunzi Decoction （SJZT） and polyamine synthesis-specific inhibitor difluoromethyornithine （DFMO） on the microvilli and polyamines （putrescine, spermidine and spermine） contents of Caco-2 human eolorectal adenoeareinoma, and further clarify the intestinal mucosal repair mechanism of Sijunzi Decoction. Methods The rats were given SJZT at a dosage of 17 g·kg^-1, twice per day for 3 days, and the control group were given the same amount of normal saline. Drug containing serum was prepared from rats＇ blood collected I h after the last administration by abdominal aorta method. Caco-2 cells were randomly divided into control group, DFMO group, SPD ＋ DFMO group, SJZT （10%, 5%, and 2.5% drug serum）＋ DFMO groups, and the correspondent treatments were applied to each group. The MTF assay was used to detect the cell proliferation, and transmission electron microscopy was used to observe the microvilli structure. The contents of polyamines in the cells were detected by high performance liquid chromatography （HPLC）. Results Compared with the control group, DFMO had a significant inhibitory effect on Caeo-2 cell proliferation, microvilli growth and polyamine contents （P 〈 0.05, P 〈 0.01 ）. However, SPD and different concentrations of SJZT can significantly promote the growth of Caco-2 cells, relieve the growth inhibition of mierovilli and increase the contents of intracellular polyamines. Conclusion The repair effects of SJZT on intestinal mucosal injury may be related to the enhancement of polyamine contents, the promotion of Caeo-2 cell proliferation, and the improvement of microvilli structure.