白术甲醇提取物对小肠上皮细胞增殖、迁移及磷脂酶C-γ1表达的影响

Effects of Methanol Extracts from Atractylodes macrocephalae Rhizoma on Small Intestinal Epithelial Cell Proliferation and Migration, and Expression of Phospholipase C-γ1

目的观察白术甲醇提取物(以下简称白术提取物)对小肠上皮细胞(IEC-6)增殖、迁移及磷脂酶C-γ1(PLC-γ1)表达的影响,旨在探讨益气健脾中药白术促进胃肠黏膜损伤修复的作用机制。方法细胞分为空白组,精脒(5μmol/L)组,白术提取物(50、100、200 mg/L)组;负荷实验设α-二氟甲基鸟氨酸[(α-difluoromethylornithine,DFMO)多胺合成抑制剂]组,精脒+DFMO组,白术提取物(50、100、200 mg/L)+DFMO组。细胞贴壁培养24 h,给予受试药培养相应时间后,采用实时细胞分析仪(Real-time Cell Analyzer,RTCA)观察白术提取物对IEC-6细胞增殖的影响;划痕法检测白术提取物对IEC-6细胞迁移数目的影响;采用荧光定量PCR法和Western blot法检测PLC-γ1 m RNA及其蛋白表达。结果与空白组比较,白术提取物对细胞增殖无明显影响(P>0.05),精脒和白术提取物(100、200 mg/L)对细胞迁移均有促进作用(P<0.01),并能增加细胞迁移过程PLC-γ1 m RNA和蛋白表达(P<0.01)。与DFMO组比较,精脒、白术提取物(100、200mg/L)能逆转DFMO所致的细胞迁移抑制及PLC-γ1 m RNA及其蛋白表达的抑制作用(均P<0.01)。结论白术提取物可通过促进多胺介导的上皮细胞迁移发挥修复胃肠黏膜损伤作用,细胞增殖不是其主要药效作用。

Objective To observe the effects of methanol extracts from Atractylodes macrocephalae Rhizoma（AMR） on the proliferation and migration of IEC-6 cell（small intestinal epithelial cells） and the expression of phospholipase C-γ1（PLC-γ1）, and to explore the mechanism of AMR（a Chinese herb capable of invigorating Pi replenishing qi） for promoting repair of gastrointestinal mucosal injury. Methods IEC-6 cells were divided into the blank group,the positive control（spermidine, SPD; 5 μmol/L） group, AMR extracts groups（50, 100, and 200 mg/L）. The alpha-difluoromethylornithine（DFMO, polyamines synthesis inhibitor） group, the SPD ＋ DFMO group, AMR extracts（50, 100,and 200 mg/L） ＋ DFMO groups were set up in stress test. IEC-6 cells were cultured by adherence for 24 h,and then treated with AMR extracts for appropriate periods of time. Effects of IEC-6 cell proliferation after action of AMR extracts were detected by Real-time Cell Analyzer（RTCA）. The effect of AMR extracts on IEC-6 cell migration number was detected using scratch method. m RNA and protein expressions of PLC-γ1 levels were detected by fluorescent quantitative polymerase chain reaction（RT-q PCR） and Western blot respectively. Results Compared with the blank group, AMR extracts showed no obvious effect on IEC-6 cell proliferation（P 〈0. 05）. But SPD and AMR extracts（100and 200 mg/L） not only promoted IEC-6 cell migration（P 〈0. 01）, but also improved m RNA and protein expressions of PLC-γ1 in the process of cell migration（P 〈0. 01）. Compared with the DFMO group, SPD and AMR extracts（100and 200 mg/L） could reverse inhibitory effects of DFMO on cell migration, and m RNA and protein expressions of PLC-γ1（all P 〈0. 01）. Conclusion AMR extracts played roles in repairing gastrointestinal mucosal injury possibly by promoting polyamine mediated intestinal epithelial cell migration, and its effect on intestinal epithelial cell proliferation was not main potentcy.