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。
目的:观察四君子汤多糖对小肠上皮IEC-6细胞迁移多胺信号通路钙离子调控的影响,探讨四君子汤促进胃肠黏膜损伤修复的作用机制。方法:在IEC-6细胞实验中,设正常对照组,阳性对照组,四君子汤多糖低、中、高剂量组(40、80、160mg/L);负荷实验则设模型组(α-二氟甲基鸟氨酸,DFMO),各用药组在加受试药同时加入DFMO;划痕法制造细胞迁移模型;相差倒置显微镜观察细胞迁移情况;HPLC法测定细胞内多胺(精脒和精胺)含量;RT-q PCR和Western Blot法分别检测瞬时受体电位通道1(TRPC1)m RNA和蛋白表达;Western Blot法检测磷脂酶C-γ1(PLC-γ1)蛋白表达;酶联免疫法检测三磷酸肌醇(IP3)含量;流式细胞仪检测细胞内游离钙离子浓度([Ca^(2+)]cyt);无钙培养条件是以不含钙离子的细胞培养液代替常规的含钙细胞培养液。结果:与正常对照组比较,四君子汤多糖各剂量组可促进细胞迁移、增加细胞内精脒和精胺含量、提高TRPC1 m RNA及蛋白表达、提高PLC-γ1蛋白表达和IP3含量、增加[Ca^(2+)]cyt(P<0.05,P<0.01);与模型组比较,四君子汤多糖各剂量组可逆转DFMO所致的细胞迁移抑制、细胞多胺含量降低、TRPC1 m RNA和蛋白表达降低、PLC-γ1蛋白表达和IP3含量降低、[Ca^(2+)]cyt降低(P<0.05,P<0.01);与正常对照组(含钙培养)比较,无钙培养可致细胞迁移抑制(P<0.01);与正常对照组(无钙培养)比较,各剂量四君子汤多糖可改善无钙培养所致的细胞迁移抑制(P<0.05,P<0.01),但不能使细胞迁移恢复正常水平。结论:四君子汤多糖促进细胞迁移与其作用于多胺调控信号通路有关,其中对Ca^(2+)调控是其关键指标。
Astragalus membranaceus(Radix Astragali, RA) and Atractylodes macrocephala(Rhizoma Atractylodis Macrocephalae, RAM) are often used to treat gastrointestinal diseases. In the present study, we determined the effects of polysaccharides extracts from these two herbs on IEC-6 cell migration and explored the potential underlying mechanisms. A migration model with IEC-6 cells was induced using a single-edged razor blade along the diameter of cell layers in six-well polystyrene plates. The cells were grown in control--media or media containing spermidine(5 μmol·L^(-1), SPD), alpha-difluoromethylornithine(2.5 mmol·L^(-1), DFMO), 4-Aminopyridine-(40 μ-mol·L^(-1), 4-AP), the polysaccharide extracts of RA or RAM(50, 100, or 200 mg·L^(-1)), DFMO plus SPD, or DFMO plus polysaccharide extracts of RA or RAM for 12 or 24 h. Next, cytosolic free Ca^(2+)([Ca^(2+)]cyt) was measured using laser confocal microscopy, and cellular polyamine content was quantified with HPLC. Kv1.1 mRNA expression was assessed using RT-qPCR and Kv1.1 and RhoA protein expressions were measured with Western blotting analysis. A cell migration assay was carried out using Image-Pro Plus software. In addition, GC-MS was introduced to analyze the monosaccharide composition of both polysaccharide extracts. The resutls showed that treatment with polysaccharide extracts of RA or RAM significantly increased cellular polyamine content, elevated [Ca^(2+)]cyt and accelerated migration of IEC-6 cells, compared with the controls(P < 0.01). Polysaccharide extracts not only reversed the inhibitory effects of DFMO on cellular polyamine content and [Ca^(2+)]cyt, but also restored IEC-6 cell migration to control level(P < 0.01 or < 0.05). Kv1.1 mRNA and protein expressions were increased(P < 0.05) after polysaccharide extract treatment in polyamine-deficient IEC-6 cells and RhoA protein expression was increased. Molar ratios of D-ribose, D-arabinose, L-rhamnose, D-mannose, D-glucose, and D-galactose was 1.0:14.1:0.3:19.9:181.3:6.3 in RA and 1.0:4.3:0.1:5.7:2.8:2.2 in
