Effect of Intestinal Flora Metabolites Deoxycholic Acid on the Proliferation and Cell Cycle of Human Umbilical Cord Mesenchymal Stem Cells
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摘要:
目的 探讨肠道菌群代谢物次级胆汁酸DCA不同浓度和不同作用时间在体外环境下对人脐带间充质干细胞(human umbilical cord mesenchymal stem cells,hUC-MSCs)增殖和细胞周期的影响。 方法 CCK8及流式细胞仪检测在含不同浓度DCA(0.00 μmol/L、1.56 μmol/L、3.13 μmol/L、6.25 μmol/L、12.50 μmol/L、25.00 μmol/L、50.00 μmol/L、100.00 μmol/L、200.00 μmol/L、400.00 μmol/L、800.00 μmol/L)干细胞培养基中培养hUC-MSCs 24 h、48 h、72 h后,干细胞增殖及细胞周期的变化。 结果 脱氧胆酸对hUC-MSCs增殖的影响体现在DCA剂量和作用时间两方面(两者交互作用,F = 6.622,P < 0.001);当浓度一定时,时间越长,细胞增殖越多;当作用时间一定时,浓度越高,细胞增殖抑制。浓度一定时,随时间的增加,细胞阻滞在G0/G1期的比例增加(P < 0.001);不同作用时间DCA对 hUC-MSCs的细胞周期表现为低浓度组细胞阻滞在G0/G1期和S期,高浓度组细胞阻滞在S期和G2/M期(P < 0.001)。 结论 体外环境下,DCA对hUC-MSCs增殖及细胞周期的影响与浓度和作用时间相关。 Abstract:Objective To investigate the influence of intestinal flora metabolites secondary bile acid DCA at different concentration and reaction time on the proliferation and cell cycle of human umbilical cord mesenchymal stem cells (hUC-MSCs) in vitro. Methods CCK8 and flow cytometry were used to detect the changes in cell proliferation and cell cycle of hUC-MSCs cultured in stem cell medium containing different concentrations of DCA (0.00 μmol/L, 1.56 μmol/L, 3.13 μmol/L, 6.25 μmol/L, 12.50 μmol/L, 25.00 μmol/L, 50.00 μmol/L, 100.00 μmol/L, 200.00 μmol/L, 400.00 μmol/L, 800.00 μmol/L) at 24h, 48h and 72h. Results The effect of deoxycholic acid on the proliferation of hUC-MSCs was both dose- and time-dependent (interaction, F = 6.622, P < 0.001).When the concentration was constant, cell proliferation increases with time; When the treatment time was fixed, the cell proliferation decreased with the increase of concentration. When the concentration was constant, the proportion of cell was arrested in G0/G1 phase increased with time (P < 0.001).The cell cycle of hUC-MSCs treated by DCA was blocked in G0/G1 and S phase in low concentration group, while S and G2/M phase in high concentration group (P < 0.001). Conclusion The effects of DCA on the proliferation and cell cycle of hUC-MSCs were both dose and time in vitro. -
近年来,课题组对姜科姜花属植物萜类成分进行了系统研究[1-6],从滇姜花、圆瓣姜花和毛姜花中分离得到一系列对多种肿瘤细胞具有显著体外细胞毒活性的萜类化合物[7-9]。一些姜科植物中的二萜类成分具有抗菌、抗肿瘤等活性[10-11]。滇姜花(Hedychium yunnanense Gangep)中的呋喃二萜Coronarin E含量较高,该成分没有细胞毒活性和抗菌活性,将其通过光敏氧化反应制备具有生物活性的丁烯酸内酯结构的二萜衍生物[12],有产率较高、选择性高、绿色环保的特点。课题组对Coronarin E经二氧化硒氧化、酰化、光敏氧化三步反应,制备两个衍生物,对其抗菌活性(抑菌圈、MIC和联合用药)及体外抗肿瘤活性进行较为深入的研究,进一步验证了二萜衍生物的生物活性,为寻找较好的药物前体提供了理论基础和科学依据。
1. 材料与方法
1.1 仪器
78-1型磁力加热搅拌器(杭州仪器电机厂);分析天平(上海第二天平仪器厂);AM-500型核磁共振波谱仪((瑞士BRUKER公司);LED灯(上海一恒科学仪器有限公司);OSB-2100 旋转蒸发仪(上海爱朗仪器有限公司);ES-315 高压蒸汽灭菌锅,(TOMY 公司);恒温培养箱(上海一恒科学仪器有限公司);SW-CJ-2FD 超净工作台(AIRTECH公司);电热恒温鼓风干燥箱(上海一恒科技有限公司)。
1.2 试剂
Coronarin E由本课题组从姜科植物滇姜花中分离得到。所用有机试剂(化学纯)和化学试剂均购自昆明市医药公司化学试剂玻璃仪器采供站,柱色谱硅胶均为青岛海洋化工厂产品,培养基配料均购自雅云生物科技有限公司。
1.3 实验方法
1.3.1 Coronarin E的SeO2氧化反应
取400 mg coronarin E、168 mg SeO2溶于5 mL干燥的二氯甲烷中,加入368 mg过氧叔丁醇,在常温下搅拌反应2 h,TLC检测原料反应完全。反应液经200~300目硅胶柱色谱分离纯化,石油醚-乙酸乙酯(80∶1~40∶1)洗脱,得到化合物1(无色油状物,见图1)300 mg,产率为71%。
1.3.2 化合物1的酰化反应
取87 mg 1-萘甲酸、0.12 mL N,N-二异丙基碳二亚胺(DIC),0.5 mg对二甲氨基吡啶(DMAP),溶于5 mL干燥过的二氯甲烷,常温搅拌10 min后,加入79 mg化合物1,搅拌反应2 h后,TLC检测反应完全,加入0.2 mL水及50 mL石油醚继续搅拌10 min,超声30 min。产物用150 mL石油醚与100 mL 70%甲醇分配,后者再用100 mL石油醚萃取,合并两次萃取的石油醚层,浓缩后经200-300目硅胶柱色谱分离,石油醚-氯仿(10∶1~5∶1)洗脱。得到化合物2(白色固体,见图2)52.7 mg,产率为44.1%。
1.3.3 化合物2的光敏氧化反应
取52.7 mg化合物2溶于10 mL吡啶中,加入1.0 mg四苯基卟啉(TPP),通入氧气并搅拌,在LED灯照射下反应2 h,TCL检测原料反应完全。溶剂蒸干,经硅胶色谱分离纯化,石油醚-乙酸乙酯(4∶1~1∶1)洗脱,得到化合物3(白色固体)和4(白色固体),见图3,分别为12 mg 和8 mg,产率分别为21.3%和14.1%。
1.3.4 化合物的抗菌活性筛选
采用滤纸片扩散法测试化合物3和4的抑菌圈直径[13-14];采用微量倍比稀释法[15-16]测定化合物3和4的最小抑菌浓度(MIC);化合物4的联合用药测试采用棋盘法,测试该成分分别与万古霉素、氨苄西林、卡那霉素3种抗生素联合用药的最小抑菌浓度[17]。
1.3.5 体外细胞毒活性测试
采用MTT法[18],测定化合物3和4对五个肿瘤细胞株的体外细胞毒活性(阳性对照采用顺铂)。
2. 结果
2.1 化合物3和4的NMR波谱数据
化合物3:1H-NMR(CD3OCD3,500 MHz)δ(ppm):0.91(3H,s,H-18),0.98(3H,s,H-19),1.19(3H,s,H-20),3.05(1H,d,J = 10.5 Hz,H-9),5.87(1H,t,J = 3.0 Hz,H-7),4.95(1H,br.s,H-17a),5.34(1H,br.s,H-17b),6.67(1H,dd,J = 16.5,10.5 Hz,H-11),5.94(1H,s,H-14),6.45(1H,d,J = 16.5 Hz,H-12),6.44(1H,s,H-16),8.98(1H,m,H-9′),7.80(1H,m,H-8′),7.36(1H,br.s,H-4′),8.51(1H,d,J = 8.1 Hz,H-3′),8.26(1H,d,J = 8.2 Hz,H-7′),8.23(1H,d,J = 8.2 Hz,H-5′),7.88(1H,d,J = 8.6 Hz,H-6′). 13C-NMR(CD3OCD3,500 MHz)δ(ppm):41.12(t,C-1),19.38(t,C-2),42.54(t,C-3),33.90(s,C-4),48.99(d,C-5),28.87(t,C-6),76.54(d,C-7),146.57(s,C-8),58.83(d,C-9),40.05(s,C-10),141.53(d,C-11),124.80(d,C-12),162.25(s,C-13),116.77(d,C-14),171.25(s,C-15),98.50(d,C-16),115.57(t,C-17),33.47(q,C-18),21.79(q,C-19),14.56(q,C-20),166.82(s,C-1′),128.88(s,C-2′),130.68(d,C-3′),125.68(d,C-4′),134.30(d,C-5′),132.00(s,C-6′),129.52(d,C-7′),126.39(d,C-8′),128.41(d,C-9′),127.15(d,C-10′),134.86(s,C-11′)。
化合物4:1H-NMR(CD3OCD3,500 MHz)δ(ppm):0.91(3H,s,H-18),0.96(3H,s,H-19),1.19(3H,s,H-20),2.97(1H,d,J = 10.0 Hz,H-9),5.81(1H,t,J = 2.5 Hz,H-7),4.89(1H,br.s,H-17a),5.32(1H,br.s,H-17b),6.97/6.99(1H,dd,J = 15.5,10.0 Hz,H-11),6.23(1H,d,J = 15.5 Hz,H-12),7.15/7.16(1H,s,H-14),6.15/6.16(1H,s,H-15),8.98(1H,m,H-9′),7.16(1H,br.s,H-4′),7.64(1H,m,H-8′),8.01(1H,d,J = 8.2 Hz,H-3′),8.16(1H,d,J = 8.2 Hz,H-7′),8.22(1H,d,J = 8.2 Hz,H-5′),7.87(1H,d,J = 8.6 Hz,H-6′). 13C-NMR(CD3OCD3,500 MHz)δ(ppm):41.18(t,C-1),19.97(t,C-2),42.59(t,C-3),33.81(s,C-4),48.99(d,C-5),29.07(t,C-6),76.95(d,C-7),146.73(s,C-8),58.78(d,C-9),39.98(s,C-10),136.91(d,C-11),122.79(d,C-12),131.00(s,C-13),144.94(d,C-14),97.21(d,C-15),170.67(s,C-16),115.20(t,C-17),33.54(q,C-18),21.94(q,C-19),14.69(q,C-20),166.71(s,C-1′),128.98(s,C-2′),130.64(d,C-3′),125.66(d,C-4′),134.93(d,C-5′),131.97(s,C-6′),129.52(d,C-7′),126.35(d,C-8′),128.29(d,C-9′),127.16(d,C-10′),134.88(s,C-11′)。
2.2 化合物3和4的抗菌活性
化合物3对两种MRSA病原菌具有一定的抗菌活性,化合物4对多种革兰氏阳性菌、革兰氏阴性菌具有明显的抗菌活性,见表1。
表 1 化合物3和4对病原菌株抑菌活性筛选结果(抑菌圈直径:mm)Table 1. The result of antimicrobial activities against pathogens ( diameter of inhibition zone:mm)病原菌株 化合物3 化合物4 金黄色葡萄球菌29213 − 10.1 MRSA 1450 8.2 10.5 MRSA 1505 10.1 10.3 MRSA 2024 − 8.2 MRSA I-20 − 10.0 MRSA I-67 − 9.8 MRSA 1957 − 8.1 MRSA 28299 − 10.5 克雷伯氏菌13883 − 10.8 粪肠球菌29212 − 9.4 白色葡萄球菌1029 − 12.0 铜绿假单胞菌PA01 − 10.2 大肠杆菌25922 − 10.0 鼠伤寒沙门氏菌χ 8956 − 17.0 鲍曼不动杆菌19606 − 13.2 枯草芽孢杆菌6633 − − 注:表中抑菌圈直径为三次测量的平均值;“−”表示无抑菌圈。革兰氏阳性菌:金黄色葡萄球菌(Staphylococcus aureus ATCC 29213),7个耐甲氧西林金黄色葡萄球菌(MRSA 1450、1505、2024、1957、28299、I-20、I-67),白色葡萄球菌(Staphylococcus albus 1029);革兰氏阴性菌:鼠伤寒沙门氏菌(Salmonella typhimurium χ 8956),铜绿假单胞菌(Pseudomonas aeruginosa PA01),大肠杆菌(Escherichia coil ATCC 25922),枯草芽孢杆菌(Bacillus subtilis ATCC 6633),鲍曼不动杆菌(Acinetobacter baumanii ATCC 19606),肺炎克雷伯氏菌(Klebsiella pneumonia ATCC 13883),粪肠球菌(Enterococcus faecalis ATCC 29212)。 2.3 化合物与抗生素的联合用药
化合物4与三种抗生素联合使用时,对MRSA病原菌株抑制活性有不同程度的协同或相加作用,见表2。
表 2 化合物4与三种抗生素的联合用药测试结果Table 2. Combination test of compound 4 with three antibiotics菌株 药物 MIC(μg/ml) 最佳抑菌点(化合物4∶抗生素) FICI 作用方式 鼠伤寒沙门氏菌χ8956 化合物4 0.25 万古霉素 0.25 0.125∶0.0625 0.75 + 氨苄西林 2 0.125∶1 1 + 卡那霉素 2 0.0625∶1 0.75 + 鲍曼不动杆菌19606 化合物4 0.5 万古霉素 0.25 0.125∶0.125 0.75 + 氨苄西林 8 0.25∶8 1.5 − 卡那霉素 4 0.25∶4 1.5 − 白色葡萄球菌1029 化合物4 0.5 万古霉素 0.125 0.125∶0.03125 0.5 ++ 氨苄西林 0.5 0.25∶0.125 0.75 + 卡那霉素 4 0.125∶1 0.5 ++ 注:1、FICI = 甲药MIC联合/甲药MIC单用 + 乙药MIC联合/乙药MIC单用,其中甲药代表化合物4,乙药代表抗生素。FICI > 1,表示两药有无关作用;0.5 < FICI≤1,表示两药有相加作用;FICI≤0.5,表示两药有协同作用。2、以“++”表示协同作用,“+”表示相加作用,“−”表示无关。 2.4 化合物3和4对5种肿瘤细胞株的细胞毒活性
化合物3对5种人类肿瘤细胞株具有显著的体外细胞毒活性;化合物4具有较弱的体外肿瘤生长抑制活性,见表3。
表 3 产物对五种肿瘤细胞株的半数生长抑制浓度IC50(μM)Table 3. The IC50 value of 3 and 4 against five tumor cell lines (μM)化合物编号 白血病HL-60 肝癌SMMC-7721 肺癌A-549 乳腺癌MCF-7 结肠癌SW480 3 2.55 2.77 1.17 2.49 1.37 4 15.71 15.62 26.49 25.13 22.87 顺铂 5.00 4.33 2.17 9.18 13.19 评价标准:无效IC50 > 40 μM;有效IC50 < 40 μM;标示下划线的为活性高于阳性对照顺铂。 3. 讨论
二萜coronarin E经三步衍生化反应,制备具有丁烯酸内酯结构单元的二萜衍生物3和4。对3和4的生物活性测试表明:化合物4对多种革兰氏阳性菌、革兰氏阴性菌有明显的抗菌活性,化合物3对两种MRSA具有一定的抗菌活性。化合物4对鼠伤寒沙门氏菌(Salmonella typhimurium χ8956)、鲍曼不动杆菌(Acinetobacter baumannii ATCC 19606)、白色葡萄球菌(Staphylococcus albus 1029)的抗菌效果显著。化合物4对鼠伤寒沙门氏菌的抗菌活性接近万古霉素,高于氨苄西林、卡那霉素;对鲍曼不动杆菌的抗菌活性高于氨苄西林、卡那霉素;对白色葡萄球菌的抗菌活性接近氨苄西林,高于卡那霉素。化合物4与三种抗生素联用时,对鼠伤寒沙门氏菌抑制活性均具有相加作用。化合物4与万古霉素、卡那霉素联用时对白色葡萄球菌抑制活性具有协同作用,与氨苄西林联用时具有相加作用。化合物4与万古霉素联用时对鲍曼不动杆菌抑制活性具有相加作用。
化合物3对5种人类肿瘤细胞株(白血病细胞株HL-60、肝癌细胞株SMMC-7721、肺癌细胞株A-549、乳腺癌细胞株MCF-7和结肠癌细胞株SW-480)均具有显著的体外细胞毒活性,超过阳性对照顺铂;化合物4具有较弱的体外肿瘤生长抑制活性。
由此可见,以姜科二萜为原料,经结构改造制备丁烯酸内酯结构单元的二萜衍生物,并从中寻找有苗头的抗菌、抗癌活性成分或先导化合物,可作为未来抗菌、抗肿瘤药物研究与开发的一个方向。
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图 1 脱氧胆酸不同浓度及不同作用时间对 hUC-MSCs 增殖的影响
A:脱氧胆酸不同浓度及不同作用时间对 hUC-MSCs 增殖的影响;B:不同浓度脱氧胆酸作用 24 h 时 hUC-MSCs增殖情况;C:不同浓度脱氧胆酸作用48 h时hUC-MSCs增殖情况;D:不同浓度脱氧胆酸作用72 h时hUC-MSCs 增殖情况。各浓度组与0.00 μmol/L浓度组比较,ns表示差异无统计学意义,*表示 P < 0.05,**表示 P < 0.01,***表示 P < 0.001,****表示 P < 0.0001。
Figure 1. Effects of DCA with different concentration and different time on the proliferation of hUC- MSCs
图 3 不同浓度及不同作用时间脱氧胆酸刺激后流式细胞技术检测 hUC-MSCs 细胞周期代表图例
A~D:24 h时脱氧胆酸0.00 μmol/L~400.00 μmol/L hUC-MSCs细胞周期分布情况;E~H:48 h时脱氧胆酸0.00 μmol/L~400.00 μmol/L hUC-MSCs细胞周期分布情况;I~L:72 h时脱氧胆酸0.00 μmol/L~400.00 μmol/L hUC-MSCs细胞周期分布情况。
Figure 3. Cell cycle of hUC-MSCs tested by flow cytometry after different concentration and different time of DCA
表 1 脱氧胆酸不同浓度及不同作用时间hUC-MSCs吸光度值(OD450nm值)(
$\bar x \pm s $ )Table 1. Absorbance (OD450nm) of hUC-MSCs at different dose and time of DCA (
$\bar x \pm s $ )脱氧胆酸浓度(μmol/L) 样本数 24 h 48 h 72 h 0.00(对照组) 45 0.61 ± 0.12 0.77 ± 0.14* 0.97 ± 0.32○* 1.56 45 0.51 ± 0.07 0.79 ± 0.19* 1.23 ± 0.24○* 3.13 45 0.54 ± 0.08 0.79 ± 0.21* 1.01 ± 0.31○* 6.25 45 0.56 ± 0.09 0.80 ± 0.16* 1.05 ± 0.33○* 12.50 45 0.55 ± 0.05 0.81 ± 0.07* 1.18 ± 0.28○* 25.00 45 0.55 ± 0.07 0.78 ± 0.13* 1.21 ± 0.20○* 50.00 45 0.57 ± 0.08 0.77 ± 0.03* 1.07 ± 0.24○* 100.00 45 0.58 ± 0.08 0.75 ± 0.16* 1.05 ± 0.23○* 200.00 44 0.58 ± 0.09 0.72 ± 0.16 0.84 ± 0.26* 400.00 45 0.50 ± 0.10 0.54 ± 0.19 0.53 ± 0.17 800.00 45 0.49 ± 0.12 0.48 ± 0.18 0.49 ± 0.18 与24 h比较,*P < 0.05;与48 h比较,○P < 0.05。 表 2 脱氧胆酸不同浓度及不同作用时间 hUC-MSCs 细胞周期比例(%)
Table 2. Cell cycle ratio of hUC-MSCs with different concentration and different time of DCA (%)
时间
(h)分期 0.00
μmol/L1.56
μmol/L25.00
μmol/L400.00
μmol/L24 G0/G1期 77.60 82.33* 83.77* 48.17* S期 16.48 17.67 16.23 51.83* G2/M期 5.91 0.00* 0.00* 0.00* 48 G0/G1期 87.07△ 92.85*△ 85.73*△ 42.99*△ S期 5.45△ 2.92*△ 14.27*△ 50.89* G2/M期 7.48△ 4.23*△ 0.00* 6.12*△ 72 G0/G1期 97.60△ 98.19*△ 98.41*△ 73.14*△ S期 2.40△ 1.81*△ 1.59*△ 22.70*△ G2/M期 0.00△ 0.00 0.00 4.16*△ 与0.00 µmol/L比较,*P < 0.0001;与24 h比较,△P < 0.0001。 -
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