摘要
目的:研究补阳还五汤及其有效部位(生物碱、苷)对凝血酶刺激的VEC表达血管活性物质和粘附分子的影响,探讨分子表达的信号转导途径,从内皮细胞环节揭示该方抗血栓形成的物质基础及作用机制。
方法:体外培养ECV-304细胞,加入凝血酶(Thr)刺激,分组给予不同浓度补阳还五汤原方、生物碱、苷;MTT法测定OD值,观察细胞活力;比色法测定LDH活性,观察细胞损伤程度;重氮化反应法测定NO含量,分光光度法检测TNOS、iNOS(NOS_2)、eNOS(NOS_3)的活性;放免法测定ET-1、RT-PCR法检测ECE mRNA;放免法测定6-酮-PGF_(1α),RT-PCR法检测PGI_2合成酶mRNA;免疫细胞化学法检测细胞粘附分子PECAM-1(CD31)、ICAM-1(CD54)、E-selectin的表达;免疫细胞化学法观察细胞PKCα的表达。体外培养ECV-304细胞,分组给予Thr、Thr+H_7、PMA、PMA+H_7、Thr+CATG、Thr+原方、Thr+生物碱、Thr+苷,观察细胞PKCα、p38、NF-κB和ERK表达的差异。
结果:①凝血酶可显著降低ECV-304细胞代谢活力,增加细胞的通透性(P＜0.05),而补阳还五汤原方和有效部位生物碱和苷对凝血酶诱导的内皮细胞活力下降和LDH漏出增加无显著影响(P＞0.05)。②凝血酶下调内皮细胞NO的分泌,降低eNOS的活性(P＜0.05);补阳还五汤原方在100mg/ml时可使内皮细胞NO分泌显著增加,在50mg/ml浓度时也可抑制凝血酶诱导的NO分泌的减少(P＜0.05),生物碱和苷对此无抑制作用(P＞0.05)。③补阳还五汤原方在100mg/ml浓度时可促进内皮细胞PGI_2合成酶mRNA的表达(P＜0.05)和PGI_2的释放,生物碱可促进PGI_2的释放(P＜0.01)。④补阳还五汤原方和生物碱、苷对凝血酶诱导的内皮细胞ET-1分泌增加无抑制作用(P＞0.05),且原方在25mg/ml、100mg/ml两种浓度时,还可使ET-1的分泌显著增加(P＜0.05);凝血酶刺激ECV-304后对内皮素转化酶(ECE)mRNA的表达无显著影响(P＞0.05),补阳还五汤及生物碱、苷对ECE mRNA表达也无显著影响(P＞0.05)。⑤凝血酶刺激后内皮细胞表达粘附分子PECAM-1、ICAM-1和E-selectin的灰度值降低(P＜0.05),提示三种粘附分子表达增强;与凝血酶组比较,补阳还五汤原方组这三种粘附分子表达的灰度值均增高(P＜0.05),生物碱和苷组PECAM-1、ICAM-1表达的灰度值增高(P＜0.05),但对于E-选择素的表达无显著影响。⑥凝血酶可促进内皮细胞PKCα的表达(P＜0.01),而高、中剂量的原方、高剂量的生物碱和三种剂量的苷均可显著抑制PKCα的表达(P＜0.01～P＜0.05)。⑦凝血酶刺激后ECV-304细胞磷酸化p38MAPK及磷酸化ERK表达无变化(P＞0.05),补阳还五汤原方及有效部位生物碱和苷对磷酸化p38MAPK及磷酸化ERK的表达亦无显著影响(P＞0.05)。⑧凝血酶刺激后ECV-304细胞NF-κB的表达无显著变化(P＞0.05),补阳还五汤原方及其有效部位亦元显著影响(P＞0.05)。
结论:补阳还五汤可促使内皮细胞分泌舒张血管物质NO和PGI_2,对缩血管物质ET-1的分泌无显著影响;补阳还五汤可下调内皮细胞粘附分子的表达,生物碱和苷可能是方中产生该作用的物质。对其机制研究表明,凝血酶激活血管内皮细胞的作用主要是通过PKC活化而介导的,p38MAPK、ERK、NF-κB信号通路并不是凝血酶活化血管内皮细胞的主要信号途径,补阳还五汤及其有效部位生物碱、苷可能通过抑制凝血酶激活PKC信号途径而发挥其抗凝血酶活化血管内皮细胞的作用。
Object:The first purpose of the study was to observe the effect of BYHW decoction and its active fractions on the expression of vasoactive substances and adhesion molecules induced by thrombin in human umbilical vein endothelium derived cell line ECV-304.The second was to research the possible action mechanism of BYHW decoction from cell signal transduction.
Method:ECV-304 stimulated with thrombin were cultured in RPMI-1640 and then given different dose of BYHWD,alkaloid and glycoside.The activities and injury degree of ECV-304 were observed with MTT measuring the OD and LDH activity.The contents of NO and the activities of TNOS, iNOS and eNOS were measured in different groups.ET-1 and 6-keto-PGF_(1α) were measured with radioimmunoassay(RIA).PGI_2 synthase mRNA and ECE mRNA were examined by semi-quantitative reverse transcription polymerase chain reaction(RT-PCR).Immunohistochemical analysis was performed to evaluate the expression of PECAM-1,ICAM-1,E-Selectin, PKCα,p38MAPK,ERK and NF-κB.
Results:The activities of ECV-304 were significantly decreased stimulated by thrombin(P＜0.05).The cells' activities in BYHWD,alkaloid and glycoside groups were not increased comparing with the thrombin group(P＞0.05).Thrombin lowered the secretion of NO and the activities of eNOS of ECV-304(P＜0.05).Compared with thrombin group,the contents of NO in BYHWD groups were increased(P＜0.05).In alkaloid groups and BYHWD groups,the level of 6-keto-PGF_(1α) and the expression of PGI_2 synthase mRNA were significantly higher than those in thrombin groups.The level of ET-1 in thrombin group was higher than in the blank group(P＜0.05),while the difference between thrombin and drug groups had no statistic value(P＞0.05).No distinct difference of the expression of ECE mRNA existed among all the groups of ECV-304(P＞0.05).Thrombin improved significantly the expression of adhesion molecules,such as PECAM-1,ICAM-1 and E-selectin in ECV-304,while drugs lowered the level(P＜0.05).Thrombin increased the expression of protein kinase C(PKC) of ECV-304,and the drugs restrained the effect(P＜0.05).The expression of p38MAPK,ERK or NF-κB showed no remarkable difference among all groups of ECV-304(P＞0.05).
Conclusion:BYHWD and its active fractions could improve VECs releasing the vasodilator material—NO and PGI_2,but couldn't significantly affect the secretion of vasoconstrictive substance—ET-1.BYHWD could reduce the increasing expression of adhesion molecules in VECs stimulated by thrombin, and alkaloid and glycoside were the possible functional components. BYHWD could prevent the activating of PKC system,and then restrain VECs' activation stimulated by thrombin.The experiment result also suggested that alkaloid and glycoside may be associated with it.The drugs including BYHWD and its main effective fractions had no influence on the expression of p38MAPK,ERK or NF-κB in VECs.It suggested that the function of BYHWD on VECs may be related to other cell signal transduction pathway.
引文
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