桂皮醛及其代谢产物肉桂酸体外抗柯萨奇病毒B_3的作用机制研究

Effects of cinnamaldehyde and its metabolite cinnamic acid on viral myocarditis caused by CVB_3

目的探讨桂皮醛(cinnamaldehyde,CA)及其代谢产物肉桂酸(cinnamic acid,CD)体外抗柯萨奇病毒B3(CVB3)的作用机制。方法用CVB3感染SD乳鼠心肌细胞,建立病毒性心肌炎(VMC)细胞模型。1)MTT法测定CA和CD对正常心肌细胞的IC50;2)MTT法测定不同剂量CA、CD对CVB3感染心肌细胞72h存活率;3)分别于接种CVB3的同时(0h)、接种前1h(-1h)和接种后1h(1h)给予100μmol/LCA、CD各100μl,观察CPE,MTT测定72h细胞存活率,并进行病毒中和抗体、免疫荧光和电镜观察。结果 1)CA对正常心肌细胞的LogIC50为-4.125,0.1～1000μmoL/L剂量的CD组心肌细胞存活率显著高于CA组(P<0.01);2)10～1000μmol/LCD组细胞存活率高于CA各组和CVB3组,心肌细胞病毒滴度差异有统计学意义(P<0.01)。100～1000μmol/LCA组病毒滴度低于CVB3组(P<0.01),细胞存活率与CVB3组比较差异无统计学意义(P>0.05);3)接种CVB3-1h、0h和1h,分别给予100μmol/LCD100μl,其病毒滴度均显著低于CVB3组(P<0.01),细胞存活率高于CA组和CVB3组(P<0.01),不同时间接种CVB3组与CD组比较差异无统计学意义(P>0.05),电镜和免疫荧光试验显示,CD对CVB3有直接灭活和抑制细胞内复制作用。接种CVB30和1h给予CA,其病毒滴度低于CVB3组(P<0.01),但细胞存活率与CVB3组比较差异无统计学意义(P>0.05)。结论 CA在体外可直接灭活CVB3,但对心肌细胞具有明显毒性,可能与其醛基结构相关。CD可直接杀灭CVB3,抑制CVB3在细胞内的复制,但对CVB3感染无预防作用。

Objective To investigate the effects of anti-CVB3 on cinnamaldehyde （CA） and its metabolite cinnamic acid （CD） in rats in vivo and in vitro. Methods A viral myocarditis （VMC） cell model was created in which primary cultured myocardial cells of Sprague-Dawley rats were infected with CVB3. 1） An MTT reduction assay was used to assess the LogIC50 value of CA and CD in normal myocardial cells. 2） The MTT reduction assay was used to assess the effects of CA and CD on the viability of CVB3-infected myocardial cells at different doses. 3） Myocardial cells were exposed to the virus （100 TCID50,0.1 ml per well） at -1,0,and 1 h and incubated with or without being treated with 100 μl CA and CD （100 μmol/L）. After culturing for 72 hours,the maximal viral CPE was determined and recorded,and then virus titers were measured in HeLa cells. An MTT test,virus-neutralizing antibody test,immunofluorescence test,and electron microscopy were performed. Results 1）The LogIC50 value of CA in inhibiting myocardial cells viability was -4.125. As shown by the MTT reduction assay,the cell viability was significantly higher with 0.1-1 000 μmol/L CD than with CA （P〈0.01）. 2） Viral titers of myocardial cells in infected rats decreased significantly in vitro with 10-1 000 μmol/L of CD in a concentration-dependent manner. The viability of myocardial cells increased significantly in comparison to rats given CA or anti-CVB3 （P〈0.01,n=3 each）.Viral titers of myocardial cells in rats given 100-1 000 μmol/L of CA were lower than those given anti-CVB3 （P〈0.05）,but the difference in the viability of myocardial cells in rats given 100-1 000 μmol/L CA was not statistically significant in comparison to rats given anti-CVB3 （P〉0.05）. 3）Viral titers of myocardial cells in infected specimens decreased significantly in vitro with 100 μl 100 μmol/L CD at -1,0,and 1 h of CVB3 treatment （P〈0.05 -0.01,n=3 each）. CD resulted in a higher viability of myocardial cells than anti-CVB3 and CA did （P〈0.01）. There was little difference between rats given CD at -1,0,and 1 h of CVB3 treatment（P〈0.05）. Electron microscopy and an immunofluorescence assay revealed that CD directly inactivated CVB3 and inhibited its replication in cells. Viral titers of myocardial cells in rats given CA at 0 h and 1 h of CVB3 treatment were lower than in rats given anti-CVB3 （P〈0.05）,but the difference in the viability of myocardial cells in rats given CA was not statistically significant in comparison to those given anti-CVB3. Conclusion CA inactivated CVB3 directly in myocardial cells with cytotoxicity that may have resulted from the unsaturated aldehydes of CA. CD may directly inactivate and inhibit CVB3 replication in cells,but it does not protect against CVB3 infection in myocardial cells.