【摘要】 目的研究大蒜新素抑制人巨细胞病毒(HCMV)复制的作用环节和机制。方法采用时间依赖的药物添加和移除实验分析大蒜新素在HCMV病毒复制周期中的作用环节。用Southern blot检测大蒜新素处理后感染细胞中HCMV DNA负荷量变化。用Northern blot和Western blot检测大蒜新素对HCMV即刻早期(ie)基因转录和翻译的影响,并与相应浓度更昔洛韦(GCV)的作用效应作比较。结果时间依赖的药物添加和移除实验发现大蒜新素只有在HCMV感染复制周期的早期存在时才能发挥对病毒增殖的抑制作用。大蒜新素对ie 1 mRNA的抑制率达30．4％-46．6％。在感染后24 h 病毒表达IE蛋白的高峰期时,大蒜新素对IE72表达的抑制率为42．6％-64．9％,对IE86表达的抑制率为50．7%- 70．6％。到感染后72 h时(子代病毒表达IE蛋白的时期),大蒜新素对IE蛋白仍然有抑制作用,对IE72的抑制率为 36.4％-49．3％,对IE86的抑制作用更为明显,抑制率达77．9％-87．7％,接近于对IE72抑制率的2倍。而GCV对一个 HCMV复制周期内的ie 1 mRNA和IE蛋白表达均无抑制作用。Southern blot结果显示,在感染后72 h(HCMV完成一个复制周期的时间点),大蒜新素和GCV均能减少HCMV DNA负荷量,两药相当剂量的抑制效应比较,差异无统计学意义(P> 0．05)。结论大蒜新素抑制HCMV ie基因的转录和翻译,进而抑制病毒的复制增殖,是其抗HCMV的重要作用机制。更多还原

【Abstract】 Objective Allitridin (diallyl trisulfide) , a main organic compound of garlic (Allium sativum) , has been reported having anti-HCMV efficacy in experiments and in clinical studies. However, its mechanism of action is still poorly defined. Our previous study demonstrated that allitridin could markedly reduce the level of HCMV immediate early antigens (flow cytometry).which are considered as being the essential regulating proteins for HCMV replication and playing an important role in the pathogenesis of HCMV associated diseases as well. This study was to investigate the action of allitridin in the replication cycle of HCMV, especially to determine its effects on the transcription and expression of viral immediate early (ie) genes. Methods The maximal tolerable concentration (MTC) of allitridin for HEL cells was determined based on the MTT assay. The inhibition of allitridin on HCMV was measured by plaque-reduction assay. To determine when allitridin acts in the HCMV replication cycle,timeof -addition and removal studies in a single viral cycle format at MOI of 2. 5 were performed. Then the effect of allitridin on the replication of HCMV genomic DNA was analyzed at 72 h past infection (after one round of viral replication) by Southern blot assay. The suppression of HCMV immediate early genes’ transcription by allitridin was measured by Northern blot at 4 h past infection. And the suppression of the translation by allitridin was assayed by Western blot at 24 h past infection (at the first round of viral replication cycle) and 72 h past infection (at the second round of viral replication cycle).respectively. All the tests were compared with those of ganciclovir control. Results MTC of allitridin for HEL cells was 9. 6μg·L-1 and the concentrations under MTC were chosen in subsequent experiments. In HCMV plaque-reduction assay, allitridin appeared as a dose-dependent inhibitory ability with EC50 value of 4. 2 μg·mL-1 (selective index, SI=16.7). Time-of-addition and time-of-removal studies showed that allitridin inhibited HCMV replication only when the drug appeared in the period from 0 h to 8 h past infection, an earlier phase of viral cycle before viral DNA synthesis, indicating the possible effect on expression of immediate early genes. Confirmed by Northern and Western blot, both immediate early gene transcription (ie 1) and IE proteins expression (IE72 and IE86) were suppressed by allitridin,but not by GCV,in a single HCMV cycle format. The inhibitory rate of allitridin on ie 1 mRNA reached 30.38% -46. 61 % with EC50 and MTC allitridin. The expression of IE72 under allitridin treatment was suppressed by 42. 6% - 64. 9% at 24 h past infection,and the expression of IE86 by 50. 7%-70. 6%. By 72 h past infection,allitridin still obviously reduced the levels of IE72 and IE86 proteins. Furthermore, allitridin showed stronger inhibition on IE86 than on IE72, with the inhibitory rate on IE86 almost as twice as that of IE72. The effects of allitridin on immediate early gene expression were different from those of ganciclovir, indicating different mechanisms of these two drugs. Decrease of viral DNA load in infected cells was also detected under allitridin treatment, probably due to an indirect consequence of the reduction in immediate early gene expression. There was no significant difference between the suppressions on viral DNA load of allitridin and ganciclovir treatment, under the concentrations used in the experiments. Conclusion Allitridin could obviously suppress the transcription and expression of HCMV ie genes,by which it further effectively inhibits HCMV replication, indicating an important mechanism of allitridin against HCMV.更多还原