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標題: Anti-coxsackievirus B3 activity and relevant mechanisms of glycyrol in Vero cells
作者: Hsin-I Chou
關鍵字: 腸病毒克沙奇B3型;甘草醇;作用機轉;coxsackievirus B3;glycyrol;mechanisms
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腸病毒克沙奇B3型 (coxsackievirus B3, CVB3) 屬於微小核醣核酸病毒科 (Picornaviridae),腸道病毒屬 (enterovirus) 之單股正向RNA病毒。主要感染嬰幼兒及孩童,嚴重時可能引起心肌炎。甘草醇 (glycyrol) 為先前由七葉一枝花 (Paris polyphylla Smith) 分離,抗腸病毒71 型之活性成分,本研究接續探討此化合物對CVB3 KMUH-92225於非洲綠猴腎細胞 (Vero) 之抑制作用及相關作用機轉。
研究結果顯示,甘草醇對CVB3之50%抑制濃度 (50% inhibitory concentration, IC50) 為1.44 ± 0.06 μg/mL,選擇指數 (selectivity index, SI) 為9.94。甘草醇對CVB3之抑制作用包括些微預防病毒感染、輕微抑制病毒吸附宿主細胞及高度抑制病毒之複製;其中,3.00 μg/mL甘草醇能顯著降低CVB3子代病毒效價達0.45-1.42 log PFU/mL;1.50 μg/mL甘草醇亦能有效抑制CVB3於Vero細胞中之VP1表現,為CVB3感染控制組之53%。顯示甘草醇對CVB3 KMUH-92225之抑制,係以抑制病毒複製為主要之作用。甘草醇對CVB3 KMUH-92225之作用機轉之結果顯示,3.00 μg/mL甘草醇顯著抑制感染病毒之宿主細胞IκB⍺降解、細胞核內NF-κB (p65) 及mitogen-activated protein kinase (MAPK) 家族 [p-p38 (phosphate-p38)、p-JNK1/2/3 (phosphate-c-Jun N-terminal kinase 1/2/3) 及p-ERK1/2 (phosphate-extracellular signal-regulated protein kinase 1/2)] 蛋白之活化,因而顯著降低下游細胞發炎物質COX-2之生成。但甘草醇未能影響感染CVB3宿主細胞引起之coxsackievirus and adenovirus receptor (CAR) 內吞作用及Ras GTPase activating protein (RasGAP) 蛋白之降解。
綜合本研究結果,甘草醇對CVB3 KMUH-92225具優異之抑制作用,係透過抑制MAPK及NF-κB路徑之活化,因而降低CVB3於宿主細胞之複製;甘草醇也可藉抑制此兩路徑之活化而有效的減少下游發炎因子COX-2蛋白生成,並減緩宿主細胞嚴重發炎反應,以避免心肌炎產生。

Coxsackievirus B3 (CVB3), a member of the genus Enterovirus, is a positive single-strand RNA virus belonging to the Picornaviridae family. CVB3 mainly infects infants and children, and leads to acute myocarditis. In our previous study, glycyrol was isolated from Paris polyphylla Smith and showed activities of anti-enterovirus 71. This study continued investigating the anti-CVB3 mechanism of glycyrol in Afrian green monkey kidney (Vero) cells.
The results showed that glycyrol had an anti-CVB3 activity of glycyrol with 1.44 ± 0.06 μg/mL of 50% inhibitory concentration (IC50). The four mechanisms of inhibiting CVB3 by glycyrol included a little bit prophylactic, slight inhibition of CVB3 adhesion to cells or virucidal, and excellent anti-viral replication effects. Glycyrol (3.00 μg/mL) significantly reduced the CVB3 progeny virus titer of 0.45-1.42 log PFU/mL and the VP1 expression in the CVB3-infected cells, in which the expression was 53% of that in CVB3-infected control. The results showed that glycyrol significantly inhibited the degradation of IκB⍺, the expression of NF-κB (p65), and the activation of mitogen-activated protein kinase (MAPK) (p38, p-JNK1/2/3 and p-ERK1/2) in the CVB3-infected cells. Additionally, COX-2, which is the downstream inflammatory factor, also decreased considerably. However, the endocytosis of coxsackievirus and adenovirus receptor (CAR) and the degradation of Ras GTPase activating protein (RasGAP) caused by CVB3-infection were not affected by glycyrol.
In conclusion, possessing the ability to block the MAPK and NF-κB pathways, glycyrol had a great effect on inhibiting CVB3 via reducing the replication in CVB3-infected cells. Moreover, reducing the synthesis of COX-2 by blocking those pathways, glycyrol might have an ability to prevent from myocarditis by reducing inflammation.
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