瑞香素体外抗肠道病毒71型的初探
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摘要
目的探讨瑞香素对肠道病毒71型(EV71)感染人恶性胚胎横纹肌肉瘤(RD)细胞的影响。方法实验设定为4组:正常细胞组指不加药物及EV71干预RD细胞;病毒对照组指感染EV71的RD细胞;瑞香素组指不同浓度(根据瑞香素最大无毒浓度设定瑞香素作用浓度为15、12. 5、10、7. 5、5、3. 75、2. 5、1. 875、1. 25、1μg/m L)的瑞香素作用于感染EV71的RD细胞;阳性药物组是指50μg/m L利巴韦林(ribavirin,RVB)作用于感染EV71的RD细胞。运用CCK8测定法、细胞病变效应(CPE)、RT-PCR方法测定瑞香素在病毒感染不同阶段的抗EV71病毒作用。结果瑞香素对RD细胞的50%细胞毒性浓度(TC50)为33. 47μg/m L,最大无毒浓度(TC0)为15μg/m L。浓度为3. 75～15μg/m L瑞香素在EV71感染期和感染后期对EV71均有较好的抑制效果,其中12. 5、15μg/m L瑞香素的治疗效果相对较佳,其病毒抑制率与正常细胞组比较差异无统计学意义(P> 0. 05)。浓度为15、10μg/m L的瑞香素在EV71感染细胞后再用药处理,能有效降低病毒载量;而以浓度为15、10、7. 5μg/m L瑞香素与EV71病毒混合作用于细胞,同样具有降低病毒载量的作用,均对EV71的复制有较好的抑制效果,差异有统计学意义(P <0. 05)。结论瑞香素能有效抑制EV71的复制,有望为开发新的抗EV71药物提供实验依据。
Objective To investigate the effect of daphnetin on RD cells infected with EV71. Methods The experiment was set up in 4 groups,normal group( RD cells),virus control group( RD cells infected with EV71),daphnetin group( RD cells infected with EV71 were treated with different concentrations of daphnetin) and positive drug group( RD cells infected with EV71 and treated with RVB). CCK8 assay,cytopathic effect( CPE) and real-time RT-PCR were used to determine the effect of daphnetin against EV71 virus at different stages of viral infection. Results The 50%cytotoxicity( TC50) of daphnetin on RD cells was 33. 47 μg/m L,and the maximum nontoxic concentration( TC0) was 15μg/m L. The daphnetin of 3. 75-15 μg/m L was used during EV71 infection and late-infection. In particular,1 daphnetin of 2. 5-15 μg/m L had a relatively good therapeutic effect,and not significantly affected the cells compared with normal cell group( P > 0. 05). Daphnetin of 15 and 10 μg/m L administered after EV71 infection significantly inhibited the virus replication,and daphnetin of 15,10 and 7,5 μg/m L administered at the time of EV71 infection also significantly inhibited the virus replication. Conclusion Daphnetin can effectively inhibit the replication of EV71 and expected to provide experimental evidence for the development of new anti-EV71 drugs.
Objective To investigate the effect of daphnetin on RD cells infected with EV71. Methods The experiment was set up in 4 groups,normal group( RD cells),virus control group( RD cells infected with EV71),daphnetin group( RD cells infected with EV71 were treated with different concentrations of daphnetin) and positive drug group( RD cells infected with EV71 and treated with RVB). CCK8 assay,cytopathic effect( CPE) and real-time RT-PCR were used to determine the effect of daphnetin against EV71 virus at different stages of viral infection. Results The 50%cytotoxicity( TC50) of daphnetin on RD cells was 33. 47 μg/m L,and the maximum nontoxic concentration( TC0) was 15μg/m L. The daphnetin of 3. 75-15 μg/m L was used during EV71 infection and late-infection. In particular,1 daphnetin of 2. 5-15 μg/m L had a relatively good therapeutic effect,and not significantly affected the cells compared with normal cell group( P > 0. 05). Daphnetin of 15 and 10 μg/m L administered after EV71 infection significantly inhibited the virus replication,and daphnetin of 15,10 and 7,5 μg/m L administered at the time of EV71 infection also significantly inhibited the virus replication. Conclusion Daphnetin can effectively inhibit the replication of EV71 and expected to provide experimental evidence for the development of new anti-EV71 drugs.
引文
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[8]Li M,Shi X,Chen F,et al. Daphnetin inhibits inflammation in the NZB/W F1 systemic lupus erythematosus murine model via inhibition of NF-κB activity:[J]. Exp Ther Med,2017,13(2):455-460.
[9]Shen L,Zhou T,Wang J,et al. Daphnetin reduces endotoxin lethality in mice and decreases LPS-induced inflammation in Raw264. 7 cells via suppressing JAK/STATs activation and ROS production[J]. Inflamm Res,2017,66(7):579-589.
[10]Shan J,Qian W,Peng L,et al. A Comparative Pharmacokinetic Study by UHPLC-MS/MS of Main Active Compounds after Oral Administration of Zushima-Gancao Extract in Normal and Adjuvant-Induced Arthritis Rats[J]. Molecules,2018,23(1):E227.
[11]刘志勇,费浩,邓明,等.瑞香素预处理对重症急性胰腺炎大鼠的抗氧化保护作用[J].微循环学杂志,2017,27(4):12-17.
[12]Lv H,Liu Q,Zhou J,et al. Daphnetin-mediated Nrf2 antioxidant signaling pathways ameliorate tert-butyl hydroperoxide(tBHP)-induced mitochondrial dysfunction and cell death[J].Free Radic Biol Med,2017,106:38-52.
[13]Mohri S,Takahashi H,Sakai M,et al. Wide-range screening of anti-inflammatory compounds in tomato using LC-MS and elucidating the mechanism of their functions[J]. PLo S One,2018,13(1):e191203.
[14]Vinayagam R,Xu B. 7,8-Dihydroxycoumarin(daphnetin)protects INS-1 pancreaticβ-cells against streptozotocin-induced apoptosis[J]. Phytomedicine,2017,24:119-126.
[15]Qi Z,Qi S,Gui L,et al. Daphnetin protects oxidative stress-induced neuronal apoptosis via regulation of MAPK signaling and HSP70 expression[J]. Oncol Lett,2016,12(3):1959-1964.
[16]Zhu HY,Gao HW,Zhang SF. 7,8-dihydroxycoumarin has a dual mechanism of action in hepatic ischemia reperfusion injury[J]. Int J Clin Exp Pathol,2015,8(9):10121-10129.
[17]Liu J,Chen Q,Jian Z,et al. Daphnetin Protects against Cerebral Ischemia/Reperfusion Injury in Mice via Inhibition of TLR4/NF-κB Signaling Pathway[J]. Biomed Res Int, 2016, 2016:2816056.
[18]王洁莹,刘玮,郑茂,等.瑞香素对CIA大鼠脾T淋巴细胞杀瘤效应的影响[J].时珍国医国药,2017,28(1):33-35.
[19]Shu K,Kuang NZ,Zhang Z,et al. Therapeutic effect of daphnetin on the autoimmune arthritis through demethylation of proapoptotic genes in synovial cells[J]. J Transl Med,2014,12:287.
[20]袁琦,侯林,崔清华,等.红景天甲醇冷浸提取物的体外抗病毒谱及抗病毒机制的研究[J].中华中医药学刊,2017,35(9):2345-2348.
[21]Li M,Yang X,Guan C,et al. Andrographolide sulfonate reduces mortality in Enterovirus 71 infected mice by modulating immunity[J]. Int Immunopharmacol,2018,55:142-150.
[22]况南珍,傅颖媛,龚淑琪,等.瑞香素对SMMC-7721细胞凋亡及Bcl-2/Bax基因mRNA表达的影响[J].广东医学,2012,33(10):1374-1377.
[23]王洁莹.瑞香素对TNF-α诱导的CIA大鼠滑膜细胞MAPK信号通路作用初探[D].南昌:南昌大学,2017.
[24]代婵,王洁莹,邓慧敏,等.瑞香素在TNF-α诱导的CIA大鼠成纤维样滑膜细胞NF-κB信号通路中的抑炎作用[J].时珍国医国药,2018,29(1):20-24.
[25]Chen X,Kuang N,Zeng X,et al. Effects of daphnetin combined with Bcl2siRNA on antiapoptotic genes in synovial fibroblasts of rats with collageninduced arthritis[J]. Mol Med Rep,2018,17(1):884-890.
[26]Liu J,Li X,Fan X,et al. Adoptive transfer of macrophages from adult mice reduces mortality in mice infected with human enterovirus 71[J]. Arch Virol,2013,158(2):387-397.
[2]王晓宇,成怡冰,宋春兰.丙种球蛋白对EV71相关重症手足口病患儿热休克蛋白70水平的影响[J].广东医学,2016,37(17):2663-2666.
[3]Yi EJ,Shin YJ,Kim JH,et al. Enterovirus 71 infection and vaccines[J]. Clin Exp Vaccine Res,2017,6(1):4-14.
[4]宋杰,胡雅洁,董少忠. EV71和CA16研究进展综述[J].生命科学,2018(3):241-247.
[5]Wang H,Li K,Ma L,et al. Berberine inhibits enterovirus 71replication by downregulating the MEK/ERK signaling pathway and autophagy[J]. Virol J,2017,14(1):2.
[6]王晓妍,王伟,孙蓉,等.参苓白术散含药血清体外抗轮状病毒的实验研究[J].山东中医杂志,2018,37(1):58-62.
[7]Dhama K,Karthik K,Khandia R,et al. Medicinal and Therapeutic Potential of Herbs and Plant Metabolites/Extracts Countering Viral Pathogens-Current Knowledge and Future Prospects[J].Curr Drug Metab,2018,19(3):236-263.
[8]Li M,Shi X,Chen F,et al. Daphnetin inhibits inflammation in the NZB/W F1 systemic lupus erythematosus murine model via inhibition of NF-κB activity:[J]. Exp Ther Med,2017,13(2):455-460.
[9]Shen L,Zhou T,Wang J,et al. Daphnetin reduces endotoxin lethality in mice and decreases LPS-induced inflammation in Raw264. 7 cells via suppressing JAK/STATs activation and ROS production[J]. Inflamm Res,2017,66(7):579-589.
[10]Shan J,Qian W,Peng L,et al. A Comparative Pharmacokinetic Study by UHPLC-MS/MS of Main Active Compounds after Oral Administration of Zushima-Gancao Extract in Normal and Adjuvant-Induced Arthritis Rats[J]. Molecules,2018,23(1):E227.
[11]刘志勇,费浩,邓明,等.瑞香素预处理对重症急性胰腺炎大鼠的抗氧化保护作用[J].微循环学杂志,2017,27(4):12-17.
[12]Lv H,Liu Q,Zhou J,et al. Daphnetin-mediated Nrf2 antioxidant signaling pathways ameliorate tert-butyl hydroperoxide(tBHP)-induced mitochondrial dysfunction and cell death[J].Free Radic Biol Med,2017,106:38-52.
[13]Mohri S,Takahashi H,Sakai M,et al. Wide-range screening of anti-inflammatory compounds in tomato using LC-MS and elucidating the mechanism of their functions[J]. PLo S One,2018,13(1):e191203.
[14]Vinayagam R,Xu B. 7,8-Dihydroxycoumarin(daphnetin)protects INS-1 pancreaticβ-cells against streptozotocin-induced apoptosis[J]. Phytomedicine,2017,24:119-126.
[15]Qi Z,Qi S,Gui L,et al. Daphnetin protects oxidative stress-induced neuronal apoptosis via regulation of MAPK signaling and HSP70 expression[J]. Oncol Lett,2016,12(3):1959-1964.
[16]Zhu HY,Gao HW,Zhang SF. 7,8-dihydroxycoumarin has a dual mechanism of action in hepatic ischemia reperfusion injury[J]. Int J Clin Exp Pathol,2015,8(9):10121-10129.
[17]Liu J,Chen Q,Jian Z,et al. Daphnetin Protects against Cerebral Ischemia/Reperfusion Injury in Mice via Inhibition of TLR4/NF-κB Signaling Pathway[J]. Biomed Res Int, 2016, 2016:2816056.
[18]王洁莹,刘玮,郑茂,等.瑞香素对CIA大鼠脾T淋巴细胞杀瘤效应的影响[J].时珍国医国药,2017,28(1):33-35.
[19]Shu K,Kuang NZ,Zhang Z,et al. Therapeutic effect of daphnetin on the autoimmune arthritis through demethylation of proapoptotic genes in synovial cells[J]. J Transl Med,2014,12:287.
[20]袁琦,侯林,崔清华,等.红景天甲醇冷浸提取物的体外抗病毒谱及抗病毒机制的研究[J].中华中医药学刊,2017,35(9):2345-2348.
[21]Li M,Yang X,Guan C,et al. Andrographolide sulfonate reduces mortality in Enterovirus 71 infected mice by modulating immunity[J]. Int Immunopharmacol,2018,55:142-150.
[22]况南珍,傅颖媛,龚淑琪,等.瑞香素对SMMC-7721细胞凋亡及Bcl-2/Bax基因mRNA表达的影响[J].广东医学,2012,33(10):1374-1377.
[23]王洁莹.瑞香素对TNF-α诱导的CIA大鼠滑膜细胞MAPK信号通路作用初探[D].南昌:南昌大学,2017.
[24]代婵,王洁莹,邓慧敏,等.瑞香素在TNF-α诱导的CIA大鼠成纤维样滑膜细胞NF-κB信号通路中的抑炎作用[J].时珍国医国药,2018,29(1):20-24.
[25]Chen X,Kuang N,Zeng X,et al. Effects of daphnetin combined with Bcl2siRNA on antiapoptotic genes in synovial fibroblasts of rats with collageninduced arthritis[J]. Mol Med Rep,2018,17(1):884-890.
[26]Liu J,Li X,Fan X,et al. Adoptive transfer of macrophages from adult mice reduces mortality in mice infected with human enterovirus 71[J]. Arch Virol,2013,158(2):387-397.