34 1 Vol 34 No 1 2015 3 Journal of South-Central University for Nationalities Nat Sci Edition Mar 2015 B 4 1 2 2 2 1 430074 2 430071 B 4 CVB 4 MTT 3 CVB 4 PCR CVB 4 mrna IC 50 = 14 10 ± 0 74 μm TI = 4 35 ± 0 60 CVB 4 mrna p < 0 05 0 ~ 4 h CVB 4 B 4 R373 A 1672-4321 2015 01-0056-06 Antiviral Effect of Emodin on Coxsakievirus B 4 in vitro Liu Zhao 1 Ma Nian 2 Zhong Yan 2 Yang Zhanqiu 2 1 Journal Editorial Department South-Central University for Nationalities Wuhan 430074 China 2 State Key Laboratory of Virology Institute of Medical Virology School of Medicine Wuhan University Wuhan 430071 China Abstract To investigate the antiviral effect of emodin on Coxsakievirus B 4 ribavirin was used as the positive control and MTT assay was used to evaluate its anti-cvb 4 effect through 3 manners including propagation inhibition direct inactivation and anti-absorption Then real-time PCR were performed to determine the effect of emodin on the mrna expression of CVB 4 and plaque forming assay were applied to detect the effect of emodin on the plaque formed rate after virus infection at different time The results indicated that emodin could not directly inactivate the viruses or block their absorption to cells but it may act as a biological synthesis inhibitor The IC 50 was detected as 14 10 ± 0 74 μm and TI were detected as 4 35 ± 0 6 Emodin could significantly reduce the mrna expression of CVB 4 gene and may act its inhibitory effect during 0 ~ 4 h postinfection p < 0 05 So emodin may act its antiviral activity of inhibiting CVB 4 replication after virus penetration in a concentration- and time-dependent manner Keywords emodin Coxsakievirus B 4 antiviral effect Coxsackie Virus B 4 Picornaviridas Enterovirus 1 1-4 5 6 1 1 0 02 g 200 μl DMSO ddh 2 O 7 8 2015-01-25 1978- E-mail 3035407@ mail scuec edu cn 2009ZX09301-014-1 81371865
1 B 4 57 2 ml 37 10 3 μm 10 mg / ml 0 04 ~ 0 06 MPa 15 100 TCID 50 CVB 4 HEp-2 min 4 2 h 12010721 1g / 409 10 3 μm 100 mg / ml 0 08 MPa 15 min 1 2 HEp-2 2% III CCTCC CVB 4 HEp-2 CPE + + + ~ + + + + 1 3 DMEM Trizol Invitrogin MTT dntp mix PCR Markers Amresco RNA AMV Reverse Transcriptase dntps SYBR GREEN qpcr Super Mix Promega DEPC Sigma CKX41 Olympus Japan OD 570 DG3022-A Rayto PCR My cycler Bio Rad USA Real-time PCR CFX96 Bio Rad USA 2 2 3 3 I II 100 μl 100 TCID 50 CVB 4 4 6 h HEp-2 2 h 37 5% CO 2 6 h 100 TCID 50 CVB 4 2 h 2% 80% MTT OD 570 /% = OD 570 100% OD 570 OD 570 2 4 Real-time PCR CVB 4 mrna 2 4 1 RNA HEp-2 24 500 μl 100 TCID 50 CVB 4 37 5% CO 2 2 1 2 h PBS 3 10 10-1 ~ 10-8 9 0 18 5 37 0 μm 50 μl 8 512 μm 37 5% CO 2 48 h Trizol 2 h 200 μl RNA CPE 9 24 h 2 4 2 cdna +++~++++ RNA 20 PBS 3 MTT μl 1 μg RNA 4 % = OD 570 / μl 5 RT Buffer 1 μl dntp 1 μl AMV Reverse OD 570 100% Transcriptase 0 4 μl Radom Primer RNase Free 2 2 dh 2 O 20 μl 25 10 min 42 60 min 94 3 min 4 4 min 96 cdna 1 HEp-2 200 μl 4 PCR 25 μl 2 5 DMSO μl cdna 9 0 μl sterile H 2 O 12 5 μl PCR Super ddh 2 O 1 199 48 h Mix 0 5 μl Primer S 0 5 μl Primer A MTT 10 OD 570 94 5 min 94 45 s 55 45 s 72 30 s 30 72 5 min 4 PCR 1%
58 34 Tab 1 1 CVB 4 GAPDH The primer sequences for CVB 4 and GAPDH / bp CVB 4 -A CVB 4 -S GAPDH-A GAPDH-S 2 4 3 5'-GTAGTCCTCCGGCCCCT-3' 5'-AATTGTCACCATAAGCAGCCA-3' 5'-TCATTGACCTCAACTACATGGTTT-3' 5'-GAAGATGGTGATGGGATTTC-3' DNA 42 ~ 45 1 2% 37 PCR μm 512 μm 37 5% CO 2 DNA CVB 4 GAPDH 3 NCBI Blast 100% PCR min 1% 10 min 2 4 4 Real-time PCR CVB 4 GAPDH PCR cdna 2 6 10 10 8 ~ 10 1 copies /μl 8 PCR 20 μl TCID 50 TC 50 sterile H 2 O 7 2 μl qpcr Super Mix 10 0 μl 0 4 μl Primer S 0 4 μl Primer A cdna 2 0 μl 95 3 min 95 10 s 55 10 s 72 30 s 39 95 10 s 65 5 s Ct CVB 4 GAPDH Ct value y log 10 RNA copies numbers / μl x 3 1 10-1 / 2 4 5 24 h MTT SPSS 2 4 1 9 0 18 5 37 0 μm CVB 4 TCID 50 10-4 713 512 μm Real-time PCR 100 TCID 50 Ct y 3 2 HEp-2 copy 10 x 2 5 OD 12 100 μl 100TCID 50 CVB 4 4 1 h 1 PBS 3 0 h 2 4 8 12 h 72 h 4% 30 % = / 100% SPSS Probit IC 50 TI = TC 50 / IC 50 One-Way ANOVA LSD 3 130 122 10-1 ~ 10-8 CVB 4 HEp-2 HEp-2 Fig 1 a ~ d 23 13 46 26 92 51 185 02 μm 1 HEp-2 The toxic effect of emodin with different concentrations on HEp-2 cells MTT 2 2
1 B 4 59 p < 0 05 TC 50 61 35 ± 4 40 μm TC 50 2642 95 ± 54 05 μm 37μM 10 μg /ml 0 5 V /V HEp-2 DMSO < 5 V /V OD DMSO 2 Fig 2 3 3 CPE +++~++++ a b HEp-2 The cytotoxic effect of emodin a and ribavirin b on HEp-2 cells CPE MTT 629 99 ± 1 64 μm TI 4 35 I ± 0 60 4 20 ± 0 70 3 3 TI p > 0 05 0 ~ 40 μm CVB 4 p < 0 05 IC 50 14 10 ± 0 74 μm Fig 3 3 a b CVB 4 The inhibitory effect of emodin a and ribavirin b on CVB 4 with different concentrations II III 4c CVB 4 GAPDH y = 0 ~ 32 141-3 762 x y = 33 090-3 709x 40 μm CPE OD 570 0 997 0 999 p > CVB 4 GAPDH 0 05 CVB 4 I CVB 4 GAPDH 3 4 copy 5 5 9 0 18 5 37 0 μm CVB 4 CVB 4 / 4 4a 4b GAPDH copies 17 10 12 76 6 67 S VC CVB 4 /GAPDH copies 28 62 9 0 18 5 37 0 μm 0 40 0 55 0 77 p
60 34 3 5 6 6 CVB 4 0 2 4 h 16 5% 20 5% 40 0% p < 0 01 p < 0 05 8 12 h p > 0 05 0 ~ 12 h p < 0 01 CVB 4 0 ~ 4 h Fig 6 6 ** p < 0 01 n = 3 CVB 4 The inhibitory effects of drug on CVB 4 at different time Fig 4 a CVB 4 b GAPDH 4 c CVB 4 GAPDH CVB 4 GAPDH Amplificatin curve and standard curve for CVB 4 and GAPDH SARS 13 < 0 01 37 μm 512 μm p > 0 05 Fig 5 5 ** p < 0 01 n = 3 CVB 4 /GAPDH The copies of CVB 4 /GAPDH under different drug treatment 4 1'3'8- -6-11 12 14 15 16 SARS-CoV S ACE2 17 3 CVB 4 CVB 4 / IC 50 TI 14 10 ± 0 74 μm 4 35 ± 0 6 TI p > 0 05 2 -ΔΔCT 18 10 CVB 4 GAPDH
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