The Effect Of α-Amanitin On The Replication Of HBV In Vitro

Objective To explore the roles ofα-amanitin in the replication ofhepatitis B virus in HepG2 2.2.15 cell lineMethods MTT assay were used to observe the cytotoxicity ofα-amanitin to the HepG 2 2.2.15 cell line. And then HepG2.2.15 cellswere cultured with different non-cellulotoxic concentration ofα-amanitin or 3TC for six days. TRFIA was adopted to determine thelevels of HBsAg and HBeAg,after the cell culture medium was collectedin the third day and the sixth day,respectively. Cells were harverested fordetermining the expression of HBsAg mRNA and HBcAg mRNA byreverse transcription PCR (RT-PCR)Results①α-amanitin has no significant cytotoxicity to HepG22.2.15 cell line in the concentration ranging from 0.0064μg/ml to0.8μg/ml,while significant cytotoxicity was found in the concentrationranging from 4μg/ml to 20μg/ml. In the third day, TC₅₀ was 10.190μg/ml,in the sixth day, TC₅₀ was 8.471μg/ml.â‘¡In the third day, HBsAg suppression rate of 3TC, 0.8μg/ml and0.016μg/ml group was 46.071%,19.881% and 15.952%,respectively.No suppression was found in other groups.In the sixth day,HBsAg suppression rate of 3TC, 0.8μg/ml and 0.016μg/ml group was55.709%,47.405% and 29.296%, respectively.No suppression was foundin other groups.â‘¢In the third day, HBeAg suppression rate of 3TC and 0.8μg/mlgroup was 31.854% and 14.159%, respectively.No suppression wasfound in other groups.In the sixth day, HBsAg suppression rate of 3TC,0.8μg/ml,0.16μg/ml and 0.032μg/ml group was 59.161%,51.524%, 45.845% and 29.086%, respectively.No suppression was found in0.0064μg/ml group.â‘£In the third day, down regulation of HBsAg mRNA expression in3TC, 0.8μg/ml and 0.16μg/ml groups was found statistical significance,comparing with the control.In the sixth day, down regulation of HBsAgmRNA expression in 3TC, 0.8μg/ml and 0.16μg/ml groups was foundstatistical significance, comparing with the control group.⑤In the third day, down regulation of HBcAg mRNA expression in3TC and 0.8μg/ml groups was found statistical significance, comparingwith the control group.In the sixth day, down regulation of HBsAgmRNA expression in 3TC, 0.8μ/ml and 0.16μg/ml groups was foundstatistical significance, comparing with the control group.Conclusionsα-amanitin has no significant cytotoxicity to HepG22.2.15 cell line in the concentration ranging from 0.0064μg/ml to0.8μg/ml,while significant cytotoxicity was found in the concentrationranging from 4μg/ml to 20μg/ml. In the third day, TC₅₀ was 10.190μg/ml,in the sixth day, TC₅₀ was 8.47μg/ml. Both the expression ofHBsAg and HBcAg mRNA and the secretion of HBsAg and HBeAg weresuppressed byα-amanitin at little cytotoxicity concentrtion in HepG22.2.15 cell line.Its suggested thatα-amanitin might inhibit theduplication of HBV-DNA,being provided therical and experimentalevidence for the further research of the pharmacological mechanism.