| Objective To probe the inhibitory effect of Longdanxiegan Chinese traditional medicine Granule on replication of herpes simplex virus (HSV-2) in Vero cells in vitro. To explore the immunological function of Longdanxiegan Granule,through studying the Dendritic cells extracted from the patients'blood by cell culture method in vitro.Methods 1) methods as following:Method I, inhibiting virus multiplication-Confluent cell monolayers in 96-well plates were infected with HSV-2 333in medium. After adsorption at 37℃for 1.5 h, residual inoculum was replaced with 0.2ml of medium containing test drug.MethodⅡ, interdicting cells infection-Cell cultures were treated with the drugmedium for 24h,washed thrice with PBS,100 TCID50 of virus were addedto give 0.1ml per well and subsequently reincubated in drug-free medium.Method III, directly killing virus-Virus were incubated in drug medium for 24h at 37℃was added to give 0.2 ml per well.2) DC in peripheral blood in patient and healthy people were cultured for five days with medium which had GM-CSF and IL-4.Flow cytometry was used to analyze surface marker of CD83 and CD86 of DC.3) the content of IL-12 in cell culture supernatant was detected by ELISA.Result 1) The results shew that the TCo and the TC50 for Longdanxiegan were 0.16 and 5.631 mg/ml, respectively. Corresponding values for ACV were 0.1 and 0.457 mg/ml, respectively.2) The Infected capability of diluted stocks of HSV-2333 strains was measured by CPE method. The results shew that the TCID50 of HSV-2 for Vero cell was 10-4/0.1ml.3) By different drug addition methods, the typical cytopathic effects in Vero cells were observed in all parallel virus controlled groups, while normal Vero cellsgroups were almost negative. By drug addition I method, the CPEs were restrained at different degrees in Longdanxiegan and ACV groups; the CPEs were only inhibited at different degrees in Longdanxiegan group under drug addition method II and 111, while the cytopathic effects were clearly visible in ACV group.4) Measured by MTT colorimetry, the mean value of A490 for the parallel virus controlled groups and normal Vero cells groups were 0.294±0.053,0.032±0.0065, respectively. By drug addition I method, compared with the values of A490 of virus controlled groups,the values of A490 of Longdanxiegan and ACV in all concentrations were almost statistically significant (P<0.05 or P<0.01).By drug addition II method, The part values of Longdanxiegan group were statistically differences compared with that of virus controlled group (P<0.05). By the last drug addition method, only the values of Longdanxiegan group was statistically significant compared with that of the virus controlled group (p<0.01).The values of acyclovir group were not statistically differences (P>0.05) under drug addition method II andⅢ. 5) The charts of dose-effect by different drug addition methods were drawn, serving drug concentrations and IR% as abscissa and y-axis respectively. Under drug addition method I, the inhibitory effects of ACV for HSV-2 became stronger with the increase of concentration, showing clear dose-effect connection. At beginning, the inhibitory effects of Longdanxiegan became stronger with the increase of concentration. After exceeding 40pg/ml, it's inhibitory effects declined with the increase of concentration. By drug addition II method, the inhibitory rates of Longdanxiegan for HSV-2 became stronger with the increase of concentration exceeding 20μg/ml. By the last drug addition method, the IR% of Longdanxiegan became weaker with the increase of concentration exceeding 80μg/ml too.6) The expression of CD83 and CD86 on DCs were higher in patients than that in healthy controls(P<0.05). After adding drug, the expression of these molecules were regulated, there were no significant differences between patients and controls(P>0.05).7) The production of IL-12 was increased obviously in control group of patient (p<0.05). After adding drug, the production of IL-12 was decreased evidently(p<0.01).Conclusion 1) Under the concentration of 0.16 mg/ml, Longdanxiegan were innocuity to Vero cells, Longdanxiegan was very safe to Vero cells.2) Multiplication of HSV-2 in Vero cells were inhibited at different degrees in Longdanxiegan and ACV groups. Only Longdanxiegan shew the activity of directly killing virus and guarding against Vero cells'infection by HSV-2 at different degree. Longdanxiegan had an activity for stronginhibition of HSV-2 at several effect points in vitro.3) The inhibitoryeffects of ACV for multiplication of HSV-2 shew clear dose-effect connection.The effects of Longdanxiegan for guarding against Vero cells'infection became stronger with the increase of concentration,but Longdanxiegan didn't show clear dose-effect connection in restraining multiplication of HSV-2 and directly killing virus.4) The compound traditional Chinese medicine has the function of immunoregulation on DC, it can make DC of healthy people have potent antigen presenting capabilities, which also can inhibit antigen presenting capabilities of DC in patient.5) The production of IL-12 was inhibited in patient and healthy subjects by compound traditional Chinese medicine,so the immune response of Thl is inhibited by compound traditional Chinese medicine,it could make the disease psoriasis vulgaris more better.
|
PDF Full Text Request