| In this paper, the antiviral effects as well as the mechanism of melittin purified from bee venom by chromatography and Shen Fengjing on HSV-1 virus were investigated. At the same time, the differences of the expression of p53 and p21 WAF1/CIP1 proteins in the brain cells of mice infected by HSV-1 were explored. The results were as follow:1. The melittin was isolated and purified through polydextran gel G-25,G-50,G-75 combined with hemolysis test from venom of honeybee (Apis mellifera lijustica) workers which was collected by QF-1 Bee Venom Collector in the top of hives. The separated component was used in next experiments after confirmed by comparing to melittin standard and protein marker in SDS-PAGE.2. The TCID50 of HSV-1 virus on Vero cell was 10-5.2 /100uL, which was tested by CPE method and calculated by the Reed-Meuench method. And the CC50 of melittin and Shen Fengjing, which were tested by MTT method, were 10-2.1mg/100uL and 10-2.4mg/100uL. In vitro test results showed that suppression rates of 1,3,6,9 times CC50 of melittin on the virus HSV-1 were 55.12%, 55.12%, 59.30% and 66.51% respectively, and the inhibition rate of drug control (Acyclovir) was 74.88%. Those of Shen Fengjing were 69.50%, 71.52%, 62.11% and 45.74% respectively, and the control was 75.34%.3. In vivo results showed that melittin and Shen Fengjing could improve the survival rate and the survival time of mice. The mice'survival rates subjected to melittin at the low, medium and high dose(1×10-3mg/mL,3×10-3mg/mL,6×10-3mg/mL) were 40%, 40% and 50% respectively, and those to Shen Fengjing were 40%, 50% and 60%, 20% higher than that of virus control group. The average mice'survival duration subjected to melittin were 11.4d, 12d, 12.7d, respectively, and those of median and high doses of melittin were significantly higher than that of the virus control group (p <0.05). Those of Shen Fengjing were 11.6d, 11.8d, and 12.8d respectively, significantly higher than that of virus control group (p <0.05). The survival time of high dose of melittin and Shen Fengjing were higher than the positive control group(ACV).4. The antiviral mechanism research of melittin and Shen Fengjing showed that HSV-1 virus couldn't directly be killed, but the adsorption, synthesis and release of HSV-1 virus were inhibited. The suppression rates of 1, 3, 6 and 9 times CC50 of melittin against adsorption of HSV-1 were 31.25%, 32.92%, 32.92% and 32.08% respectively. The inhibitive percentages caused by Shen Fengjing against virus adsorption were 29.17%, 31.25%, 32.08% and 32.08% respectively. The inhibition rates of melittin against viral synthesis were 29.05%, 29.91%, 31.69% and 33.06% respectively. Those of Shen Fengjing against synthesis were 23.12%, 23.49%, 24.25% and 23.72% respectively. The suppression rates of the four concentrations of melittin against the release of virus were 29.31%, 18.37%, 13.28% and 10.70% respectively, and as for Shen Fengjing groups they were 42.89%, 33.77%, 31.32% and 30.67% respectively.5. Mice were injected with 0.05 mL 100LD50 HSV-1 virus, Melittin and Shen Fengjing was injected for the following two days, then immunohistochemical tests were conducted 4 days after virus injection. The results showed that the protein p21 WAF1/CIP1 positive rates of the normal test group, the virus control group, low , median and high doses of melittin group, low, medium and high doses of Shen Fengjing group were 100%, 2.1%±0.6%, 14.2%±0.4%, 28.5%±0.6%, 33.8%±0.8%, 13.7%±0.5%, 22.3%±0.4% and 29.4%±0.6% respectively. The protein p53 positive cells rates were 3.5%±0.2, 40.2%±0.5%, 31.2%±0.5%, 22.4%±0.4%, 20.5%±0.3%, 27.3%±0.6%, 20.9%±0.3% and 14.1%±0.2% respectively.Melittin purified by chromatogram methods and Shen Fengjing had the antiviral effects on HSV-1 virus in vivo and in vitro by inhibiting the virus adsorption, synthesis and release. And the expression of p21 WAF1/CIP1 and p53 proteins and the cell cycle were adjusted and DNA damage by HSV-1 was protected.The content of bee venom in Shen Fengjing is about 0.1 mg / mL, and the content of melittin is about 0.05 mg / mL. The melittin in 100uL 1CC50 Shen Fengjing and melittin solution are about 0.0002 mg and 0.0079mg. The concentration of melittin tested is 40 times higher than that of Shen Fengjing. There were no significant differences between Shen Fengjing and melittin in the ability of in vivo and in vitro viral resistance to HSV-1, the inhibition against viral adsorption, synthesis and release, and the effects on the positive expression rates of protein p53 and p21. The reasons maybe as followings: The Chinese medicine in Shen Fengjing inhibited the virus releasing too, they might change the conformation of melittin so the antiviral effects were improved. This enhancement effects provided new theoretic basis on pharmacodynamic mechanism for the development and utilization of melittin and Shen Fengjing. |
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