An Experimental Study Of Resveratrol Anti-Friend Murine Acquired Immunodefiency Syndrome (MAIDS)

ObjectivesThe objective of this research topic is to investigate the effective compound of Giant knotweed Rhizome against the anti-Friend Murine Acquired Immunodefiency Syndrome (Friend-MAIDS) of reservatrol (RES) in vivo and discuss its anti-viral mechanism. We aim to develop an effective traditional Chinese herbal anti-viral monome and provide reliable experimental data for clinical application.Experiment Summary1. Transfer Friend murine leukemia virus (FLV) culture and measure its viral infection dose (ID₅₀).2. RES inhibitory effect of splenomegalia caused by the FLV.3. Preliminary investigation of the influence of RES on the cellular immunological function of FLV infected mice.4. The effect of RES on the hematology index of FLV infected mice.5. Using RT-PCR to evaluate the effect of the FLV-gag gene mRNA expression of FLV infected mice treated with RES.Methods 1. Female BALB/c mice were divided into 5 groups randomly with 4 mice in each group. Tap water thawed the FLV. Mice were injected with 0. 5ml into the peritoneal cavity after disinfected with alcohol and iodine tincture. 14 days after the FLV injection, the mice were weighed and sacrificed. The spleens weighing >1g were obtained and grinded. Then they were mixed with sterile saline to make a spleen homogenate. Simultaneously, a double antibiotics (10,000U/mL) was created by adding benzylpenicillin and estreptomicina. The final concentration of the double antibiotics was 500 U/ml. This suspension was centrifuged frozen and the supernatants were extracted creating a 10% splenic suspension. The spleen suspension was applied to challenge mice. This procedure was repeated twice. After transferring the culture twice, toxicity was enhanced and the the mice adaptation cell strain had been successfully prepared. This solution was diluted 10-fold in a medium at five concentrations of 10^-1, 10^-2, 10^-3, 10^-4, 10^-5.This was used to estimate the virus titer. 20 Female BALB/c mice were divided into 5 groups, with 4 mice in each group. The mice were challenged with 0.5ml injections into the peritonealcavity with the 5 concentrations of spleen suspension as described previously. 14 days after the FLV injection, the mice were weighed and killed. The spleens were weighed and the splenic index was estimated. A healthy control group was also established. A splenic index in which the mean of normal group exceeded the standard deviation by 3 fold was positive. The dilution of the FLV was measured using the REED-MUENCH method.2. The FLV was thawed using tap water and diluted to 100 ID50 by DMEM and preserved in ice water, female BALB/c mice were divided randomly into 5 groups. These groups consisted of the positive control group (AZT control group), normal control group, virus control group, RES large dosage group and RES small dosage group. Except for the normal control group, the other mice were disinfected and challenged with 0. 5ml FLV injection into the peritoneal cavity. Drugs were administered beginning on the next day. After 21 days of consecutive drug administration, the mice were weighed and killed. Mice were not fed for at least 4 hours before killing. The spleens were removed and washed twice with 0. 9% saline. After the water was absorbed with bibulous paper the spleens were weighed. Then the splenic index was calculated and the splenic index inhibition rate was estimated according to the standard formula. Spleen tissue from identical locations were selected, fixated with 10% neutral formalin, imbedded with paraffin, sliced at 5u m and dyed with H. E.3. FLV was thawed with tap water, diluted to 100 ID50 by DMEM, and conserved in ice water. Female BALB/c mice were divided randomly into 5 groups. The five groups included a positive control group, a normal healthy control group, a virus control group, a RES large dosage group and a RES small dosage group. Except for the normal control group, the other mice were disinfected and challenged with 0.5ml FLV injections into the peritoneal cavity. Drugs were administered from the next day for 21 consecutive days. The challenged mice were observed. 21 days after infection, 50u L of blood from the eyeballs was sampled whereupon the mice were killed. CD3/CD4/CD8 monoclonal antibodies were respectively diluted to the concentration of 0.125 ng/10ul by ditilled water according to the product instruction; 50 U L anticoagulated blood was added in-to testtubes, CD3/CD4/CD8 monoclonal antibody into three tubes one by one, and mixed completely. Put it at the dark place to incubate 20-30 minutes at room temperature. Added 250 u 1 hemolysin (IX) into every tube, mixed completely, placed at the dark place 10 minutes accurately. Centrifugated it 5 minutes at the rate of 1200r/min, then pilled out the supernatant, lml PBS washed and mixed completely, added 1% paraformaldehyde into every tube to estimate and analysis every time take 10000 cells, and statisticed the result.4. FLV was thawed using tap water and diluted by DMEM to a concentration of 0.5mL/drop containing 100 ID50FLV and conserved in ice water. Female BALB/c mice were divided randomly into 5 groups which consisted of a positive control group, a normal control group, a virus control group, a RES large dosage group and a small dosage group. Each group had 6-8 mice. Except for the healthy control group, the other mice were disinfected and challenged with 0. 5ml injection of FLV into the peritoneal cavity. Administration of drugs began from the next day for 21 consecutive days. After 21 days, 500 p L blood was sampled from the eyeballs. Bloodsamples was sent to the clinical laboratory of the first affiliated hospital of Guangzhou TCM University to measure the hematology index and observe the effect of RES on the hemograms of the FLV infected mice.5. 50mg of spleen tissue from the splenic index experiment was selected and the total RNA was extracted by Trizol. The RNA was reverse transcripted and a specific gag gene primer was used to amplify the RNA by PCR. The PCR products were stained by EB. They then underwent electrophoresis using 2% gelose gel in the TBE electrophoresis buffer. The electrophoregram analysed the demi-ratio to evaluate the influence of RES on the FLV-gag gene mRNA expression of the FLV infected mice.Results1. The virus titer test ensured that FLV ID50 was 4.0 and its dilution of the FLV was 100 fold.2. After 21 days post FLV peritoneal infection, the weight of the mice rarely changed. The splenic indices of the AZT group and RES large dosage group were 96. 82% and 30.35% respectively, compared with the virus control group, P<0. 01, P<0. 05 showing significant statistical difference. This indicated that both the lOOmg/kg dose of AZT and 20mg/kg dose of RES could inhibit splenomegalia caused by FLV. The effect of AZT was notable, while the small dosage of RES had little effect. Histopathological results showed that the AZT control group and the RES large dosage group could improve splenic pathological change to some degree, while the RES small dosage group was ineffective.3. 4 days after infection with FLV, the mice showed raised fur, curled or huddled body posturing, lackluster fur, activity and appetite reduction, while the mice of the AZT and RES large dosage groups had marked activity, were well-developed and good physical ability compared to the virus control group. The thymus index of the RES group was 0. 24 compared with 0. 12 of the virus control group (P<0. 05) showing significant difference. Regarding the lowering of the thymus index caused by FLV infection, there was noted protection against the damage to the immunological function caused by FLV. CTL analysis showed that the percentage of CD3+ cells, CD4+cells and CD8+ cells of the RES large dosage group was 39.41%, 30. 96% and 11. 76% respectively. This was compared to the percentage of CD3+ cells, CD4+ cells and CD8+ cells of the virus control group (22.05%, 15.53% and 7.31%), p<0.05. This result indicated that large dosage RES can enhance the percentage of CD4+ T lymphocytes and the percentage of CD8+ T lymphocytes in peripheral blood. It also has a marked effect in adjusting the CD4:CD8 ratio to a normal range.4. Hematology index results: compared with the virus infected control group, WBC of large dosage RES group mice was reduced by 23. 22%, RBC by 10. 62%, Hb by 13.81% and PLT by 32. 90%. The WBC of the small dosage RES group mice was enhanced by 38. 12%, RBC reduced by 3. 69%, Hb reduced by 7. 02 % and PLT reduced by 18.92%.The WBC of the AZT group mice was reduced by 82.67%, RBC by 21.60%, Hb by 5.20 % and PLT by 35.24%. There was statistical significance regarding the difference in WBC and ALT between the AZT and other groups. This indicates that AZT could...