| Recently, sexual transmission has become the main way of global AIDS virus (HIV) transmission, searching for safe and effective female-controlled vaginal and rectal microbicides is an urgent need for China. Deoxycholytyosine(DCT) is a derivative of Cholic acid(CA) and retains the possible pharmacophore of CA and modify the structure by using amino acid in the side chain part.In the present study, we examined the efficacy and safety of DCT by the contraceptive efficacy study, targets of the contraception study and the vaginal mucous membrane irritation studies. We also discussed the mechanism or the targets of spermicidal activities.Contraceptive efficacy study include in vitro spermicidal test, in vivo spermicidal test and rabbit contraceptive test to evaluate the contraceptive efficacy of DCT. In vitro spermicidal tests, we mixed sperm and DCT together to test the viability change with the increase of time and DCT concentration which was called the dose-time response relationship test. Sperm movement parameters were tested in the meantime by CASA.Then DCT was washed out and tested whether sperm viability would recover. The results showed that there was dose-time response relationship between DCT concentration, response time and sperm viability. DCT had significant inhibitive effect on sperm movement parameters and motility parameters. Meanwhile, sperm viability did not change too much when removing DCT.For in vivo spermicidal test and rabbit contraceptive test, sixty female estrus rabbits were divided into 6 groups randomly. The rabbits were administered with test substances through vagina, and then mated with male rabbits(1:1)after 10 minutes.At 5,10,15 and 20 minutes after mating, semen samples were obtained from vagina to observe sperm motility using phase contrast microscope. Results showed that no mobile sperms were found in rabbits’vagina of the 12.50 mg DCT gel group and the 25.00 mg DCT gel group. About 60 min after in vivo spermicidal test, the above rabbits were administrated with the same test substances and then mated with male rabbits (1:1) after 10 minutes. The number of pups was recorded and the infertile rate was calculated.Results showed that the infertile rates of middle-dose and high-dose DCT gel group were 100%, which were similar to that of N-9 jelly group.Targets of the contraception studies included membrane integrity test, apotosis test, acrosome test, mitochondrial test as well as observing the ultrastructure spermatozoon with EM. Quantitative and qualitative assessment of semen stained with SYBR-14/PI was determined using FCM and fluorescence microscope. Results showed that the ratio of alive sperm in DCT 0.25,0.5, and 1.0 g·L-1 were (31.26±8.35)%, (13.26±5.21)% and (0.74±0.93)% and had obvious dose-effect relationships(P<0.05). The acrosomal status was tested by fluorescence microscope using FITC-PSA/PI. Results showed that sperms in DCT group the fluorescence intensity in the acrosome zone decreased and even disappeared which indicated that DCT had obvious damage on sperm acrosome. Quantitative and qualitative assessment of semen stained with JC-1 was determined using FCM and fluorescence microscope. Results showed in DCT group, the JC-1 reagent dispersed through the entire cells and fluoresce green and the red fluorescence intensity decreased as the increase of DCT concentration.Sperm mitochondrial membrane potential(MMP) were significant different among groups(P< 0.05). Apoptosis test results showed that the ratio of late apoptotic sperm in DCT 0.25,0.5, and 1.0 g·L-1 were (11.92±1.19)%、 (30.09±6.29)% and(75.55±6.30)%, which also had obvious dose-effect relationship and had significant difference compared with the control group(P<0.05). Proportion of early-stage apoptosis sperm increased with the increase of DCT at low concentration(<0.5 mg/ml), but decreased at high DCT concentration.Electron microscopy (SEM) results showed that sperms cultured in DCT solution were seriously damaged, the plasma and acrosome membrane were incomplete or be dissolved, the tail had many perforation and even rupture.For vaginal mucosa irritation study, nine pigs were divided into three groups randomly, which were gel base group, N-9 jelly group and DCT jel group. Intravaginal gel application and CVL fluid collection was performed during the non-estrus stage of the 21-day porcine reproductive cycle. Nine pigs in subgroups of 3 were administered 5 ml of a gel formulation per day for 7 days, CVL fluid was collected similarly before and after gel application using 80 ml of sterile phosphate buffered saline (PBS). To examine the lymphocytic cellular profiles and cytokine levels following intravaginal exposure to base control gel, DCT gel and N-9 jelly. CVL fluid was collected at 24 and 72 hours postapplication and tested by a FACScan flow cytometer. Following euthanization, the genital area was examined macroscopically and the genital tract was retrieved and the vagina was excised and fixed in 10% neutral-buffered formalin for microscopic evaluation. CD45 immunostaining was used to evaluate the vaginal tissues for the presence of inflammatory cells. When compared with N-9 jelly, the proportion of neutrophile granulocyte and CD45+cells was much lower and had no significant difference with the control group.Endpoint histology of vaginal mucosa HE staining showed it had no obvious damage (5.5±2.7) on mucosal epithelium, lamina propria, blood vessels and surrounding tissue in DCT gel group. It had no significant difference (3.3±1.6) compared to the control group, but it has significant difference (20.5±3.5, P<0.05) compared to N-9 jelly group. Compared to the control group, DCT gel group vaginal mucosa CD45 expression of vaginal mucosa in DCT gel group was not significantly different (P> 0.05), and significantly increased in N-9 jelly group (P<0.05)... |
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