Therapeutic Effect of Bushen Huoxue Recipe(补肾活血方) on Autoimmune Premature Ovarian Failure Mice Established by Immunization with Recombinant Porcine Zona Pellucida 4 Antigen

Objective： To investigate the efficacy and mechanism of Bushen Huoxue Recipe （补肾活血方, BHR） in the treatment of mudne autoimmune premature ovarian failure （POF）. Methods： The recombinant porcine zona pellucida 4 （pZP4） was expressed in E. coli BL21 （DE3） strain within prokaryotic plasmid pET28a （＋）, purified by Ni-affinity chromatography and verified by Western blot. Murine autoimmune POF model was established by immunization with pZP4 of female BALB/c mice. Fifty POF mice were randomly divided into 5 groups, which were respectively given low （3.75 mg/kg）, moderate （7.5 mg/kg）, and high dose （15.0 mg/kg） of BHR by gastrogavage once daily for 20 days, with 17-13-estradiol （0.13 mg/kg） and normal saline as positive and negative control. Estrous cycles were analyzed through vaginal smears, serum estradiol （E2） levels, and anti-pZP4 antibody titers were detected by ELISA. The proliferative responses in vitro of spleen lymphocytes to pZP4 antigen restimulation were measured by [3H]-thymidine incorporation, and the histomorphology changes of ovary were evaluated by optical microscope. ]Results： The purified pZP4 was visible as a single lane with 14.4 kD in SDS-PAGE and Western blot. The murine POF model with lengthening estrous cycles, decreased levels of serum E2, high titers of serum anti-pZP4 antibody, and reduced ovarian follicles and corpus lutea were established by immunization with recombinant pZP4. Treatment with moderate and high dosage BHR significantly increased ovarian follicles and reduced the proliferation of spleen lymphocytes to the pZP4 antigen of POF mice （P〈0.05）. However, only the high dosage BHR administration significantly improved the estrous cycles, elevated the serum E2 levels （P〈0.01）, and decreased the serum anti-pZP4 antibody titers of model mice （P〈0.05）. Conclusions： The recombinant pZP4 could evoke the antigen-specific immune response in mice and induce the autoimmune ovarian injury. It has been demonstrated that BHR was able to increase the serum E2 levels and protect ovarian functions from the autoimmune injury in murine POF model.

Effect of Bushen Yiqi Huoxue Recipe on Placental Vasculature in Pregnant Rats with Fetal Growth Restriction Induced by Passive Smoking

Interactions of vascular endothelial growth factor （VEGF） with receptors VEGFR1/Fltl and VEGFR2/Flk1, and those of angiopoietins （Ang-1, Ang-2） with receptor Tie2 play important roles in placental angiogenesis. This study investigated vascular morphology and expression of these angiogenic factors in rat placenta on the day 15, 18, 21 of gestation （D 15, D 18 and D21）. The rats were randomly assigned into 3 groups： normal group, model group [fetal growth restriction （FGR） model], and Bushen Tqi Huoxue （BYHR） recipe treatment group （BYHR group, the pregnant rats with FGR were treated with BYHR recipe）. Morphological analysis indicated that during initial villous formation, fetal nucle- ated erythrocytes （FNEs） appeared in maternal blood sinus （MBS）. Subsequently, FNEs were sur- rounded by endothelial cells to form fetal capillary （FC） and then by trophoblast cells to form villi. As pregnancy proceeded, FC density increased progressively with increasing endothelial identification staining （EIS） in normal and BYHR groups. Whereas, villous formation was suppressed, normal in- crease in FC density was impaired and EIS was weakened in model group. Quantitative PCR analysis showed that VEGF and Flkl mRNA increased over gestation in all groups, indicating that VEGF might play a pivotal role in FC growth during late gestation. VEGF mRNA was increased on D15, while de- creased on D21 in model group as compared with normal group and BYHR group. Immunohistochemi- cally, Ang-2 protein was highly expressed in FNEs, gradually disappeared as villi matured, and decreased over gestation in all groups, indicating that Ang-2 might play a pivotal role in villous formation, which was further supported by decreased Ang-2 mRNA and protein expression in model group on D 15. Ang-1 mRNA, Tie2 mRNA and Ang-1/Ang-2 ratio increased from D15 to D18 in all groups as placenta matured. Ang-1 mRNA, Tie2 mRNA and Ang-1/Ang-2 ratio were decreased on D18 in model group as compared with normal and BYHR groups, indicating delayed maturity of FGR placenta. Alterations in angiogenic factors may result in altered placental vasculature and cause placental insufficiency. BYHR recipe could balance the angiogenic factors to promote the formation and maturation of FGR placental vasculature.