title: Therapeutic effect of Bushen Huoxue recipe on autoimmune premature ovarian failure mice established by immunization with recombinant porcine zona pellucida 4 antigen.
abstract: OBJECTIVE
  To investigate the efficacy and mechanism of Bushen Huoxue Recipe (, BHR) in the treatment of murine 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-β-estradiol (0.13 mg/kg) and normal saline as positive and negative control. Estrous cycles were analyzed through vaginal smears, serum estradiol (E) 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 [(3)H]-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 E 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 E levels and protect ovarian functions from the autoimmune injury in murine POF model.
authors.affiliation: Laboratory for Reproductive Immunology, Hospital and Institute of Obstetrics and Gynecology, Fudan University Shanghai Medical College, Shanghai, China.
authors.forename: Chuan-ling
authors.initials: CL
authors.lastname: Tang
authors.forename: Fang
authors.initials: F
authors.lastname: Li
authors.forename: Lan
authors.initials: L
authors.lastname: Sun
authors.forename: Da-jin
authors.initials: DJ
authors.lastname: Li
mesh_terms: D000818:Animals; D004195:Disease Models, Animal; D004365:Drugs, Chinese Herbal / Q000494:pharmacology / Q000627:therapeutic use*; D004527:Egg Proteins / Q000276:immunology* / Q000302:isolation & purification; D005260:Female; D007114:Immunization; D007121:Immunocompetence / Q000187:drug effects; D008562:Membrane Glycoproteins / Q000276:immunology* / Q000302:isolation & purification; D051379:Mice; D008807:Mice, Inbred BALB C; D010053:Ovary / Q000187:drug effects / Q000276:immunology / Q000473:pathology; D016649:Primary Ovarian Insufficiency / Q000188:drug therapy* / Q000276:immunology* / Q000473:pathology; D011956:Receptors, Cell Surface / Q000276:immunology* / Q000302:isolation & purification; D011994:Recombinant Proteins / Q000276:immunology* / Q000302:isolation & purification; D034421:Sus scrofa; D000072216:Zona Pellucida Glycoproteins
chemical_list: D004365:Drugs, Chinese Herbal; D004527:Egg Proteins; D008562:Membrane Glycoproteins; D011956:Receptors, Cell Surface; D011994:Recombinant Proteins; D000072216:Zona Pellucida Glycoproteins; C527851:bushen huoxue