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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

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Abstract

Objective

To investigate the efficacy and mechanism of Bushen Huoxue Recipe (补肾活血方, 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 [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 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.

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Authors and Affiliations

  1. Laboratory for Reproductive Immunology, Hospital and Institute of Obstetrics and Gynecology, Fudan University Shanghai Medical College, Shanghai, 200011, China

    Chuan-ling Tang  (唐传玲) & Da-jin Li  (李大金)

  2. Jiangsu Kanion Pharmaceutical Co., Ltd., Lianyungang, Jiangsu Province, 222001, China

    Fang Li  (李 芳) & Lan Sun  (孙 兰)

  3. Department of Obstetrics and Gynecology, the Affiliated Hospital, Hainan Medical College, Haikou, 570102, China

    Da-jin Li  (李大金)

Authors
  1. Chuan-ling Tang  (唐传玲)
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  2. Fang Li  (李 芳)
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  3. Lan Sun  (孙 兰)
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  4. Da-jin Li  (李大金)
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Corresponding author

Correspondence to Da-jin Li  (李大金).

Additional information

Supported by National Key Research Program of China (No. 2006CB944007), National and Shanghai Leading Academic Discipline Project (No. 211XK22), Program for Outstanding Medical Academic Leader (to LI Da-jin)

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Tang, Cl., Li, F., Sun, L. et al. 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. Chin. J. Integr. Med. 19, 439–445 (2013). https://doi.org/10.1007/s11655-012-1025-y

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  • DOI: https://doi.org/10.1007/s11655-012-1025-y

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