Abstract: BACKGROUND: Vitrification causes cryoinjury to ovarian tissue. The protective effect and molecular mechanism of Ban’s Culuan Zhuyun Decoction on vitrified ovarian tissue urgently need to be further elucidated.  
OBJECTIVE: To investigate the effect and molecular mechanism of Ban’s Culuan Zhuyun Decoction on vitrified mouse ovarian tissue.  
METHODS: (1) Pre-vitrification experiment: Sixty female Kunming mice, 6-8 weeks old, were randomly divided into control group I, model group I, and low, medium-, and high-dose Ban’s Culuan Zhuyun Decoction groups. After continuous administration for 21 days, ovarian tissues were collected. Ovaries in the control group I were fresh, while those in the other groups were vitrified for 7 days. (2) Post-vitrification experiment: Ovarian tissues were collected from another 60 female Kunming mice, 6-8 weeks old. Ovaries in the control group II were fresh. The remaining ovaries, after vitrification for 7 days, were randomly divided into model group II and low-, medium-, and high-dose Ban’s Culuan Zhuyun Decoction-containing serum groups, and then cultured in vitro at 37 °C for 1 hour in medium containing 15% blank rat serum or different doses of Ban’s Culuan Zhuyun Decoction-containing serum. Ovarian morphology was observed by hematoxylin-eosin staining. Ovarian cell apoptosis was detected by TUNEL assay. The levels of ferrous ion glutathione, malondialdehyde, and glutathione peroxidase 4 were measured by colorimetric methods. The mRNA and protein expression levels of solute carrier family 7 member 11, tumor suppressor protein p53, and glutathione peroxidase 4 were detected by RT-qPCR and western blot, respectively.  
RESULTS AND CONCLUSION: (1) Compared with control groups I and II, model groups I and II showed disorganized and partially absent ovarian granulosa cells, a significant increase in TUNEL-positive cell rate, ferrous ion content, malondialdehyde content, and tumor suppressor protein p53 mRNA and protein expression levels, as well as a significant reduction in glutathione content, glutathione peroxidase 4 content, and solute carrier family 7 member 11 and glutathione peroxidase 4 mRNA and protein expression levels (P < 0.05). (2) Compared with model groups I and II, Ban’s Culuan Zhuyun Decoction and its drug-containing serum reduced the number of atretic follicles in ovarian tissue, significantly decreased the TUNEL-positive cell rate, ferrous ion content, malondialdehyde content, and tumor suppressor protein p53 mRNA and protein expression levels, and significantly increased glutathione content, glutathione peroxidase 4 content, and solute carrier family 7 member 11 and glutathione peroxidase 4 mRNA and protein expression levels (P < 0.05). Therefore, Ban’s Culuan Zhuyun Decoction and its drug-containing serum can improve the morphological structure of ovarian tissue and follicles in vitrified mice, inhibit ovarian cell apoptosis and ferroptosis, thereby alleviating cryoinjury in ovarian tissue. Its mechanism may be related to inhibiting the abnormal activation of the tumor suppressor protein p53/solute carrier family 7 member 11/glutathione peroxidase 4 signaling pathway.  

Key words: Ban’s Culuan Zhuyun Decoction, ovarian tissue vitrification, cryoinjury, ferroptosis, fertility preservation, tumor suppressor protein p53, solute carrier family 7 member 11 (SLC7A11), glutathione peroxidase 4, signaling pathway