Abstract: BACKGROUND: Ovarian tissue vitrification cryopreservation is one of the important methods for preserving fertility. Tanshinone IIA has various pharmacological activities, including anti-oxidation, inhibition of inflammatory response, and reduction of apoptosis, but its role as an additive for vitrification cryoprotection of ovarian tissue is still unclear.
OBJECTIVE: To explore the protective effect of tanshinone IIA on vitrification cryopreservation of mouse ovarian tissue. 
METHODS: Twenty-five 6-week-old female KM mice were randomly selected and their ovarian tissues were randomly divided into five groups, with 10 ovaries per group. The fresh group was not cryopreserved. The frozen control group used vitrification cryoprotectant. The 0.5, 2.5, and 5 µmol/L tanshinone IIA groups used vitrification cryoprotectant containing 0.5, 2.5, and 5 µmol/L tanshinone IIA, respectively, and were cryopreserved in liquid nitrogen. After 3 days of storage, the cryopreserved tubes were taken out and thawed. The ovarian tissue and follicle morphology of each group were observed by hematoxylin-eosin staining, and the normal follicle morphology and survival rate were analyzed. The levels of superoxide dismutase, catalase, malondialdehyde, tumor necrosis factor-α, interleukin-1β, and interleukin-17 in the ovary were detected by enzyme-linked immunosorbent assay. RT-qPCR and western blot assay were used to detect the mRNA and protein expressions of nuclear factor E2-related factor 2 (Nrf2) and heme oxygenase 1 (HO-1) in the mouse ovary. 
RESULTS AND CONCLUSION: (1) Compared with the fresh group, the frozen control group had abnormal morphology of follicles at all levels in the ovary, decreased follicle survival rate (P < 0.05), decreased superoxide dismutase and catalase activities (P < 0.05); the levels of malondialdehyde, and tumor necrosis factor α, interleukin 1β, and interleukin 17 were all increased (P < 0.05), and the mRNA and protein expressions of Nrf2 and HO-1 were decreased (P < 0.05). (2) Compared with the frozen control group, different concentrations of tanshinone IIA could improve the morphology of follicles at all levels in the ovary, increase the survival rate of follicles, enhance the activities of superoxide dismutase and catalase, and reduce the levels of malondialdehyde, tumor necrosis factor α, interleukin 1β, and interleukin 17, increased the mRNA and protein expression of Nrf2 and HO-1 in a concentration-dependent manner, with 5 µmol/L tanshinone IIA having the most significant effect. (3) The results show that tanshinone IIA may reduce the oxidative stress level and inflammatory response of mouse ovarian tissue by mediating the Nrf2/HO-1 signaling pathway, thereby alleviating the reproductive damage caused by vitrification cryopreservation of mouse ovaries.

Key words: tanshinone IIA, mouse ovaries, cryopreservation, oxidative stress, nuclear factor E2-related factor 2 (Nrf2), heme oxygenase 1 (HO-1), engineered tissue construction