| Objective: To explore the effect and mechanism of Jixiang Ankun Pill on the repair of ovarian function in mice with ovarian premature failure(POF)induced by chemotherapy drug cyclophosphamide(CTX),and provide a theoretical basis for the clinical use of Jixiang Ankun Pill in the treatment of POF.Methods: Fifty healthy female mice with normal estrous cycle were selected,and 10 mice were randomly selected as a blank control group.They were injected intraperitoneally with 8㎎ / ㎏ distilled water for 30 days per day,and the remaining 40 mice were injected intraperitoneally with CTX at a dose of 8 ㎎ / ㎏ for 30 days continuously according to literature reports and preliminary experimental results to construct a POF model.During this period,vaginal exfoliated cells were smeared to check the estrous cycle,observe the general condition of the mice,and record the weight changes.Forty CTX model mice were randomly divided into four groups: model group(10 mice),low dose drug treatment group(10 mice),medium dose drug treatment group(10 mice),and high dose drug treatment group(10 mice).Administration was conducted 24 hours after the completion of modeling: the blank control group and model group were given physiological saline for 14 consecutive days,the low dose treatment group was given low concentration Jixiang Ankun pill solution(0.026 g/ml)for 14 consecutive days,the medium dose treatment group was given medium concentration Jixiang Ankun pill solution for 14 consecutive days(0.052 g/ml),and the high dose treatment group was given high concentration Jixiang Ankun pill solution(0.078 g/ml)for 14 consecutive days.Blood samples were taken the next day after administration,and serum estradiol(E2),follicle stimulating hormone(FSH),and anti Mullerian hormone(AMH)levels were measured by ELISA;After blood collection,the mice were killed,and bilateral ovaries were taken.One ovarian tissue was stained with HE.The morphological changes of the ovarian tissue were observed under the light microscope,and the number of primitive follicles,primary follicles,mature follicles,and atresia follicles were counted;Western blot method was used to detect the changes in the levels of phosphatidylinositol 3 kinase(PI3K),threonine kinase(AKT),and rapamycin target protein(m TOR)in the ovarian tissue of the other side.The relevant data were statistically analyzed.Results:1.The estrous cycle in the blank control group was normal,while the estrous cycle in the modeling group was disordered 30 days after intraperitoneal injection of cyclophosphamide.A POF mouse model was successfully constructed.2.Serum sex hormone levels: Compared with control group mice,the serum E2 and AMH levels in model group mice decreased(3.11 ± 0.35 vs 5.60 ± 0.90,P<0.05;172.98 ± 27.72 vs 263.56 ± 19.54,P<0.05),while FSH levels increased(36.78 ± 2.99 vs 25.22 ± 4.71,P<0.05),with statistically significant differences;In the low dose treatment group,the E2 value was 160.39 ± 12.44,FSH value was 32.62 ± 2.61,and AMH value was 3.46 ± 0.38,respectively.Compared with the model group,the E2 and FSH levels decreased,while AMH levels increased,but there was no statistical significance;The E2 values in the medium dose treatment group and the high dose treatment group were 191.89 ± 19.42 and 200.85 ± 28.01,respectively,which were significantly higher than those in the model group(P<0.05);The FSH values were 29.99 ±4.95 and 26.53 ± 2.37,respectively,which were significantly lower than those in the model group(P<0.05);The AMH values were 4.34 ± 0.38 and 4.22 ± 0.36,respectively,which were significantly higher than those of the model group(P<0.05).3.Follicle count: Compared with the control group,the number of primitive follicles in the model group decreased(76.20 ±7.40 vs 103.40 ± 7.44),the number of primary follicles decreased(57.20 ± 6.02 vs 75.40 ±4.93),the number of mature follicles decreased(56.40 ± 4.16 vs 70.40 ± 5.68),and the number of atresia follicles increased(43.80 ± 3.27 vs 31.00 ± 3.39),with significant differences(P<0.05).In the low dose treatment group,the number of primary follicles and primary follicles were 75.40 ± 5.86 and 61.00 ± 9.57,respectively;The number of mature follicles(59.20 ± 2.86)increased compared to the model group,but the difference was not statistically significant.The number of atresia follicles(40.40 ± 3.51)decreased compared to the model group,and the difference was not statistically significant;The number of primary follicles(83.00 ± 4.18,90.60 ± 4.04),primary follicles(64.00 ± 3.54,69.20 ± 6.94),and mature follicles(62.60 ± 3.21,65.80 ± 3.70)in the medium and high dose treatment groups increased compared to the model group,while the number of atresia follicles(37.60 ± 3.85,34.20 ± 5.17)decreased compared to the model group,with statistically significant differences(P<0.05).4.Western blot protein hybridization results: Compared with the control group,the protein expression levels of PI3K(0.63 ± 0.02 vs 0.96 ± 0.04),Akt(0.63 ± 0.03 vs0.91 ± 0.02),and m TOR(0.61 ± 0.02 vs 0.92 ± 0.02)in the model group decreased significantly(P<0.05);The protein expression levels of PI3K(0.72 ± 0.02,0.79 ± 0.01,0.88 ±0.02),Akt(0.73 ± 0.02,0.79 ± 0.02,0.84 ± 0.02),m TOR(0.68 ± 0.02,0.74 ± 0.02,0.79 ±0.02)in the low,medium,and high dose Jixiang Ankun Pills treatment groups were higher than those in the model group,with statistical significance(P<0.05).Conclusion:1.Successfully constructed a mouse model of chemotherapy induced premature ovarian failure.2.Jixiang Ankun Pill can effectively repair the ovarian function of POF mice.3.Jixiang Ankun Pill may repair mouse ovarian function by upregulating the expression of key proteins PI3 K,Akt,and m TOR in the PI3K/Akt/m TOR signaling pathway. |
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