| Objective The aim of this study was to elucidate the mechanism of action of the liver-sparing and kidney-tonifying method in the treatment of premature ovarian failure (POF) rats by transcriptomics.
METHODS Thirty SD rats were assigned to the blank group (n=10) and modelling group (n=20) by random number table method. No intervention was applied to the blank group, and the POF rat model was established and evaluated in the modelling group by using the method of chronic unpredictable mild stimulation in combination with cyclophosphamide chemical induction. After the model was established, the rats in the modelling group were divided into the model group and the treatment group according to the random number table method, with 10 animals in each group. The liver-sparing and kidney-tonifying group was given 13.55 g/(kg-d) by gavage with the Liver-Sparing and Kidney-Tonifying Formula, while the blank group and the modelling group were given saline gavage with a dosage of 10 ml/(kg-d) for a 4-week period of intervention. The weight fluctuation of animals in each group was monitored during the experiment, and the morphological changes of ovarian tissue were observed by hematoxylin-eosin (HE) staining, and serum follicle stimulating hormone (FSH) and estradiol (E2) levels were detected by enzyme-linked immunosorbent assay (ELISA), and differentially expressed genes (DEGs) were screened by RNA-Seq.
Results At the end of modelling, compared with the blank group, the body weight of rats in the modelling group was significantly lower, the serum FSH level was significantly higher, and the serum E2 level was significantly lower (P＜0.01) suggesting that modelling was successful. After drug intervention, compared with the blank group, the ovarian tissue of the model group was reduced in size, the internal structure was damaged, the number of immature atretic follicles increased, and the number of mature follicles was less. Compared with the model group, the ovarian tissue in the treatment group was larger in size, the number of atretic follicles was reduced, and more follicles of all levels were visible; serum FSH was reduced (p<0.01) and serum E2 content was increased (p<0.05). The transcriptome GO functional enrichment analysis of the blank group vs. the model group showed that DEGs were mainly enriched in pathways related to chronic stress and oxidative stress regulation. A total of 81 DEGs were identified in the transcriptome analysis of the model group vs. the treatment group (screening conditions: FDR< 0.005 and Fold Change> 1.5), and the GO functional enrichment analysis showed that DEGs were mainly enriched in redox pathways. Among them, significantly up-regulated oncogenes (Tk1, Kif20b, Micall2, ENPP3, MAFK) were present in the model group.
Conclusion Liver-sparing and kidney-tonic method may improve the environment of follicular development by suppressing oncogenes through redox function. This study revealed the molecular network of liver-sparing and kidney-tonic method in regulating ovarian function at the transcriptome level, which provides a scientific basis for the treatment of POF by traditional Chinese medicine. |