Can vildagliptin protect against radiation-induced premature ovarian failure? Insights into the AMPK and AKT signaling pathways

Abstract

Background: Among the detrimental side effects caused by radiotherapy in young females is the ovarian damage, eventually causing premature ovarian failure (POF). While many signaling pathways contribute to the pathogenesis of POF, to date no sufficient data exist on the AMPK and AKT signaling pathways in irradiated ovaries. Both AMPK and AKT play crucial roles in the process of folliculogenesis. Vildagliptin (vilda) is a dipeptidyl peptidase-4 inhibitor with modulatory effect on both AMPK and AKT. Therefore, our study aimed to investigate the biochemical changes that occur in the AMPK/AKT signaling pathway, and the effect of co-administration of vildagliptin in radiation-induced POF.

Methods: Female Sprague-dawley rats were randomly divided into four groups: control, radiation, radiation + vilda, or vilda alone groups. Vilda was administered orally once/day, and on the 10th day of the experiment, radiation and radiation + vilda group rats were subjected to 3.2 Gy of whole-body gamma irradiation. Behavioral activity was assessed on the 13th day of the experiment. On day 14 of the experiment, all rats were euthanized. Serum samples were collected, and ovaries were dissected for histological and biochemical analyses.

Results: Irradiation of female rats resulted in increased locomotor hyperactivity, impaired memory, and ovarian damage as evidenced by the marked histopathological deterioration. Additionally, irradiation led to a significant decrease in body weight gain, gonadosomatic index, and serum estradiol level. Further, it caused a significant increase in serum AMH, phosphorylated AMPK, phosphorylated AKT, cytoplasmic Nrf2 expression and phosphorylated CREB levels. Co-administration of vilda exhibited neuroprotective effects, preserved the ovarian histological architecture but failed to preserve the primordial follicle pool in irradiated rats.

Conclusion: In conclusion, AMPK/AKT signaling pathway is upregulated in radiation-induced POF. It possibly contributes to POF pathogenesis by accelerating the activation of primordial follicles, hence leading to their premature depletion. Coadministration of vilda can protect the ovaries and temporarily preserve its endocrine function; however, it does not sustain the ovarian reproductive capacity due to the early depletion of the pool of primordial follicles. Women undergoing radiotherapy should be cautious with the use of AKT-activating drugs.

Keywords: AKT; AMPK; CREB; Ionizing radiation; Ovaries; Vildagliptin.

Fig. 1

Experimental design and timeline

Fig. 2

Effect of vildagliptin co-administration on radiation-induced behavioral changes. (A) Locomotor activity. (B) SAP% in Y-maze test. Data are presented as mean ± S.D (n = 8), a; significantly different from control group at p < 0.05, b; significantly different from radiation group at p < 0.05 using one-way ANOVA followed by Tukey-Kramer as a post-hoc test. (C) Passive avoidance test; Step-through latency time in training session. (D) Passive avoidance test; Step-through latency time in test session. Passive avoidance test data are presented as mean ± S.D (n = 8), a; significantly different from control group at p < 0.05, b; significantly different from radiation group at p < 0.05 using Kruskal-Wallis test followed by Dunn’s post-hoc test

Fig. 3

Effect of vildagliptin co-administration on radiation-induced histological alterations of the ovarian tissue using H & E staining. (A) Control ovaries showing normal histological architecture, ovarian follicles in various stages of development (black arrows) (x 200). (B) Ovaries from vilda-alone treated rats showing normal histological architecture, ovarian follicles in various stages of development (black arrows) (x 200). (C) Irradiated ovaries showing hyperplasia of ovarian connective tissue, marked dilatation of ovarian blood vessels, and leukocytic infiltration (blue arrows) (x 200). (D) Irradiated ovaries showing pyknosis and degradation of granulosa cell layers of graafian follicles and disorganization of internal thecal layer (red arrows) (x 400). (E) Ovaries from IR + vilda rats where mild oedema of interstitial tissue and mild congestion of ovarian blood vessels were observed (black asterisk) (x 200). (F) Ovaries from IR + vilda showing a mature graafian follicle with a centrally-located oocyte and normal organization of granulosa cells (red asterisk) (x 400)

Fig. 4

Morphometric analysis of ovarian follicles population. (A) Primordial follicles percentage. (B) Preantral and antral follicles count. (C) Healthy follicles percentage. (D) Atretic follicles percentage. Data are presented as mean ± S.D (n = 5), a; significantly different from control group at p < 0.05, b; significantly different from radiation group at p < 0.05 using one-way ANOVA followed by Tukey-Kramer as a post-hoc test

Fig. 5

Effect of vildagliptin co-administration on radiation-induced hormonal changes. (A) Serum estradiol level. (B) Serum AMH level. Data are presented as mean ± S.D (n = 5), a; significantly different from control group at p < 0.05, b; significantly different from radiation group at p < 0.05 using one-way ANOVA followed by Tukey-Kramer as a post-hoc test

Fig. 6

Immunohistochemical detection of Nrf2 expression (x 400). (A, B) Control and vilda alone groups showed minimal immunostaining, respectively. (C) Ovaries from irradiated rats showed extensive brown immunostaining (increased cytoplasmic Nrf2). (D) Vilda co-administration resulted in a marked reduction in cytoplasmic Nrf2. (E) Quantitative image analysis for Nrf2 immunostaining was expressed as optical density (O.D.). (F) Scoring of the immunostaining considering both the staining intensity and the percentage of positive cells

Fig. 7

Effect of vilda co-administration on radiation-induced changes in AMPK/AKT/CREB signaling pathway. (A) Ovarian tissue level of phosphorylated AMPK. (B) Ovarian tissue level of phosphorylated AKT. (C) Ovarian tissue level of phosphorylated CREB. Data are presented as mean ± S.D., a; significantly different from control group at p < 0.05 using one-way ANOVA, b; significantly different from radiation group at p < 0.05 using one-way ANOVA followed by Tukey-Kramer as a post-hoc test