Complex Interplay between Autophagy and Oxidative Stress in the Development of Endometriosis

Abstract

Endometriosis (Endo) is a chronic gynecological disease. This paper aimed to evaluate the modulation of autophagy, oxidative stress and apoptosis with Açai Berries in a rat model of endometriosis. Endometriosis was induced with an intraperitoneal injection of minced uterus tissue from a donor rat into a recipient one. The abdominal high-frequency ultrasound (hfUS) analysis was performed at 7 and 14 days from the endometriosis induction to evaluate the growth of the lesion during the experiment. Seven days from the induction, once the lesions were implanted, an Açai Berry was administered daily by gavage for the next seven days. At the end of the experiment, the hfUS analysis showed a reduced lesion diameter in animals given the Açai Berry. A macroscopical and histological analysis confirmed this result. From the molecular point of view, Western blot analyses were conducted to evaluate the autophagy induction. Samples collected from the Endo group showed impaired autophagy, while the Açai Berry administration inhibited PI3K and AKT and ERK1/2 phosphorylation and promoted autophagy by inactivating mTOR. Additionally, Açai Berry administration dephosphorylated ATG1, promoting the activity of the ATG1/ULK1 complex that recruited Ambra1/Beclin1 and Atg9 to promote autophagosome nucleation and LC3II expression. Açai Berry administration also restored mitophagy, which increased Parkin cytosolic expression. The Açai Berry increased the expression of NRF2 in the nucleus and the expression of its downstream antioxidant proteins as NQO-1 and HO-1, thereby restoring the oxidative imbalance. It also restored the impaired apoptotic pathway by reducing BCL-2 and increasing BAX expression. This result was also confirmed by the TUNEL assay. Overall, our results displayed that Açai Berry administration was able to modulate autophagy, oxidative stress and apoptosis during endometriosis.

Keywords: apoptosis; autophagy; endometriosis; mitophagy; oxidative stress.

Figure 1

Analysis of endometriotic-like lesions development: High-frequency ultrasound analysis (hfUS) at 7 days from the endometriosis induction: Endo (A), Endo + Açai Berry (B), lesion diameter (C) and lesion number (D). hfUS analysis at 14 days from the endometriosis induction: Endo (E), Endo + Açai Berry (F), lesion diameter (G) and lesion number (H). A p-value of less than 0.05 was considered significant. *** p < 0.001 vs. Endo.

Figure 1

Analysis of endometriotic-like lesions development: High-frequency ultrasound analysis (hfUS) at 7 days from the endometriosis induction: Endo (A), Endo + Açai Berry (B), lesion diameter (C) and lesion number (D). hfUS analysis at 14 days from the endometriosis induction: Endo (E), Endo + Açai Berry (F), lesion diameter (G) and lesion number (H). A p-value of less than 0.05 was considered significant. *** p < 0.001 vs. Endo.

Figure 2

Analysis of Açai Berry administration on macroscopical and histological analysis: Macroscopic analysis: Endo (A), Endo + Açai Berry (B), lesion area (C) and lesion volume (D). Histological analysis: Endo (E), Endo + Açai Berry (F) and histopathologic score (G). A p-value of less than 0.05 was considered significant. ** p < 0.01 vs. Endo, *** p < 0.001 vs. Endo.

Figure 2

Analysis of Açai Berry administration on macroscopical and histological analysis: Macroscopic analysis: Endo (A), Endo + Açai Berry (B), lesion area (C) and lesion volume (D). Histological analysis: Endo (E), Endo + Açai Berry (F) and histopathologic score (G). A p-value of less than 0.05 was considered significant. ** p < 0.01 vs. Endo, *** p < 0.001 vs. Endo.

Figure 3

Analysis of Açai Berry administration on PI3K/AKT/ERK1/2 pathways. Western blot analysis of PI3K (A), pAKT (B), p-ERK1/2 (C) and p-mTOR (D) expression. A p-value of less than 0.05 was considered significant. ** p < 0.01 vs. Endo, *** p < 0.001 vs. Endo.

Figure 4

Analysis of Açai Berry administration on the autophagosome formation. Western blot analysis of pATG1 (A), AMBRA1 (B), BECLIN (C), ATG9 (D) and LC3II (E) expression. A p-value of less than 0.05 was considered significant. *** p < 0.001 vs. Endo.

Figure 5

Analysis of Açai Berry administration on the Pink1 and Parkin expression. Western blot analysis of PINK1 (A) and PARKIN (B) mitochondrial expression and PARKIN (C) cytoplasmic expression. A p-value of less than 0.05 was considered significant. ** p < 0.01 vs. Endo, *** p < 0.001 vs. Endo.

Figure 6

Analysis of Açai Berry administration on prooxidative alterations. Western blot analysis of NRF2 (A) nuclear expression, HO-1 (B) and NQO-1 (C) cytosolic expression, GSH levels (D), SOD activity (E) and MDA (F) levels. A p-value of less than 0.05 was considered significant. ** p < 0.01 vs. Endo, *** p < 0.001 vs. Endo.

Figure 7

Analysis of Açai Berry administration on apoptosis impairment: Western blot analysis of BCL-2 (A) and BAX (B) expression. TUNEL assay: Endo (C), Endo + Açai Berry (D) and TUNEL-positive cells (E). A p-value of less than 0.05 was considered significant. ** p < 0.01 vs. Endo, *** p < 0.001 vs. Endo.