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

Female Sprague–Dawley rats were employed in this study. The University of Messina Review Board for animal care (OPBA) approved this study. All animal experiments complied with the new Italian and EU regulations (D.Lgs 2014/26, EU Directive 2010/63). Rats were randomly distributed into two groups, donor or recipient, and endometriosis was induced as already described [ 43 ]. To establish similar estrogen levels among the rats, donor animals were administered 10 IU pregnant mare serum gonadotropin. After 41 h, the rats were euthanized and the uterus was removed. Tissue was minced with scissors in a 1.5 mL centrifuge tube containing PBS. Tissue from all the donors was pooled, and the equivalent of one uterus/500 uL of PBS was intraperitoneally injected along the midventral line of the recipient animals. Endometriosis was allowed to develop for seven days. The success rate for the lesion development was 70% [ 44 ]. The rats were randomized and assigned to the following groups ( n = 12): (1) Endo group: rats were subjected to experimental endometriosis and vehicle (saline) was administered by a gavage on the 7th day and for the next 7 days. (2) Endo + Açai Berry group: rats were subjected to experimental endometriosis as described and an Açai Berry (200 mg/kg) was orally administered on the 7th day and for the next 7 days. (3) Sham group: rats were injected intraperitoneally with 500 uL of PBS instead of endometrial tissue, and a vehicle (saline) was administered on the 7th day and for the next 7 days. Endo group: rats were subjected to experimental endometriosis and vehicle (saline) was administered by a gavage on the 7th day and for the next 7 days. Endo + Açai Berry group: rats were subjected to experimental endometriosis as described and an Açai Berry (200 mg/kg) was orally administered on the 7th day and for the next 7 days. Sham group: rats were injected intraperitoneally with 500 uL of PBS instead of endometrial tissue, and a vehicle (saline) was administered on the 7th day and for the next 7 days. The Açai Berry dose was based on previous studies [ 45 ]. In order to evaluate the effect of the Açai Berry administration on the endometriotic-like lesions, the rats were sacrificed 14 days after the induction. Thereafter, a laparotomy was performed to collect the endometriotic implants for further analyses. Ultrasonographic exams were performed using the Esaote MYLAB OMEGA VET on anesthetized rats (2% isoflurane) positioned in dorsal recumbency. An abdominal B-mode was performed with a High Frequency Linear array (4–15 MHz) transducer [ 46 ]. Longitudinal and transverse scanning planes were employed for the evaluation of different abdominal structures. Endometriotic lesions were fixed in a formaldehyde solution and were embedded in Paraplast [ 47 , 48 ]. Tissue slides were stained with H&E and were evaluated using a Leica DM6 microscope (Leica Microsystems SpA, Milan, Italy). A histological analysis was performed using a double-blind procedure. Histopathological scores were assigned according to the formula P (persistence of epithelial cells in the explants) × I (intensity of glands), as already described [ 30 ]. The lesion volume was calculated according to the formula V = (length × width2) × 0.5 [ 49 ]. Apoptosis was analyzed with a TUNEL assay using an in situ cell death detection kit (Roche 11684795910) [ 50 , 51 , 52 ]. Western blots were performed as already described to obtain either cytosolic and mitochondrial [ 53 ] or cytosolic and nuclear [ 54 ] protein fractions. The specific primary antibodies anti-Beclin (sc-48381, Heidelberg, Germany), anti-mTOR (Cell Signaling, 2972, Milan, Italy), anti-p-mTOR (sc-293089, Heidelberg, Germany), anti-p-AKT (sc-293125, Heidelberg, Germany), anti-AKT (Invitrogen AHO1112, London UK), anti-LC3 II (Sigma Aldrich, ABC232, Milan, Italy), anti-AMBRA1 (Abcam, Ab69501, Cambrige, UK), anti-IP3K (sc-1637), anti-BCL-2 (sc-7382, Heidelberg, Germany), anti-PARKIN (sc-32282, Heidelberg, Germany), anti-BAX (sc-7480, Heidelberg, Germany), anti-NQO1 (sc-32793, Heidelberg, Germany), anti-HO1 (sc-136960, Heidelberg, Germany), anti-PINK1 (sc-517353, Heidelberg, Germany), anti-NRF2 (sc-365949, Heidelberg, Germany), anti-p-ERK (sc-7383, Heidelberg, Germany), anti-ATG9 (cell signaling 13509, Milan, Italy), anti-ERK (sc-514302, Heidelberg, Germany), anti-p-ATG1 (Bioss, BS-3464R, Cambrige, UK) and anti-ATG1 (Sigma, A7481, Cambrige, UK) were mixed in a 5% w/v nonfat dried milk solution and were incubated at 4 °C overnight. Blots were incubated with a peroxidase-conjugated goat antirabbit IgG (Jackson Immuno Research) or a peroxidase-conjugated bovine antimouse IgG secondary antibody for 1 h at room temperature [ 55 , 56 ]. To confirm the equal amounts of protein, filters were also incubated with the antibody against β-ACTIN (sc-47778), COXIV (ab14744) and HISTONE 3 (ab1791). Signals were detected with an enhanced chemiluminescence detection system reagent (Super-Signal West Pico Chemiluminescent Substrate) [ 57 , 58 ]. The relative expression of the protein bands was quantified using densitometry with Bio-Rad ChemiDoc XRS software, #1708265 [ 59 ]. Images of the blot signals were imported to analysis software (Image Quant TL, Amersham Biosciences, Freiburg, Germany, v2003) [ 60 , 61 ]. Lipid peroxidation was evaluated with the TBARS test by reading the MDA levels at 535 nm [ 54 , 62 ]. SOD activity was evaluated as already described [ 47 , 63 ] and is expressed as U/g protein [ 64 ]. GSH levels were determined using a microplate reader at 412 nm [ 65 , 66 ]. All the values are expressed as mean ± standard error of the mean of N observations. The results were analyzed with a t-test when comparing the two groups, and we used the t-test and the Kolmogorov–Smirnov test to analyze the normal distribution of the data (Prism 8 for macOS version 8.2.1 (279)). A p -value of less than 0.05 was considered significant. * p < 0.05 vs. Endo, ** p < 0.01 vs. Endo, *** p < 0.001 vs. Endo.

Endometriosis is a chronic disease of the endometrium [ 1 , 2 , 3 ]. The abnormal infiltration and growth of stromal cells and endometrial epithelial cells causes the formation of masses and nodules [ 2 , 4 ]. These lesions induce dysmenorrhea, chronic pelvic pain and infertility [ 5 , 6 ]. Actual endometriosis affects 30 to 50% of women in menopause and 15% of women of reproductive age [ 7 ]. The most accepted theories that explain the invasion and implantation of endometrial tissue are the ectopic presence of endometrial stem cells [ 8 ], retrograde menstrual reflux [ 9 ] and defects in the immune system [ 10 ]. Evidence from the literature shows a dysregulated antioxidant/pro-oxidant balance and an increased proinflammatory microenvironment in endometrial lesions [ 11 ]. Recently, increasing importance has been assigned to the autophagic pathway in the induction of the endometriosis [ 12 , 13 , 14 ]. It is the major constitutive pathway for the degradation of cytoplasmic organelles and long-lived proteins in eukaryotic cells [ 15 , 16 ]. This catabolic pathway mediates both the targeted and nonspecific sequestration of macromolecules and cellular organelles, promoting the recycling of useful metabolites and permitting the degradation of cellular constituents in lysosomes [ 17 ]. Autophagy can be deleterious to the cell when its activation is too extensive, and it can induce cell death. Differently, a basal autophagic response acts as a survival and housekeeping mechanism that maintains cellular homeostasis in physiological conditions and contributes to overcoming the stressful conditions induced by both extracellular and intracellular stimuli, including reduced nutrient supply, hypoxia, invasion of microorganisms, oxidative stress and therapeutic stress [ 18 , 19 , 20 ]. Autophagy is also responsible for the elimination of damaged or aged organelles. Mitophagy or mitochondrial autophagy is the selective mechanism to remove the dysfunctional mitochondria [ 21 ]. Indeed, autophagy shows a key role in inducing cell death by promoting caspase-dependent apoptosis in homeostatic conditions [ 22 , 23 ]. For instance, the autophagic machine has important roles in the process of differentiation, growth, cell immunity, tissue remodeling and environmental adaptation [ 24 , 25 ]. In normal endometrial cells, the induction of autophagy exercises proapoptotic effects [ 26 ]. Meanwhile, ectopic endometrial cells showed a reduced autophagic pathway compared with the normal endometrium [ 27 ]. The autophagic pathway was impaired in the endometriotic-like lesions of the mice, and the autophagic markers were altered as compared to the control [ 28 ]. Strongly associated with autophagy, apoptosis is one of the main impaired pathways during endometriosis because it contributes to the survival of the ectopic cells and the growth of the lesions [ 29 ]. Thus, several papers described that autophagic and apoptotic activators would reduce the development of this pathology by reducing the growth of the lesions [ 30 ]. Recently, increased interest has been developed for the nutritional properties and medicinal uses of Açai Berries [ 31 , 32 , 33 ]. They are an Amazonian fruit produced by the Euterpe oleracea palm. For millennia, it has been used by Indians as a natural mixture to treat many diseases [ 34 , 35 , 36 , 37 ]. Açai Berries, in fact, contain many biologically active phytochemicals including quercetin, luteolin, delphinidin, cyanidin, malvidin and pelargonidin [ 38 ]. Several studies report that Açai Berries have neuroprotective, anti-inflammatory and antioxidant properties [ 38 , 39 , 40 , 41 , 42 ]. Recently, the modulation of the autophagic pathway by the Açai Berry supplementation was reported [ 33 ]. However, more data are required to confirm the beneficial effect of Açai Berries. In this paper, we employed a well-consolidated endometriosis model to investigate the effects of Açai Berry administration and the molecular pathway involved.

Ultrasonographic exams were employed to monitor the development of the pathology at seven and fourteen days from the induction. A pelvic ultrasound showed endometriotic-like lesions in the inner surface of the peritoneal cavity in both groups at seven days from the induction ( Figure 1 A,B). This analysis was conducted to control the establishment of the pathology before the Açai Berry administration. No differences were detected in diameter ( Figure 1 C) and lesions number ( Figure 1 D). After this control was applied, the Açai Berry was administered for the next 7 days. Fourteen days from the induction, the ultrasonographic exams showed that the Endo group had an increased lesion diameter ( Figure 1 E,G) as compared to the Endo + Açai Berry group ( Figure 1 F,G). The same number of lesions was detected in both groups ( Figure 1 H). Fourteen days after the rats developed endometriosis, an induction laparotomy was performed in both groups and lesions were harvested. The macroscopic analysis ( Figure 2 A,B) was in line with the ultrasonographic exams. The endometriosis lesions collected from the Endo group showed a higher volume ( Figure 2 C) and area ( Figure 2 D) that than collected from the Endo + Açai Berry group. The histopathological analysis showed that the Açai Berry administration changed the lesion morphology. Lesions harvested from the Endo group presented characteristic glands and stroma ( Figure 2 E,G), while the Açai Berry administration reduced the histopathological score ( Figure 2 F,G). A Western blot analysis was employed to evaluate the modulation of the autophagic pathway induced by the Açai Berry administration. Samples collected from the Endo group showed an elevated PI3K expression ( Figure 3 A) and an increased phosphorylation of AKT ( Figure 3 B), ERK ( Figure 3 C) and mTOR ( Figure 3 D). Differently, in the samples harvested from the Endo + Açai Berry group, the PI3K expression ( Figure 3 A) decreased, as did the pAKT ( Figure 3 B), p-ERK1/2 ( Figure 3 C) and the p-mTOR ( Figure 3 D) levels. To further evaluate the autophagosome formation, we checked the phosphorylation of the ATG1/ULK1 complex and the expression of the downstream proteins. The samples collected from the Endo group showed elevated ATG1 phosphorylation ( Figure 4 A) and low AMBRA1 ( Figure 4 B), BECLIN ( Figure 4 C), ATG9 ( Figure 4 D) and LC3II ( Figure 4 E) expressions. The Açai Berry administration reduced Atg1 phosphorylation ( Figure 4 A) and increased the expression of AMBRA1 ( Figure 4 B), BECLIN ( Figure 4 C), ATG9 ( Figure 4 D) and LC3II ( Figure 4 E). To investigate mitophagy induction, we investigated the cytoplasmic and mitochondrial expression of PINK1 and PARKIN. The samples collected from the Endo group showed elevated PINK1 ( Figure 5 A) and PARKIN ( Figure 5 B) expressions in the mitochondria, while Parkin expression was reduced in the cytosol ( Figure 5 C). The Açai Berry administration reduced PINK1 ( Figure 5 A) and PARKIN ( Figure 5 B) mitochondrial expressions, while the PARKIN cytoplasmic expression was increased ( Figure 5 C). In order to evaluate the oxidative alterations, the NRF2 pathway was examined. A Western blot analysis showed a low nuclear NRF2 expression ( Figure 6 A) and low cytosolic HO-1 ( Figure 6 B) and NQO-1 ( Figure 6 C) expression in the samples collected from the Endo group. Conversely, the Açai Berry administration increased the NRF2 nuclear expression ( Figure 6 A) and the cytosolic expression of the downstream proteins ( Figure 6 B,C). The lesions collected from the Endo group also showed low GSH levels ( Figure 6 D) and SOD ( Figure 6 E) activity, while lipid peroxidation was found to be elevated ( Figure 6 F). The Açai Berry administration increased the GSH levels ( Figure 6 D) and SOD activity ( Figure 6 E) and reduced lipid peroxidation ( Figure 6 F). The samples collected from the Endo group showed an impaired apoptotic pathway ( Figure 7 ). A Western blot analysis revealed an elevated BCL-2 ( Figure 7 A) and low BAX ( Figure 7 B) expression in the Endo group. The Endo + Açai Berry group showed a reduced BCL-2 ( Figure 7 A) and increased Bax ( Figure 7 B) expression. These results were confirmed with a TUNEL analysis where the number of TUNEL-positive cells strongly increased in the Endo + Açai Berry group ( Figure 7 D,E) as compared to the Endo group ( Figure 7 C,E).

Endometriosis is a chronic disease with intricate molecular mechanisms. Açai Berries have important antioxidant, anti-inflammatory and neuroprotective proteins that have the ability to modulate the autophagic pathway in many diseases [ 38 , 39 , 40 , 41 , 42 ]. This paper aimed to evaluate the molecular mechanisms regulated by Açai Berry supplementations during endometriosis. The pathology was induced and monitored with an hfUS analysis. The Açai Berry supplementation reduced the lesion area, volume and diameter. From the molecular point of view, the endometrial microenvironment was characterized by dysregulated autophagic, oxidative balance and apoptotic pathways [ 33 , 67 , 68 ]. Many papers described that autophagy is suppressed in endometriotic cells by the PI3K/AKT/ERK1/2 pathways that positive regulate the expression of mTOR, which is the major modulator of autophagy [ 69 , 70 , 71 , 72 , 73 ]. Açai Berry administration inhibited PI3K and AKT and ERK1/2 phosphorylation and promoted autophagy by inactivating mTOR. mTOR has a central role in the regulation of cell growth and autophagy [ 74 ]. Inhibiting mTOR Açai Berry administration dephosphorylated ATG1, which promoted the activity of the ATG1/ULK1 complex. The ATG1/ULK1 complex recruits other proteins, including AMBRA1/BECLIN1 and ATG9, to promote autophagosome nucleation [ 75 ]. Additionally, Açai Berry supplementation increased the expression of AMBRA1 and BECLIN-1, which promotes the autophagic pathway transforming LC3I into its membrane-bound form of LC3-II [ 76 ]. These findings showed the role of the Açai Berry administration in the management of the autophagic pathway in endometriosis. Although autophagy was initially considered a nonselective process, accumulating evidence has shown the presence of specific pathways for the degradation of damaged organelles [ 77 , 78 ]. Recent papers already described the role of mitochondrial autophagy in endometriosis [ 30 ]. Currently, PARKIN and PINK1 are the most well-studied proteins involved in the mechanism [ 79 ]. In the functional mitochondria PINK1, a serine/threonine kinase is continuously degraded by matrix-processing peptidase [ 80 , 81 ]. It is a sensor of organelle damage and an initiator of mitophagy [ 82 ]. In depolarized mitochondria, it accumulates in the outer mitochondrial membrane [ 83 ] and recruits PARKIN, a cytosolic E3 ubiquitin ligase [ 84 ]. PARKIN is a cytosolic protein that is recruited by PINK1 in impaired mitochondria. PARKIN-labeled mitochondria are polyubiquitinated [ 85 ]. The phospho-ubiquitin chain further recruits autophagy receptor proteins, triggering the formation of autophagosomes for degradation. The Açai Berry administration restored this organelle-specific autophagy by facilitating the removal of damaged mitochondria through mitophagy. Mitochondria are the major source of ROS; therefore, when the mitophagy function is impaired and unfunctional mitochondria are not removed properly, they increase ROS production, which aggravates tissue injury [ 86 , 87 , 88 , 89 ]. The endometrial microenvironment is, in fact, characterized by a dysregulated oxidative balance [ 24 ]. The NRF2 signaling controls the transactivation of several cytoprotective genes and is one on the most important regulatory pathways in defending cells from ROS [ 90 ]. Physiologically bound to its inhibitor KEAP1, NRF2 is usually polyubiquitinated by the E1 ligase complex and is degraded [ 33 , 91 ]. A dysregulated oxidative balance, which is characteristic of the disease, breaks the KEAP1-NRF2 link and allows for the NRF2 translocation into the nucleus [ 92 ]. Here it binds the antioxidant response elements (ARE), promoting the expression of cytoprotective genes with antioxidant and detoxifying roles [ 91 ]. The samples collected from the animals administered with Açai Berries showed an increased NRF2 nuclear expression as well the cytosolic expression of the cytoprotective proteins NQO-1 and HO-1. Well in line with the literature, where elevated lipid peroxidation and ROS were found in ectopic endometrium biopsies, Açai Berry supplementation restored the oxidative imbalance. Increased oxidative stress and dysregulated autophagic pathways result in impaired apoptosis. It has been recently shown that the induction of autophagy has a proapoptotic effect on normal human endometrial cells [ 93 ]. While the overactivation of autophagy and apoptosis has been identified as damaging in many pathologies, during endometriosis they extern cytoprotective properties [ 94 , 95 ]. Indeed, they are tightly regulated by common signals [ 96 , 97 , 98 ]. Our results demonstrated the proapoptotic effect of the Açai Berry administration, which reduced the expression of the proapoptotic protein Bax and the antiapoptotic protein BCL2. This result was confirmed by the TUNEL assay, where apoptotic cells were identified by the terminal deoxynucleotidyl transferase (TdT)-mediated addition of labeled (X) de-oxyuridine triphosphate nucleotides (X-dUTPs) to the 3′-OH end of DNA strand breaks. Overall, our result showed the role of the Açai Berry administration on the management of endometriosis, describing the modulation of the autophagy, oxidative stress and apoptosis.

Overall, this paper showed the key role of autophagy, oxidative stress and apoptosis in the development of endometriosis. Our results showed that Açai Berries modulate the PI3K/AKT/ERK1/2 pathways, thereby reducing the expression of mTOR and promoting the autophagy. Indeed, the Açai Berries facilitated the removal of damaged mitochondria through the activation of mitophagy and restored the oxidative imbalance and the impaired apoptosis.