Hidrox® and Endometriosis: Biochemical Evaluation of Oxidative Stress and Pain

Endometriosis is a gynecological and painful condition affecting women of reproductive age. It is characterized by dysfunctional endometrium-like implants outside of the uterine cavity. The purpose of this study was to evaluate the effects of Hidrox®, an aqueous extract of olive pulp containing hydroxytyrosol, on endometriotic lesions associated with pro-oxidative alterations and pain-like behaviors. Endometriosis was induced by intraperitoneal injection of uterine fragments, and Hidrox® was administered daily. At the end of the 14-day treatment, behavioral alterations were assessed and hippocampal tissues were collected. Laparotomy was performed, and the endometrial implants were harvested for histological and biochemical analysis. Hidrox® treatment reduced endometriotic implant area, diameter and volumes. Vehicle-treated rats showed lesional fibrosis, epithelial–mesenchymal transition and fibroblast–myofibroblast transdifferentiation, angiogenesis and pro-oxidative alterations in the peritoneal cavity. Hidrox® treatment reduced the aniline blue-stained area, α-smooth muscle actin (α-sma) and CD34 positive expressions. Moreover, it reduced mast cell recruitment into the lesions, myeloperoxidase activity and lipid peroxidation and increased superoxide dismutase (SOD) activity and glutathione levels in the endometrial explants. In the peritoneal fluid, Hidrox® treatment reduced interleukin (IL)-1β, IL2, IL6, tumor necrosis factor-α (TNF-α) and vascular endothelial grow factor (VEGF) levels increased by the disease. Hidrox® administration also reduced peripheral and visceral sensibility as shown by the behavioral tests (open field test, hot plate test, elevated plus maze test and acetic-acid-induced abdominal contractions). Animals treated with Hidrox® also showed reduced blood–brain barrier permeability and mast cell infiltration in the hippocampus, as well as astrocyte and microglia activation and brain oxidative status restoring brain-derived neurotrophic factor (BDNF) protein expression and increasing Nuclear factor erythroid 2-related factor 2 (Nfr2) nuclear translocation. In conclusion, Hidrox® displayed potential ameliorative effects on endometriotic implants and related pain-induced behaviors due to its potent antioxidative properties.


Experimental Protocol
Animals were randomly divided into two groups, donor or recipient, and endometriosis was established as already described [26]. To stimulate similar estrogen levels, donor rats were intraperitoneally injected with 10 international units (IU) pregnant mare serum gonadotropin to induce similar estrogen levels between various animals. The animals were euthanized 41 h later by CO 2 asphyxia. A midline incision was performed, and the uterus was removed and minced with scissors. Tissue from all donors was pooled, and the recipient animals were injected intraperitoneally with the equivalent of tissue from one uterus in 500 µL of phosphate buffered saline (PBS) along the midventral line. Endometriosis was allowed to develop for seven days.

Experimental Groups
Rats were randomized and assigned to the following groups (n = 20): (1) Vehicle group: Rats were subjected to experimental endometriosis as described above, and vehicle (saline) was administered by gavage on the 7th day and for the next 7 days. (2) Hidrox ® group: Rats were subjected to experimental endometriosis as described above, and Hidrox ® (10 mg/kg) was administered by gavage on the 7th day and for the next 7 days. (3) Sham group: Rats were injected intraperitoneally with 500 µL of PBS without endometrial tissue, and vehicle (saline) was administered by gavage on the 7th day and for the next 7 days.
The dose of Hidrox ® was based on previous experiments [25,27]. In order to evaluate endometriotic lesions, rats were sacrificed at 14 days after endometriosis induction [26].
Brain tissues were harvested and laparotomy was performed to collect the endometriotic implants.
Implants were excised from both groups, measured [28,29] and processed for histological and biochemical studies.

Open Field Test
Locomotor activity and exploratory behavior were measured using a squared open field area [29,30]. After 1 min of habituation, each rat was placed into a corner of the area and observed for 5 min. For cleaning the apparatus after each analysis, a solution of 20% ethanol was used. The parameters recorded were: number of animal crossings with four legs (spontaneous locomotion), entries in central square and time spent in the central square (in seconds).

Hot Plate
Hot plate test was used to evaluate pain threshold to thermal stimuli [31,32]. Rats were allowed to walk on the hot plate (53.0 ± 0.1 • C) for up to 45 s.

Elevated Plus Maze Test
The elevated plus maze apparatus [33,34]

Acetic-Acid-Induced Abdominal Contractions
The animals received an intraperitoneal injection of 0.6% acetic acid, and the number of acid-induced writhes was observed for 20 min, starting 5 min after administration [35]. A writhe was defined as a contraction of the abdomen following a stretch of the hind limbs.

Determination of Reduced Glutathione Levels
The levels of reduced glutathione (GSH) were determined in endometriosis lesions and hippocampi to evaluate the endogenous antioxidant defenses. GSH levels were determined using a microplate reader at 412 nm and expressed as ng/mg wet tissue [36,37].

Measurement of Lipid Peroxidation
Lipoperoxidation was estimated in endometriosis lesions and hippocampi using the thiobarbituric acid reactive substances (TBARS) test [38,39]. The levels of malondialdehyde (MDA) were determined using a microplate reader at 535 nm and expressed as µmol/mg wet tissue.

Measurement of Superoxide Dismutase (SOD) Activity
In endometriosis lesions and hippocampi determination of SOD activity was performed according to a previously described method [40][41][42]. SOD activity (U/µg protein) was determined using a microplate reader at 560 nm.

Histological Examination
For histopathological investigations, endometriosis lesions were fixed at room temperature in buffered formaldehyde solution (10% in PBS); histological sections were stained with H&E and evaluated using a Leica DM6 microscope (Leica Microsystems SpA, Milan, Italy) equipped with a motorized stage and associated with Leica LAS X Navigator software (Leica Microsystems SpA, Milan, Italy) [47]. Histopathologic scores were evaluated with the formula P (persistence of epithelial cells in the explants) × I (intensity of glands) as described previously [48]: P: 3 = well-preserved epithelial layer, 2 = moderately preserved epithelium with leukocyte infiltrating, 1 = poorly preserved epithelium (occasional epithelial cells only), and 0 = no epithelium; I: from 0 (no glands) to 3 (abundant glands). Additionally, lesion volume was calculated according to the formula: V = (length × width 2 ) × 0.5. [48]. The degree of fibrosis was evaluated by the Masson trichrome staining performed according to the manufacturer's protocol (Bio-Optica, Milan, Italy) [49,50]. Mast cell analyses were performed by toluidine blue staining [51].

Statistical Evaluation
All values are expressed as mean ± standard error of the mean (SEM) of N observations. For in vivo studies, N represents the number of animals used. The results were analyzed by t-test when comparing two groups while we used the one-way ANOVA followed by a Bonferroni post hoc test for multiple comparisons. A p-value of less than 0.05 was considered significant. * p < 0.05 vs. sham, # p < 0.05 vs. vehicle, ** p < 0.01 vs. sham, ## p < 0.01 vs. vehicle, *** p < 0.001 vs. sham, ### p < 0.001 vs. vehicle.

Effect of Hidrox ® Treatment on Lesion Size in Endometriosis
At 14 days of induction, all animals from the vehicle and Hidrox ® groups displayed endometriosis lesions, while sham animals did not show any implants. Even the groups did not show different cyst numbers ( Figure 1C); the area ( Figure 1D), diameter ( Figure 1E) and volume ( Figure 1F) were smaller in Hidrox ® treated animals ( Figure 1B) compared to the vehicle ( Figure 1A). Histologically, endometriotic lesions from vehicle-treated rats showed abundant stromal structure and endometrial-type glands ( Figure 1G,I). Hidrox ® administration reduced the histopathological marks of endometriosis ( Figure 1H,I).

Effect of Hidrox ® Treatment on Fibrosis and Angiogenesis Associated with Endometriosis
As the advanced stages of endometriosis lesion development are associated with a high degree of tissue fibrosis and increased neovascularization events, we first analyzed the number of collagen fibers, the expression of α-sma as markers for fibrosis. Further, the angiogenesis was evaluated by the abundance of hematopoietic CD34+ stem cells and VEGF expression. The degree of fibrosis was evaluated by Masson's trichrome staining and α-sma immunolocalization. The collagen fibers were significantly reduced by Hidrox ® treatment ( Figure 2B,C) as compared to the vehicle-treated animals (Figure 2A,C). αsma immunolocalization was weakest in lesions from Hidrox ® administered animals ( Figure 2E,F) as compared to the vehicle rats ( Figure 2D,F). Positive immunostaining for CD34 and VEGF was detected in the endometriotic lesions from vehicle-treated rats ( Figure 2G,I,J,L), which was decreased by the Hidrox ® treatment ( Figure 2H,I,K,L). For the macroscopic analyses, n = 20 animals from each group were employed. For the histological analyses, n = 5 animals from each group were employed. A p-value of less than 0.05 was considered significant. # p < 0.05 vs. vehicle, ### p < 0.001 vs. vehicle.

Effect of Hidrox ® Treatment on Hyperproliferation and Anti-Apoptosis
Important characteristics of endometriosis are hyperproliferation and inhibited apoptosis. We evaluated, using immunohistochemical analysis of the expression of the cell proliferation marker (Ki67) and by Western blot analysis, the anti-apoptotic protein Bcl-2 and the pro-apoptotic protein Bax expressions in the endometriosis lesions. Elevated Ki67 expressions were detected in samples collected from vehicle-treated rats ( Figure 3A,C), while Hidrox ® administration reduced Ki67 positive staining ( Figure 3B,C). Western blot analysis showed elevated Bcl-2 expression and low Bax expression in tissues harvested from vehicle-treated rats, while Hidrox ® treatment reduced Bcl-2 ( Figure 3D,F) and increased Bax levels ( Figure 3E,F).

Effect of Hidrox ® Treatment on Hyperproliferation and Anti-apoptosis
Important characteristics of endometriosis are hyperproliferation and inhibited apoptosis. We evaluated, using immunohistochemical analysis of the expression of the

Effect of Hidrox ® Treatment on Mast Cell Number and on Biochemical Parameters
Several papers described the key role of inflammatory cell recruitment at the lesion site during endometriosis and the impaired oxidant-antioxidant balance during the pathology.
Toluidine blue staining showed elevated mast cell number in lesions harvested from vehicle-treated rats ( Figure 4A,C). Hidrox ® treatment reduced mast cell infiltration into the cysts ( Figure 4B,C). Levels of GSH and MDA and MPO and SOD activities were determined in endometrial explants. Treatment with Hidrox ® resulted in a significant reduction of MPO activity ( Figure 4D) and MDA levels ( Figure 4E) compared to the vehicle-treated rats. Moreover, Hidrox ® administration increased GSH levels ( Figure 4F) and SOD activity ( Figure 4G).

Effect of Hidrox ® Treatment on Cytokine Expressions in Lesions and Peritoneal Fluid
It has been described that in patients affected by endometriosis, peritoneal fluid directly reflects the changes of the local microenvironment. Thus, inflammatory cytokines in peritoneal fluid were focused. In peritoneal fluid of Hidrox ® treated rats, the levels of IL-1β ( Figure 5A), IL2 ( Figure 5B), IL6 ( Figure 5C), TNF-α ( Figure 5D) and IL-10 ( Figure 5E) were increased as compared to the sham animals. Moreover, the analysis conducted on the lesions confirmed the same trend: elevated levels of IL-1β ( Figure 5F), IL2 ( Figure 5G), IL6 ( Figure 5H), TNF-α ( Figure 5I) and IL-10 ( Figure 5J

Effect of Hidrox ® Treatment on Cytokine Expressions in Lesions and Peritoneal Fluid
It has been described that in patients affected by endometriosis, peritoneal fluid directly reflects the changes of the local microenvironment. Thus, inflammatory cytokines in peritoneal fluid were focused. In peritoneal fluid of Hidrox ® treated rats, the levels of  Figure 5D) and IL-10 ( Figure  5E) were increased as compared to the sham animals. Moreover, the analysis conducted on the lesions confirmed the same trend: elevated levels of IL-1β ( Figure 5F), IL2 ( Figure  5G), IL6 ( Figure 5H), TNF-α ( Figure 5I) and IL-10 ( Figure 5J) were detected. Hidrox ® administration reduced in both peritoneal fluid and lesions reduced IL-1β ( Figure 5A,E), IL2 ( Figure 5B,F), IL6 ( Figure 5C,G), TNF-α ( Figure 5D,H) and IL-10 ( Figure 5E,J) levels.

Effect of Hidrox ® Treatment on Pain Sensitivity Threshold
Deep endometriosis induced behavioral alterations in vehicle-treated rats. In the open field test, vehicle-treated animals showed reduced spontaneous locomotion ( Figure 6A), number of entries in the central square ( Figure 6B) and time spent in it ( Figure 6C). Hidrox ® treatment ameliorated locomotor activity and exploratory behavior ( Figure 6A-C). In the elevated plus maze test, vehicle-treated rats showed reduced number of entries in closed and open arms ( Figure 6D), % of open entries ( Figure 6E) and the % of time in open arms ( Figure 6F). Hidrox ® administration ameliorated all these parameters ( Figure 6D-F).
Vehicle-treated rats displayed a significant increase in the number of writhes, which were reduced by Hidrox ® treatment ( Figure 6G). In the hot plate test, it was observed a significant reduction in the latency to pain reaction in the vehicle-treated, which was reduced by the Hidrox ® treatment ( Figure 6H).

Effect of Hidrox ® Treatment on Tight Junctions Neuroinflammation
Tight junctions, mainly occludin and claudin-5, are important factors responsible for blood-brain barrier integrity. To further explore the impact on BBB integrity, we evaluated the level of occludin and claudin-5 by immunohistochemistry staining and Western blot.

Effect of Hidrox ® Treatment on Neuroinflammation
Further, it has been shown that a significant increase in mast cell number in the hippocampus is a sensor of brain injury and related to the stress-mediated neuroinflammation. Thus, we evaluated mast cell infiltration and Iba1 and GFAP expression. Toluidine blue staining showed increased mast cell infiltration in hippocampi from vehicle-treated rats ( Figure 8B,D), as compared to the sham rats ( Figure 8A,D). Animals treated with Hidrox ® showed reduced mast cell infiltration ( Figure 8C,D). Western blot analysis showed a significant increase in Iba1 ( Figure 8E,G) and GFAP ( Figure 8F,G) expression in hippocampi tissues from vehicle-treated rats as compared to the sham tissues. Tissues harvested from Hidrox ® -treated rats showed reduced expression of both neuroinflammatory markers ( Figure 8E-G).

Effect of Hidrox ® Treatment on Oxidative Hippocampal Alterations
Some evidence described the impaired brain oxidative status of rats subjected to endometriosis. Levels of GSH and MDA and MPO and SOD activities were determined in hippocampi. Western blot analyses were performed to evaluate BDNF and Nrf2 expressions. Vehicle-treated rats showed a significant increase in MPO activity and lipid peroxidation as compared to the sham animals. Treatment with Hidrox ® resulted in a significant reduction of MPO activity ( Figure 9A) and MDA levels ( Figure 9B). Moreover, Hidrox ® administration increased GSH levels ( Figure 9C) and SOD activity ( Figure 9D), as compared to the vehicle-treated rats. Western blot analysis showed a significant decrease in BDNF protein levels compared to the sham animals. Hidrox ® administration restored its expression ( Figure 9E,F). Moreover, hippocampi from Hidrox ® -treated animals showed increased Nrf2 nuclear translocation compared to the tissues harvested from vehicle-treated rats ( Figure 9G,H). Differently, cytosolic expression of Nrf2 was decreased by Hidrox ® administration as compared with vehicle-treated rats ( Figure 9I,J). rats ( Figure 8B,D), as compared to the sham rats ( Figure 8A,D). Animals treated with Hidrox ® showed reduced mast cell infiltration ( Figure 8C,D). Western blot analysis showed a significant increase in Iba1 ( Figure 8E,G) and GFAP ( Figure 8F,G) expression in hippocampi tissues from vehicle-treated rats as compared to the sham tissues. Tissues harvested from Hidrox ® -treated rats showed reduced expression of both neuroinflammatory markers ( Figure 8E-G).

Discussion
To our best knowledge, the induction and progression of endometriosis requires a proinflammatory environment, increased angiogenesis, changes in the epigenetic and structural elements and oxidative stress [60][61][62].
In particular, the oxidative damage, the elevation of inflammatory cytokines and the mast cell activation are considered a decisive step in the pathophysiology of endometriosis [63].
Our study demonstrated that Hidrox ® effectively decreased cyst diameter, area and volume and modified cyst morphology.
Advanced endometriotic lesions are characterized by widespread adhesions and fibrosis associated with pelvic morbidity, such as chronic pelvic pain and infertility [64]. Hidrox ® administration reduced collagen deposition and α-sma-positive myofibroblast in lesional stroma near the glandular epithelium showing reduced fibrosis. Angiogenesis is assumed to be a prerequisite for the formation and development of endometriosis [65,66]. Our results show that Hidrox ® treatment caused a reduction in CD34 and VEGF expression in the implants. Moreover, Hidrox ® administration was able to manage hyperproliferation and apoptosis during endometriosis. Many researchers found Ki67 and Bcl-2 overexpression and Bax downregulation in the gland and stroma of endometriotic loci [67]. Hidrox ® administration counteracted hyperproliferation and restored the apoptotic pathway in the lesions. From the histological point of view, mast cell involvement in endometriosis is well described [68,69]. The reduced number and degranulation of mast cells in endometriotic lesions from the Hidrox ® -treated group relate to suppressed neuropathic pain and release of inflammatory mediators [70].
Several papers displayed a significant suppression of the production of the antioxidant defense, such as SOD activity, and increment of oxidized lipoproteins in the peritoneal microenvironment in women with endometriosis [71,72]. This rise in SOD activity occurred in response to the oxidative stress and to the high amount of ROS as an adaptive cell reaction accompanied by the decreased GSH and increased MDA levels. In this inflammatory condition, the innate immune system activates the phagocytic cells as shown by the increased MPO activity in the endometriosis rats [73]. MPO is a key enzyme of the innate immune system that produces oxidant radicals that can covalently alter proteins and lipids [73]. Indeed, Hidrox ® treatment by its antioxidant activity normalized the imbalance in oxidant-antioxidant activity in endometriotic rats as shown by the restored GSH levels, decreased SOD and MPO activity and lipid peroxidation.
Studies conducted on patients with endometriosis showed increased levels of cytokines in both peritoneal fluid and ectopic lesions [74]. Anti-inflammatory cytokines are a class of immunoregulatory molecules that regulate the development of pro-inflammatory cytokines. Anti-inflammatory cytokines limit the potentially harmful effects of prolonged or excessive inflammatory responses under physiological conditions. Anti-inflammatory mediators in immune-mediated diseases may have inadequate control over pro-inflammatory behaviors under pathological conditions, or they may overcompensate and suppress the immune response, raising the risk of systemic infection. IL-6, IL-1β and TNF-α support adhesion of endometrial cells to the peritoneum, and TNF-α stimulates the proliferation of ectopic tissue. IL-4 and IL-10 family proteins are the main Th2 anti-inflammatory cytokines. Several lines of evidence indicate that the Th2 immune response is associated with endometriosis [75,76]. Some evidence demonstrated that anti-inflammatory cytokines, in particular IL-10 [77,78], are sharply increased in peritoneal fluid and the ectopic endometrium of women with endometriosis. Hidrox ® administration reduced levels of IL-2, IL-1β, TNF-α, IL-6 and IL-10 in the peritoneal fluid and endometriotic loci compared to the vehicle-treated group. Thus, Hidrox ® treatment significantly restored the pro-inflammatory microenvironment.
Our experimental conditions showed an interesting relationship between the growth of the implants, the inflammatory microenvironment and the development of pain-like symptoms. As stated previously, a severe local inflammatory and hemorrhagic response occurs at the beginning of the endometriosis establishment. In particular, inflammation grows exponentially with the size of the cyst and invasion of peritoneal organs [79].
It was recently shown that endometriosis exacerbated inflammatory manifestations and altered pain threshold [80]. In the present paper, we investigated rat pain perception based on different tests for peripheral and visceral sensibility, respectively, at the development of the endometriosis model. In particular, in accordance with literature showing decreased mechanical and thermal pain threshold in rats and mice in a similar model of endometriosis [81,82], our results show that endometriosis rats presented increased visceral sensitivity. Animals subjected to endometriosis and treated with Hidrox ® displayed reduced thermal and mechanical hyperalgesia and pain sensitivity.
Regarding vulnerability to pain, endometriosis is related to central and peripheral pain sensitization [83]. Initially, tissue injury and inflammation sensitized in the peripheral nociceptive system, producing a decreased pain threshold and an increased sensory input to the central nervous system. With these continuous stimuli, because of long-term central adaptations in the process called central sensitization, central behavior may become independent of any peripheral inputs [84]. One of the crucial brain regions involved in the affective and cognitive consequence of neuropathic pain is the hippocampus [85]. Patients with endometriosis displayed abnormal connectivity in the hippocampus and in their afferences to the frontoinsular and somatosensory cortex [86]. This brain area, in fact, is involved in the transition from acute to chronic pain [87]. Further, it has been reported that a significant increase in mast cell number in the hippocampus is a sensor of brain injury and related to the stress-mediated neuroinflammation [88,89]. Mast cells act by amplifying neuroinflammation through microglial and astrocytes activation [90]. They are the resident immune cells in the brain and play a pivotal role in immune surveillance of the central nervous system (CNS).
Whereby the mechanism employed by mast cells to transit the brain capillary endothelium remains to be fully characterized, several investigations support the hypothesis of the disruption of the blood-brain barrier [91]. Well in line with the literature, we observed occludin and claudin-5 protein changes in the brain harvested from the endometriosis rats. Additionally, our findings show increased mast cell degranulation and Iba1 and GFAP expression in endometriosis rat hippocampus. Hidrox ® treatment was able to reduce degradation of tight junction proteins and mast cell infiltration in the hippocampus. Therefore, Hidrox ® reduced microglial and astrocytes activation.
Additionally, ROS overproduction by microglia is suggested to be a main cause of neuronal damage and dysfunction [92][93][94] inducing derangement of neuronal redox signaling circuits or direct oxidative damage [95,96]. Multiple evidence supports the role of oxidative stress in the progress of endometriosis [13,14,97]. A recent study evaluated the brain oxidative status of rats subjected to endometriosis [98]. In particular, oxidative alterations in the hippocampus of endometriosis rats have been described. Here, we displayed that endometriosis induced a pronounced oxidative imbalance in rat hippocampus, as evidenced by the reduced levels of the endogenous antioxidant GSH, the increased SOD activity and the augmented lipid peroxidation.
Additionally, the induction of an immune oxidative environment in the endometriosis rat hippocampus is one of the underlying mechanisms of the pain sensitization observed. Hidrox ® administration was able to restore the oxidative balance in rat hippocampus subjected to endometriosis by managing GSH levels, SOD and MPO activity and lipid peroxidation. We propose that Hidrox ® antioxidant and anti-inflammatory activity would contribute to the reduced pain-like symptoms.
In accordance with these immuno-oxidative findings, it has been demonstrated that an increased oxidative status in the hippocampus is related to reduced BDNF levels [99]. It is a neurotrophic factor that regulates synaptic plasticity and brain neurogenesis. Moreover, BDNF controls the Nrf2 translocation into the nucleus, which is a transcription factor responsible for the activation of several antioxidant defenses [100]. Hidrox ® treatment by reducing the persistent state of oxidative stress in the hippocampus of endometriosis animals was able to restore BDNF levels which would be a central mechanism underlying the pro-oxidative and behavioral changes in endometriosis. As already described [25,101], Hidrox ® increased Nfr2 nuclear translocation which in turn activates several genes with cytoprotective function restoring the redox homeostasis.

Conclusions
In conclusion, our results show that Hidrox ® is a very effective antioxidant and a powerful anti-inflammatory agent. Therefore, our hypothesis is that Hidrox ® carries out its action through the modulation of the oxidant/anti-oxidant balance, the reduction of the hyperproliferation and angiogenesis leading to smaller lesion sizes. Hidrox ® administration by restoring oxidative balance in the hippocampus, a crucial mood-regulating region of the brain, also involved in the processing of nociception, relieves endometriosis-associated pain. Informed Consent Statement: Not applicable.

Data Availability Statement:
The data presented in this study are available on request from the corresponding author.

Conflicts of Interest:
Roberto Crea is the president of Oliphenol LLC and owns a patent for the Hidrox compound. The other authors declare no conflict of interest.