{"paper_id":"f1f800b0-b91c-4931-8119-1c1422653524","body_text":"Endometriosis, a chronic condition in which the endometrium, which usually provides the inner facing of the uterus, sprouts in other areas, normally on the bowel, ovaries, bladder, rectum, and pelvic lining. Depending on the stage of the disease, it could lead to dysmenorrhoea, infertility and chronic recurring pelvic pain in billions of women of reproductive age [ 1 ]. Retrograde menstruation is the generally accepted mechanism underlying the pathogenesis of endometriosis [ 2 ]. This mechanism was originally proposed in 1927, whereby endometrial fragments migrate from the fallopian tubes into the peritoneal cavity during menstruation. Once the endometrial debris becomes ectopic, adhesion needs to occur in order to initiate the development of lesions and the induction of endometriosis. While the mechanisms underlying this process remain unclear, it is considered that immune dysfunction and the subsequent inability to effectively clear these fragments enables endometrial lesions to form in the peritoneal cavity [ 3 ]. From the histological point of view, epithelial cells and stroma are capsuled in surrounding tissue and show extensive fibrosis and smooth muscle metaplasia. Lesions are characterized by invasiveness and mobility, fibroblast–myofibroblast differentiation and epithelial–mesenchymal transition. Recent findings underline the importance of the oxidative imbalance and inflammatory responses both at the lesion site and in the peritoneum and the related chronic pain state. These proinflammatory microenvironment includes inflammatory cytokines/chemokines, prostaglandins, growth factors (GR) and reactive oxygen species (ROS). In peritoneal fluid of patients, increased levels of protein oxidative stress markers, tumor necrosis factor-α (TNF-α), prostaglandin E2 (PGE 2 ) and interleukin (IL) IL-1β and IL-8 were found [ 4 , 5 , 6 , 7 ]. These mediators produce activation of sensory nerve and nociceptive pathways [ 4 , 8 , 9 ], proposing inflammatory mechanisms may be critical in endometriosis associated pain [ 10 , 11 ]. Further retrograde menstruations increase extra-uterine debris and lesions. The increased inflammatory answer within the peritoneum activates sensory nerves to induce chronic pelvic pain [ 12 ]. Moreover, stimulation of sensory afferent nerves leads to the recruitment of mast cells and consequent release of the previously mentioned proinflammatory mediators, which contributes to establishing a positive feedback loop called “neurogenic inflammation” [ 13 ]. The activation of peripheral nerve endings translates the stimuli to the spinal cord inducing central sensitization. Cannabinoid receptor and cannabinoids are potential targets for pain and inflammation [ 14 , 15 , 16 ]. Δ9-tetrahydrocannabinol (THC) and cannabidiol (CBD) are the primary compounds contained in  Cannabis sativa , although it contains more approximately 80 different cannabinoids [ 17 ]. CBD, structurally related to THC, is a non-psychoactive compound with therapeutic potential for inflammation, cancer and neuropathic pain [ 18 , 19 , 20 ]. Recent papers discussed the effects of CBD consumption in endometriosis focusing the attention on the pelvic pain and related symptoms [ 21 , 22 ]. CBD appears to be effective across all reported symptoms, with a noted propensity for inhaled delivery due to the potential increased speed of onset of effects versus the slower onset of oral products, particularly for pelvic pain. Conversely, oral forms appeared to be superior for the less reported mood and gastrointestinal categories. Whilst topical products demonstrated a good effect on pain, due to a very small data set, caution should be exercised in interpreting or extrapolating from this data.\nBasic and/or clinical studies have shown that cannabidiol has multidirectional properties, such as antioxidant, anti-inflammatory [ 23 ], immunomodulatory, antiarthritic, anticonvulsant, neuroprotective [ 24 ], precognitive [ 25 ], anti-anxiety, antipsychotic and anti-proliferative, among others [ 25 ]. Thus, CBD possesses wide therapeutic potential, which includes e.g., hypertension [ 26 , 27 ], epilepsy, neurodegenerative diseases (multiple sclerosis, Alzheimer’s, Parkinson’s and Huntington’s diseases) [ 25 , 28 ], neuropsychiatric disorders (depression, anxiety disorders, schizophrenia, post-traumatic stress disorder, autistic spectrum disorders) [ 25 , 29 ], gastrointestinal disorders (nausea and vomiting, inflammatory bowel diseases, irritable bowel syndrome) [ 30 ], rheumatic diseases [ 26 ], graft versus host disease and cancer [ 28 , 31 ]. However, most of these indications require further investigation to confirm clinical effectiveness.\nThis paper was aimed to evaluate the molecular properties of CBD in an in vivo model of endometriosis underlying its regulatory impact on the pathways involved in the pathology.\n\nAt the end of the experiment, pelvic ultrasound was employed to evaluate the presence of the endometriomas. The evaluation included both anterior and posterior pelvic compartments to evaluate the different endometriosis locations. Lesions were detected in the inner surface of the peritoneal cavity in both vehicle ( Figure 1 A) and CBD ( Figure 1 B) groups. The lesions of both groups appeared flat or nodular, like a plaque. Lesions from the CBD group appeared smaller and more superficially attached to the peritoneal cavity. The macroscopic analysis ( Figure 1 C,D) confirmed the hfUS examination. The two groups did not differ for the lesions number ( Figure 1 E), but diameter ( Figure 1 F), volume ( Figure 1 G) and area ( Figure 1 H) were significantly smaller in lesions harvested from the vehicle group ( Figure 1 C), as compared to the one harvested from the CBD group ( Figure 1 D). The histological analysis displayed that CBD administration also changed lesions morphology ( Figure 1 K). Lesions from vehicle treated rats showed characteristic endometrial glands and stroma ( Figure 1 I), which were significantly reduced in the histopathological marks of endometriosis ( Figure 1 J,K).\nThe anti-oxidant effects of CBD administration were evaluated by biochemical and Western blot analysis. The thiobarbituric acid reactive substances (TBARS) test showed an increased lipid peroxidation in lesions harvested from vehicle treated rats, while tissues harvested from CBD rats showed a reduced membrane peroxidation ( Figure 2 A). Moreover, CBD administration restored the endogenous cellular defense mechanisms increasing glutathione (GSH) levels ( Figure 2 B) and superoxide dismutase (SOD) activity ( Figure 2 C). Western blot analysis showed increased nicotinamide adenine dinucleotide phosphate (NADPH) oxidases (Nox) 1 ( Figure 2 D) and Nox-4 ( Figure 2 E) expressions in lesions from vehicle treated rats while CBD administration significantly reduced their expressions.\nCBD administration also showed important anti-fibrotic effects. Masson trichrome staining showed a reduction of collagen fibers in lesions from CBD treated rats ( Figure 3 B,C), as compared to the vehicle one ( Figure 3 A,C). Well in line with the staining, Western blot analysis confirmed this anti-fibrotic effect as showed by the reduced expression of matrix metallopeptidase 9 (MMP-9) ( Figure 3 D), inducible nitric oxide synthase (iNOS) ( Figure 3 E) and transforming growth factor-β (TGF-β) ( Figure 3 F) in CBD tissues, as compared to the vehicle treated rats.\nThe anti-inflammatory effects of CBD were assessed by Western blot analysis. Increased nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor alpha (Ikb-α) ( Figure 4 A) and reduced cyclooxygenase-2 (COX2) ( Figure 4 B) cytosolic expressions were found in samples harvested from CBD rats, as compared with the vehicle one. Additionally, a nuclear factor kappa light chain enhancer of activated B cells (NF-kB) expression was found to be reduced in the harvested lesions from CBD administered rats, as compared to the vehicle rats ( Figure 4 C). Endometriosis induced an increased inflammatory state in peritoneal fluid, as compared to the sham group. IL-1β ( Figure 4 D), TNF-α ( Figure 4 E) and PGE 2  ( Figure 4 F) levels were found to be reduced in the peritoneal fluids of rats treated with CBD.\nFurthermore, it has been shown that a significant increase in mast cell number in the spinal cord of rats subjected to endometriosis and treated with vehicle ( Figure 5 B,D), as compared to sham animals ( Figure 5 A,D). CBD administration strongly reduced mast cells recruitment in the spinal cord ( Figure 5 C,D). The reduced mast cells infiltration also relates with the decreased expression of the neurogenic mediators c-FOS ( Figure 5 E) and nerve growth factor (NGF) ( Figure 5 F) in the spinal cord of rats treated with CBD, as compared to the vehicle animals. Additionally, immunohistochemical analysis showed increased glial fibrillary acidic protein (GFAP) and ionized calcium binding adaptor molecule 1 (Iba-1) expression in the spinal cord harvested from vehicle treated rats ( Figure 5 H,J,L respectively), as compared to the sham animals ( Figure 5 G,J,K,L respectively). Tissues harvested from CBD-treated rats showed reduced expression of both neuroinflammatory markers ( Figure 5 I,J,M,N).\nEndometriosis is associated with behavioral alterations-pain induced. CBD administration ameliorated exploratory behavior and locomotor activity, as compared to the vehicle treated rats ( Figure 6 A–C). Additionally, rats treated with CBD and tested in the elevated plus maze test showed a reduced number of entries in closed and open arms ( Figure 6 D), % of open entries ( Figure 6 E) and the % of time in open ( Figure 6 F), as compared to the vehicle treated animals. Vehicle treated animals showed increased sensitivity to the acetic-acid-induced abdominal contractions ( Figure 6 G) and to thermal stimuli ( Figure 6 H), which were significantly reduced by CBD administration ( Figure 6 G,H).\n\nMolecular targets, pharmacokinetics, and safety and abuse liability of CBD have been widely discussed [ 32 ]. It is the primary non-addictive and non-euphorizing compound of cannabis and displayed therapeutic potential for healing a wide range of disorders including chronic inflammation [ 33 ], chronic pain [ 22 , 29 ], epilepsy [ 34 , 35 ], CNS disorders [ 36 , 37 , 38 ] and neuropsychiatric disorders [ 39 ]. Additionally, previous authors evaluated the effects of CBD in endometriosis [ 22 , 40 , 41 , 42 ].\nThis paper showed that the anti-oxidant, antifibrotic and anti-inflammatory activities of CBD would be useful to counteract the development of endometriosis and related chronic pain. CBD administration strongly reduced cyst diameter, volume and area. In particular, it was able to modify lesion morphology, reducing epithelial glands and stroma.\nFrom the molecular point of view, endometriosis is characterized by nitrosative and oxidative stress [ 43 ]. ROS produces free radicals and oxidizing agent that induce a cascade of reactions that produce pro-inflammatory mediators and growth factors that may stimulate the pathogenesis of fibrosis [ 44 ]. These ROS are released by the electron transport chain reactions and enzymes like COXs, oxidoreductases, peroxisomal oxidases, lipoxygenases and NADPH oxidases are attributed to ROS synthesis [ 45 ]. CBD reduced lipid peroxidation and restored the oxidative equilibrium in the endogenous cellular defense as showed by the SOD activity and the GSH levels in the lesions. Additionally, CBD reduced Nox-1 and Nox-4 expressions, enzymes that play a key role in the synthesis of H 2 O 2  and O 2 .  [ 46 ]. Nox-4 overexpression has also been related with TGF-β expression and fibrosis [ 47 ]. Masson trichrome staining showed a significant reduction of collagen deposition in animals treated with CBD. The reduced fibrosis was also confirmed by the downregulation of the MMP-9, iNOS and TGF-β expression, which were significantly increased in endometriotic lesions. The increased oxidative stress and fibrosis are accompanied with an increased inflammation [ 43 ]. A peritoneal inflammation is established when the uterus fragments implanted in the peritoneum, leading to the degranulation of mast cells, production of ROS, PGs, chemokines and cytokines in the peritoneal fluid. Increased inflammatory actors in the endometrium, peritoneal fluid and serum of patients have been recently described in several studies and defined as lesion inflammation [ 48 , 49 , 50 ]. CBD administration reduced lesion inflammation as displayed by the increased Ikb-α and reduced COX2 cytosolic expressions and reduced NF-kB nuclear localization in the harvested lesions. Moreover, it strongly reduced peritoneal inflammation, as showed by the decreased TNF-α, PGE 2  and IL-1β levels in the peritoneal fluid.\nThe immune response activated by the inflamed peritoneum may also contribute to directly activate nerve endings and induce positive feedback, further increasing proinflammatory mediator production. This neurogenic inflammation intensifies the painful stimuli transduced to the spinal cord, inducing chronic pelvic pain and central sensitization [ 51 , 52 ].\nMast cells play a key role in this contest. They could migrate from the nerve fibers, where they reside, to the spinal cord, to modulate neural activity and nociception [ 53 ]. Mast cell degranulation leads to the release of neuro-sensitizing and pro-inflammatory mediators [ 54 , 55 , 56 ]. CBD was able to down regulate mast cells migration and degranulation microglia in the spinal cord and in turn the expression levels of the pain-related pro-inflammatory mediators c-FOS and NGF. Mast cells infiltration also relates with the increased neuroinflammation and glial activation, which were assessed by Iba-1 and GFAP expressions. CBD administration significantly prevented both microgliosis and astrogliosis in spinal cord tissues. Well in line with these data, CBD administration from the behavioral point of view strongly reduced visceral sensitization altered pain threshold.\nThe current research has some limitations. The employed endometriosis model was applied by transplanting normal rat uterine tissue into the abdominal cavity of another rat. It does not accurately represent the pathogenesis of human endometriosis [ 57 ]. However, rat models have a long history of being widely used in endometriosis research and have also been validated as a model that depicts the pathology dynamics [ 58 ]. In future experiments, it would be interesting to study the lesions for a longer period of time.\n\nSprague–Dawley rats (8–10 weeks old) (Envigo, Milan, Italy) were used in this research. The University of Messina Review Board for animal care (OPBA) approved the study (897/2021-PR). All animal experiments agree with the new Italian regulations (D.Lgs 2014/26), EU regulations (EU Directive 2010/63).\nAnimals were randomly assigned to two groups, donor or recipient. Donor rats were injected intraperitoneally with 10 IU PMSG to induce similar estrogen levels between various animals. The animals were euthanized 41 h later by progressive CO 2  asphyxia. The uterus was removed through a midline incision and washed in PBS before extrauterine tissue, including ovary and oviduct, was removed under a dissecting microscope. A longitudinal incision was made from one horn to the other. Tissue was then transferred to a 1.5-mL centrifuge tube containing fresh PBS and minced with dissecting scissors [ 52 ]. Minced tissue from all donors was pooled, and the volume was adjusted to the equivalent of one uterus/500 µL of PBS. Recipient rats were injected intraperitoneally with the equivalent of tissue from one uterus in 500 µL of PBS (1:1 donor/recipient ratio) along the midventral line using an 18-gauge needle. The tissue was injected intraperitoneally to allow the development of lesion in the intraperitoneal region. The disease was allowed to establish for 7 days.\nRecipient animals were randomly divided into the following groups ( n  = 30):  (1) Endometriosis group: animals were subjected to the experimental protocol as already described, and vehicle (ethanol/Tween 80/0.9% saline (3:1:16)) was orally administered on the 7th day and for the next 7 days; (2) Endometriosis+CBD group: animals were subjected to the experimental protocol as already described, and CBD at the dose of 10 mg/Kg was orally administered on the 7th day and for the next 7 days; (3) Sham group: animals were subjected to the same experimental protocol but they were intraperitoneally injected with the equivalent volume of phosphate buffered saline (PBS) along the midventral line instead of endometrial tissue.\nEndometriosis group: animals were subjected to the experimental protocol as already described, and vehicle (ethanol/Tween 80/0.9% saline (3:1:16)) was orally administered on the 7th day and for the next 7 days;\nEndometriosis+CBD group: animals were subjected to the experimental protocol as already described, and CBD at the dose of 10 mg/Kg was orally administered on the 7th day and for the next 7 days;\nSham group: animals were subjected to the same experimental protocol but they were intraperitoneally injected with the equivalent volume of phosphate buffered saline (PBS) along the midventral line instead of endometrial tissue.\nCBD dosage was chosen based on previous experimental works [ 59 , 60 ].\nFourteen days from the endometriosis induction abdominal high-frequency ultrasound (hfUS) examination and behavioral analyses were performed. Animals were then euthanized by progressive CO 2  asphyxia and laparotomy was performed to evaluate and collect the endometrial lesions. Additionally, an L4–L6 area of spinal cord tissues was harvested to evaluate the pain-related pro-inflammatory mediators expression.\nThe hair in the ventral portion of the abdomen was carefully clipped from a point approximately 1 cm cranial to the xyphoid cartilage to the caudal-most part of the pubis. Alcohol and coupling gel were applied to the skin. To minimize interoperatorvariability, all ultrasonographic examinations were performed by the same operator (FM). Ultrasonographic examinations were performed using an Esaote MYLAB OMEGA VET (Esaote Italia, Milan, Italy) on sedated rats positioned in dorsal recumbency. Abdominal B-mode was performed with a High Frequency Linear array (4–15 MHz) transducer. The transverse and longitudinal scanning planes were used for evaluation of different abdominal structures. During the procedure, animals were anesthetized by 2% isoflurane. Measurements were performed offline (Esaote workstation) by a reader blinded to the condition of the rat [ 61 ].\nA squared open field area was employed to evaluate exploratory behavior and locomotor activity [ 62 , 63 ]. After 1 min of conditioning, each rat was observed for 5 min starting from a corner of the area. After each test, the apparatus was cleaned with a solution of 20% ethanol. The recorded parameters were: spontaneous locomotion, identified as number of animal crossings with four legs, number of entries in central square and time spent in the central square (in seconds).\nThe answer to thermal stimuli was evaluated by the hot plate test. A hot surface (53.0 ± 0.1 °C) was employed and a cut of 45 s was established [ 64 ].\nThe elevated plus maze apparatus employed [ 65 ] is composed of a central square connected to two closed arms and two open arms. Each rat was evaluated for 5 min. After each test, the apparatus was cleaned with a solution of 20% ethanol. For cleaning the apparatus after each analysis, a solution of 20% ethanol was used. The number of entries open arms and the time spent in it were reported as the %.\nThe animals were tested for 20 min after an intraperitoneal injection of 0.6% acetic acid and the number of acid-induced writhes was recorded [ 52 ].\nReduced GSH levels were evaluated in endometriosis lesions using a microplate reader at 412 nm as already described [ 66 , 67 ].\nThe TBARS test was employed to evaluate the lipoperoxidation. The levels of MDA were assessed using a microplate reader at 535 nm as already described [ 68 , 69 ].\nSOD activity was determined as already shown and expressed as U/g protein [ 54 , 70 , 71 ].\nIL-1β, TNF-α and PGE 2  and levels were determined in peritoneal fluids using an ELISA kit (BioLegend, San Diego, CA, USA; R&D Systems, Milan, Italy) [ 72 , 73 ].\nFor the histological analysis, endometriosis explants were fixed in buffered formaldehyde solution, dehydrated and embedded in Paraplast [ 74 ]. Tissue slides were stained with H&E and evaluated using a Leica DM6 microscope (Leica Microsystems SpA, Milan, Italy) [ 75 ]. Histopathologic scores were evaluated with the formula P (persistence of epithelial cells in the explants) × I (intensity of glands) as already described [ 76 ]. 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). Lesion volume was calculated according to the formula: V = (length × width 2 ) × 0.5. [ 76 ]. Explants fibrosis was evaluated by the Masson trichrome staining (Bio-Optica, Milan, Italy) [ 77 , 78 ]. Mast cell analyses were performed by Toluidine blue staining employed to evaluate mast cells recruitment [ 79 ].\nImmunohistochemical localization of anti-GFAP (sc-33673) or anti-iba-1 (sc-32725) was performed in the spinal cord as already described [ 80 , 81 ]. All sections were incubated with the primary antibodies, then washed with PBS and treated as previously reported [ 82 , 83 ]. Stained sections were observed using a Leica DM6 microscope (Leica Microsystems SpA, Milan, Italy). The histogram profile is related to the positive pixel intensity value obtained [ 84 ].\nLesion samples and spinal cord tissues were homogenized and Western blots were performed as already described [ 85 , 86 ]. Specific primary antibody anti-Nox-1 (PA5-103220), anti-Nox-4 (PA5-72816), anti-MMP-9 (sc-13520), anti-iNOS (sc-7271), anti-TGF-β (sc-130348), anti-Ikb-α (sc-1643), anti-NF-kB (sc-8008), anti-COX2 (sc-376861), anti-c-FOS (sc-166940) and anti-NGF (sc-32300) was mixed in 5%  w / v  nonfat dried milk solution and was incubated overnight. Afterward, blots were incubated with peroxidase-conjugated bovine anti-mouse IgG secondary antibody or peroxidase conjugated goat ant-irabbit IgG (Jackson Immuno Research, Milan, Italy) for 1 h at room temperature [ 62 ]. Membranes were also blotted with the antibody against β-actin or lamin B1. Signals were detected with enhanced chemiluminescence detection system reagent (Super-SignalWest Pico Chemiluminescent Substrate) [ 87 ]. The relative expression of the protein bands was quantified by densitometry with Bio-Rad ChemiDoc XRS software (Bio-Rad, Milan, Italy) and standardized to β-actin or lamin B1 levels. Images of blot signals were imported to analysis software (v2003, Image Quant TL).\nAll 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.\n\nIn conclusion, this paper focuses the attention on the CBD effect on endome-triosis, using an in vivo model. The data collected underlined its role in the upregulation of the ROS scavenging (SOD) enzymes and endogenous antioxidant systems (GSH) and downregulation of ROS producing (Nox) enzymes. These downregulations also link with anti-inflammatory and anti-proliferative and effects at the lesion size and in the peritoneal fluids. CBD administration also reduces the neurogenic inflammation by decreasing mast cells infiltration and degranulation in the spinal cord. Moreover, it reduced expression of the related neuro-sensitizing mediators, leading to a reduction of the astrocytes and mi-croglia activation. All these effects definitely are responsible of the reduced pain behavior and visceral sensitization.","source_license":"CC0","license_restricted":false}