Non-Hormonal Therapy for Endometriosis Based on Angiogenesis, Oxidative Stress and Inflammation

Endometriosis, a prevalent benign condition, impacts approximately 10% of women in their reproductive years ( 1 ). It is characterized by the presence of endometriallike tissue outside the uterus, which includes stromal fibroblasts, epithelial cells, nerves, immune cells, and vascular/perivascular cells. Patients with endometriosis often suffer from dysmenorrhea, dyspareunia, pain during defecation, dysuria and chronic pelvic pain, as well as infertility, which significantly reduces their quality of life. The selection of the treatment approach is determined by several factors including the patient's age at the time of diagnosis, the stage of the disease, the symptoms experienced by the patient, their priorities and expectations, plans for fertility, safety considerations, occurrence of potential side effects, tolerability, and cost ( 2 ). Considering that the current endometriosis treatments are associated with recurrence of pain and disease despite the treatment of the disease and have many side effects, it seems that non-hormonal treatments and compounds that target angiogenic processes, oxidative stress, and inflammation may be particularly useful for loss of progression of endometriotic lesions ( 3 ). The aim of this study is to review and evaluate non-hormonal treatments for endometriosis, with a focus on key biological processes such as angiogenesis, oxidative stress, and inflammation. While several theories have been proposed as to how endometriosis develops, the etiology of the disease process is still debated. The proliferation of endometrial cells and the induction of an inflammatory response are the common denominator of all the most prevalent ideas, which include coelomic metaplasia, stem cells, Sampson's hypothesis of retrograde menstruation, and lymphatic/ hematogenous spread ( 4 ). Benign metastasis theory states that the cause of endometriosis lesions is lymphatic/hematogenous dissemination of endometrial tissue. In studies, it was found that lymph flows from the body of the uterus into the ovary, which supports the role of the lymphatic system in the development of ovarian endometriosis. In addition, it has been found that in 6-7% of lymphadenectomies, lymph nodes contain endometrial tissue ( 5 ). This finding has also been shown in a model of induced endometriosis. Histological reports that show the presence of endometriosis lesions in places far from the uterus, such as bone, lung and brain, are considered strong evidence in support of the theory of benign metastasis ( 6 ). In the treatment of endometriosis, control of pain symptoms and prevention of recurrence after surgery in the framework of long-term treatment strategies should be considered as the main goal, however, current treatment options for endometriosis are not satisfactory. Primary treatment of endometriosis is limited to hormonal treatments ( 7 ). Existing drugs (eg, combined oral contraceptive pills, progestins and gonadotropinreleasing hormone (GnRH) analogs, oral antagonists, and letrozole) disrupt ovarian function and are not therapeutically effective. Actually, there is a chance that using anti-estrogen medications will have serious negative effects, such the patient developing osteoporosis and their depression getting worse. However, it has been demonstrated that a substantial recurrence incidence follows surgical excision of endometriosis lesions ( 8 ). Therefore, patients with endometriosis urgently need non-hormonal treatments that increase the regression of endometriosis lesions without causing severe side effects and affecting fertility. On the other hand, although the cause of endometriosis is still debated, there is a general consensus that several biological processes such as angiogenesis and vasculogenesis, oxidative stress, and inflammation, contributes to its complex pathophysiology. Accordingly, it seems that non-hormonal therapies and compounds that simultaneously target these processes may be particularly suitable for the prevention and eradication of endometriotic lesions. Selecting the most suitable protein targets for pharmacological therapy of endometriosis requires an understanding of the key biochemical processes involved in its development. We go over a few of the key molecular pathways that lead to the development of illness below. Like other tumor cells, endometriotic lesions also need dense blood vessels and new blood sources to transport oxygen, nutrients, eliminate catabolites, and maintain fluid balance for their creation, survival, and proliferation, and in fact, angiogenesis is not only important for the development of the disease, but also plays an important role in the development of endometriosis through the creation of a vascular network ( 9 ). This issue has been confirmed by increasing the expression of angiogenic factors, especially in active lesions. In addition, angiogenesis is essential for normal uterine function. Research has indicated that the eutopic endometrium in women with endometriosis has the potential to produce angiogenic factors ( 10 ). These factors can cause the abnormal growth of invasive cells in various areas outside the uterus, such as the ovaries, pelvic peritoneum, intestine, and diaphragm. This growth occurs in a hormonal and immune environment that is favorable for the development of endometriosis. Vascular endothelial growth factor (VEGF), a key angiogenic factor, plays a significant role in promoting angiogenesis. This glycoprotein stimulates the growth and movement of endothelial cells, enhances vascular permeability, and contributes to the formation of new blood vessels in lesions and endometriosis tissue ( 11 ). Peritoneal macrophages and implanted tissue are responsible for producing VEGF. Additionally, its concentration rises in peritoneal fluid, serum, ovarian endometrioma, and both eutopic and ectopic endometrium in individuals with endometriosis. Therefore, it can be concluded that the use of drugs that have the ability to inhibit angiogenesis can be a promising approach in the treatment of endometriosis ( 12 ). Oxidative stress refers to an imbalance between the productions of reactive oxygen species (ROS) and the availability of antioxidants. ROS is a by-product of cellular metabolism, and when its production exceeds a certain level, it can cause damage to the structure and function of cells. In fact, excessive ROS production is a major contributor to cell death. Interestingly, oxidative stress has been found to stimulate the proliferation of endometrial and tumor cells, suggesting that ROS acts as a second messenger in cell proliferation ( 13 , 14 ). The cause of the proliferative response observed in the follicular fluid (FF) of women with endometriosis was previously unknown. However, it has been discovered that the induction of oxidative stress in the FF is responsible for this response. It is believed that the FF may transmit this oxidative stress into the peritoneal fluid after ovulation, which then leads to an increased proliferation of ectopic endometrial cells. Numerous studies have examined the antioxidant balance of oxidants in the blood, peritoneal fluid, FF, and tissue environment of endometriosis patients ( 15 ). The levels of ROS in the serum and FF of individuals with endometriosis are significantly higher compared to those in the control group. Biomarkers of lipid oxidation, including conjugated diene/triene, malondialdehyde, and oxidized low-density lipoproteins, are elevated in both the peritoneal fluid and serum of endometriosis patients. Furthermore, several studies have demonstrated that individuals with endometriosis have lower concentrations of antioxidant components such as vitamin A, vitamin C, vitamin E, and superoxide dismutase (SOD) in their serum and FF ( 16 ). The study conducted by Nasiri et al. ( 3 ) revealed that women diagnosed with endometriosis exhibited elevated levels of lipid peroxides (LPO) and decreased levels of total antioxidant capacity (TAC) in both their FF and blood serum, in comparison to women in the control group. These findings indicate that oxidative stress plays a significant role in the progression of endometriosis, and suggest that the utilization of antioxidants could potentially ameliorate the symptoms associated with this condition. Endometriosis has many characteristics of an immune disorder. The development of endometriosis lesion along with the secretion of excess cytokines in the angiogenic microenvironment creates an external chemotactic gradient ( 17 ). The levels of macrophages and their secreted products, including growth factors, cytokines, and angiogenic factors, are altered in the peritoneal fluid of women with endometriosis. Research has shown an increase in the expression of VEGF, and its receptors, as well as pro-inflammatory cytokines such as interleukin-6 (IL-6), IL-8, and IL-1 in these patients compared to those without the condition ( 18 ). Pro-inflammatory cytokines like IL-1 and tumor necrosis factor-alpha (TNF-α) promote angiogenesis and are produced by macrophages in response to inflammatory stimuli ( 19 ). Research has shown that the activation of genes involved in angiogenesis and tissue remodeling, such as nuclear factor-κB (NF-κB), is responsible for the development of endometriosis. TNF-α, an angiogenic cytokine, plays a role in promoting the production of other cytokines and the proliferation of endometriotic stromal cells in patients with endometriosis ( 20 ). It seems that these pro-inflammatory cytokines and angiogenic factors contribute to the formation of ectopic lesions, leading to the development of endometriosis ( 21 ). In fact, the levels of inflammatory cytokines and angiogenesis factors are elevated in patients with endometriosis, and these levels are influenced by the presence of endometriosis, resulting in an increased number of ectopic lesions. Studies show that the incidence of immune disorders is significantly increased in patients with endometriosis, especially autoimmune diseases and celiac disease ( 22 ). In general, it can be said that immunotherapy may be a promising approach and an effective method in the treatment of this disease. According to recent research, several hormonal medications, including selective progesterone receptor modulators and aromatase inhibitors, have not proven to be effective in treating endometriosis ( 23 ). Consequently, it would appear that non-hormonal substances that concurrently address oxidative stress, inflammation, and angiogenic processes would be more appropriate for the avoidance and removal of endometriosis lesions. Based on the findings of several research studies to date, non-hormonal therapies may be categorized into three subgroups: natural substances, immune modulators, and anti-angiogenic medicines ( 24 ). Rapamycin is a widely used drug that has antifungal, immunosuppressive, and antitumor activities. In a study, it was determined that rapamycin as an anti-angiogenic agent plays a role in the treatment of endometriosis in a way that leads to the regression of endometriosis lesions with a significant reduction in the size of the lesions in a short period of 14 days. In fact, this study showed that this drug, by inhibiting angiogenesis, leads to a decrease in the density of small vessels and newly formed microvascular networks in endometriosis lesions, and it was found that treatment with rapamycin leads to a decrease in the expression of VEGF, which is one of the strongest angiogenic growth factors ( 25 ). As a result, rapamycin medication might provide a novel therapeutic strategy for endometriosis' anti-angiogenic management. The use of drugs such as rapamycin, whose side effects are not fully understood, it is preferable to assess the likelihood of their efficacy using in silico approaches before to prescription to patients. Nowadays, it's common practice to employ in silico techniques to lessen the adverse effects of medications and unidentified chemicals ( 26 ). Xanthohumol, a prenylated flavonoid found in hops, has been identified as a potent cancer-inhibiting compound. It targets multiple cellular mechanisms, leading to decreased proliferation and increased apoptosis in various cancer cell lines. Furthermore, xanthohumol exhibits anti-inflammatory properties by inhibiting NFκB signaling and reducing the expression of inflammatory cytokines such as IL-1. Studies have also demonstrated its ability to inhibit VEGF secretion in both acute and chronic myelogenous leukemia cell lines ( 27 ). This suggests that xanthohumol effectively targets the angiogenic process. Studies have shown that xanthohumol inhibits the angiogenesis process and leads to a significant reduction in the vascularization of endometriosis lesions. The Rudzitis-Auth study showed that xanthohumol inhibited cell proliferation in microvessels of endometriosis lesions without inducing endothelial cell apoptosis, which may be due to direct inhibition of VEGF signaling. On the other hand, xanthohumol is able to scavenge ROS, including hydroxyl and peroxyl radicals. In addition, it reduces the expression of VEGF in endometrial cells by inhibiting oxidative stress. Furthermore, it has been shown that xanthohumol may inhibit the proliferation and growth of endometriotic lesions by inhibiting Phosphoinositide 3-kinase (PI3-K) signaling in endometriotic lesions ( 28 ). It was also found that xanthohumol may affect the secretory activity of glandular epithelium. In addition, consistent with previous studies, it was found that the efficacy of xanthohumol treatment was not essentially dependent on the location of endometriosis lesions within the peritoneal cavity. In addition to the analysis of endometriosis lesions, the effect of xanthohumol treatment on the uterus and ovaries was investigated to assess whether xanthohumol may cause severe side effects in reproductive organs. This is not unlikely, because these organs have high angiogenic and proliferative activity. Interestingly, it was found that the uterine horns and ovaries of both groups of animals treated with xanthohumol and control animals showed normal histomorphology and no functional differences were observed in terms of angiogenesis and cell proliferation. These results were consistent with the study of Hussong et al. ( 29 ) who reported that even oral administration of xanthohumol at doses higher than 100 mg/kg body weight did not cause any adverse effects on fertility and embryo development in rats. These results show that xanthohumol can be useful for the treatment of endometriosis by inhibiting the growth and angiogenesis of endometriosis lesions without affecting the reproductive organs. Calligonum comosum, known as Escanbil, has antioxidant, anti-inflammatory and anti-cancer effects ( 30 ). In the study of Kiani et al. ( 1 ), it was shown for the first time that Calligonum comosum is also effective in inhibiting angiogenesis and due to its functional profile, it can be a promising option for the treatment of endometriosis. For the in vivo tests, they employed this last Calligonum comosum complete extract (CCTE). It was demonstrated that in mice endometriosis lesions, this plant’s extract inhibits growth, cyst formation, angiogenesis, and immune cell infiltration ( 31 ). This plant's potent chemicals work by blocking the enzymes lipoxygenase (LOX-5) and cyclooxygenase (COX-2). In fact, by preventing inflammation and angiogenesis, CCTE can actually mitigate the illness ( 1 ). Resveratrol ( 3 , 5 , 4ʹ-trihydroxy-trans-stilbene) is a naturally synthesized polyphenolic compound found in some fruits such as grapes, berries and nuts. There are many studies showing the anti-neoplastic, anti-inflammatory, antioxidant and anti-angiogenic effects of resveratrol ( 31 ). In an experimental model of endometriosis, the antiangiogenic and anti-inflammatory effect of resveratrol was confirmed by showing a decrease in serum levels of VEGF and MCP1 in endometriosis implants. In addition, resveratrol inhibits the formation of new blood vessels by significantly reducing the expression of VEGF in a mouse model of endometriosis ( 32 ). Experimental evidence shows that resveratrol effectively inhibits inflammatory responses induced by Sirtuin 1 (SIRT1) pathway in endometrial stromal cells (ESCs). Recently, a study by Khodarahmian et al showed that resveratrol reduces the expression of VEGF and TNF-α as angiogenic and inflammatory markers ( 33 ). Therefore, based on these results and considering the pro-apoptotic, antiinflammatory, antioxidant and anti-angiogenic effects of resveratrol, this drug may be a suitable treatment for endometriosis patients. Curcumin is the main polyphenol isolated from turmeric. Curcumin’s anti-tumor, anti-mutagenic, anti-metastatic, anti-inflammatory, antioxidant, anti-angiogenic, wound healing, hypoglycemic, antimicrobial and hormoneregulating properties have been confirmed by numerous studies ( 34 ). Based on the results of a laboratory study, it seems that curcumin leads to the improvement of folliculogenesis in endometriosis. In addition, curcumin prevents the development and spread of endometriosis by reducing estrogen production ( 35 ). Many animal studies have reported a reduction in endometriotic implant size or delayed growth after curcumin administration ( 36 ). Human studies have reported the effects of curcumin in downregulating the VEGF signaling pathway and reducing serum CA125 and prostaglandins (PGE2) levels ( 37 ). Therefore, curcumin seems to have potential benefits as a dietary supplement and pharmacological agent for the prevention or treatment of endometriosis. Quercetin, also known as 3, 3′, 4′, 5, 7-pentahydroxyflavone, is a flavonol that can be found in various vegetables and fruits, including onions, cauliflower, lettuce, apple skin, and chili peppers. It has been found to possess properties that can inhibit cell proliferation and induce cell cycle arrest in endometriosis cells. Additionally, quercetin has been observed to trigger apoptosis by generating reactive oxygen species, causing DNA fragmentation, and disrupting mitochondrial membrane potential ( 38 ). Furthermore, an animal study has demonstrated its anti-estrogenic and progesteronic effects ( 39 ). Therefore, quercetin may have a beneficial impact on the healing and treatment of endometriosis. N-acetylcysteine (NAC), which is the acetylated form of the amino acid cysteine, is naturally found in certain vegetables like onion and garlic. It has been found to have an inhibitory effect on the growth of cancer cells ( 40 ). Research conducted in laboratories has shown that the mechanism of action of NAC does not involve causing cell death or inducing a specific toxic effect. Instead, it is related to the differentiation pathway, which includes activating specific molecular mechanisms that result in a change in the rate of cell proliferation. Ultimately, this leads to a decrease in cell proliferation and a decrease in cell locomotory behavior. Additionally, NAC has anti-inflammatory properties ( 41 ). Animal studies have demonstrated that NAC significantly reduces the average size of lesions, as well as the levels of TNF-α in the serum and peritoneum, and the levels of the inflammatory enzyme COX-2. This could potentially explain the observed decrease in the dimensions of endometriomas ( 42 ). In fact, reducing the expression of COX-2 decreases the amount of estrogen in the ectopic endometrial tissue by reducing the production of PGE2, which is a potent stimulator of aromatase. Researchers reported a reduction in the size of endometrioma in women who took NAC and concluded that NAC can effectively help treat endometriosis and prevent its recurrence and the possible reason for that is the effect of NAC on cell signaling and the activity of proteins involved in proliferation. In another study, the combined effect of NAC, alpha lipoic acid and bromelain was a significant reduction of pain in women with endometriosis ( 43 ). In a human study recently conducted by Asgari et al. ( 44 ), it was determined for the first time that the addition of NAC to low dose contraceptive treatment has a similar effect in reducing the recurrence rate of endometrioma and pelvic pain compared to low dose contraceptives alone, and it was suggested that in subsequent studies and in order to increase the effectiveness of this treatment method, the duration of drug use should be increased. Alpha-lipoic acid (α-LA) is a substance that is naturally found in spinach, broccoli and tomatoes and is also synthesized in the human body. When α-LA is taken orally, it is absorbed from the intestinal villi and converted in liver cells to dihydrolipoic acid (DHLA), a powerful antioxidant. In a study conducted in an experimental model of endometriosis, it was found that oxidative stress, endometrial implant volume and histopathological scores were lower in the group treated with α-LA compared to the control group ( 2 ). Other studies have shown that regular use of α-LA significantly improves endometriosis-related pain symptoms such as dyspareunia, dysmenorrhea, and chronic pelvic pain, as well as increases quality of life and sexual function ( 2 , 45 ). α-LA can be a good option for the treatment of endometriosis, especially in patients who mainly suffer from endometriosis pain. Vitamin C is a water-soluble vitamin that is naturally found in citrus fruits, tomatoes, potatoes, red and green peppers, kiwi, broccoli, and strawberries. Intravenous injection of vitamin C leads to the inhibition of endometriotic implant induction and facilitating the regression of endometriotic implant volume, which is due to the anti-inflammatory and anti-angiogenic effects of vitamin C. Also, vitamin C significantly reduces the volume and weight of endometriosis cysts in a dosedependent manner ( 46 ). In an experimental model, in the group receiving vitamin C, the volume of the endometriosis implant was significantly lower than the control group ( 47 ). Human studies on the effects of vitamin C on endometriosis are few, a randomized controlled trial showed that vitamin C led to an increase in the level of vitamin C in FF, however, it had no effect on markers of oxidative stress ( 48 ). Due to its antioxidant, anti-inflammatory and anti-angiogenic effects, vitamin C can be considered to prevent the growth of endometriotic implants, especially in patients with high oxidative stress. Vitamin E, which can be found in vegetable oils, nuts, seeds, fruits, and vegetables, is composed of tocopherols and tocotrienols. When taken orally, vitamin E is absorbed by the intestinal villi and transported to the liver through chylomicrons. Alpha-tocopherols are carried into the bloodstream after binding to specific proteins. Alphatocopherol, also known as vitamin E, helps prevent lipid peroxidation and oxidative stress ( 2 ). Research has shown that patients with endometriosis tend to have lower levels of vitamin E ( 2 , 49 ). It appears that individuals with endometriosis have higher levels of lipid peroxidase and lower levels of selenium, SOD, and vitamin E compared to healthy individuals ( 2 ). Patients with moderate to severe endometriosis have lower levels of vitamin E and glutathione compared to those with minimal to mild endometriosis, suggesting that the reduction in the antioxidant system is related to the severity of the disease. It is reported by investigators that patients with endometriosis who received antioxidant therapy combined with vitamin C and E experienced significant reductions in chronic pelvic pain, dysmenorrhea, dyspareunia, and inflammatory parameters IL-6 and MCP-1 in peritoneal fluid. Another study showed that levels of malondialdehyde (MDA) and lipid hydroperoxides (LOOH), indicators of oxidative stress in plasma and peritoneal fluid, were significantly lower after six months of treatment with vitamins C and E. Vitamin E has an antioxidant effect, particularly on lipid peroxidation. However, it may be more effective to use a combination of vitamin E and vitamin C supplements to alleviate pain. Vitamin D is a fat-soluble vitamin that is naturally found in fatty fish such as salmon, tuna, and mackerel, as well as in liver, egg yolk, and cheese. It is mainly present in the form of vitamin D3 and its metabolite 25(OH)D3. Vitamin D, also known as 1,25-dihydroxyvitamin-D3, plays a crucial role in immune modulation and affects cell differentiation and proliferation. There is a connection between Vitamin D deficiency, endometriosis, and autoimmune diseases ( 50 ). Furthermore, the presence of vitamin D receptors and vitamin D metabolizing enzymes has been observed in the ovaries and endometrium of both healthy women and those with endometriosis. These receptors and enzymes are also found in immune cells. This suggests that vitamin D plays a role in the local immune response within these tissues ( 51 ). Vitamin D has been shown to decrease levels of both pro-inflammatory cytokines (such as TNF-α, IL-2, IL-6) and antiinflammatory cytokines [like transforming growth factorbeta (TGF-β), IL-4], indicating its potential to alleviate symptoms of chronic inflammatory and autoimmune conditions ( 52 ). Research on animal models has shown that vitamin D supplementation leads to a decrease in IL-6 levels in peritoneal fluid ( 53 ). A recent study conducted on the status of vitamin D in patients with endometriosis revealed that women diagnosed with endometriosis had lower levels of vitamin D compared to the control group. Furthermore, the study found a negative correlation between the severity of endometriosis and vitamin D levels ( 54 ). Omega-3 Polyunsaturated fatty acids (PUFAs) are rich in omega-3 fatty acids (FAs). The anti-proliferative, antiangiogenic, anti-inflammatory and anti-apoptotic effects of omega-3 PUFAs have been demonstrated in several studies ( 55 ). Fish oil contains an omega-3 polyunsaturated fatty acid called Eicosapentaenoic acid (EPA), which stops arachidonic acid from converting to PGE2 and leukotrienes (LTB4) ( 56 ). Animal studies have reported a reduction in pain associated with endometriosis after omega-3 supplementation ( 57 ). In addition to improving symptoms, PUFAs also play a role in preventing disease pathogenesis ( 58 ). Also, adding fish oil to the diet of endometriosis models reduced the size of lesions caused by endometriosis ( 59 ). EGCG is a plant compound found especially in green tea. Epigallocatechin-3-gallate (EGCG) has been shown to inhibit VEGF expression and estrogen-dependent activation and proliferation of endometrial cells in vitro ( 60 ). It also significantly reduces the number and volume of endometriotic implants, inhibits cell proliferation, reduces vascularity, and increases apoptosis ( 61 ). A study conducted on mouse models of endometriosis showed that EGCG significantly reduced the growth of endometrial implants and lesion size and weight, and inhibited angiogenesis and induced apoptosis in the lesion ( 62 ). Another animal study reported that EGCG down-regulates the VEGF signaling pathway and down-regulates VEGF expression, thereby inhibiting angiogenesis in endometriotic implants ( 63 ). There are no human studies on the effects of EGCG on endometriosis. Aspirin (Asp) is a synthesized chemical compound that is one of the most effective drugs in treating fever, inflammation and pain in patients with very few side effects. In addition, it has been found that Asp has the ability to inhibit fibrogenesis, reduce the epithelialmesenchymal transition (EMT), reduce the capacity of cell migration/invasion, and delay apoptosis ( 64 ). In the study of Nasiri et al. ( 65 ), for the first time, the effect of Asp on the expression of inflammatory factors, proliferation, invasion, adhesion and migration capacity of eutopic ESCs (EuESCs) obtained from endometrial biopsy of patients with severe endometriosis (stage IV) was investigated in comparison to control ESCs (CESCs). Asp, as an efficient drug for the treatment of several types of cancer, probably acts through regulation of NF-κB signaling and simultaneous inhibition of proliferation and regulation of apoptosis ( 66 ). It has been found that Asp leads to the inhibition of NF-κB mainly by inhibiting the activity of IKK. It is a factor that separates NF-κB from its inhibitory factor, IKB, and leads to NF-κB activation ( 67 ). This evidence indicates the effectiveness of Asp in controlling endometriosis. Metformin, which was discovered in 1922, is widely used as an anti-diabetic medication. Recent research has shown that diabetic patients treated with metformin have a lower risk of developing cancer compared to those taking other antidiabetic drugs. Additionally, combining metformin with chemotherapy agents such as carboplatin, cisplatin, doxorubicin, and paclitaxel has been found to enhance the effectiveness of these drugs by increasing their cytotoxicity. Metformin has also been investigated as a potential treatment for cancer ( 68 ). Studies have demonstrated that metformin can reduce the migration and proliferation of ESCs in a time-dependent manner. It has been shown to decrease the expression of VEGF, Hypoxia-inducible factor (HIF), MMP-2, and MMP-9, while increasing the expression of TIMP in ECT-ESCs. These effects may be attributed to metformin's antiinflammatory properties, which can help mitigate the inflammatory processes associated with endometriosis lesions. Inflammation in endometriosis can stimulate angiogenesis and contribute to the progression of the condition through elevated VEGF levels ( 69 ). A study demonstrated that the expression of angiogenic factors such as VEGF can be reduced by metformin, leading to an improvement in sensitivity to progesterone through an increase in the expression of the progesterone receptor ( 70 ). Yilmaz et al. ( 71 ) found that metformin treatment resulted in a notable decrease in VEGF and MMP-9 levels, an increase in TIMP levels, and regression of endometriotic implants. Clinical trial results on endometriosis patients indicated that metformin significantly reduced dysmenorrhea, pelvic pain, and serum VEGF levels ( 72 ). Based on its ability to lower inflammation, inhibit angiogenesis, and impede cell proliferation, it can be inferred that metformin holds potential as a treatment for endometriosis. Pentoxifylline (PTX), a methylxanthine with known hemorological activity, has been used for many years in the treatment of peripheral vascular disease. Several studies show the anti-inflammatory, antioxidant and antiangiogenic effect of this drug ( 73 ). In an animal model, PTX is able to induce regression of endometriotic tissue without causing a hypoestrogenic state ( 74 ). In another animal study, it was found that pentoxifylline, in addition to reducing the number and size of endometriosis lesions, leads to a decrease in the expression of the angiogenic factor VEGF-C and its receptor flk-1 ( 75 ). In another animal study, Perello et al. ( 73 ) reported that PTX significantly reduced the number and size of endometriosis lesions, decreased the expression of inflammatory cytokines (IL-1b, IL-6, and TNF-α) and increased the expression of the anti-inflammatory cytokine IL-10. In addition, it leads to a decrease in VEGF expression and angiogenesis. There are few randomized, controlled trials in humans evaluating the efficacy of PTX after surgical treatment of endometriosis ( 76 ). Therefore, it seems that this drug can potentially be used in new treatment methods for endometriosis. However, more human studies are needed. The drug research and development (R&D) for endometriosis has been painfully slow ( 77 ). Nowadays, the use of computational methods has been very effective in drug discovery for the treatment of multifactorial diseases ( 78 , 79 ). According to scattered studies in the field of identifying effective drugs in the treatment of endometriosis, the use of in silico methods is very effective in increasing the speed and accuracy to discover drugs in the treatment of this disease ( 80 ).

Considering that the current endometriosis treatments are associated with recurrence of pain and disease despite the treatment of the disease and have many side effects, it seems that non-hormonal treatments and compounds that target angiogenic processes, oxidative stress, and inflammation may be particularly useful for loss of progression of endometriotic lesions. It is important to note that studies in this field often have limitations, such as experimental study design and low sample size. In addition, one should keep in mind that these drugs may have different mechanisms of actions, also there are still questions and there is no consensus about on dosing and recommendation guidelines. Using in silico methods can help to choose more accurately FDAapproved drugs and herbal compounds for the treatment of endometriosis.