Endometriosis is a chronic, estrogen-dependent inflammatory disease affecting approximately 10% of reproductive-aged women. Despite its high prevalence, the pathophysiology remains incompletely understood, and current treatment options are limited by side effects and recurrence rates. In this study, we established a surgically induced endometriosis model in rats and demonstrated that bromelain treatment significantly improved endocannabinoid levels and ameliorated oxidative stress markers, highlighting its potential as a multi-target therapeutic candidate in endometriosis.

The surgical autotransplantation model used in this study, first described by Vernon and Wilson in 1985, remains the most widely employed experimental approach for studying endometriosis pathophysiology and therapeutic interventions ^8,9 . Our biochemical findings demonstrated significant reductions in serum 2-AG and anandamide (AEA) levels following endometriosis induction, consistent with clinical observations in women with endometriosis ¹⁰ . Andrieu et al. reported significantly lower peritoneal fluid concentrations of AEA and 2-AG in affected women and identified a negative correlation between endocannabinoid levels and pain intensity, suggesting that endocannabinoid deficiency may contribute to pain sensitization mechanisms in endometriosis ¹¹ .

The distinct biological roles of AEA and 2-AG warrant consideration. AEA preferentially activates CB1 receptors involved in nociceptive pathways, whereas 2-AG acts as a full agonist at both CB1 and CB2 receptors and plays a role in immune modulation ^11,12 . The concurrent reduction of both mediators observed in our model suggests a broader dysregulation of the ECS rather than selective impairment of a single signaling axis.

Emerging evidence implicates the ECS in angiogenesis and cellular survival. While CB1 activation may promote neovascularization, CB2 signaling appears to exert inhibitory effects on aberrant angiogenesis ¹³ . Reduced endocannabinoid tone may contribute to lesion persistence through impaired regulation of inflammatory and survival pathways.

Consistent with previous literature, our results demonstrated significant oxidative stress in the EMS group, characterized by decreased SOD and GSH levels and elevated MDA concentrations ¹⁴ . These findings reinforce the concept that redox imbalance is a central component of endometriosis pathophysiology and may interact with ECS dysregulation to perpetuate inflammation and lesion persistence.

Recent clinical discourse has questioned the routine surgical management of endometriomas. The Reproductive BioMedicine Online (RBMO) review by Urman et al. ¹⁵ emphasizes that surgical intervention should be carefully individualized due to risks including diminished ovarian reserve, recurrence, and procedural complications. In parallel, hormonal therapies, although effective in symptom control, are associated with adverse effects such as bone mineral density loss, mood changes, and contraceptive consequences that may be undesirable in women seeking fertility ¹⁶ . Nonsteroidal anti-inflammatory drugs (NSAIDs) offer symptomatic relief but do not modify disease progression.

Beyond conventional medical therapies, several antioxidant and anti-inflammatory compounds have demonstrated beneficial effects in experimental endometriosis models. These studies provide a broader context for understanding the potential role of oxidative stress modulation as a therapeutic strategy in endometriosis. For example, resveratrol has been shown to inhibit vascularization and cell proliferation through modulation of VEGF and matrix metalloproteinases and to reduce oxidative stress by enhancing antioxidant enzyme activity while decreasing MDA levels ^17–19 . Similarly, curcumin exhibits anti-inflammatory and anti-angiogenic properties in endometriosis models through NF-κB and COX-2 inhibition ^20,21 . Omega-3 polyunsaturated fatty acids have also shown promise in reducing endometriosis-associated pain and inflammation through modulation of prostaglandin synthesis ²² . More recently, platelet-rich plasma (PRP) has emerged as a potential therapeutic approach, with experimental studies showing reduced lesion size and inflammation in rat models of endometriosis ^23,24 .

Bromelain treatment significantly improved endocannabinoid levels and antioxidant capacity in our study. These beneficial effects can be attributed to bromelain's multifaceted pharmacological properties. Kansakar et al. ⁶ described bromelain's ability to modulate NF-κB, MAPK, and COX-2 pathways, thereby reducing inflammatory cytokine production. Additionally, bromelain exerts antioxidant effects through free radical scavenging and upregulation of endogenous defense systems. Paksoy et al. ⁷ demonstrated reduced oxidative stress indices in a rat ischemia-reperfusion model following bromelain administration.

Although previous studies have investigated bromelain in combination with other antioxidant agents such as N-acetyl cysteine (NAC) and alpha-lipoic acid (LA), data on its isolated use in endometriosis are lacking. In an experimental study, the combined administration of NAC, LA, and bromelain demonstrated significant anti-inflammatory and proapoptotic effects, including reduced cyst number and size as well as suppression of endothelial activation markers such as VCAM-1 ²⁵ . Similarly, clinical evidence from the LEAP study showed that an antioxidant preparation containing NAC, LA, and bromelain significantly improved endometriosis-associated pelvic pain and reduced the need for analgesics ²⁶ . These findings suggest a potential synergistic interaction between antioxidant compounds in modulating inflammation and oxidative stress pathways.

However, unlike these combination therapies, the present study specifically evaluates bromelain as a single agent. Because these studies evaluated bromelain as part of a combination therapy, their findings cannot be directly extrapolated to bromelain monotherapy. To the best of our knowledge, this is the first study demonstrating that isolated bromelain administration can improve both endocannabinoid balance and oxidative stress parameters in an experimental endometriosis model. These findings provide preclinical evidence supporting bromelain as a potential non-hormonal therapeutic strategy targeting multiple interconnected mechanisms, including ECS dysfunction, oxidative stress, and inflammation, without the adverse effects associated with hormonal therapies or surgical interventions.

However, several limitations must be acknowledged. First, although the rat model is widely used, it does not fully recapitulate human menstrual physiology or immune complexity. Second, formal blinding was not implemented during group allocation, treatment administration, or outcome assessment, which may have introduced a risk of observer-related bias. Third, the sham procedure consisted of laparotomy without uterine tissue transplantation and therefore may not have fully controlled for all effects related to surgical manipulation and inflammation. Fourth, histological confirmation of endometriotic lesions and quantitative assessment of lesion burden were not available for the present analysis, limiting the morphological characterization of the experimental model and the ability to correlate biochemical findings with disease severity. We also did not evaluate behavioral pain parameters, which would enhance translational interpretation. In addition, the relatively small sample size may have limited statistical power to detect modest effect sizes and increased the risk of type II error. Furthermore, optimal dosing strategies and long-term safety profiles remain to be defined. Esterase or enzyme inhibitors were not used during blood collection and processing; therefore, ex vivo degradation of endocannabinoids cannot be completely excluded. Furthermore, endocannabinoid levels were measured using ELISA rather than liquid chromatography–tandem mass spectrometry (LC-MS/MS), which is considered the gold-standard analytical method for quantification.

Finally, the precise molecular mechanisms underlying bromelain's effects on ECS enzymes (fatty acid amide hydrolase [FAAH] and monoacylglycerol lipase [MAGL]), receptor expression, and related signaling pathways warrant further investigation. Future studies should incorporate behavioral assessments, histological quantification, bromelain-only control groups, and mechanistic analyses to better characterize the isolated effects of bromelain and clarify its therapeutic positioning. Comparative and combination studies with established medical treatments would further delineate its role within the evolving endometriosis management paradigm.