{"paper_id":"5f5e3658-3619-4888-a60d-01f1261fe056","body_text":"Endometriosis is a benign estrogen-dependent gynecological disorder in which endometrial\ntissue grows outside the uterus. Endometriosis affects about 2 to 22% of reproductive-age\nwomen; and 40 to 60% of women who have painful menstruation, as well as 25 to 50% of\ninfertile women ( Yang  et al.,  2004 ).\nDespite its well-established pathophysiology, substantial morbidity and healthcare\nexpenditures are associated with endometriosis, and the specific reason is largely unclear\n( Viganò  et al.,  2004 ).\nHowever, oxidative stress - an imbalance between the radical species and endogenous\nantioxidant molecules has been fingered as a culprit in the pathogenesis of endometriosis,\nresulting in a peritoneal cavity inflammatory response ( Augoulea  et al.,  2009 ).\nReactive oxygen species (ROS) are inflammatory mediators that control cell growth and\nelicit harmful consequences during normal oxygen metabolism ( Jena  et al.,  2023 ). Cells have developed various antioxidant\nsystems to limit ROS production and cell damage, including up-regulating antioxidant\nmolecules such as superoxide dismutase, catalase, and glutathione peroxidase activities\n( Jena  et al.,  2023 ). However,\noxidative stress occurs when the balance between ROS production and antioxidant defense is\ndisrupted ( Lousse  et al.,  2012 ).\nThis is in line with the fact that macrophages, erythrocytes, and apoptotic endometrial\ntissue that are transplanted into the peritoneal cavity by retrograde menstruation are\nrecognized inducers of oxidative stress; hence, peritoneal generation of ROS is involved in\nthe endometriotic condition ( Lousse  et al., \n2012 ).\nSevere endometriosis distorts pelvic anatomy, lowers fertility, and causes changes to the\neutopic endometrium, implying that the endometriotic implants communicate with the native\nendometrium in some way ( Rocha  et al., \n2023 ). The influence of specific anomalies in the eutopic endometrium of women with\nendometriosis on fertility, particularly in moderate illness, is now the focus of exciting\nnews in endometriosis research. Changes in the endometrium of women also contribute to the\ndisease’s pathophysiology and proclivity to deteriorate or recur. The condition manifests\nitself in a variety of ways, from acyclic pelvic discomfort to infertility ( Rocha  et al.,  2023 ).\nDue to the toxicity of synthetic antioxidants used in medicines, natural antioxidants\nderived from plant species, such as flavonoids, have recently received a lot of attention\n( Juntachote & Berghofer, 2005 ) due to their\nability to halt free radical production ( Ahmadi &\nShadboorestan, 2016 ). Hesperidin is a polyphenolic chemical that occurs naturally\nin fruits and vegetables. Citrus fruits principal flavonoid, hesperidin, may be extracted in\nsignificant concentrations from the rinds of several citrus species ( Wilmsen  et al.,  2005 ). Hesperidin’s antioxidative\nproperties include substantial reducing power, metal chelating, and radical (hydrogen\nperoxide, superoxide, and hydroxyl) scavenging abilities ( Wilmsen  et al.,  2005 ).\nOn the other hand, physical exercise is known for its positive effects on human antioxidant\ndefenses ( Bouzid  et al.,  2018 ).\nEngaging in moderate exercise in an active lifestyle has been shown to help alleviate\noxidative stress ( Baltaci  et al., \n2016 ). The beneficial effects of exercise include releasing myokines, cytokines,\ninterleukins, and other peptides to prevent inflammatory disorders like endometriosis ( Golbidi  et al.,  2012 ). The prevalence of\nendometriosis has caused infertility and painful menstrual periods in reproductive women;\nthereby having a significant impact on their physical, mental, and social well-being. To\ndate, there is no established cure for endometriosis, and most current medical treatments\nare not suitable for long-term use due to their side-effects. Therefore, this study aims to\nevaluate the effect of physical exercise with/without hesperidin consumption on the\nendometriosis-induced oxidative stress markers in female rabbits.\n\nThe study used adult female New Zealand rabbits of size 1.5 – 2.0 kg maintained under\nregulated conditions of ambient humidity, temperature, and light for 14 days, receiving\nwater and food  ad libitum.  The university’s Ethics Committee approved all\nanimal handling procedures.\nThe rabbits were subjected to a running program for 4 days to become familiarized with\nthe treadmills under constant supervision ( Such  et\nal.,  2008 ), and those that did not adequately run on the treadmill\nwere excluded from the study. Thereafter, under sterile and strict antisepsis conditions\nin the laboratory, the animal’s abdominal fur was carefully shaved to get a clean cut of\nthe abdominal skin. The anesthetic agent (35 mg/kg of ketamine and 2 mg/kg diazepam) was\nadministered. The pelvic cavity was opened by a median longitudinal incision of\napproximately 4 cm at a distance of 4 cm from the pubis. A segment of about 2 cm of the\nuterine horn was resected, and the horn was closed with Vicryl 6.0. The resected uterine\nportion was immersed inside normal saline to avoid desiccation of the tissue and then cut\nlongitudinally to obtain a 10 by 10 mm fragment from the tissue. The fragment was sutured\nto the peritoneal wall at the junction where two blood vessels meet, and the endometrial\ntissue was faced directly to the abdominal cavity using Vicryl 6.0 suture. The abdominal\nincision was then closed layer by layer using Vicryl 2.0 suture. Diclofenac injection was\ngiven to the animal after surgery to relieve pain ( Rosa-e-Silva  et al.,  2010 ).\nThe rabbits were grouped into five groups (n=6) as follows: Group 1 - Normal control\n(NC); Group 2 - untreated endometriotic rabbits (ENDO); Group 3 - endometriotic rabbit\ntreated with hesperidin (ENDO + HESP); Group 4 – physically exercised endometriotic rabbit\n(ENDO + EXER); Group 5 - physically exercised endometriotic rabbit treated with hesperidin\n(ENDO + HESP+ EXER). The animals were allowed to recover from surgery for 5 days.\nHesperidin solution was prepared and orally administered to group 3 (ENDO + HESP) and 5\n(ENDO + HESP + EXER) rabbits at a dose of 50 mg/kg every day for 2 weeks ( Melekoglu  et al.,  2018 ). The animals\nin groups 4 (ENDO + EXER) and 5 (ENDO + HESP + EXER) were placed on a treadmill for two\nweeks. The treadmill was set at a speed of 1.2 m/s for the first day, and the rabbits were\nplaced on it for 10 min. The treadmill’s speed and time for exercise were later increased\nto 2m/s for 20 min.\nThe hesperidin administration and physical exercise protocols were done concurrently for\ntwo weeks. Thereafter, the animals were anaesthetized with ketamine, and cervical\ndislocation was done. Grafted endometriosis tissue. The endometrial-implanted tissue in\nthe rabbits was collected and separately homogenized in 0.1M phosphate buffer (pH 7.4) in\na laboratory homogenizer. The homogenate was centrifuged, and the supernatant was used to\nestimate biochemical assays.\nThe harvested tissues were fixed in Bouin’s fluid for histological analysis. The uterus\nof the control and the experimental groups as well as the ectopic surgically grafted\nendometrial tissue together with portions of the peritoneal walls were also harvested for\nanalysis. The fixed tissue was dehydrated with alcohol, cleared with xylene, and embedded\nin paraffin wax. The tissue was cut to produce 4–5 µm sections using a\nmicrotome, fixed on the slides, stained with hematoxylin and eosin (H and E), and then\nviewed under a light microscope (Olympus/3H -Tokyo, Japan).\nROS level was estimated as equivalent to H 2 O 2 , according to the\nmethod reported by  Oboh  et al. \n(2018)  using the reagent n-n-diethyl-para-phenylenediamine (DEPPD).\nTBARS levels in the endometrial tissue homogenates of experimental rabbits were carried\nout using the described method by  Ohkawa  et\nal.  (1979) .\nThe formed nitrous acid diazotises sulphanilamide, and the product, coupled with N-(1\n1-naphthyl) ethylenediamine, was determined in the acid medium and the presence of\nnitrate. The resulting azo dye, which has a bright reddish-purple colour, was measured\nat 570 nm.\nThis assay was carried out using Sinha’s method ( Sinha,\n1972 ) with a dichromate (acetic acid) solution and measured at 620 nm for 3 min\nat 30-second intervals.\nThe determination of SOD activity in the uterus was based on the inhibition of the\nradical superoxide reaction with adrenalin, as described by  Misra & Fridovich (1972) .\nGlutathione peroxidase was determined by the method described by  Rotruck  et al.  (1973)  using Ellman reagents, while\nGST was carried out using 30 mM GSH as described by Mannervik and Guthenberg method\n( Mannervik & Guthenberg, 1981 ).\nTotal glutathione (GSH) was determined using Ellman’s method ( Ellman, 1959 ).\nThe MAO activity was measured using the modified method of Ademosun and Oboh ( Ademosun & Oboh, 2014 ).\nAll data were expressed as mean ± standard error of the mean (SEM). One-way\nanalysis of variance (ANOVA) was used to analyze the differences between the groups with\nthe aid of GraphPad Prism 5.0 Software (GraphPad Software Inc., San Diego, CA). Followed\nby the post hoc Tukey’s test,  p <0.05 represented a significant\ndifference in both analyses.\n\nThe Normal control group showed the typical uterine histology. There are three distinct\nlayers: the endometrium, myometrium, and perimetrium. The endometrium’s surface epithelium\nis pseudo-columnar and encloses a centrally placed lumen. The underlying stroma is cellular\nand composed of ovoid cells with scanty cytoplasm. The endometrial stroma has numerous\nendometrial glands dispersed within it and is well demarcated from the myometrium, as\nevidenced by the fascicular nature of the myometrium. The myometrium comprises smooth muscle\ncells containing one or two elongated eosinophilic nuclei. Surrounding the myometrium is a\nserous layer known as the perimetrium ( Fig. 1 ). The\nperitoneal wall of the normal control group was sectioned for the purpose of comparison to\nthe ectopic endometrial tissues, and it was found to be characterized by parallel\nlongitudinal fibers with myocytes containing one or two elongated nuclei ( Fig. 2 ). The Endometriosis group presents the histological\ncharacteristics of endometriosis defined by the presence of glands, small arteries,\nendometrial cysts, endometrial stroma, and endometrial epithelium ( Fig. 2 ). The animal placed on the exercise only possessed cystic\ndilatations of the endometrial glands, which arise from intra-glandular hemorrhage.\nEndometrial glands, fibro-adipose tissue, and stroma are also present centrally. Peripheral\nto these, there are numerous surrounding macrophages ( Fig.\n2 ). The hesperidin, as well as hesperidin plus exercise groups, present\nendometriosis histology characterized by obvious endometrial glands, hemorrhage, and\nendometrial cysts ( Fig. 2 ). The endometriosis in\nexercise only, hesperidin, and hesperidin plus exercise groups are poorly formed compared to\nthat in the untreated endometriosis group.\nFigure 1 Shows the uterine histology of the control group (H&E; x40, x100, x400). M\n-myometrium; P - perimetrium; E - endometrium; L – _lumen; Blue arrows – _surface\nepithelium of the endometrium; Yellow arrow – _endometrial glands; Black arrows –\n_nuclei of endometrial stroma cells; Black dotted arrows – _hemorrhage.\nShows the uterine histology of the control group (H&E; x40, x100, x400). M\n-myometrium; P - perimetrium; E - endometrium; L – _lumen; Blue arrows – _surface\nepithelium of the endometrium; Yellow arrow – _endometrial glands; Black arrows –\n_nuclei of endometrial stroma cells; Black dotted arrows – _hemorrhage.\nFigure 2 Shows the histology of the uterus and peritoneal wall of the control groups (H&E;\nx40, x100, x400) and the endometriotic tissue of the experimental groups (H&E;\nx40, x100). M - myometrium; P - perimetrium; E - endometrium; L - _lumen; F -\nFasciculi; Long yellow arrows – _nuclei of muscle cells; Blue arrows – _surface\nepithelium of the endometrium; Short yellow arrows – _endometrial glands; Black arrows\n– _nuclei of endometrial stroma cells. White arrows – _hemorrhage/artery; *represents\nendometrial cyst; Red arrowhead - fibro-adipose tissue.\nShows the histology of the uterus and peritoneal wall of the control groups (H&E;\nx40, x100, x400) and the endometriotic tissue of the experimental groups (H&E;\nx40, x100). M - myometrium; P - perimetrium; E - endometrium; L - _lumen; F -\nFasciculi; Long yellow arrows – _nuclei of muscle cells; Blue arrows – _surface\nepithelium of the endometrium; Short yellow arrows – _endometrial glands; Black arrows\n– _nuclei of endometrial stroma cells. White arrows – _hemorrhage/artery; *represents\nendometrial cyst; Red arrowhead - fibro-adipose tissue.\nAn increased level of ROS was observed in the untreated ENDO group. Subsequently, the\nanimal was treated with HESP and EXER, and there was a decrease in ROS level. However, those\ntreated with HESP+EXER therapy exhibited a remarkably low level of ROS at par with that of\nNC ( Fig. 3A ). TBAR levels were significantly higher in\nthe untreated ENDO group than in the NC group. Meanwhile, the group treated with HESP or\nEXER had little or no difference in the TBAR level. ENDO rats treated with combined HESP\nplus EXER had a close value level of TBAR ( p >0.05) to the NC’s TBAR\nlevel ( Fig. 3B ). NO levels were significantly increased\nin the ENDO group compared to the NC group. The ENDO rabbit treated with EXER had a reduced\nNO level, while the treatment with HESP only possessed a reduced NO value relative to NC.\nThe HESP+EXER group indicated a suitable level of NO with respect to NC ( Fig. 3C ).\nFigure 3 a. Reactive oxygen species (ROS), b. Thiobarbituric acid reactive species and c.\nnitric acid (NO) levels in the homogenate of the uterus tissue of an endometriotic\nrabbit (n = 6). Values represent mean standard deviation (SD) and are statistically\ndifferent at: * p <0.05, ** p <0.01,\n*** p <0.001  vs.  normal control;\n # p <0.05,  ## p <0.01,\n ### p <0.001  vs.  Endo;\n** p <0.05  vs.  ENDO + EXER. Key: ENDO =\nEndometriosis; HESP = Hesperidin; Excer = Exercise.\na. Reactive oxygen species (ROS), b. Thiobarbituric acid reactive species and c.\nnitric acid (NO) levels in the homogenate of the uterus tissue of an endometriotic\nrabbit (n = 6). Values represent mean standard deviation (SD) and are statistically\ndifferent at: * p <0.05, ** p <0.01,\n*** p <0.001  vs.  normal control;\n # p <0.05,  ## p <0.01,\n ### p <0.001  vs.  Endo;\n** p <0.05  vs.  ENDO + EXER. Key: ENDO =\nEndometriosis; HESP = Hesperidin; Excer = Exercise.\nThe ENDO condition reduced catalase activity, but an extended treatment in another group\nwith HESP and EXER described an improved catalase status ( Fig.\n4A ). This attribute can be due to the presence of HESP and EXER. Though treatment\nwith HESP had no significant difference from EXER treatment, the combined HESP+EXER therapy\npossesses the greatest increase in catalase activity. SOD activity, as shown in  Fig. 4B  in a combined treatment of HESP+EXER, exhibited a\nsignificant increase that was greater than even the NC. The group treated with EXER alone\nshowed an improved SOD activity, significantly higher than the group treated with HESP\nalone. The ENDO condition decreased GPx activity ( Fig.\n4C ), but the treatment with HESP and EXER improved GPx activity. Perhaps treatment\nwith EXER has no significant difference compared to HESP treatment, but the combined use of\nHESP+EXER therapy indicated the most suitable level of GPx activity. The ENDO condition\nreduced the activity of glutathione–s-transferase ( Fig.\n4D ) relative to NC, but the treatment group with HESP and EXER exhibited improved\nGST activity. The treatment with HESP is slightly higher than that with EXER, but the\ncombined treatment of HESP+EXER showed the most increased GST activity, almost at the same\nlevel as NC.\nFigure 4 a. Catalase, b. Superoxide dismutase (SOD); c. glutathione peroxidase (GPx) and d\nglutathione–S-Transferase (GST) activities in the homogenate of uterus tissue of\nendometriotic rabbit (n = 6). Values represent mean standard deviation (SD) and are\nstatistically different at: * p <0.05, ** p <0.01,\n*** p <0.001  vs.  normal control;\n* p <0.05,  ## p <0.01,\n ### p <0.001 vs. Endo; ** p <0.05\n vs.  ENDO + EXER. Key: ENDO = Endometriosis; HESP = Hesperidin;\nExcer = Exercise.\na. Catalase, b. Superoxide dismutase (SOD); c. glutathione peroxidase (GPx) and d\nglutathione–S-Transferase (GST) activities in the homogenate of uterus tissue of\nendometriotic rabbit (n = 6). Values represent mean standard deviation (SD) and are\nstatistically different at: * p <0.05, ** p <0.01,\n*** p <0.001  vs.  normal control;\n* p <0.05,  ## p <0.01,\n ### p <0.001 vs. Endo; ** p <0.05\n vs.  ENDO + EXER. Key: ENDO = Endometriosis; HESP = Hesperidin;\nExcer = Exercise.\nThe combined effect of HESP+EXER as a therapy for ENDO tissue on GSH level is presented in\n Figure 5 , showing a significant increase in GSH\nlevel. The group treated with HESP alone also showed an improved level of GSH compared with\nthe group treated with EXER alone, which had a lower GSH level. The ENDO group exhibited a\nreduced level of GSH. The result of monoamine oxidase (MAO) activity in endometriosis tissue\n(lesion) shows that there was a significant increase ( Fig.\n6 ) of MAO activity. MAO activity was high in the untreated ENDO group. However,\nthere is a close-range activity of MAO between the ENDO + HESP group and the ENDO + EXER\ngroup. There is a drastic decrease in MAO activity in the group treated with HESP + EXER\n( Fig. 1 ).\nFigure 5 Glutathione (GSH) level in an endometriotic rabbit’s homogenate of the uterus tissue\n(n = 6). Values represent mean standard deviation (SD) and are statistically different\nat: * p <0.05, ** p <0.01  vs. \nnormal control; * p <0.05,  ## p <0.01\n vs.  Endo; * p <0.05 vs. ENDO + EXER Key: ENDO =\nEndometriosis; HESP = Hesperidin; Excer = Exercise.\nGlutathione (GSH) level in an endometriotic rabbit’s homogenate of the uterus tissue\n(n = 6). Values represent mean standard deviation (SD) and are statistically different\nat: * p <0.05, ** p <0.01  vs. \nnormal control; * p <0.05,  ## p <0.01\n vs.  Endo; * p <0.05 vs. ENDO + EXER Key: ENDO =\nEndometriosis; HESP = Hesperidin; Excer = Exercise.\nFigure 6 Monoamine oxidase activity in the homogenate of the endometriotic rabbit’s uterus\ntissue (n = 6). Values represent mean standard deviation (SD) and are statistically\ndifferent at: * p <0.05, *** p <0.01\n vs.  normal control;  ## p <0.01,\n ### p <0.001  vs.  Endo;\n* p <0.05  vs.  ENDO + HESP,\nº p <0.05  vs.  ENDO + EXER. Key: ENDO =\nEndometriosis; HESP = Hesperidin; Excer = Exercise.\nMonoamine oxidase activity in the homogenate of the endometriotic rabbit’s uterus\ntissue (n = 6). Values represent mean standard deviation (SD) and are statistically\ndifferent at: * p <0.05, *** p <0.01\n vs.  normal control;  ## p <0.01,\n ### p <0.001  vs.  Endo;\n* p <0.05  vs.  ENDO + HESP,\nº p <0.05  vs.  ENDO + EXER. Key: ENDO =\nEndometriosis; HESP = Hesperidin; Excer = Exercise.\n\nThe endometriosis medical therapy options available for clinical use are ineffective and\nhave significant side effects. No convincing evidence suggests one treatment over another\n( Dunselman  et al.,  2014 ). Natural\nantioxidants and effective therapies for endometriosis are currently being researched. Due\nto oxidative stress involvement in the pathogenesis of endometriosis, effective antioxidant\ntherapies with little or no side effects are being developed ( Melekoglu  et al.,  2018 ). This study investigates the\neffect of hesperidin and physical exercise on a rabbit model of surgically induced\nendometriosis.\nSignificant histological changes were observed in the endometriotic lesions. Destruction of\nthe endometrium was more evident in the columnar epithelial layer and glands. Prominent\nhemorrhage, vascular congestion, necrosis, inflammatory cell infiltration, cystically\ndilated glands, and significant blood cell accumulation in the lumen of endometrial lesions\nwere detected in the endometrial lesion layers. All histological parameters were improved\nafter hesperidin and exercise administration ( Fig.\n1 ).\nROS are byproducts of normal cellular metabolism that play important roles in signaling\npathways, including intracellular signal transmission, metabolism, proliferation, and\napoptosis. ROS are formed over time in response to long-term environmental stress, which can\ncause considerable damage to cell structure and function ( Fleury  et al.,  2002 ). This biological reduction of molecular\noxygen is a source of reactive oxygen species (ROS), including the primary free radical\nsuperoxide anion (O 2 •_ ) and the major non-free radical hydrogen\nperoxide (H 2 O 2 ) ( Zorov  et\nal.,  2014 ). The result showed that ROS was prominent in untreated ENDO\nrabbits, but the animals treated with hesperidin revealed a low level of ROS. The combined\ntherapy of HESP+EXER significantly decreases ROS level ( Fig.\n2 ). This is consistent with the study of  Liu\n et al.  (2017) , which revealed that HESP could block the\nproduction of free radicals.\nUptake of oxygen, generation of lipid radicals, and rearrangement of double bonds in\nunsaturated and polyunsaturated fatty acids are all part of the lipid peroxidation process\n( Dasgupta & Klein, 2014 ). TBARS is a lipid\nperoxidation end product, malondialdehyde, a reactive aldehyde formed by lipid peroxidation\nof polyunsaturated fatty acids. This study revealed that TBARS was high in the tissue of\nuntreated ENDO rabbits. However, the combined therapy of exercise and hesperidin alleviates\nTBARS level ( Fig. 3B ). This is in line with the study\nof  Homayouni  et al.  (2017) , which\nshowed that hesperidin supplement alleviates oxidative DNA damage and lipid\nperoxidation.\nNitric oxide (NO) is a vasodilator produced by the nitric oxide synthase (NOS) enzyme, from\n L -arginine ( Najafi  et al., \n2012 ), but when combined with superoxide (O 2 ), it produces\nperoxynitrite, a hazardous radical with adenomyosis, a form of endometriosis that affects\nthe myometrium ( Jena  et al.,  2023 ).\nHigh levels of NO negatively influence fertility in several ways: toxic embryos, inhibit\nimplantation, and affect contractile activity in the oviduct ( Osborn  et al.,  2002 ). Our research showed that the\nuntreated endometriosis group’s NO level was high. Still, treatment with HESP and/or\nexercise showed a complementary decrease.\nStill, it was pronounced in the combined treatment of hesperidin and exercise ( Fig. 3C ). Previous studies indicated that hesperidin\nattenuated NO production in mice macrophage cell lines ( Sakata  et al.,  2003 ) due to its antioxidant, anti-inflammatory,\nanti-proliferative, and anti-carcinogenic properties ( Parhiz\n et al.,  2014 ). In this study, both physical exercise and\nhesperidin exhibited antioxidative potentials.\nThe antioxidant enzyme catalase (CAT) is found in practically all biological tissues,\nwhereby its breakdown of hydrogen peroxide to water and molecular oxygen uses either iron or\nmanganese as a cofactor, thereby completing the detoxification process ( Ighodaro & Akinloye, 2018 ). From  Fig. 4A , the catalase activity in the endometriosis group was\nsignificantly lower compared to NC, thus indicating oxidative stress ( Sourial  et al.,  2014 ). The combined treatment of\nhesperidin and exercise increased catalase activity relative to the untreated endometriosis\ngroup. Antioxidant supplements such as hesperidin induce maturity and growth of follicles by\nlowering oxidative stress, activating enzymes such as CAT, promoting the development of\nfollicles and embryonal development, and improving fertilization rate; thereby preventing\nreproductive diseases such as unexplained infertility and endometriosis ( Wang  et al.,  2017 ).\nSOD is an important endogenous antioxidant enzyme that acts as a first line of defense\nagainst free radical species (Perhiz  et al.,  2014). The present study\nindicates that aerobic exercise is a good modulator of SOD activity in the body. However,\nits combination with hesperidin raises the enzyme activity above normal control ( Fig. 4B ). The study by  Yan\n& Spaulding (2020)  found that endurance (aerobic) exercise increased the SOD\nactivity in the aorta of mice and human plasma. Exercise training increases the amount of\nSOD protein in peripheral organs such as the heart, kidneys, liver, heart, lung, and adipose\ntissue. These data support the hypothesis that exercise increases SOD expression in skeletal\nmuscle, the body’s biggest organ, resulting in increased extracellular antioxidant defense\nin the circulation and peripheral tissues as a molecular transducer of the benefits of\nexercise to health and disease ( Yang & Spaulding,\n2020 ).\nGlutathione peroxidase (GPx) is an intracellular enzyme that breaks down hydrogen peroxide\nto water and lipid peroxides to their corresponding alcohols ( Gill & Tuteja, 2010 ). GPx is particularly important in suppressing\nlipid peroxidation; thereby protecting cells from oxidative stress ( Gill & Tuteja, 2010 ). From  Fig\n3C  of the result, the GPx activity was greatly reduced in the endometriosis group\ncompared to the NC group. However, the combined treatment of HESP+EXCER significantly\nincreased GPx activity. Physical activity improves antioxidant defenses and lowers lipid\nperoxidation levels in adults and aged individuals. The exercise-induced ROS generation\nresults in increased activity of enzymatic antioxidants, which then leads to increased\nresistance to oxidative challenges, including a wide variety of oxidative stress-related\ndiseases such as diabetes, mitochondrial myopathies, and endometriosis, among others ( Baltaci  et al.,  2016 ).\nGlutathione–s-transferase (GST) is a subfamily of phase II detoxification enzymes that uses\nGSH as a co-factor to derivatize cellular electrophiles of various origins, such as\nxenobiotics and endogenous reactive metabolites; thereby providing yet another important\nmechanism of cell protection against harmful electrophiles ( Scirè  et al.,  2019 ). The results from  Fig. 4D  showed an increase in GST activity in animals with endometriosis\ntreated with hesperidin and the combination of hesperidin plus exercise, yielding a higher\nlevel of enzyme activity. In the study of  Melekoglu\n et al.  (2018) , oxidative stress markers were significantly\nhigher in the endometriosis group, and increased antioxidant activity was observed in the\nendometriotic foci of rats treated with hesperidin and nerolidol.\nGlutathione (GSH) is a co-substrate of GPx, which allows peroxides (hydrogen and lipid\nperoxides) to be reduced and GSSG to be produced. NADPH-reducing equivalents and glutathione\ndisulfide reductase catalysis are then used to convert GSSG to 2GSH ( Gaucher  et al.,  2018 ). The present study revealed that\nthe GSH level was significantly low in the untreated ENDO group, and the combined therapy of\nHESP+EXCER improved GSH level.  Scutiero  et\nal.  (2017)  reported the role of oxidative stress in the development and\nprogression of endometriosis; and therapeutic approaches involving flavonoids to prevent the\nformation of endometriosis. Thiols are the most abundant antioxidants in the body,\naccounting for most total antioxidants, and playing an important role in the defense against\nradical species ( Prakash  et al., \n2009 ). Glutathione is made up of intracellular and extracellular thiols that are\neither free (oxidized or reduced glutathione) or attached to proteins ( Prakash  et al.,  2009 ). Aside from its involvement in\nfree radical defense, GSH is involved in detoxification, signal transduction, apoptosis, and\na variety of other molecular processes. The increased level of GSH reduced the risk of\nendometriosis and infertility ( Prakash  et\nal.,  2009 ). The result from  Figure\n5  proves that endometriosis reduces the expression of GSH, but it gets improved\nwhen treated with hesperidin or exercise, and a significant increment when treated with the\ncombination of exercise and hesperidin.\nThe monoamine oxidase (MAO), which is found in the mitochondrial membrane, has been\ndemonstrated to be a key cause of oxidative stress ( Adefegha\n et al.,  2021 ). It uses flavin adenine dinucleotide as a cofactor\nto catalyze the oxidative deamination of a range of monoamines ( Henriquez  et al.,  2006 ). Untreated endometriosis group\nexpressed higher levels of MAO, therefore increasing the chances of infertility. However,\ninduced animal groups placed on exercise showed a decrease in the level of MAO, while the\ngroup treated with HESP+EXCER had reduced MAO activity ( Fig.\n6 ), indicating the potential of combination therapy in the treatment of\nendometriosis and possible decrease of infertility. Our findings agree with the study of\n Campos  et al.  (2013)  who reported\nthe impact of exercise on MAO; an important source of ROS in the mitochondrial membrane\n( Campos  et al.,  2013 ).\n\nThis study ascertains that exercise improved the antioxidant status, while the combination\nof hesperidin and exercise have therapeutic effects in endometriosis.","source_license":"public-domain-us","license_restricted":false}