{"paper_id":"91a0eaca-778f-419e-b635-7b97952dab2a","body_text":"Adenomyosis, like endometriosis, is a prevalent gynecologic condition, and the\npathogenesis of adenomyosis is poorly understood. 1 , 2  A recent paper revealed that\ntissue injury and repair is shown to be the most reasonable explanation for the\nemergence of the adenomyosis because the etiology of archimetrosis is linked to the\nevolution of the stratum vasculare. 3  Adenomyosis exhibits many\nsimilarities to endometriosis in terms of estrogen reliance, symptomology,\nprogesterone resistance, and various molecular abnormalities, but it differs in risk\nfactors, onset age, and etiology. 4 , 5  Our current understanding of\nthe mechanisms causing adenomyosis-related pain is severely poor. As a result,\nadenomyosis treatment has proven challenging. 6  Hysterectomy has been used as a\npreferred therapy for severe symptomatic adenomyosis. 7 , 8  Medical treatment such as\nchronic usage of oral contraceptives, high-dose progestins, selective estrogen\nreceptor modulators, aromatase inhibitors, and gonadotrophin receptor hormone\nagonists can temporarily reverse adenomyosis and alleviate symptoms. 4 , 9  A novel therapy for adenomyosis\nis clearly an unmet medical need that has yet to be satisfied.\nWe previously demonstrated that treatment of mice with induced adenomyosis using\nandrographolide, a nuclear factor κB (NF-κB) inhibitor, extracted from the medicinal\nherb Andrographis, or levo-tetrahydropalmatine, an extraction from analgesia, was\nable to decrease uterine contractility, reduce myometrial infiltration, and mitigate\ngeneralized hyperalgesia in mice with adenomyosis. 10 , 11\nResveratrol is a polyphenol commonly found in various berries, grape skins, and\ndeep-colored fruits and vegetables. It has been demonstrated to exert its beneficial\neffects (i.e., anti-cancer, anti-oxidation, or anti-inflammation) via modulating a\nvariety of pathways, making it a multitargeted treatment for chronic\nillnesses. 12 - 14  In addition\nto its antiangiogenic effect, as shown in cancer research fields, the resveratrol\nhas various other favorable features. It has been reported that resveratrol can\nsuppress DNA methylation and deacetylase activity through inhibition of DNA\nmethyltransferases (DNMTs), methyl-CpG-binding domain protein 2, and pan-histone\ndeacetylase. 15  Resveratrol was able to activate progesterone receptor\nisoform B and inhibit NF-κB activation and cyclooxygenase-2 expression, leading to\nblocked cell proliferation of ectopic endometrial tissues. 16 , 17  Notably, numerous preclinical\nstudies have proven that resveratrol might be a potential agent for the prevention\nand treatment of endometriosis.\nWe hypothesized that resveratrol might suppress the progression of adenomyosis\nthrough modulating genes that are typically elevated in endometriosis and\nadenomyosis. This research was conducted to prove this hypothesis.\n\nPregnant ICR mice at the gestational age of around 15–16 days were acquired from\nShanghai Laboratory Animal Corporation (Shanghai, China). Each mouse was kept in\na single cage for the remainder of the gestation, delivery, and nursing period.\nTheir pups were sexed one day after birth, and all the female pups were chosen\nfor this study. The dam and her offspring were housed in the same cage until\nthey were weaned. Experiments were approved by the ethics committee of Wenzhou\nMedical University.\nAdenomyosis was stimulated in female neonatal mice by orally administering\n1 mg/kg tamoxifen suspended in a mixture of condensed milk/peanut oil/lecithin\nat a 3:2:0.2 ratio by volume at a dosage volume of 5 μl/g bodyweight from\npost-delivery day 2 to day 5. In contrast, the control neonate mice were given a\nsolvent without tamoxifen. These female mice were weaned and separated from\ntheir dams when they reached the age of 3 weeks. Starting at four-week age, all\nmice were given a hotplate test at a four-week interval. At sixteen (16)-week of\nage, all tamoxifen-treated female mice were randomly divided into 3 groups. The\nuntreated group of mice received the vehicle. Another two groups were\nintraperitoneally (i.p.) given either low-dose resveratrol (25 mg/kg) or\nhigh-dose resveratrol (50 mg/kg) daily for 3 weeks. After the final hotplate\ntest and body weight measurement were performed, the blood samples were\ncollected. All mice were sacrificed by perfusing formalin into heart. Each\nmouse’s uterus was weighed, and the uterine horn was collected.\nThe Immunohistochemistry (IHC) method was described previously. 18  Briefly,\nthe ectopic endometrium tissues were embedded in the optimal cutting temperature\n(OCT) compound. For each block, serial 6 μm slices were performed on a cryostat.\nThe sections were immersed in block buffer (goat anti-mouse serum) for 15 mins\nbefore being incubated with primary antibody overnight at 4°C. After rinsing\nwith phosphate-buffered saline (PBS), the sections were incubated with secondary\nantibodies for one hour before being washed with PBS and mounted. The primary\nantibodies were purchased from Abcam.\nThe fibers in endometriotic lesions were quantified using Masson trichrome\nstaining. The OCT embedded endometrium tissues were stained with Trichrome Stain\nKit (Connective Tissue Stain) (ab150686) (Abcam, Shanghai, China) according to\nthe manufacturer’s instruction. Image Pro-Plus v6.0 was used to calculate the\nareas of the blue collagen fiber layer compared to the overall ectopic\nendometrium area.\nFollowing the manufacturer’s instruction, the plasma levels of hyaluronic acid\n(HA), high mobility group box 1 (HMGB1), and osteopontin were measured using HA\n(R&D Systems, Minneapolis, MN), HMGB1 (MyBioSource, San Diego, CA),\nosteopontin (R&D Systems), and ELISA kits, respectively.\nData were presented as mean ± standard deviation (SD) with scatter plots. The\ndifference between different groups was calculated by Student’s t test with\nWelch’s post hoc test, or one-way analysis of variance (ANOVA) with a Tukey’s\nmultiple tests. P values of less than .05 were considered statistically\nsignificant.\n\nThe body weight of mice was monitored at 4 weeks, 8 weeks, and 12 weeks, before\nand after resveratrol treatment. The results in  Figure 1(A)-1(E)  showed that the body\nweight in mice with adenomyosis (tamoxifen-treated group) was significantly and\nconstantly lower than that in control normal mice at all time points.\nFurthermore, the uterine weight was markedly higher in mice with adenomyosis\nthan in control mice ( Figure\n1(F) ). These results of body weight and uterine weight shrinkage in\nmice with adenomyosis were consistent with our previous publications, partially\nconfirming the successful establishment of mice with adenomyosis. The mice with\nadenomyosis were subjected to vehicle, resveratrol treatment (25 mg/kg) or\n(50 mg/kg) starting at week 16 until the end of the experiment. We observed that\nthe body weight and uterine weight of mice that received resveratrol treatment\n(25 or 50 mg/kg) were considerably reduced when compared with untreated mice and\ncontrol mice, suggesting resveratrol treatment at doses of 25 mg/kg and 50 mg/kg\nmight exert unfavorable effects on mice with adenomyosis, leading to reduced\nbody weight and uterine weight. Figure 1. Effects of\nresveratrol treatment at the doses of 25 or 50 mg/kg on the body\nweight at indicated time point and uterine weight of the mice. Body\nweight was measured at week 4 (A), week 8 (B), week 12 (C), before\ntreatment (D), and after treatment (E). (F) Uterine weight was\ndetermined after the experiment. The data are mean ± SD with scatter\nplots. Control group: n = 10; untreated group: n = 10; 50 mg/kg\nresveratrol group: n = 9; 25 mg/kg resveratrol group: n = 10.\n* P  < .05, ** P  < .01,\n*** P  < .001 vs control group;\n # P  < .05,\n ## P  < .01,\n ### P  < .001 vs untreated\ngroup.\nEffects of\nresveratrol treatment at the doses of 25 or 50 mg/kg on the body\nweight at indicated time point and uterine weight of the mice. Body\nweight was measured at week 4 (A), week 8 (B), week 12 (C), before\ntreatment (D), and after treatment (E). (F) Uterine weight was\ndetermined after the experiment. The data are mean ± SD with scatter\nplots. Control group: n = 10; untreated group: n = 10; 50 mg/kg\nresveratrol group: n = 9; 25 mg/kg resveratrol group: n = 10.\n* P  < .05, ** P  < .01,\n*** P  < .001 vs control group;\n # P  < .05,\n ## P  < .01,\n ### P  < .001 vs untreated\ngroup.\nThe hotplate latency of mice was measured at 4 weeks, 8 weeks, and 12 weeks,\nbefore and after resveratrol treatment. Compared with control normal mice, the\nhotplate latency was significantly decreased in the mice with adenomyosis at\nweeks 4, 8, 12, and 16 (before treatment) ( Figure 2(A)-2(D) ). After 3 weeks of\nresveratrol treatment, the hotplate latency was notably upregulated compared to\nthe vehicle treatment ( Figure\n2(E) ). Figure 2. Effects of resveratrol treatment at\nthe doses of 25 or 50 mg/kg on the hot plate latency at indicated\ntime point in the mice. The hot plate experiments were conducted at\nweek 4 (A), week 8 (B), week 12 (C), before treatment (D) and after\ntreatment (E). The data are mean ± SD with scatter plots. Control\ngroup: n = 10; untreated group: n = 10; 50 mg/kg resveratrol group:\nn = 9; 25 mg/kg resveratrol group: n = 10. * P  <\n.05, *** P  < .001 vs control group;\n ### P  < .001 vs untreated\ngroup.\nEffects of resveratrol treatment at\nthe doses of 25 or 50 mg/kg on the hot plate latency at indicated\ntime point in the mice. The hot plate experiments were conducted at\nweek 4 (A), week 8 (B), week 12 (C), before treatment (D) and after\ntreatment (E). The data are mean ± SD with scatter plots. Control\ngroup: n = 10; untreated group: n = 10; 50 mg/kg resveratrol group:\nn = 9; 25 mg/kg resveratrol group: n = 10. * P  <\n.05, *** P  < .001 vs control group;\n ### P  < .001 vs untreated\ngroup.\nMasson staining and hematoxylin and eosin (H&E) staining were conducted using\nectopic endometrium tissues from four groups of mice. As shown in  Figures 3(A) and 3(B) ,\nthe extent of lesional fibrosis was dramatically upregulated in mice with\nadenomyosis compared with control normal mice and was considerably reduced after\nbeing treated with resveratrol at 25 mg/kg and 50 mg/kg. Figure 3. Effects of\nresveratrol treatment at the doses of 25 or 50 mg/kg on the\nhistopathology and fibrosis in the mice. (A) H&E staining of the\nectopic endometrium tissues in the mice, and the histopathological\nscores in the four experimental groups. (B) Masson staining of the\nectopic endometrium tissues in the mice, and the fibrosis scores in\nthe four experimental groups. The data are mean ± SD with scatter\nplots. Control group: n = 10; untreated group: n = 10; 50 mg/kg\nresveratrol group: n = 9; 25 mg/kg resveratrol group: n = 10.\n* P  < .05, *** P  < .001 vs\ncontrol group;  ### P < .001 vs untreated\ngroup.\nEffects of\nresveratrol treatment at the doses of 25 or 50 mg/kg on the\nhistopathology and fibrosis in the mice. (A) H&E staining of the\nectopic endometrium tissues in the mice, and the histopathological\nscores in the four experimental groups. (B) Masson staining of the\nectopic endometrium tissues in the mice, and the fibrosis scores in\nthe four experimental groups. The data are mean ± SD with scatter\nplots. Control group: n = 10; untreated group: n = 10; 50 mg/kg\nresveratrol group: n = 9; 25 mg/kg resveratrol group: n = 10.\n* P  < .05, *** P  < .001 vs\ncontrol group;  ### P < .001 vs untreated\ngroup.\nOur previous studies have shown that upregulation of several biomarkers [i.e.,\nHMGB1, p-p65, osteopontin, receptor for advanced glycation end-products (RAGE),\ninterleukin (IL)-33, and proliferating cell nuclear antigen (PCNA)] was revealed\nin the endometrium tissues of patients with endometriosis. 19  However,\ntheir expression in endometrium tissues from mice with adenomyosis was unknown.\nHence, in this study, we tested the expression of HMGB1, IL-33, osteopontin,\nPCNA, p-p65, RAGE, and toll-like receptor 4 (TLR4) in the endometrium tissues of\nfour groups of mice using IHC staining ( Figure 4 ). Not surprisingly, we observed\nthat when compared to the endometrium tissues from control normal mice, the\nexpression of HMGB1, IL-33, osteopontin, PCNA, p-p65, RAGE, and TLR4 were\nsubstantially upregulated in the endometrium tissues from mice with adenomyosis\n( Figure 5(A)-5(G) ).\nResveratrol treatment at 25 mg/kg and 50 mg/kg were able to reduce the\nexpression levels of HMGB1, IL-33, osteopontin, PCNA, p-p65, RAGE, and TLR4 in\nthe endometrium tissues, suggesting that resveratrol treatment might mitigate\nthe progression of adenomyosis through downregulation of these\nadenomyosis-related gene expressions. The high dose (50 mg/kg) of resveratrol\nexhibited a more potent gene expression inhibition effect than the low dose\n(25 mg/kg) of resveratrol. Figure 4. Representative immunohistochemistry\nstaining of the HMGB1, IL-33, Ostepontin, PCNA, p-p65, RAGE, and\nTLR4 in the endometrium tissues in the mice. Figure\n5. Immunohistochemistry staining results to\nindicate the staining levels of the HMGB1 (A), IL-33 (B), Ostepontin\n(C), PCNA (D), p-p65 (E), RAGE (F), TLR4 (G) in the mice. The data\nare mean ± SD with scatter plots. Control group: n = 10; untreated\ngroup: n = 10; 50 mg/kg resveratrol group: n = 9; 25 mg/kg\nresveratrol group: n = 10. * P  < .05,\n** P  < .01, *** P  < .001\nvs control group;  # P  < .05,\n ## P  < .01,\n ### P  < .001 vs untreated\ngroup.\nRepresentative immunohistochemistry\nstaining of the HMGB1, IL-33, Ostepontin, PCNA, p-p65, RAGE, and\nTLR4 in the endometrium tissues in the mice.\nImmunohistochemistry staining results to\nindicate the staining levels of the HMGB1 (A), IL-33 (B), Ostepontin\n(C), PCNA (D), p-p65 (E), RAGE (F), TLR4 (G) in the mice. The data\nare mean ± SD with scatter plots. Control group: n = 10; untreated\ngroup: n = 10; 50 mg/kg resveratrol group: n = 9; 25 mg/kg\nresveratrol group: n = 10. * P  < .05,\n** P  < .01, *** P  < .001\nvs control group;  # P  < .05,\n ## P  < .01,\n ### P  < .001 vs untreated\ngroup.\nWe recently reported that plasma HMGB1, HA, and osteopontin serve as promising\nbiomarkers for endometriosis. 20  Whether they were able to\nbe used as potential biomarkers for adenomyosis was not addressed. Therefore, we\nmeasured the plasma HMGB1, HA, and osteopontin levels from four groups of mice\nusing ELISA assay. The results in  Figure 6(A)-6(C)  depicted that the\nplasma HMGB1, HA, and osteopontin levels were remarkably enhanced in mice with\nadenomyosis compared to control normal mice. Intriguingly, administration of\nresveratrol at 25 mg/kg and 50 mg/kg exerted similar effects on the\ndownregulation of plasma HMGB1, HA, and osteopontin levels in mice with\nadenomyosis. Figure\n6. Plasma concentrations of HA (A), HMGB1 (B), and\nOstepontin (C) in the blood of the experimental mice, determined by\nELISA assays. The data are mean ± SD with scatter plots. Control\ngroup: n = 10; untreated group: n = 10; 50 mg/kg resveratrol group:\nn = 9; 25 mg/kg resveratrol group: n = 10. *** P \n< .001 vs control group;  ### P  <\n.001 vs untreated group.\nPlasma concentrations of HA (A), HMGB1 (B), and\nOstepontin (C) in the blood of the experimental mice, determined by\nELISA assays. The data are mean ± SD with scatter plots. Control\ngroup: n = 10; untreated group: n = 10; 50 mg/kg resveratrol group:\nn = 9; 25 mg/kg resveratrol group: n = 10. *** P \n< .001 vs control group;  ### P  <\n.001 vs untreated group.\n\nIn this study, we have shown that starting just four weeks after the induction, the\nmice displayed a significantly higher body weight gain rate and progressive\ndeterioration of hotplate latency when compared to control normal mice without the\ninduction. Furthermore, mice with adenomyosis exhibited extensive fibrosis in the\nectopic endometrium, as demonstrated by Masson staining and H&E staining. In\ncontrast, no fibrotic tissue was observed in the ectopic endometrium of control\nnormal mice. These results were consistent with our several previous publications,\nconfirming the successful establishment of a mouse model of adenomyosis. 17 - 22  Remarkably, we further\nrevealed that compared with the untreated group, resveratrol treatment was capable\nof enhancing hotplate latency and reducing fibrosis in the ectopic endometrium of\nthe mice with adenomyosis, although the restoration effect of resveratrol was not\ncomplete as compared to the control normal mice. Surprisingly, the resveratrol\ntreatment resulted in substantially reduced body weight and uterine weight as\ncompared to either the control group or the untreated group, raising potential\nunfavorable safety concerns regarding resveratrol to mice and humans. 23 , 24\nOur previous study reported the significant upregulation of HMGB1, TLR4, RAGE, p-p65,\nPCNA, IL-33, and osteopontin in endometriotic tissues from mice with endometriosis\nand these results were further confirmed in ovarian endometriomas from patients with\nendometriosis. 20  Interestingly, our results illustrated that the expression\nlevels of HMGB1, TLR4, RAGE, p-p65, PCNA, IL-33, and osteopontin in endometriotic\ntissues from mice with adenomyosis were significantly higher than those in\nendometriotic tissues from normal mice, confirming that adenomyosis exhibits many\nsimilarities to endometriosis. Importantly, resveratrol treatment, especially high\ndose, potently suppressed the expression of HMGB1, TLR4, RAGE, p-p65, PCNA, IL-33,\nand osteopontin in endometriotic tissues from mice with endometriosis, suggesting\nresveratrol might decrease the progression of adenomyosis via reducing these gene\nexpression. Because activation of RAGE, IL-33, p-p65, PCNA, and osteopontin play\ncritical roles in promoting cell proliferation of osteoclasts and fibroblasts, and\nimmune cell activation and infiltration. 25 - 30  The resveratrol-induced\ninhibition of these gene expression might result in reduced proliferation of\nosteoclasts and fibroblasts and suppressed myometrial infiltration, leading to\nattenuating the progression of adenomyosis.\nHA is a glycosaminoglycan generated by numerous mesenchymal cells and tumor cells,\nand it is critically important in the recruitment and activation of inflammatory\ncells. 31  Osteopontin is reported to be involved in the development of\nangiotensin II-induced fibrosis. Osteopontin has been shown to interact with a\nvariety of extracellular matrices (e.g., collagen and fibronectin), implying a\npotential function in matrix organization and stability. 32  HMGB1 has a diverse set of\nimmunological functions, including the activation of cell proliferation,\ndifferentiation, and cytokine secretion. 33 , 34  Accumulating evidence has\nlinked HMGB1 to liver fibrosis. As potential indicators, the plasma concentration of\nHA, HMGB1, and osteopontin have been associated with fibrosis. 35 , 36  We previously\ndemonstrated that plasma HA, HMGB1, and osteopontin could be promising biomarkers\nfor detecting endometriosis. 37  In line with this finding, we\nalso observed upregulation of plasma HA, HMGB1, and osteopontin concentration in\nmice with adenomyosis. Resveratrol treatment significantly reduced the plasma HA,\nHMGB1, and osteopontin concentrations, further confirming that resveratrol can slow\nthe progression of adenomyosis.\nThere are several limitations should be noted. First, the adenomyosis-related gene\nexpression is not explored here. Second, the protective effects of resveratrol in\nother adenomyosis models established by different methods could be verified. Last,\nit would be of much clinical relevance and importance if the effects of resveratrol\nin clinical patients with adenomyosis are studied.\n\nThe current study further confirms many previous reports that the induction of\nadenomyosis in mice results in body weight loss and uterine weight gain, reduced\nhotplate latency, and progressive ectopic endometrium fibrosis. The underlying\nmolecular mechanism might be attributed to the upregulation of several cell\nproliferation- and immune-regulation-associated genes, such as HMGB1, TLR4, RAGE,\np-p65, PCNA, IL-33, and osteopontin. We proved that administration of resveratrol\nwas able to attenuate the progression of adenomyosis as manifested by increasing\nhotplate latency, decreasing fibrosis status of endometrium, and restoring cell\nproliferation- and immune-regulation-associated gene expression levels in\nendometrium and plasma. However, it also should be noted that resveratrol treatment\nreduced the body and uterine weight, which may be its side effects in the treatment\nof adenomyosis.","source_license":"CC0","license_restricted":false}