{"paper_id":"c9d6acd7-463f-4283-997d-a1c21b4d7819","body_text":"LI M Y , WANG Y W, SHAO Y T, et al. Effects of acupuncture and moxibustion on UHRF1  and DNMT1 in ectopic endometrium of  \nrats with endometriosis. J Acupunct Tuina Sci, 2024, 22(3): 204-213  \nDOI: https://doi.org/10.1007/s11726-023-1412-2 \n● 204 ●︱© The author(s) for open access article 2023    \nBasic Study \n \nEffects of acupuncture and moxibustion on UHRF1 \nand DNMT1 in ectopic endometrium of  \nrats with endometriosis     \n针灸对子宫内膜异位症大鼠异位内膜组织UHRF1和DNMT1的影响 \nLI Mingyang (李铭旸)1,2, WANG Yanwen (王延文)3, SHAO Yanting (邵燕婷)1, SUN Yichun (孙祎纯)1, HU Jiawei (胡佳玮)1,  \nGAO Yuan (高源)1, LI Yuran (李语然)1, WU Chuting (吴楚婷)1, ZHANG Chunyan (张春雁)1 \n1 Yueyang Hospital of Integrated Traditional Chinese and Wester n Medicine, Shanghai University of Traditional Chinese Medicine,  \nShanghai 200437, China  \n2 Shanghai Minhang District Meilong Community Health Service Center, Shanghai 201108, China \n3 Longhua Hospital, Shanghai University of Traditional Chinese Medicine, Shanghai 200032, China \n \nAbstract  \nObjective: To observe the effects of acupuncture and moxibustion on ubiquitin-like containing PHD and RING finger \ndomains 1 (UHRF1) and DNA methyltransferase 1 (DNMT1) in ectopic endometrium of rats with endometriosis (EMS).  \nMethods: Forty Sprague-Dawley rats were randomly divided into a sham operation group with 10 rats and a \nmodel-building group with 30 rats according to body mass.  EMS rat models were established in the model-building \ngroup and then were divided into a model group, an acupuncture and moxibustion group, and a progesterone group, \nwith 10 rats in each group. All rats were fixed by a fixator. The sham operation group and the model group were given \nnormal saline by gavage. The acupuncture and moxibustion group received acupuncture at Xuehai (SP10) and \nSanyinjiao (SP6), moxibustion at Guanyuan (CV4), and gavage of normal saline. The progesterone group was given the \nmixed liquid made of dydrogesterone and normal saline by gavage. After 28 d of treatments, the three diameters \n(length, width, and height) of EMS rats’ ectopic cysts were measured, the cyst volumes were calculated, the volumes \nbefore intervention were subtracted, and the difference values were used to evaluate the growth of ectopic cysts. \nUHRF1 and DNMT1 mRNA and protein levels in normal endometrium, eutopic endometrium, and ectopic endometrium \nwere detected by real-time polymerase chain reaction and immunohistochemistry. \nResults:\n There was no significant difference in the ectopic cyst volume difference between the acupuncture and \nmoxibustion group and the progesterone group (P>0.05), but they were smaller than that of the model group ( P<0.05). \nThe levels of UHRF1 and DNMT1 mRNA and protein in the ec topic endometrium of the mo del group were lower than \nthose in the normal endometrium ( P<0.05). The levels of DNMT1 mRNA and UHRF1 protein in the eutopic \nendometrium of the model group were lower than those in the normal endometrium ( P<0.05). The levels of UHRF1 \nmRNA and protein and the level of DNMT1 protein in the ectopic endometrium of the acupuncture and moxibustion \ngroup were higher than those in the model group ( P<0.05), and the level of  UHRF1 mRNA was higher than that in the \nprogesterone group ( P<0.05). The level of DNMT1 mRNA in the eutopic endometrium of the acupuncture and \nmoxibustion group was higher than that in the model group ( P<0.05). The levels of UH RF1 and DNMT1 mRNA and \nprotein in the acupuncture and moxibustion group were insignificantly different from those in the normal \nendometrium (P>0.05).  \nConclusion:\n Acupuncture and moxibustion may up-regulate the levels of UHRF1 mRNA and UHRF1 and DNMT1 \nproteins in the ectopic endometrium to the normal level so as to reduce the volume of ectopic cysts and cure EMS in \nrats. \nKeywords: Acupuncture Therapy; Moxibustion Therapy; Acupuncture-moxibustion Therapy; Endometriosis; UHRF1; DNMT1; \nRats     \n【摘要】目的 : 观察针灸对子宫内膜异位症 (EMS) 泛素样PHD和含环指结构域蛋白 1(UHRF1) 和DNA 甲基转移酶 1 \n(DNMT1)的影响。方法: 将40只Sprague-Dawley大鼠按体质量随机分为假手术组10只和造模组30只。 造模组大鼠复 \n \nCo-first Authors: LI Mingyang, M.M., resident physician; WANG Yanwen, \nbachelor, technician in charge \nJoint Corresponding Authors: WU Chuting, M.M., resident physician.  \nE-mail: wuchutingkk@163.com; ZHANG Chunyan, M.D., chief physician, \nmaster student supervisor. E-mail: zh_ch_yan@163.com \n\nJ. Acupunct. Tuina. Sci. 2024, 22(3): 204-213  \n© The author(s) for open access article 2023︱● 205 ● \n制EMS模型后随机分为模型组、针灸组和孕酮组, 每组 10 只。固定器固定各组大鼠。假手术组和模型组予以生理\n盐水灌胃; 针灸组予以针刺血海和三阴交, 艾灸关元和生理盐水灌胃; 孕酮组给予地屈孕酮生理盐水混悬液灌胃。 治\n疗 28 d后, 测量大鼠异位囊肿三径(长、宽及厚度)并计算体积, 减掉干预前异位囊肿体积, 取差值评估异位囊肿生长\n情况; 实时荧光定量聚合酶链反应和免疫组织化学法检测正常子宫内膜、在位及异位内膜组织UHRF1、DNMT1 mRNA\n和蛋白水平。结果: 针灸组和孕酮组异位囊肿体积差值无统计学差异(P>0.05), 但均小于模型组异位囊肿体积差值\n(P<0.05)。模型组异位内膜组织UHRF1、DNMT1 mRNA和蛋白水平低于正常子宫内膜组织(P<0.05); 模型组在位内膜组\n织DNMT1 mRNA 以及UHRF1 蛋白水平低于正常子宫内膜组织(P<0.05); 针灸组异位内膜组织UHRF1 mRNA 及蛋白水\n平、DNMT1 蛋白水平高于模型组异位内膜组织(P<0.05), 其中UHRF1 mRNA水平高于孕酮组(P<0.05); 针灸组在位内膜\n组织DNMT1 mRNA水平高于模型组在位内膜组织(P<0.05); 针灸组异位内膜组织UHRF1、DNMT1 mRNA和蛋白水平与\n正常子宫内膜比较无统计学差异(P>0.05)。结论: 针灸可能通过上调异位内膜组织中UHRF1 mRNA和UHRF1、DNMT1\n蛋白水平, 使其趋于正常子宫内膜水平, 从而达到缩小异位囊肿体积, 治疗大鼠子宫内膜异位症的效应。 \n【关键词】针刺疗法; 灸法; 针灸疗法; 子宫内膜异位症; 泛素样PHD和含环指结构域蛋白1;  DNA甲基转移酶 1; 大鼠 \n【中图分类号】R2-03        【文献标志码】A \n \nEndometriosis (EMS) refers to the occurrence, \ngrowth, and infiltration of the endometrium (glands and \nstroma) outside the uterine cavity, which leads to \nrepeated bleeding, pain, infertility, nodules, or cysts. It \nis a common disease in women of reproductive age\n[1], \nand it is an estrogen-dependent chronic inflammatory \ndisease\n[2]. Estrogen plays a crucial role in EMS through \nestrogen receptors in two subtypes: estrogen receptor \nα (ERα) and estrogen receptor β (ERβ). In comparison \nwith normal endometrium, ER α mRNA and protein \nlevels are significantly lower in ectopic endometrium, \nwhile ER β is higher\n[3-6]. ER β affects ER α during the \npathogenesis of EMS[7]. ERβ may play a key role in the \ndevelopment of endometriosis, but the specific \nmechanism of ERβ up-regulation is still unclear. Studies \nhave found that the hypomethylation of cytosine- \nphosphate-guanine (CpG) islands in the ER β gene \npromoter region may be related to the high ERβ levels \nin endometriosis tissues\n[8]. DNA methyltransferase 1 \n(DNMT1) is the main DNA methyltransferase in \nmammals. It restores hemimethylated sites on CpG \nislands to fully methylated status after DNA replication, \nthereby silencing transcription with no expression of \ngenes\n[9-10]. Domestic and foreign studies have found \nthat the DNMT1 expression level in the ectopic and \neutopic endometrium of EMS patients is significantly \nlower than that in normal endometrium\n[11-12]. \nSemi-methylation is a special state of DNA that can be \nrecognized by ubiquitin-like containing PHD and RING \nfinger domains 1 (UHRF1), guiding DNMT1 to target \nDNA replication forks, thereby ensuring stable \ninheritance of DNA methylation\n[13-14]. It is speculated \nthat UHRF1 may be one of the influencing factors of \nabnormal DNMT1 expression in endometriosis. \nTherefore, this study established an EMS rat model and \ninvestigated the expression of UHRF1 and DNMT1 in the \nnormal endometrium, eutopic endometrium of EMS \nrats, and ectopic endometrium to explore the possible \nmechanism of acupuncture and moxibustion treatment \nfor EMS. \n \n \n1 Materials and Methods \n  \n1.1 Materials  \n1.1.1 Animals \nForty 6-8 week s female Sprague-Dawley r a t s  w i t h  a  \nbody mass of (200±20) g were provided by the \nExperimental Animal Center of Yueyang Hospital of \nIntegrated Traditional Chinese and Western Medicine, \nShanghai University of Traditional Chinese Medicine \n[License No. SYXK (Hu) 2018-0040]. The rats were \nproduced by Shanghai SLAC Laboratory Animal Co., Ltd., \nChina [Production License No. SCXK (Hu) 2022-0004]. \nThe rats were housed under controlled conditions: \ntemperature (20±2) ℃, relative humidity (50%±10%), \nlight/dark cycle 14 h/10 h, free access to food and water, \nand high-pressure sterilized padding and drinking water. \nAfter acclimatization, the rats were screened for estrus \nby vaginal smears. The animal experiments were \napproved by the Animal Ethics Committee of Yueyang \nHospital of Integrated Traditional Chinese and Western \nMedicine, Shanghai University of Traditional Chinese \nMedicine (Ethics Approval No. YYLAC-2021-134-1). \n1.1.2 Appliance and reagents \nFiliform needles (0.22 mm in diameter and 25 mm in \nlength, Lot No. 210224, Su zhou Medical Appliance \nFactory, China); moxa sticks (Lot No. 20220506/10, \nNanyang Hanyi Moxa Co., Ltd., China); anti-UHRF1 \nantibody (Cat. No. ab194236, Abcam, USA); anti- \nDNMT1 antibody (Cat. No . ab188453, Abcam, USA); \nHRP-conjugated secondary antibody, buffer, DAB \nchromogenic agent, and high-pH antigen retrieval \nsolution (Lot No. 20078526, No. 20076962, No. \n20074026, No. 20079714, Dako, Denmark); chloroform \n(Lot No. 20161026, Sinopharm, China); iTaq\nTM Universal \nSYBR® Green Supermix and iScriptTM cDNA synthesis kit \n(Cat. No. 1708891, No. 1725124, Bio-Rad, USA); tissue \nRNA purification kit (Cat. No. B0004DP , EZBioscience, \nUSA).  \n1.1.3 Equipment \nASP300S tissue dehydrator, HISTOCORE ARCADIA H \ntissue embedding machine, RM2235 rotary tissue slicer,  \n\nJ. Acupunct. Tuina. Sci. 2024, 22(3): 204-213  \n● 206 ●︱© The author(s) for open access article 2023 \nDMi8 inverted microscope (Leica, Germany); \nAUTOSTAINER LINK48 automated immune- \nhistochemistry staining system (Dako, Denmark); 7500 \nfluorescence quantitative polymerase chain reaction \n(PCR) instrument (Life Technologies, USA); Veriti \ngradient PCR instrument and NanoDrop One \nspectrophotometer (Thermo Fisher Scientific Inc., USA). \n1.2 Grouping and modeling \nA total of 40 rats were randomly divided into a sham \noperation group with 10 rats and a model-building \ngroup with 30 rats.  According to the previous research \nof our team\n[5], rats in the model-building group in estrus \nwere selected by vaginal smear examination and were \nsubjected to surgical modeling according to the method \nof JONES R C\n[15]. We operated on the rats under sterile \nconditions, fixed the anesthetized rats to the operating \ntable, shaved hair on the abdomen of the rats, and \nperformed routine disinfection. Then, a 2-3 cm long \nlongitudinal incision was made at 1 cm above the pubic \nsymphysis, and the abdominal cavity was opened. The \nleft uterine was found, and local blood vessels and the \nsegment of the uterus to be excised were ligated. Then, \nthe left uterine was removed, placed in normal saline, \nand longitudinally cut open to separate the \nendometrium from the muscular layer. The \nendometrium was cut into 5 mm × 5 mm fragments, \nand one corner was fixed with a 5-0 silk suture with the \nepithelium facing the ovary, uterine ligament, or \nperitoneum. Rats in the sham operation group \nunderwent a sham operation using the same incision \nmethod. After 21 d, the modeled rats were explored by \nlaparotomy to observe the growth of ectopic cysts, and \nsome cysts were taken for histopathological observation \nto confirm the success of the EMS model. After the \nmodel was established successfully, the model-building \ngroup was randomly divided into a model group, an \nacupuncture and moxibustion group, and a \nprogesterone group, with 10 rats in each group. \n1.3 Interventions \nRats in the sham operation group and the model \ngroup were fixed and given normal saline by oral gavage. \nRats in the acupuncture and moxibustion group were \nfixed and received acupuncture and moxibustion \ntreatment and gavage of normal saline. The \nprogesterone group was fixed and given a suspension of \ndydrogesterone and normal saline by gavage. \nAcupuncture and moxibustion treatment: The rats \nwere fixed in a supine position, and Xuehai (SP10), \nSanyinjiao (SP6), and Guanyuan (CV4) were selected. \nThe point selection method and location for Sanyinjiao \n(SP6) and Guanyuan (CV4) consulted the Nomenclature \nand Location of Acupuncture Points for Laboratory \nAnimals. Part 2: Rat\n[16]. The point selection method and \nlocation for Xuehai (SP10) were based on Experimental \nAcupuncture Science [17]. The filiform needles were \ninserted perpendicularly into the unilateral Xuehai \n(SP10) and Sanyinjiao (SP6) for 2 mm. A moxa cone of \n15 mm × 20 mm was placed over Guanyuan (CV4), 3 cm \naway from the skin surface, and ignited for moxibustion. \nTreatment lasted 10 min per session, once daily for one \nside points, with the other side treated on the following \nday. \nGavage: The equivalent dose of dydrogesterone for \nr a t s  w a s  c a l c u l a t e d  a s  0 . 9 4  m g / ( k g · b w )  a c c o r d i n g  t o  \nDose Conversion Among Different Animals and Healthy \nVolunteers in Pharmacological Study\n[18], and the gavage \nv o lu me  w as  c a l cul a t ed  as  10  m L / ( kg · b w)  b as ed  o n  th e \nrat body mass. Before gavage, the prepared suspension \nwas mixed thoroughly by inverting the container. \nTreatment course: Once a day for 28 d. \n1.4 Detection indicators and methods  \n1.4.1 Measurement of ectopic cyst size \nAfter treatment, the estrous cycle of rats was \ndynamically monitored, and the rats were euthanized \nduring estrus. We collected the normal endometrium, \nectopic endometrium, and eutopic endometrium from \nthe rats. After laparotomy, we measured the length, \nwidth, and height of ectopic cysts with a vernier caliper \nand calculated the volumes of the cysts by the formula \nV = 0.52 × a × b × c (a, b, and c represent the length, \nwidth, and height of the cyst, respectively)\n[19]. The \nvolumes of ectopic cysts were subtracted from the \nvolumes after modeling, and the differences in the \nvolumes of ectopic cysts were taken to observe the \ngrowth of ectopic cysts and evaluate the effect of \ntreatment intervention. \n1.4.2 Detection of UHRF1 and DNMT1 mRNA levels in \nrat endometrium  \nTotal RNA was extracted from normal endometrium, \nectopic endometrium, and eutopic endometrium of rats \nusing chloroform and the EZ BTM RNA purification kit. \nThe concentration and purity of the RNA samples were \ndetermined. Then cDNA was synthesized using the \niScript\nTM cDNA synthesis kit. Fluorescence quantitative \nPCR was performed with GAPDH as the housekeeping \ngene. The levels of UHRF1 and DNMT1 mRNA in rat \nendometrium of each group were detected. The \namplification primers were synthesized by Sangon \nBiotech (Shanghai) Co., Ltd., China, and the primer \nsequences are shown in Table 1. The reaction \nconditions were as follows: pre-denaturation at 95 ℃ \nfor 30 s, denaturation at 95 ℃ for 15 s, annealing and \nextension at 60 ℃ for 60 s, 40 cycles in total. Three \nreplicate wells were set up for each group. The relative \nlevels of the target genes were calculated using the \n2\n-ΔΔCt method.  \n1.4.3 Detection of UHRF1 and DNMT1 protein level in \nrat endometrium  \nNormal endometrium, ectopic endometrium, and \neutopic endometrium of rats were fixed in 10% neutral \nformalin solution, dehydrated, embedded in paraffin, \n\nJ. Acupunct. Tuina. Sci. 2024, 22(3): 204-213  \n© The author(s) for open access article 2023︱● 207 ● \nand sectioned continuously at 4 μm. The levels of \nUHRF1 and DNMT1 proteins in the endometrium were \ndetected by immunohistochemistry. UHRF1 and \nDNMT1 antibodies were diluted at 1:100, and antigen \nretrieval was performed. DAB staining was performed, \nfollowed by counterstaining with hematoxylin. UHRF1 \nand DNMT1 were both positively expressed in the cell \nnucleus. The positive results of UHRF1 and DNMT1 \nwere judged according to the histochemistry score \n(H-score)\n[20], as described in our team’s previous study[5]. \nTwo pathologists independen tly scored the staining \nintensity and the percentage of positive cells. The \nstaining intensity was classified as 0, 1\n+, 2+, or 3 +. The \npercentage of positive cells was estimated to range \nfrom 0% to 100%. The scores were multiplied and \nadded to obtain a total score, which ranged from 0 \n(negative) to 300 (strongly positive). \n \nTable 1  Primer sequences for real-time polymerase chain reaction \nPrimer name Base sequence (5 ′→3′) Product length/bp \nUHRF1 \nForward primer CAACTGCTTTGCTCCCATCAATGAC 25 \nReverse primer TCCTCCCT CTTGCTGTTCTCCTTC 24 \nDNMT1 \nForward primer GGATTCCACCAAGCAGGCATCTC 23 \nReverse primer CCACCACACAGCATCTCCACATC 23 \nGAPDH \nForward primer GACATGCCGCCTGGAGAAAC 20 \nReverse primer AGCCCAGGATGCCCTTTAGT 20 \nNote: UHRF1=Ubiquitin-like containing PHD and RING finger domains 1; DNMT1=DNA methyltransferase 1. \n \n1.5 Statistical methods  \nThe statistical program used was SPSS version 26.0. \nNormally distributed cont inuous variables were \nexpressed as mean ± standard deviation  ( x ±s), and \none-way analysis of varian ce was used for multiple \ngroup comparisons. If the variance was homogeneous, \nthe least significant difference t-test was used, and if \nthe variance was heterogeneous, the Dunnett T3 test \nwas used. For variables that did not follow a normal \ndistribution, the median combined with the \ninterquartile range was used to express the data, and \nnonparametric tests were used for group comparisons. \nAll tests were two-sided, α=0.05 was used as the test \nlevel, and P<0.05 indicated that the difference was \nstatistically significant. \n \n2 Results  \n \n2.1 Comparison of the ectopic cyst volume in EMS rats  \nUpon dissection of the model rats, ectopic cysts \nformed. We took partial ectopic cysts at random. \nHistopathological observation showed typical \nendometrial interstitial and glandular structures, thin \nepithelial areas, irregular arrangement of interstitial, \nnumerous glands, abundant glandular ducts, abundant \nand uneven distribution of blood vessels, and \ninflammatory cell infiltration in the interstitial space, \nand the cyst was confirmed to be ectopic endometrium. \nThese findings validated the successful establishment of \nEMS rat models. The length, width, and height of \nectopic cysts in each group of rats were measured by \nvernier caliper. The difference in the volume of ectopic \ncysts was calculated (after intervention minus before \nintervention). See Figure 1. \n \n \nNote: A) Ectopic cyst formation and measurement of the three \ndiameters; B) pathological observation of ectopic cysts (×400). \nFigure1  Establishment of endometriosis rat models \n \nThe results showed that the acupuncture and \nmoxibustion group and the progesterone group had \nsmaller volume differences of ectopic cysts than the \nmodel group ( P<0.05), and there was no statistically \nsignificant difference in the volume difference of \nectopic cysts between the acupuncture and \nmoxibustion group and the progesterone group \n(P>0.05). See Figure 2. \n2.2 Comparison of the UHRF1 mRNA and protein \nlevels in rat endometrium  \nReal-time (RT)-PCR was used to detect the mRNA \nlevels of UHRF1 in the normal endometrium, ectopic \nendometrium, and eutopic endometrium of rats. The \nresults showed that the UHRF1 mRNA level in the \nnormal endometrium (1.00±0.46) was higher than that \nin the ectopic endometrium of the model group and the \nprogesterone group ( P<0.05), while there was no \nnotable difference compared with the ectopic \n\nJ. Acupunct. Tuina. Sci. 2024, 22(3): 204-213  \n● 208 ●︱© The author(s) for open access article 2023 \nendometrium of the acupuncture and moxibustion \ngroup (P>0.05). The UHRF1 mRNA level in the ectopic \nendometrium of the acupuncture and moxibustion \ngroup was higher than that of the model group and the \nprogesterone group ( P<0.05), while there was no \nsignificant difference between the model group and the \nprogesterone group (P>0.05). There was no significant \ndifference in the UHRF1 mRNA level among the groups \nin the eutopic endometrium (P>0.05). See Table 2. \nImmunohistochemistry was used to detect the level \nof UHRF1 protein in the normal endometrium, ectopic \nendometrium, and eutopic endometrium of rats. \nUHRF1 protein was mainly expressed in the nucleus of \nglandular epithelial and stromal cells. The H-score was \nused to analyze the protein staining of tissues semi- \nquantitatively. See Figure 3. \n \nNote: Compared with the model group, 1) P<0.05.  \nFigure 2  Comparison of the volume difference of ectopic   \ncysts among groups after intervention \n \n \nTable 2  Comparison of the UHRF1 mRNA level in normal, ectopic, and eutopic endometrium of rats (x ±s) \nGroup n Normal endometrium Ectopic endometrium Eutopic endometrium \nSham operation  10 1.00±0.46 / / \nModel  10 /   0.01±0.01 1)  0.95±0.95 \nAcupuncture and moxibustion 10 /     1.20±0.29 2)3) 0.04±0.04 \nProgesterone  10 /     0.10±0.15 1)2)  0.31±0.31 \nNote: /  represents no value; compared w ith the sham operation group, 1) P<0.05; compared with the model group, 2) P<0.05; \ncompared with the progesterone group, 3) P<0.05.  \n \n \n  \n  \n \n  \n  \n  \nNote: A) Normal endometrium of the sham operation group; B) ectopic endometrium of the model group; C) ectopic endometrium of the \nacupuncture and moxibustion group; D) ectopic endometrium of the progesterone group; E) eutopic endometrium of the model group;           \n F) eutopic endometrium of the acupuncture and moxibustion group; G) eutopic endometrium of the progesterone group.  \nFigure 3  Level of UHRF1 in normal, ectopic, and eutopic endometrium of rats (immunohistochemistry) \n \nThe results showed that the level of UHRF1 protein in \nthe normal endometrium was h i g h e r  t h a n  t h a t  i n  t h e  \nectopic endometrium and eutopic endometrium of the \nmodel group ( P<0.05), lower than that in the eutopic \nendometrium of the progesterone group (P<0.05), but \nno significant difference compared with the ectopic \nendometrium of the acupuncture and moxibustion \ngroup (P>0.05). In the ectopic endometrium, the level \nof UHRF1 protein in the acupuncture and moxibustion \ngroup and the progesterone group was significantly \nhigher than that in the model group ( P<0.05). In the \neutopic endometrium, the level of UHRF1 protein in the \nprogesterone group was significantly higher than that in \nthe model group (P<0.05). See Table 3. \n2.3 Comparison of the DNMT1 mRNA and protein \nlevels in rat endometrium \nRT-PCR was used to detect the DNMT1 mRNA level in \nnormal, ectopic, and eutopic endometrium of rats. The \n\nJ. Acupunct. Tuina. Sci. 2024, 22(3): 204-213  \n© The author(s) for open access article 2023︱● 209 ● \nresults showed that the DNMT1 mRNA level in the \nnormal endometrium was higher than that in the \nectopic and eutopic endometrium of the model group, \nas well as in the euto pic endometrium of the \nacupuncture and moxibustion group and the \nprogesterone group (P<0.05). There were no significant \ndifferences between various groups in the ectopic \nendometrium (P>0.05). In the eutopic endometrium, \nthe DNMT1 mRNA level in the acupuncture and \nmoxibustion group was higher than that in the model \ngroup (P<0.05). See Table 4. \nImmunohistochemical staining was used to detect \nthe DNMT1 protein level in normal, ectopic, and \neutopic endometrium of rats. The DNMT1 protein was \nmainly expressed in the nucleus of glandular epithelial \nand stromal cells. The H-score was used to analyze the \nprotein staining of tissue s semi-quantitatively. See \nFigure 4. \nThe results showed that the DNMT1 protein level in \nthe normal endometrium was h i g h e r  t h a n  t h a t  i n  t h e  \nectopic endometrium of the model group ( P<0.05). In \nthe ectopic endometrium, the DNMT1 protein level in \nthe acupuncture and moxibustion group was \nsignificantly higher than that in the model group \n(P<0.05), and there was no significant difference \nbetween the acupuncture and moxibustion group and \nthe progesterone group ( P>0.05). In the eutopic \nendometrium, the DNMT1 protein level in the model \ngroup and the acupuncture and moxibustion group was \nhigher than that in the progesterone group ( P<0.05). \nSee Table 5. \n \nTable 3  Comparison of the UHRF1 protein level in normal, ectopic, and eutopic endometrium of rats (x ±s) \nGroup n Normal endometrium Ectopic endometrium Eutopic endometrium \nSham operation  10 58.33±7.64 / / \nModel  10 /   18.33±8.39 1)  23.67±3.06 1) \nAcupuncture and moxibustion 10 /   45.00±5.00 2) 88.33±18.93 \nProgesterone  10 /    53.33±7.64 2)    145.00±18.03 1)2) \nNote: / represents no value; compared with the sham operation group, 1) P<0.05; compared with the model group, 2) P<0.05.  \n \nTable 4  Comparison of the DNMT1 mRNA level in normal, ectopic, and eutopic endometrium of rats (x ±s) \nGroup n Normal endometrium Ectopic endometrium Eutopic endometrium \nSham operation  10 1.00±0.12 / / \nModel  10 /   0.03±0.01 1) 0.00±0.00 1) \nAcupuncture and moxibustion 10 / 0.94±0.33   0.01±0.00 1)2) \nProgesterone  10 / 0.46±0.58 0.00±0.00 1) \nNote: / represents no value; compared with the sham operation group, 1) P<0.05; compared with the model group, 2) P<0.05.  \n \n \n  \n  \n \n  \n  \n  \nNote: A) Normal endometrium of the sham operation group; B) ectopic endometrium of the model group; C) ectopic endometrium of the \nacupuncture and moxibustion group; D) ectopic endometrium of the progesterone group; E) eutopic endometrium of the model group;  \nF) eutopic endometrium of the acupuncture and moxibustion group; G) eutopic endometrium of the progesterone group.  \nFigure 4  Level of DNMT1 in normal, ectopic, and eutopic endometrium of rats (immunohistochemistry) \n \n\nJ. Acupunct. Tuina. Sci. 2024, 22(3): 204-213  \n● 210 ●︱© The author(s) for open access article 2023 \nTable 5  Comparison of the DNMT1 protein levels in normal, ectopic, and eutopic endometrium of rats (x ±s) \nGroup n Normal endometrium Ectopic endometrium Eutopic endometrium \nSham operation  10 70.00±15.00 / / \nModel  10 /   28.33±7.64 1)  89.67±34.65 \nAcupuncture and moxibustion 10 /     98.33±20.21 2)  106.67±20.21 3) \nProgesterone  10 /   60.00±13.23    48.33±12.58 2) \nNote: /  represents no value; compared w ith the sham operation group, 1) P<0.05; compared with the model group, 2) P<0.05; \ncompared with the progesterone group, 3) P<0.05.  \n \n3 Discussion  \n \nTo date, the pathogenesis of endometriosis is not \nclear. Since the beginning of the 20th century, \nSAMPSON J A\n[21] proposed the classic “theory of \nretrograde menstruation and transplantation”, most \nwomen of childbearing age experience retrograde \nmenstruation\n[22], and only a few develop endometriosis. \nA wealth of evidence suggests that estrogen levels are \nelevated in ectopic tissues\n[23]. The increase in estrogen \nproduction, impaired estrogen metabolism, and altered \nexpression of estrogen receptors are the reasons for \nlocal estrogen excess that leads to the pathology of \nendometriosis\n[24]. Currently, hormone therapy remains \nthe most commonly used conservative treatment for \nendometriosis patients. The existing treatments that \ninterfere with the estrogen pathway aim to induce a \nstate of menopause or pseudo-pregnancy to block \nmenstruation. They can control the pain but have side \neffects, do not prevent or cure the disease, and often \nlead to treatment interruption by patients\n[19,25-26]. \nAlthough surgical treatment can remove the lesions, \npain may recur or persist if the ectopic lesion is not \ncompletely excised\n[27-28]. Therefore, exploring the \npathogenesis and treatment of endometriosis is of \ngreat significance for guiding clinical diagnosis and \ntreatment. \nThe research on stromal cells in endometriosis has \nfound that compared to normal endometrium, the low \nmethylation of the ER β promoter region leads to \nabnormal elevation of ERβ mRNA and protein levels\n[8]. \nIn most DNA promoter regions in mammals, there are \nCpG islands. Gene silencing can occur through CpG \nisland methylation. Abnormal DNA methylation often \noccurs in the promoter regions of transcription \nfactors\n[29-30]. DNMT1 plays a necessary role in \nmaintaining methylation stability [31]. Abnormal low \nexpression of DNMT1 in EMS may lead to epigenetic \nabnormalities in patients and participate in the \ndevelopment of EMS\n[12]. The estrogen-DNMT1 pathway \ninduces the methylation of RUNX3 and promotes \nproliferation and invasion activity in eutopic \nendometrial stromal cells\n[32]. Studies have reported that \nDNMT1 and ERβ are related, and 5-Aza-CdR induces cell \napoptosis and inhibits HT29 colon cancer cell growth by \ndownregulating DNMT1 and upregulating ERα and ERβ \ngene expression\n[33]. Disulfiram inhibits cell proliferation \nin prostate cancer and other cancers by re-expressing \nERβ and tumor suppressor genes, including DNMT1\n[34]. \nUHRF1 is a multi-domain protein that includes a \nubiquitin-like domain, tandem Tudor (TTD) domains, a \nplant homeodomain (PHD), a SET and RING-associated \n(SRA) domain, and a RING domain\n[35]. It is an auxiliary \nfactor required for maintaining DNA methylation. At the \nmolecular level, UHRF1 senses the presence of \nhemimethylated DNA through its SRA domain and it \nmay simultaneously recognize the binding of histone \nH3K9me2/3 and DNMT1 through its TTD domain\n[36-37], \nwhich is the structural basis for mediating DNMT1 \nrecruitment to DNA replication forks\n[38]. Dysregulation \nof DNMT1 and UHRF1 has been found in the malignant \ntransformation and progression of different types of \ncancer\n[39]. After DNMT1 and UHRF1 expression is \nreduced, a large number of tumor suppressor genes are \nre-expressed\n[39-41]. However, there have been no studies \non UHRF1 in EMS. In our study, the DNMT1 mRNA and \nprotein levels in the ectopic endometrium were lower \nthan those in the normal endometrium, consistent with \nthe reports of VEENA K V, et al\n[11] and YANG J, et al[12]. \nMoreover, we first discovered that UHRF1 mRNA and \nprotein levels in the ectopic endometrium of the model \ngroup were lower than those in the normal \nendometrium. Therefore, it is speculated that UHRF1 \nand DNMT1 may jointly participate in the occurrence \nand development of EMS. Professor LANG J H\n[42] \nproposed the “eutopic endometrial determinant”, \nholding that the primary cause of whether \nendometrium fragments in the blood reflux of different \npeople (with or without endometriosis) can adhere, \ninvade, and grow is the difference in the eutopic \nendometrium. In our study, the DNMT1 mRNA and \nprotein levels and UHRF1 protein level in the eutopic \nendometrium of the model group were lower than \nthose in the norm al endometrium, suggesting that \nDNMT1 and UHRF1 may participate in the disease’s \ndevelopment through eutopic endometrium. \nEndometriosis can be classified as “abdominal \nmasses”, “dysmenorrhea”, “menstrual disorders”, \n“infertility”, and other traditional Chinese medicine \ndiseases based on clinical manifestations. The main \npathogenesis is blood stasis, and the main location of \nthe disease is the uterus. It is closely related to the liver, \n\nJ. Acupunct. Tuina. Sci. 2024, 22(3): 204-213  \n© The author(s) for open access article 2023︱● 211 ● \nspleen, and kidney organs. Acupuncture and \nmoxibustion treatment for endometriosis has definite \ntherapeutic effects and minimal adverse reactions. Its \nmain function is circulating blood, transforming stasis, \nand regulating the liver, spleen, and kidney. In clinical \npractice, needling at Xuehai (SP10) and Sanyinjiao (SP6) \nand moxibustion at Guanyuan (CV4) are often used to \ntreat EMS patients. Sanyinjiao (SP6) is the crossing point \nof the Spleen Meridian of  Foot-Taiyin, the Liver \nMeridian of Foot-Jueyin, and the Kidney Meridian of \nFoot-Shaoyin, and is connected to the Conception \nVessel, Thoroughfare Vessel, and the uterus. It has the \nfunctions of tonifying and nourishing the spleen, liver, \nand kidney, regulating the balance of Yin and Yang of \norgans, and regulating the circulation of Qi and blood in \nthe Thoroughfare and Conception Vessels. Guanyuan \n(CV4) is a crossing point of three foot-Yin meridians and \nConception Vessel and is known as the “place where \nwomen store blood”. Acupuncture and moxibustion at \nthis point can stimulate the point, and the heat \ngenerated by burning moxa sticks can enter the \nabdomen, enhancing the functions of circulating blood, \nresolving masses, and consolidating basis. Xuehai (SP10), \nalso known as the “sea of twelve meridians” , is a point \non the Spleen Meridian of Foot-Taiyin and an important \npoint for treating gynecolo gical diseases with the \nfunctions of tonifying Qi and blood. Our team has long \nbeen committed to clinical and experimental research \non acupuncture and moxibustion treatment of EMS and \nhas found that acupuncture and moxibustion can \nup-regulate the expression of ERα, which is abnormally \nlow in the eutopic and ectopic endometrium, and \ndown-regulate the expression of ER β, which is \nabnormally high in the eutopic and ectopic \nendometrium\n[5]. In our study, acupuncture-moxibustion \ntreatment was chosen because it not only includes the \neffect of needling on harmonizing meridians but also \nexerts the effect of moxibustion on warming meridians \nand dissipating cold. The study found that acupuncture \nand moxibustion can reduce the size of ectopic cysts, \nfurther confirming the inhibitory effect of acupuncture \nand moxibustion treatment on EMS lesions. The \nexperiment chose the commonly used positive control \ndrug, dydrogesterone, which has been shown to \nalleviate the symptoms of endometriosis, cause atrophy \nof ectopic endometrium, and inhibit the development \nof EMS lesions\n[43]. Our study found that acupuncture- \nmoxibustion and dydrogesterone had similar effects in \nreducing the size of ectopic cysts. Acupuncture and \nmoxibustion can up-regulate the UHRF1 mRNA and \nprotein levels and DNMT1 protein level in the ectopic \nendometrium, suggesting that acupuncture and \nmoxibustion may inhibit the growth of ectopic lesions \nby up-regulating the abnormally low levels of UHRF1 \nmRNA and protein and DNMT1 protein in the ectopic \nendometrium to reach the levels of normal \nendometrium. However, this study only focused on the \nrelative mRNA and protein levels of UHRF1 and DNMT1. \nFurther research is needed to explore whether UHRF1 \nparticipates in the pathogenesis of EMS by regulating \nthe methylation of ERβ through DNMT1. \n \nReceived: 10 May 2023/Accepted: 18 July 2023 \n \nReferences \n \n[1] Chinese Obstetricians and Gynecologists Association, \nCooperative Group of Endometriosis, Chinese Society of \nObstetrics and Gynecology, Chinese Medical Association. \nGuideline for the diagnosis a nd treatment of endometriosis \n(third edition). Zhonghua Fu Chan Ke Za Zhi, 2021, 56(12): \n812-824. \n[2] BULUN S E, YILMAZ B D, SISON C, MIYAZAKI K, \nBERNARDI L, LIU S, KOHLMEIER A, YIN P, MILAD M, \nWEI J. Endometriosis. Endocr Rev, 2019, 40(4): 1048-1079. \n[3] SMUC T, PUCELJ M R, SINKOVEC J, HUSEN B, \nTHOLE H, LANISNIK RIZNER T.  Expression analysis of \nthe genes involved in estradiol and progesterone action in \nhuman ovarian endometriosis. 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Effect of acupuncture and moxibustion with \ntraditional Chinese medicine  in regulating endometrial \nConflict of Interest \nThe authors declare that there is no potential conflict of \ninterest in this article. \nAcknowledgments \nThis work was supported by the Rising-star Program of \n“Shanghai 2022 Science and Technology Innovation Action \nPlan”: Yangfan Special Project (上海市 2022 年度“科技\n创新行动计划”启明星培育 - 扬帆专项 , No. \n22YF1444200); Health Industry C linical Research Project \nof Shanghai Municipal Health Commission (上海市卫生健\n康委员会卫生行业临床研究专项 , No. 202240054); \nXinglin Scholar of Shanghai University of Traditional \nChinese Medicine ( 上海中医药大学“杏林学者” ); \nShanghai Shenkang Hospital Development Center to \nPromote Municipal Hospital Clinical Skills and Clinical \nInnovation Ability Three-year Action Plan Project (上海申\n康医院发展中心促进市级医院临床技能与临床创新能\n力三年行动计划项目, No. SHDC2020CR3093B). \nStatement of Human and Animal Rights \nThis study had been approved by the Animal Ethics \nCommittee of Y ueyang Hospital of Integrated Traditional \nChinese and Western Medicine, Shanghai University of \nTraditional Chinese Me dicine (Approval No.  \nYYLAC-2021-134-1). 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