Effects of acupuncture and moxibustion on UHRF1 and DNMT1 in ectopic endometrium of rats with endometriosis

Objective: To observe the effects of acupuncture and moxibustion on ubiquitin-like containing PHD and RING finger domains 1 (UHRF1) and DNA methyltransferase 1 (DNMT1) in ectopic endometrium of rats with endometriosis (EMS).

Forty Sprague-Dawley rats were randomly divided into a sham operation group with 10 rats and a model-building group with 30 rats according to body mass. EMS rat models were established in the model-building group and then were divided into a model group, an acupuncture and moxibustion group, and a progesterone group, with 10 rats in each group. All rats were fixed by a fixator. The sham operation group and the model group were given normal saline by gavage. The acupuncture and moxibustion group received acupuncture at Xuehai (SP10) and Sanyinjiao (SP6), moxibustion at Guanyuan (CV4), and gavage of normal saline. The progesterone group was given the mixed liquid made of dydrogesterone and normal saline by gavage. After 28 d of treatments, the three diameters (length, width, and height) of EMS rats’ ectopic cysts were measured, the cyst volumes were calculated, the volumes before intervention were subtracted, and the difference values were used to evaluate the growth of ectopic cysts. UHRF1 and DNMT1 mRNA and protein levels in normal endometrium, eutopic endometrium, and ectopic endometrium were detected by real-time polymerase chain reaction and immunohistochemistry.

There was no significant difference in the ectopic cyst volume difference between the acupuncture and moxibustion group and the progesterone group (P>0.05), but they were smaller than that of the model group ( P<0.05). The levels of UHRF1 and DNMT1 mRNA and protein in the ec topic endometrium of the mo del group were lower than those in the normal endometrium ( P<0.05). The levels of DNMT1 mRNA and UHRF1 protein in the eutopic endometrium of the model group were lower than those in the normal endometrium ( P<0.05). The levels of UHRF1 mRNA and protein and the level of DNMT1 protein in the ectopic endometrium of the acupuncture and moxibustion group were higher than those in the model group ( P<0.05), and the level of UHRF1 mRNA was higher than that in the progesterone group ( P<0.05). The level of DNMT1 mRNA in the eutopic endometrium of the acupuncture and moxibustion group was higher than that in the model group ( P0.05).

Acupuncture and moxibustion may up-regulate the levels of UHRF1 mRNA and UHRF1 and DNMT1 proteins in the ectopic endometrium to the normal level so as to reduce the volume of ectopic cysts and cure EMS in rats.

Acupuncture Therapy; Moxibustion Therapy; Acupuncture-moxibustion Therapy; Endometriosis; UHRF1; DNMT1; Rats 【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 (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 Co-first Authors: LI Mingyang, M.M., resident physician; WANG Yanwen, bachelor, technician in charge Joint Corresponding Authors: WU Chuting, M.M., resident physician. E-mail: [email protected]; ZHANG Chunyan, M.D., chief physician, master student supervisor. E-mail: [email protected] J. Acupunct. Tuina. Sci. 2024, 22(3): 204-213 © The author(s) for open access article 2023︱● 205 ● 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 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 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 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 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 (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 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 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 组织DNMT1 mRNA水平高于模型组在位内膜组织(P0.05)。结论: 针灸可能通过上调异位内膜组织中UHRF1 mRNA和UHRF1、DNMT1 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。 【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 【中图分类号】R2-03 【文献标志码】A Endometriosis (EMS) refers to the occurrence, growth, and infiltration of the endometrium (glands and stroma) outside the uterine cavity, which leads to repeated bleeding, pain, infertility, nodules, or cysts. It is a common disease in women of reproductive age [1], and it is an estrogen-dependent chronic inflammatory disease [2]. Estrogen plays a crucial role in EMS through estrogen receptors in two subtypes: estrogen receptor α (ERα) and estrogen receptor β (ERβ). In comparison with normal endometrium, ER α mRNA and protein levels are significantly lower in ectopic endometrium, while ER β is higher [3-6]. ER β affects ER α during the pathogenesis of EMS[7]. ERβ may play a key role in the development of endometriosis, but the specific mechanism of ERβ up-regulation is still unclear. Studies have found that the hypomethylation of cytosine- phosphate-guanine (CpG) islands in the ER β gene promoter region may be related to the high ERβ levels in endometriosis tissues [8]. DNA methyltransferase 1 (DNMT1) is the main DNA methyltransferase in mammals. It restores hemimethylated sites on CpG islands to fully methylated status after DNA replication, thereby silencing transcription with no expression of genes [9-10]. Domestic and foreign studies have found that the DNMT1 expression level in the ectopic and eutopic endometrium of EMS patients is significantly lower than that in normal endometrium [11-12]. Semi-methylation is a special state of DNA that can be recognized by ubiquitin-like containing PHD and RING finger domains 1 (UHRF1), guiding DNMT1 to target DNA replication forks, thereby ensuring stable inheritance of DNA methylation [13-14]. It is speculated that UHRF1 may be one of the influencing factors of abnormal DNMT1 expression in endometriosis. Therefore, this study established an EMS rat model and investigated the expression of UHRF1 and DNMT1 in the normal endometrium, eutopic endometrium of EMS rats, and ectopic endometrium to explore the possible mechanism of acupuncture and moxibustion treatment for EMS. 1 Materials and Methods 1.1 Materials 1.1.1 Animals Forty 6-8 week s female Sprague-Dawley r a t s w i t h a body mass of (200±20) g were provided by the Experimental Animal Center of Yueyang Hospital of Integrated Traditional Chinese and Western Medicine, Shanghai University of Traditional Chinese Medicine [License No. SYXK (Hu) 2018-0040]. The rats were produced by Shanghai SLAC Laboratory Animal Co., Ltd., China [Production License No. SCXK (Hu) 2022-0004]. The rats were housed under controlled conditions: temperature (20±2) ℃, relative humidity (50%±10%), light/dark cycle 14 h/10 h, free access to food and water, and high-pressure sterilized padding and drinking water. After acclimatization, the rats were screened for estrus by vaginal smears. The animal experiments were approved by the Animal Ethics Committee of Yueyang Hospital of Integrated Traditional Chinese and Western Medicine, Shanghai University of Traditional Chinese Medicine (Ethics Approval No. YYLAC-2021-134-1). 1.1.2 Appliance and reagents Filiform needles (0.22 mm in diameter and 25 mm in length, Lot No. 210224, Su zhou Medical Appliance Factory, China); moxa sticks (Lot No. 20220506/10, Nanyang Hanyi Moxa Co., Ltd., China); anti-UHRF1 antibody (Cat. No. ab194236, Abcam, USA); anti- DNMT1 antibody (Cat. No . ab188453, Abcam, USA); HRP-conjugated secondary antibody, buffer, DAB chromogenic agent, and high-pH antigen retrieval solution (Lot No. 20078526, No. 20076962, No. 20074026, No. 20079714, Dako, Denmark); chloroform (Lot No. 20161026, Sinopharm, China); iTaq TM Universal SYBR® Green Supermix and iScriptTM cDNA synthesis kit (Cat. No. 1708891, No. 1725124, Bio-Rad, USA); tissue RNA purification kit (Cat. No. B0004DP , EZBioscience, USA). 1.1.3 Equipment ASP300S tissue dehydrator, HISTOCORE ARCADIA H tissue embedding machine, RM2235 rotary tissue slicer, J. Acupunct. Tuina. Sci. 2024, 22(3): 204-213 ● 206 ●︱© The author(s) for open access article 2023 DMi8 inverted microscope (Leica, Germany); AUTOSTAINER LINK48 automated immune- histochemistry staining system (Dako, Denmark); 7500 fluorescence quantitative polymerase chain reaction (PCR) instrument (Life Technologies, USA); Veriti gradient PCR instrument and NanoDrop One spectrophotometer (Thermo Fisher Scientific Inc., USA). 1.2 Grouping and modeling A total of 40 rats were randomly divided into a sham operation group with 10 rats and a model-building group with 30 rats. According to the previous research of our team [5], rats in the model-building group in estrus were selected by vaginal smear examination and were subjected to surgical modeling according to the method of JONES R C [15]. We operated on the rats under sterile conditions, fixed the anesthetized rats to the operating table, shaved hair on the abdomen of the rats, and performed routine disinfection. Then, a 2-3 cm long longitudinal incision was made at 1 cm above the pubic symphysis, and the abdominal cavity was opened. The left uterine was found, and local blood vessels and the segment of the uterus to be excised were ligated. Then, the left uterine was removed, placed in normal saline, and longitudinally cut open to separate the endometrium from the muscular layer. The endometrium was cut into 5 mm × 5 mm fragments, and one corner was fixed with a 5-0 silk suture with the epithelium facing the ovary, uterine ligament, or peritoneum. Rats in the sham operation group underwent a sham operation using the same incision method. After 21 d, the modeled rats were explored by laparotomy to observe the growth of ectopic cysts, and some cysts were taken for histopathological observation to confirm the success of the EMS model. After the model was established successfully, the model-building group was randomly divided into a model group, an acupuncture and moxibustion group, and a progesterone group, with 10 rats in each group. 1.3 Interventions Rats in the sham operation group and the model group were fixed and given normal saline by oral gavage. Rats in the acupuncture and moxibustion group were fixed and received acupuncture and moxibustion treatment and gavage of normal saline. The progesterone group was fixed and given a suspension of dydrogesterone and normal saline by gavage. Acupuncture and moxibustion treatment: The rats were fixed in a supine position, and Xuehai (SP10), Sanyinjiao (SP6), and Guanyuan (CV4) were selected. The point selection method and location for Sanyinjiao (SP6) and Guanyuan (CV4) consulted the Nomenclature and Location of Acupuncture Points for Laboratory Animals. Part 2: Rat [16]. The point selection method and location for Xuehai (SP10) were based on Experimental Acupuncture Science [17]. The filiform needles were inserted perpendicularly into the unilateral Xuehai (SP10) and Sanyinjiao (SP6) for 2 mm. A moxa cone of 15 mm × 20 mm was placed over Guanyuan (CV4), 3 cm away from the skin surface, and ignited for moxibustion. Treatment lasted 10 min per session, once daily for one side points, with the other side treated on the following day. Gavage: The equivalent dose of dydrogesterone for r 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 Dose Conversion Among Different Animals and Healthy Volunteers in Pharmacological Study [18], and the gavage v 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 rat body mass. Before gavage, the prepared suspension was mixed thoroughly by inverting the container. Treatment course: Once a day for 28 d. 1.4 Detection indicators and methods 1.4.1 Measurement of ectopic cyst size After treatment, the estrous cycle of rats was dynamically monitored, and the rats were euthanized during estrus. We collected the normal endometrium, ectopic endometrium, and eutopic endometrium from the rats. After laparotomy, we measured the length, width, and height of ectopic cysts with a vernier caliper and calculated the volumes of the cysts by the formula V = 0.52 × a × b × c (a, b, and c represent the length, width, and height of the cyst, respectively) [19]. The volumes of ectopic cysts were subtracted from the volumes after modeling, and the differences in the volumes of ectopic cysts were taken to observe the growth of ectopic cysts and evaluate the effect of treatment intervention. 1.4.2 Detection of UHRF1 and DNMT1 mRNA levels in rat endometrium Total RNA was extracted from normal endometrium, ectopic endometrium, and eutopic endometrium of rats using chloroform and the EZ BTM RNA purification kit. The concentration and purity of the RNA samples were determined. Then cDNA was synthesized using the iScript TM cDNA synthesis kit. Fluorescence quantitative PCR was performed with GAPDH as the housekeeping gene. The levels of UHRF1 and DNMT1 mRNA in rat endometrium of each group were detected. The amplification primers were synthesized by Sangon Biotech (Shanghai) Co., Ltd., China, and the primer sequences are shown in Table 1. The reaction conditions were as follows: pre-denaturation at 95 ℃ for 30 s, denaturation at 95 ℃ for 15 s, annealing and extension at 60 ℃ for 60 s, 40 cycles in total. Three replicate wells were set up for each group. The relative levels of the target genes were calculated using the 2 -ΔΔCt method. 1.4.3 Detection of UHRF1 and DNMT1 protein level in rat endometrium Normal endometrium, ectopic endometrium, and eutopic endometrium of rats were fixed in 10% neutral formalin solution, dehydrated, embedded in paraffin, J. Acupunct. Tuina. Sci. 2024, 22(3): 204-213 © The author(s) for open access article 2023︱● 207 ● and sectioned continuously at 4 μm. The levels of UHRF1 and DNMT1 proteins in the endometrium were detected by immunohistochemistry. UHRF1 and DNMT1 antibodies were diluted at 1:100, and antigen retrieval was performed. DAB staining was performed, followed by counterstaining with hematoxylin. UHRF1 and DNMT1 were both positively expressed in the cell nucleus. The positive results of UHRF1 and DNMT1 were judged according to the histochemistry score (H-score) [20], as described in our team’s previous study[5]. Two pathologists independen tly scored the staining intensity and the percentage of positive cells. The staining intensity was classified as 0, 1 +, 2+, or 3 +. The percentage of positive cells was estimated to range from 0% to 100%. The scores were multiplied and added to obtain a total score, which ranged from 0 (negative) to 300 (strongly positive). Table 1 Primer sequences for real-time polymerase chain reaction Primer name Base sequence (5 ′→3′) Product length/bp UHRF1 Forward primer CAACTGCTTTGCTCCCATCAATGAC 25 Reverse primer TCCTCCCT CTTGCTGTTCTCCTTC 24 DNMT1 Forward primer GGATTCCACCAAGCAGGCATCTC 23 Reverse primer CCACCACACAGCATCTCCACATC 23 GAPDH Forward primer GACATGCCGCCTGGAGAAAC 20 Reverse primer AGCCCAGGATGCCCTTTAGT 20 Note: UHRF1=Ubiquitin-like containing PHD and RING finger domains 1; DNMT1=DNA methyltransferase 1. 1.5 Statistical methods The statistical program used was SPSS version 26.0. Normally distributed cont inuous variables were expressed as mean ± standard deviation ( x ±s), and one-way analysis of varian ce was used for multiple group comparisons. If the variance was homogeneous, the least significant difference t-test was used, and if the variance was heterogeneous, the Dunnett T3 test was used. For variables that did not follow a normal distribution, the median combined with the interquartile range was used to express the data, and nonparametric tests were used for group comparisons. All tests were two-sided, α=0.05 was used as the test level, and P<0.05 indicated that the difference was statistically significant. 2 Results 2.1 Comparison of the ectopic cyst volume in EMS rats Upon dissection of the model rats, ectopic cysts formed. We took partial ectopic cysts at random. Histopathological observation showed typical endometrial interstitial and glandular structures, thin epithelial areas, irregular arrangement of interstitial, numerous glands, abundant glandular ducts, abundant and uneven distribution of blood vessels, and inflammatory cell infiltration in the interstitial space, and the cyst was confirmed to be ectopic endometrium. These findings validated the successful establishment of EMS rat models. The length, width, and height of ectopic cysts in each group of rats were measured by vernier caliper. The difference in the volume of ectopic cysts was calculated (after intervention minus before intervention). See Figure 1. Note: A) Ectopic cyst formation and measurement of the three diameters; B) pathological observation of ectopic cysts (×400). Figure1 Establishment of endometriosis rat models The results showed that the acupuncture and moxibustion group and the progesterone group had smaller volume differences of ectopic cysts than the model group ( P0.05). See Figure 2. 2.2 Comparison of the UHRF1 mRNA and protein levels in rat endometrium Real-time (RT)-PCR was used to detect the mRNA levels of UHRF1 in the normal endometrium, ectopic endometrium, and eutopic endometrium of rats. The

showed that the UHRF1 mRNA level in the normal endometrium (1.00±0.46) was higher than that in the ectopic endometrium of the model group and the progesterone group ( P<0.05), while there was no notable difference compared with the ectopic J. Acupunct. Tuina. Sci. 2024, 22(3): 204-213 ● 208 ●︱© The author(s) for open access article 2023 endometrium of the acupuncture and moxibustion group (P>0.05). The UHRF1 mRNA level in the ectopic endometrium of the acupuncture and moxibustion group was higher than that of the model group and the progesterone group ( P0.05). There was no significant difference in the UHRF1 mRNA level among the groups in the eutopic endometrium (P>0.05). See Table 2. Immunohistochemistry was used to detect the level of UHRF1 protein in the normal endometrium, ectopic endometrium, and eutopic endometrium of rats. UHRF1 protein was mainly expressed in the nucleus of glandular epithelial and stromal cells. The H-score was used to analyze the protein staining of tissues semi- quantitatively. See Figure 3. Note: Compared with the model group, 1) P<0.05. Figure 2 Comparison of the volume difference of ectopic cysts among groups after intervention Table 2 Comparison of the UHRF1 mRNA level in normal, ectopic, and eutopic endometrium of rats (x ±s) Group n Normal endometrium Ectopic endometrium Eutopic endometrium Sham operation 10 1.00±0.46 / / Model 10 / 0.01±0.01 1) 0.95±0.95 Acupuncture and moxibustion 10 / 1.20±0.29 2)3) 0.04±0.04 Progesterone 10 / 0.10±0.15 1)2) 0.31±0.31 Note: / represents no value; compared w ith the sham operation group, 1) P<0.05; compared with the model group, 2) P<0.05; compared with the progesterone group, 3) P<0.05. Note: A) Normal endometrium of the sham operation group; B) ectopic endometrium of the model group; C) ectopic endometrium of the acupuncture and moxibustion group; D) ectopic endometrium of the progesterone group; E) eutopic endometrium of the model group; F) eutopic endometrium of the acupuncture and moxibustion group; G) eutopic endometrium of the progesterone group. Figure 3 Level of UHRF1 in normal, ectopic, and eutopic endometrium of rats (immunohistochemistry) The results showed that the level of UHRF1 protein in the normal endometrium was h i g h e r t h a n t h a t i n t h e ectopic endometrium and eutopic endometrium of the model group ( P<0.05), lower than that in the eutopic endometrium of the progesterone group (P0.05). In the ectopic endometrium, the level of UHRF1 protein in the acupuncture and moxibustion group and the progesterone group was significantly higher than that in the model group ( P<0.05). In the eutopic endometrium, the level of UHRF1 protein in the progesterone group was significantly higher than that in the model group (P<0.05). See Table 3. 2.3 Comparison of the DNMT1 mRNA and protein levels in rat endometrium RT-PCR was used to detect the DNMT1 mRNA level in normal, ectopic, and eutopic endometrium of rats. The J. Acupunct. Tuina. Sci. 2024, 22(3): 204-213 © The author(s) for open access article 2023︱● 209 ●

showed that the DNMT1 mRNA level in the normal endometrium was higher than that in the ectopic and eutopic endometrium of the model group, as well as in the euto pic endometrium of the acupuncture and moxibustion group and the progesterone group (P0.05). In the eutopic endometrium, the DNMT1 mRNA level in the acupuncture and moxibustion group was higher than that in the model group (P<0.05). See Table 4. Immunohistochemical staining was used to detect the DNMT1 protein level in normal, ectopic, and eutopic endometrium of rats. The DNMT1 protein was mainly expressed in the nucleus of glandular epithelial and stromal cells. The H-score was used to analyze the protein staining of tissue s semi-quantitatively. See Figure 4. The results showed that the DNMT1 protein level in the normal endometrium was h i g h e r t h a n t h a t i n t h e ectopic endometrium of the model group ( P<0.05). In the ectopic endometrium, the DNMT1 protein level in the acupuncture and moxibustion group was significantly higher than that in the model group (P0.05). In the eutopic endometrium, the DNMT1 protein level in the model group and the acupuncture and moxibustion group was higher than that in the progesterone group ( P<0.05). See Table 5. Table 3 Comparison of the UHRF1 protein level in normal, ectopic, and eutopic endometrium of rats (x ±s) Group n Normal endometrium Ectopic endometrium Eutopic endometrium Sham operation 10 58.33±7.64 / / Model 10 / 18.33±8.39 1) 23.67±3.06 1) Acupuncture and moxibustion 10 / 45.00±5.00 2) 88.33±18.93 Progesterone 10 / 53.33±7.64 2) 145.00±18.03 1)2) Note: / represents no value; compared with the sham operation group, 1) P<0.05; compared with the model group, 2) P<0.05. Table 4 Comparison of the DNMT1 mRNA level in normal, ectopic, and eutopic endometrium of rats (x ±s) Group n Normal endometrium Ectopic endometrium Eutopic endometrium Sham operation 10 1.00±0.12 / / Model 10 / 0.03±0.01 1) 0.00±0.00 1) Acupuncture and moxibustion 10 / 0.94±0.33 0.01±0.00 1)2) Progesterone 10 / 0.46±0.58 0.00±0.00 1) Note: / represents no value; compared with the sham operation group, 1) P<0.05; compared with the model group, 2) P<0.05. Note: A) Normal endometrium of the sham operation group; B) ectopic endometrium of the model group; C) ectopic endometrium of the acupuncture and moxibustion group; D) ectopic endometrium of the progesterone group; E) eutopic endometrium of the model group; F) eutopic endometrium of the acupuncture and moxibustion group; G) eutopic endometrium of the progesterone group. Figure 4 Level of DNMT1 in normal, ectopic, and eutopic endometrium of rats (immunohistochemistry) J. Acupunct. Tuina. Sci. 2024, 22(3): 204-213 ● 210 ●︱© The author(s) for open access article 2023 Table 5 Comparison of the DNMT1 protein levels in normal, ectopic, and eutopic endometrium of rats (x ±s) Group n Normal endometrium Ectopic endometrium Eutopic endometrium Sham operation 10 70.00±15.00 / / Model 10 / 28.33±7.64 1) 89.67±34.65 Acupuncture and moxibustion 10 / 98.33±20.21 2) 106.67±20.21 3) Progesterone 10 / 60.00±13.23 48.33±12.58 2) Note: / represents no value; compared w ith the sham operation group, 1) P<0.05; compared with the model group, 2) P<0.05; compared with the progesterone group, 3) P<0.05. 3 Discussion To date, the pathogenesis of endometriosis is not clear. Since the beginning of the 20th century, SAMPSON J A [21] proposed the classic “theory of retrograde menstruation and transplantation”, most women of childbearing age experience retrograde menstruation [22], and only a few develop endometriosis. A wealth of evidence suggests that estrogen levels are elevated in ectopic tissues [23]. The increase in estrogen production, impaired estrogen metabolism, and altered expression of estrogen receptors are the reasons for local estrogen excess that leads to the pathology of endometriosis [24]. Currently, hormone therapy remains the most commonly used conservative treatment for endometriosis patients. The existing treatments that interfere with the estrogen pathway aim to induce a state of menopause or pseudo-pregnancy to block menstruation. They can control the pain but have side effects, do not prevent or cure the disease, and often lead to treatment interruption by patients [19,25-26]. Although surgical treatment can remove the lesions, pain may recur or persist if the ectopic lesion is not completely excised [27-28]. Therefore, exploring the pathogenesis and treatment of endometriosis is of great significance for guiding clinical diagnosis and treatment. The research on stromal cells in endometriosis has found that compared to normal endometrium, the low methylation of the ER β promoter region leads to abnormal elevation of ERβ mRNA and protein levels [8]. In most DNA promoter regions in mammals, there are CpG islands. Gene silencing can occur through CpG island methylation. Abnormal DNA methylation often occurs in the promoter regions of transcription factors [29-30]. DNMT1 plays a necessary role in maintaining methylation stability [31]. Abnormal low expression of DNMT1 in EMS may lead to epigenetic abnormalities in patients and participate in the development of EMS [12]. The estrogen-DNMT1 pathway induces the methylation of RUNX3 and promotes proliferation and invasion activity in eutopic endometrial stromal cells [32]. Studies have reported that DNMT1 and ERβ are related, and 5-Aza-CdR induces cell apoptosis and inhibits HT29 colon cancer cell growth by downregulating DNMT1 and upregulating ERα and ERβ gene expression [33]. Disulfiram inhibits cell proliferation in prostate cancer and other cancers by re-expressing ERβ and tumor suppressor genes, including DNMT1 [34]. UHRF1 is a multi-domain protein that includes a ubiquitin-like domain, tandem Tudor (TTD) domains, a plant homeodomain (PHD), a SET and RING-associated (SRA) domain, and a RING domain [35]. It is an auxiliary factor required for maintaining DNA methylation. At the molecular level, UHRF1 senses the presence of hemimethylated DNA through its SRA domain and it may simultaneously recognize the binding of histone H3K9me2/3 and DNMT1 through its TTD domain [36-37], which is the structural basis for mediating DNMT1 recruitment to DNA replication forks [38]. Dysregulation of DNMT1 and UHRF1 has been found in the malignant transformation and progression of different types of cancer [39]. After DNMT1 and UHRF1 expression is reduced, a large number of tumor suppressor genes are re-expressed [39-41]. However, there have been no studies on UHRF1 in EMS. In our study, the DNMT1 mRNA and protein levels in the ectopic endometrium were lower than those in the normal endometrium, consistent with the reports of VEENA K V, et al [11] and YANG J, et al[12]. Moreover, we first discovered that UHRF1 mRNA and protein levels in the ectopic endometrium of the model group were lower than those in the normal endometrium. Therefore, it is speculated that UHRF1 and DNMT1 may jointly participate in the occurrence and development of EMS. Professor LANG J H [42] proposed the “eutopic endometrial determinant”, holding that the primary cause of whether endometrium fragments in the blood reflux of different people (with or without endometriosis) can adhere, invade, and grow is the difference in the eutopic endometrium. In our study, the DNMT1 mRNA and protein levels and UHRF1 protein level in the eutopic endometrium of the model group were lower than those in the norm al endometrium, suggesting that DNMT1 and UHRF1 may participate in the disease’s development through eutopic endometrium. Endometriosis can be classified as “abdominal masses”, “dysmenorrhea”, “menstrual disorders”, “infertility”, and other traditional Chinese medicine diseases based on clinical manifestations. The main pathogenesis is blood stasis, and the main location of the disease is the uterus. It is closely related to the liver, J. Acupunct. Tuina. Sci. 2024, 22(3): 204-213 © The author(s) for open access article 2023︱● 211 ● spleen, and kidney organs. Acupuncture and moxibustion treatment for endometriosis has definite therapeutic effects and minimal adverse reactions. Its main function is circulating blood, transforming stasis, and regulating the liver, spleen, and kidney. In clinical practice, needling at Xuehai (SP10) and Sanyinjiao (SP6) and moxibustion at Guanyuan (CV4) are often used to treat EMS patients. Sanyinjiao (SP6) is the crossing point of the Spleen Meridian of Foot-Taiyin, the Liver Meridian of Foot-Jueyin, and the Kidney Meridian of Foot-Shaoyin, and is connected to the Conception Vessel, Thoroughfare Vessel, and the uterus. It has the functions of tonifying and nourishing the spleen, liver, and kidney, regulating the balance of Yin and Yang of organs, and regulating the circulation of Qi and blood in the Thoroughfare and Conception Vessels. Guanyuan (CV4) is a crossing point of three foot-Yin meridians and Conception Vessel and is known as the “place where women store blood”. Acupuncture and moxibustion at this point can stimulate the point, and the heat generated by burning moxa sticks can enter the abdomen, enhancing the functions of circulating blood, resolving masses, and consolidating basis. Xuehai (SP10), also known as the “sea of twelve meridians” , is a point on the Spleen Meridian of Foot-Taiyin and an important point for treating gynecolo gical diseases with the functions of tonifying Qi and blood. Our team has long been committed to clinical and experimental research on acupuncture and moxibustion treatment of EMS and has found that acupuncture and moxibustion can up-regulate the expression of ERα, which is abnormally low in the eutopic and ectopic endometrium, and down-regulate the expression of ER β, which is abnormally high in the eutopic and ectopic endometrium [5]. In our study, acupuncture-moxibustion treatment was chosen because it not only includes the effect of needling on harmonizing meridians but also exerts the effect of moxibustion on warming meridians and dissipating cold. The study found that acupuncture and moxibustion can reduce the size of ectopic cysts, further confirming the inhibitory effect of acupuncture and moxibustion treatment on EMS lesions. The experiment chose the commonly used positive control drug, dydrogesterone, which has been shown to alleviate the symptoms of endometriosis, cause atrophy of ectopic endometrium, and inhibit the development of EMS lesions [43]. Our study found that acupuncture- moxibustion and dydrogesterone had similar effects in reducing the size of ectopic cysts. Acupuncture and moxibustion can up-regulate the UHRF1 mRNA and protein levels and DNMT1 protein level in the ectopic endometrium, suggesting that acupuncture and moxibustion may inhibit the growth of ectopic lesions by up-regulating the abnormally low levels of UHRF1 mRNA and protein and DNMT1 protein in the ectopic endometrium to reach the levels of normal endometrium. However, this study only focused on the relative mRNA and protein levels of UHRF1 and DNMT1. Further research is needed to explore whether UHRF1 participates in the pathogenesis of EMS by regulating the methylation of ERβ through DNMT1. Received: 10 May 2023/Accepted: 18 July 2023

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Effect of acupuncture and moxibustion with traditional Chinese medicine in regulating endometrial Conflict of Interest The authors declare that there is no potential conflict of interest in this article. Acknowledgments This work was supported by the Rising-star Program of “Shanghai 2022 Science and Technology Innovation Action Plan”: Yangfan Special Project (上海市 2022 年度“科技 创新行动计划”启明星培育 - 扬帆专项 , No. 22YF1444200); Health Industry C linical Research Project of Shanghai Municipal Health Commission (上海市卫生健 康委员会卫生行业临床研究专项 , No. 202240054); Xinglin Scholar of Shanghai University of Traditional Chinese Medicine ( 上海中医药大学“杏林学者” ); Shanghai Shenkang Hospital Development Center to Promote Municipal Hospital Clinical Skills and Clinical Innovation Ability Three-year Action Plan Project (上海申 康医院发展中心促进市级医院临床技能与临床创新能 力三年行动计划项目, No. SHDC2020CR3093B). 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