l-arginine promotes angio-osteogenesis to enhance oxidative stress-inhibited bone formation by ameliorating mitophagy.

Background: Osteoporosis is one of the most common bone diseases in middle-aged and elderly populations worldwide. The development of new drugs to treat the disease is a key focus of research. Current treatments for osteoporosis are mainly directed at promoting osteoblasts and inhibiting osteoclasts. However, there is currently no ideal approach for osteoporosis treatment. l-arginine is a semi-essential amino acid involved in a number of cellular processes, including nitric production, protein biosynthesis, and immune responses. We previously reported that l-arginine-derived compounds can play a regulatory role in bone homeostasis. Purpose: To investigate the specific effect of l-arginine on bone homeostasis. Methods: Mildly aged and ovariectomized mouse models were used to study the effects of l-arginine on osteogenesis and angiogenesis, assessed by micro-computed tomography and immunostaining of bone tissue. The effect of l-arginine on osteogenesis, angiogenesis, and adipogenesis was further studied in vitro using osteoblasts obtained from cranial cap bone, endothelial cells, and an adipogenic cell line. Specific methods to assess these processes included lipid staining, cell migration, tube-forming, and wound-healing assays. Protein and mRNA expression was determined for select biomarkers. Results: We found that l-arginine attenuated bone loss and promoted osteogenesis and angiogenesis. l-arginine increased the activity of vascular endothelial cells, whereas it inhibited adipogenesis in vitro. In addition, we found that l-arginine altered the expression of PINK1/Parkin and Bnip3 in the mitochondria of osteoblast-lineage and endothelial cells, thereby promoting mitophagy and protecting cells from ROS. Similarly, l-arginine treatment effectively ameliorated osteoporosis in an ovariectomized mouse model. Conclusion: l-arginine promotes angio-osteogenesis, and inhibits adipogenesis, effects mediated by the PINK1/Parkin- and Bnip3-mediated mitophagy. The Translational Potential of this Article: L-arginine supplementation may be an effective adjunct therapy in the treatment of osteoporosis.

Urolithin B reduces cartilage degeneration and alleviates osteoarthritis by inhibiting inflammation.

Osteoarthritis is the most prevalent degenerative joint disease reported worldwide. Conventional treatment strategies mainly focus on medication and involve surgical joint replacement. The use of these therapies is limited by gastrointestinal complications and the lifespan of joint prostheses. Hence, safe and efficacious drugs are urgently needed to impede the osteoarthritis progression. Urolithin B, a metabolite of ellagic acid in the gut, exhibits anti-inflammatory and antioxidant properties; however, its role in osteoarthritis remains unclear. In this study, we demonstrated that urolithin B efficiently inhibits the inflammatory factor-induced production of matrix metalloproteinases (MMP3 and MMP13) in vitro and upregulates the expression of type II collagen and aggrecan. Urolithin B alleviates cartilage erosion and osteophyte formation induced by anterior cruciate ligament transections. Moreover, urolithin B inhibits the activation of the NF-κB pathway by reducing the phosphorylation of Iκb-α and the nuclear translocation of P65. In summary, urolithin B significantly inhibits inflammation and alleviates osteoarthritis. Hence, urolithin B can be considered a potential agent suitable for the effective treatment of osteoarthritis in the future.

Restrained Mitf-associated autophagy by Mulberroside A ameliorates osteoclastogenesis and counteracts OVX-Induced osteoporosis in mice.

Bone and mineral metabolism homeostasis accounts for the maintenance of normal skeletal remodeling. However, with aging and changes in hormone levels, over-activated osteoclasts disrupt homeostasis, induce osteoporosis, and even cause osteoporotic fractures, leading to an enormous economic burden. Despite the rapid development of pharmacological therapy for osteoporosis, safer and more effective treatments remain to be explored. Here, we demonstrate that Mulberroside A (Mul-A), a natural component extracted from mulberry bark and branches, effectively suppresses osteoclastogenesis in vitro and counteracts bone loss caused by ovariectomy (OVX). The mechanism underlying this effect involves the repression of autophagic flux during osteoclastogenesis by Mul-A, which can be attributed to the restrained expression of microphthalmia-related transcription factor (Mitf) and its nuclear translocation. Importantly, Mitf overexpression partially reverses the inhibitory effects of Mul-A on autophagy and osteoclastogenesis. Moreover, applying two autophagy agonizts, rapamycin and Torin 1, attenuates the osteoclastogenic regulatory role of Mul-A. Collectively, our study demonstrates that Mul-A damages osteoclast differentiation and ameliorates osteoporosis caused by estrogen deficiency by modulation of Mitf-associated autophagy, indicating its therapeutic potential against osteoporosis.

USP7 Inhibits Osteoclastogenesis via Dual Effects of Attenuating TRAF6/TAK1 Axis and Stimulating STING Signaling.

Ubiquitination is a reversible post-translational modification implicated in cell differentiation, homeostasis, and organ development. Several deubiquitinases (DUBs) decrease protein ubiquitination through the hydrolysis of ubiquitin linkages. However, the role of DUBs in bone resorption and formation is still unclear. In this study, we identified DUB ubiquitin-specific protease 7 (USP7) as a negative regulator of osteoclast formation. USP7 combines with tumor necrosis factor receptor-associated factor 6 (TRAF6) and inhibits its ubiquitination by impairing the Lys63-linked polyubiquitin chain. Such impairment leads to the suppression of receptor activator of NF-κB ligand (RANKL)-mediated nuclear factor-κB (NF-κB) and mitogen-activated protein kinases (MAPKs) activation without affecting TRAF6 stability. USP7 also protects the stimulator of interferon genes (STING) against degradation, inducing interferon-ß (IFN-ß) expression in osteoclast formation, thereby inhibiting osteoclastogenesis cooperatively with the classical TRAF6 pathway. Furthermore, USP7 inhibition accelerates osteoclast differentiation and bone resorption both in vitro and in vivo. Contrarily, USP7 overexpression impairs osteoclast differentiation and bone resorption in vitro and in vivo. Additionally, in ovariectomy (OVX) mice, USP7 levels are lower than those in sham-operated mice, suggesting that USP7 plays a role in osteoporosis. Altogether, our data reveal the dual effect of USP7-mediated TRAF6 signal transduction and USP7-mediated protein degradation of STING in osteoclast formation.

Netrin-1 mediates nerve innervation and angiogenesis leading to discogenic pain.

Objective: Discogenic low back pain (LBP) is associated with nociceptive nerve fibers that grow into degenerated intervertebral discs (IVD) but the etiopathogenesis of disease is not fully understood. The purpose of this study was to clarify the role of Netrin-1 in causing discogenic LBP. Methods: The level of nociceptive nerve innervation was examined in disc degenerative patients and rat needle-punctured models by immunohistochemistry. Nucleus pulposus (NP) cells were isolated from IVD tissues of rats and induced degeneration by interleukin-1ß (IL-1ß) or tumor necrosis factor α (TNFα). The candidate genes related to neuron outgrowth and migration were selected by Next-generation sequencing (NGS). CRISPR/Cas9 was used to knockdown Netrin-1 in NP cells. The impact of Netrin-1 on nerve innervation were evaluated with P2X2、NF200 staining and microfluidics assay. Meanwhile the CD31 staining and transwell assay were used to evaluate the impact of Netrin-1 in angiogenesis. The proteins and RNA extracted from NP cells related to catabolism and anabolism were examined by western blot assay and RT-qPCR experiment. ChIP and luciferase experiments were used to assess the intracellular transcriptional regulation of Netrin-1. Further, a needle-punctured rat model followed by histomorphometry and immunofluorescence histochemistry was used to explore the potential effect of Netrin-1 on LBP in vivo. Results: The level of nerve innervation was increased in severe disc degenerative patients while the expression of Netrin-1 was upregulated. The supernatants of NP cells stimulated with IL-1ß or TNFα containing more Netrin-1 could promote axon growth and vascular endothelial cells migration. Knocking down Netrin-1 or overexpressing transcription factor TCF3 as a negative regulator of Netrin-1 attenuated this effect. The needle-punctured rat model brought significant spinal hypersensitivity, nerve innervation and angiogenesis, nevertheless knocking down Netrin-1 effectively prevented disc degeneration-induced adverse impacts. Conclusion: Discogenic LBP was induced by Netrin-1, which mediated nerve innervation and angiogenesis in disc degeneration. Knocking down Netrin-1 by CRISPR/Cas9 or negatively regulating Netrin1 by transcription factor TCF3 could alleviate spinal hypersensitivity. The translational potential of this article: This study on Netrin-1 could provide a new target and theoretical basis for the prevention and treatment for discogenic back pain.

Corrigendum: Oxymatrine attenuates osteoclastogenesis via modulation of ROS-mediated SREBP2 signaling and counteracts ovariectomy-induced osteoporosis.

[This corrects the article DOI: 10.3389/fcell.2021.684007.].

Pexmetinib suppresses osteoclast formation and breast cancer induced osteolysis via P38/STAT3 signal pathway.

Breast cancer metastases to the bone can lead to a series of bone-related events that seriously affect the quality of life. Pexmetinib, a novel p38 mitogen-activated protein kinase (p38) inhibitor that has been evaluated in phase I clinical trials for myelodysplastic syndrome, but the effects of Pexmetinib on breast cancer induced osteolysis haven't been explored. Here, we found that Pexmetinib inhibited receptor activator of nuclear factor-κB ligand-induced osteoclast formation and bone resorption in vitro. Pexmetinib suppressed p38-mediated signal transducer and activator of transcription 3 (STAT3), which direct regulated transcription of the nuclear factor of activated T cells 1 (NFATc1), leading to reduced osteoclast formation. Moreover, Pexmetinib exerted anti-tumor effects in breast cancer cells in vitro via suppressing p38-mediated STAT3 activation and matrix metalloproteinases (MMPs) expression. Furthermore, Pexmetinib suppressed breast cancer-associated osteolysis in vivo. These results suggest that Pexmetinib may be a promising drug for the treatment of breast cancer-induced osteolysis.

USP1 inhibition suppresses the progression of osteosarcoma via destabilizing TAZ.

Mutations and altered expression of deubiquitinating enzymes (DUBs) profoundly influence tumor progression. Ubiquitin-specific protease 1 (USP1) is a well-characterized human DUB reportedly overexpressed in and associated with maintaining the mesenchymal stem cell status of osteosarcoma (OS); however, the potential mechanisms of USP1 in OS remain poorly understood. In this study, we identified that USP1 directly interacts with Transcriptional Co-Activator With PDZ-Binding Motif (TAZ) in OS cell lines, and with mechanistic analysis indicating that the anti-OS effects of USP1 inhibition could be partially attributed to TAZ instability, with its reduced nuclear accumulation responsible for a subsequent decrease in the expression of downstream genes associated with the Hippo signaling pathway. Moreover, pharmacological inhibition USP1 by ML323 presented the similar effects on Hippo signaling pathway and suppressed OS growth and metastasis both in vitro and in vivo. Taken together, our results revealed a novel molecular mechanism underlying the function of USP1 in OS and a potential role of ML323 as a therapeutic strategy for the clinical treatment of OS.

Mechanical force promotes dimethylarginine dimethylaminohydrolase 1-mediated hydrolysis of the metabolite asymmetric dimethylarginine to enhance bone formation.

Mechanical force is critical for the development and remodeling of bone. Here we report that mechanical force regulates the production of the metabolite asymmetric dimethylarginine (ADMA) via regulating the hydrolytic enzyme dimethylarginine dimethylaminohydrolase 1 (Ddah1) expression in osteoblasts. The presence of -394 4 N del/ins polymorphism of Ddah1 and higher serum ADMA concentration are negatively associated with bone mineral density. Global or osteoblast-specific deletion of Ddah1 leads to increased ADMA level but reduced bone formation. Further molecular study unveils that mechanical stimulation enhances TAZ/SMAD4-induced Ddah1 transcription. Deletion of Ddah1 in osteoblast-lineage cells fails to respond to mechanical stimulus-associated bone formation. Taken together, the study reveals mechanical force is capable of down-regulating ADMA to enhance bone formation.

The SFRP1 Inhibitor WAY-316606 Attenuates Osteoclastogenesis Through Dual Modulation of Canonical Wnt Signaling.

Osteoporosis, a noteworthy age-related disease induced by imbalanced osteogenesis and osteoclastogenesis, is a serious economic burden on both individuals and society. Small molecule drugs with dual effects on both bone resorption and mineralization are pressingly needed. Secreted frizzled-related protein 1 (SFRP1), a well-known extracellular repressor of canonical Wnt signaling, has been reported to regulate osteogenesis. Global SFRP1 knockout mice show significantly elevated bone mass. Although osteoclasts (OCs) express and secrete SFRP1, the role of SFRP1 produced by OCs in osteoclastogenesis and osteoporosis remains unclear. In this work, the levels of SFRP1 were found to be increased in patients with osteoporosis compared with healthy controls. Pharmacological inhibition of SFRP1 by WAY-316606 (WAY)- attenuated osteoclastogenesis and bone resorption in vitro. The expressions of OC-specific genes were suppressed by the SFRP1 inhibitor, WAY. Mechanistically, both extracellular and intracellular SFRP1 could block activation of the canonical Wnt signaling pathway, and WAY reverse the silent status of canonical Wnt through dual effects, leading to osteoclastogenesis inhibition and osteogenesis promotion. Severe osteopenia was observed in the ovariectomized (OVX) mouse model, and WAY treatment effectively improved the OVX-induced osteoporosis. In summary, this work found that SFRP1 supports OC differentiation and function, which could be attenuated by WAY through dual modulation of canonical Wnt signaling, suggesting its therapeutic potential. © 2021 American Society for Bone and Mineral Research (ASBMR).

Oxymatrine Attenuates Osteoclastogenesis via Modulation of ROS-Mediated SREBP2 Signaling and Counteracts Ovariectomy-Induced Osteoporosis.

Osteoporosis, mainly caused by osteoclast-induced bone resorption, has become a major health problem in post-menopausal women and the elderly. Growing evidence indicates that inhibiting osteoclastogenesis is an efficient approach to develop alternative therapeutic agents for treating osteoporosis. In this study, we identified the potential regulating role of Oxymatrine (OMT), a quinazine alkaloid extracted from Sophora flavescens with various therapeutic effects in many diseases, on osteoclastogenesis for the first time. We found that OMT attenuated RANKL-induced osteoclast formation in both time- and dose-dependent manners. Further, OMT significantly suppressed RANKL-induced sterol regulatory element-binding protein 2 (SREBP2) activation and the expression of the nuclear factor of activated T cells 1 (NFATc1). Moreover, OMT inhibited the generation of RANKL-induced reactive oxygen species (ROS), and the upregulation of ROS could rescue the inhibition of SREBP2 by OMT. More importantly, ovariectomy (OVX) mouse model showed that OMT could effectively improve ovariectomy (OVX)-induced osteopenia by inhibiting osteoclastogenesis in vivo. In conclusion, our data demonstrated that OMT impaired ROS mediated SREBP2 activity and downstream NFATc1 expression during osteoclastogenesis, suppressed OVX-induced osteopenia in vivo, which suggested that OMT could be a promising compound for medical treatment against osteoporosis.

An Antioxidant Sesquiterpene Inhibits Osteoclastogenesis Via Blocking IPMK/TRAF6 and Counteracts OVX-Induced Osteoporosis in Mice.

Excessive bone resorption induced by increased osteoclast activity in postmenopausal women often causes osteoporosis. Although the pharmacological treatment of osteoporosis has been extensively developed, a safer and more effective treatment is still needed. Here, we found that curcumenol (CUL), an antioxidant sesquiterpene isolated from Curcuma zedoaria, impaired receptor activator of nuclear factor-κB (NF-κB) ligand (RANKL)-induced osteoclastogenesis in vitro, whereas the osteoblastogenesis of MC3T3-E1 cells was not affected. We further demonstrated that CUL treatment during RANKL-induced osteoclastogenesis promotes proteasomal degradation of TRAF6 by increasing its K48-linked polyubiquitination, leading to suppression of mitogen-activated protein kinases (MAPKs) and NF-κB pathways and the production of reactive oxygen species (ROS). We also showed that inositol polyphosphate multikinase (IPMK) binds with TRAF6 to reduce its K48-linked polyubiquitination under RANKL stimulation. Concurrently, IPMK deficiency inhibits osteoclast differentiation. The binding between IPMK and TRAF6 blocked by CUL treatment was found in our study. Finally, we confirmed that CUL treatment prevented ovariectomy (OVX)-induced bone loss in mice. In summary, our study demonstrates that CUL could impair the stability of TRAF6 enhanced by IPMK and suppress excessive osteoclast activity in estrogen-deficient mice to treat osteoporosis. © 2021 American Society for Bone and Mineral Research (ASBMR).

Oxidative stress abrogates the degradation of KMT2D to promote degeneration in nucleus pulposus.

Nucleus pulposus (NP) degeneration plays pivotal roles in intervertebral disc degeneration. The effect and mechanism of oxidative stress and epigenetics in NP degeneration is still unclear. We performed this study to evaluate the function of oxidative stress in NP and to explore the potential mechanism of ROS induced expression of matrix metalloproteinases (MMPs). We tested four methyltransferases, KMT2A, KMT2B, KMT2C and KMT2D in human NP samples, only KMT2D was significantly up-regulated in the severe degeneration samples. Knockdown of Kmt2d by siRNA significantly down-regulated the expression levels of catabolic enzymes including Mmp3, Mmp9 and Mmp13. Moreover, an interaction between KMT2D and ubiquitination was confirmed, and the application of H2O2 abrogated this process. Co-IP assay confirmed that H2O2 induced the phosphorylation of KMT2D to block the ubiquitination degradation, which was mainly mediated by phosphorylation of p38/MAPK. Further investigation suggested that ROS induced the alteration in levels of methylation is linked to H3K4me1 and H3K4me2, but not me3. However, usage of OICR-9429 (OICR) also suppressed the expression levels of Mmp3, Mmp9 and Mmp13. In an ex vivo model, application of OICR-9429 (OICR) also attenuated the degeneration of NP according to the H&E and Safranin-O/Fast Green staining assay, and the protein levels of MMP3, MMP9 and MMP13 were down-regulated, as well. In conclusion, we approved that oxidative stress induced ROS production promote the process of NP degeneration by enhancing KMT2D mediated transcriptional regulation of matrix degeneration related genes during NP degeneration.

Magnetic natural composite Fe3O4-chitosan@bentonite for removal of heavy metals from acid mine drainage.

Magnetic Fe3O4-chitosan@bentonite (Fe3O4-CS@BT) composites using natural materials were synthesized and used to remediate acid mine drainage (AMD) for heavy metal removal. It was found that the composites had a good magnetism, high stability and good performance in removal of Cr(VI) in the synthetic aqueous solutions. The thermodynamic parameters showed that Cr(VI) adsorption onto Fe3O4-CS@BT was an exothermic and spontaneous process. The adsorption kinetics was well described by a pseudo-second-order model, and the adsorption isotherms fit the Langmuir model with a maximum adsorption capacity of 62.1 mg/g at 25 °C. Solution pH was a key factor for adsorption and the optimized pH was 2.0. Fe3O4-CS@BT could be easily recycled through an external magnetic field and the adsorption capacity reduced by only 3% after five consecutive adsorption-desorption processes. In addition, Fe3O4-CS@BT was found to be an excellent adsorbent for actual AMD remediation containing Cd, Cr, Cu, Fe Zn, Ni and Pb, suggesting that the Fe3O4-CS@BT was an effective and economical adsorbent for the treatment of AMD.

Initiation timing effect of levothyroxine treatment on subclinical hypothyroidism in pregnancy.

The aim of this study is to estimate the timing impact on levothyroxine replacement among pregnant women with subclinical hypothyroidism (SCH). Ninety-eight pregnant women diagnosed as SCH in the first trimester were randomly divided into three groups: Group A, instantly initiated levothyroxine after diagnosis; Group B, administrated treatment in the second trimester, and Group C, received no prescription. Incidence of pregnancy complications and pregnancy outcomes were compared among three groups and subgroup analysis were performed stratified with TPO status in Group B. Group A exhibited lower rate of pregnancy complications (9.7%) and adverse outcome (3.2%) than Group B (41.9% and 32.3%) and Group C (64.5% and 38.7%). But the late initiation treatment group shared a comparable complication and maternal outcome with untreated women (p = .075 and .596, respectively). After stratified with TPOAb status in Group B, TPOAb+ women experienced a remarkable lower complication (14.2%) and adverse outcome rate (7.1%) compared with negative subjects (64.7% and 45%, respectively). Our data suggest that levothyroxine administrated in the first trimester was associated with decreased risk of adverse obstetric event. Additionally, pregnant women with TPOAb positive could also benefit from thyroid hormone therapy even initiated in the second trimester.

Lengthy one-dimensional magnetite (Fe3O4) sub-microfibers with excellent electrochemical performance.

One dimensional Fe3O4 sub-microfibers with an average diameter of about 920nm and length of about 25µm were prepared by hydrothermal synthesis in the presence of sodium citrate (Na3cit). The physicochemical properties were characterized by X-ray powder diffraction (XRD), Fourier transform infrared spectroscopy (FTIR), transmission electron microscopy (TEM), and scanning electron microscopy (SEM). It was found that Fe3O4 crystal nuclei were generated from different Fe2+-citrate complexes under hydrothermal conditions and Na3cit as a capping agent promoted the formation of Fe3O4 microfibers. The Fe3O4 nanocrystals grew along the (110) axis on the exposed (111) facet, and then further formed microfibers via an oriented attachment mechanism during the collisions. Polyvinylpyrrolidine (PVP) was more conducive to microfibers growth in comparison to polyethyleneglycol 4000 (PEG-4000), cetyltrimethylammonium bromide (CTAB) and sodium dodecyl sulfate (SDS). The electrochemical measurements show that the Fe3O4 sub-microfibers had a specific capacitance of 117.6Fg-1 with good cycling performance.

Diagnostic value of positron emission tomography (PET) and PET/computed tomography in recurrent/metastatic ovarian cancer: A meta-analysis.

AIM: The study was conducted to assess and compare the overall diagnostic value of positron emission tomography (PET) alone and PET/computed tomography (CT) for the detection of recurrent/metastatic ovarian cancer. METHODS: A systematic literature search in PubMed, EMBASE and Chinese literature databases (CNKI, VIP and Wanfang) up to August 2014 was performed to identify relevant articles. The diagnostic performance of PET alone or PET/CT was assessed in terms of the following evaluation indexes: summary sensitivity, specificity, positive and negative likelihood ratio, diagnostic odds ratio and area under the curve summary receiver operating characteristics. RESULTS: Sixty-four studies with 3722 patients were included in this meta-analysis. Favorable results were obtained for both PET/CT and PET alone with similar sensitivity, specificity, positive and negative likelihood ratios, diagnostic odds ratio and area under the curve in diagnosing recurrent/metastatic ovarian cancer (P > 0.05). However, the sensitivity of PET/CT was significantly higher than PET alone in European patients. CONCLUSIONS: Both PET and PET/CT have promising diagnostic value in recurrent/metastatic ovarian cancer. The diagnostic performance of PET/CT may be superior to PET alone in European patients.

Transvaginal mesh repair for pelvic organ prolapse.

AIM: We compared outcomes for traditional vaginal surgery with uterosacral ligament suspension (USLS) versus transvaginal mesh repair of pelvic organ prolapse (POP). METHODS: We performed a retrospective chart review of pelvic organ prolapse cases that occurred between February 2007 and December 2011. Success rates for traditional vaginal repair plus USLS and transvaginal mesh repair were determined. RESULTS: As compared with traditional surgery (n=81), using mesh (n=90) for stage III or IV prolapse resulted in higher rates of successful treatment. However, mesh repair also resulted in increased intraoperative blood loss and postoperative adverse events, such as pain and dyspareunia. CONCLUSION: Traditional vaginal repair plus USLS was an improvement on traditional surgery, resulting in a higher anatomical success rate. On the other hand, both intraoperative and postoperative complications in mesh repair could be reduced by protective factors such as the surgeon's experience.

Dysmenorrhea and its severity are associated with increased uterine contractility and overexpression of oxytocin receptor (OTR) in women with symptomatic adenomyosis.

OBJECTIVE: To evaluate uterine contractility, oxytocin receptor (OTR) expression in myometrial smooth muscle cells (MSMCs) derived from uterine tissues from women with and without adenomyosis correlate OTR expression with uterine contractility and dysmenorrhea severity, and see whether trichostatin A (TSA) and andrographolide inhibit OTR expression. DESIGN: Laboratory study using human tissues. SETTING: Academic hospital. PATIENT(S): Twenty patients (cases) with histologically confirmed adenomyosis and 10 (controls) with leiomyomas, cervical dysplasia, and cancer but no adenomyosis or endometriosis. INTERVENTION(S): Dysmenorrhea severity was scored by Visual Analog Scale. Uterine tissue samples were collected during hysterectomy. Myometrial smooth muscle cells derived from tissue samples were cultured and OTR protein levels were measured. The effect of TSA (0.5 or 1 µM) and andrographolide (15 or 30 µM) on OTR expression was evaluated. MAIN OUTCOME MEASURE(S): Visual Analog Scale scores, and contractile amplitude and frequency. The OTR protein levels in MSMCs were quantified by Western blot analysis. RESULT(S): The contractile amplitude and OTR expression levels were significantly higher in cases than in controls. Dysmenorrhea Visual Analog Scale scores correlated positively with contractile amplitude and OTR expression level. Both TSA and andrographolide dose-dependently inhibit OTR expression in MSMC. CONCLUSION(S): Oxytocin receptor overexpression in MSMCs may be responsible for increased uterine contractility and adenomyosis-associated dysmenorrhea. Both histone deacetylase inhibitors and andrographolide are therapeutically promising.