Suppression of FPR2 expression inhibits inflammation in preeclampsia by improving the biological functions of trophoblast via NF-κB pathway.

PURPOSE: The dysfunction of trophoblast during inflammation plays an important role in PE. Formyl peptide receptor 2 (FPR2) plays crucial roles in the development of inflammation-associated disease. This present study aimed to explore the effect of FPR2 on a trophoblast cellular model of preeclampsia. METHODS: The expression of FPR2 in placenta was detected by immunohistochemical staining and western blotting. Transfection of siRNA was used to knockdown FPR2 in HTR-8/SVneo cells. Inflammatory cytokines were detected by ELISA. CCK8, Transwell, wound healing, FACS and tube formation assays were performed to observe the abilities of cell proliferation, migration, invasion, apoptosis and angiogenesis. Western blotting was implemented to clarify that NF-κB signaling pathway was downstream of FPR2. RESULTS: The expression levels of FPR2 were higher in placental tissues of patients with PE. Knockdown of FPR2 expression by siFPR2 or inhibition of its activity by WRW4 decreased the release of proinflammatory cytokines in HTR8/SVneo cells treated with LPS. Knockdown of FPR2 expression or inhibition of its activity further reversed the LPS-induced attenuation of the proliferation, migration, invasion and angiogenesis and increase in apoptosis in HTR8/SVneo cells. Moreover, the NF-κB signaling pathway was activated in both placental tissues of patients with PE and LPS-treated HTR8/SVneo cells. However, the activation was attenuated when FPR2 was knocked down or inhibited. CONCLUSION: Suppression of FPR2 expression alleviated the effects of inflammation induced by LPS on trophoblasts via the NF-κB signaling pathway, which provided a novel and potential strategy for the treatment of PE.

A Comprehensive cis-eQTL Analysis Revealed Target Genes in Breast Cancer Susceptibility Loci Identified in Genome-wide Association Studies.

Genome-wide association studies (GWASs) have identified more than 150 common genetic loci for breast cancer risk. However, the target genes and underlying mechanisms remain largely unknown. We conducted a cis-expression quantitative trait loci (cis-eQTL) analysis using normal or tumor breast transcriptome data from the Molecular Taxonomy of Breast Cancer International Consortium (METABRIC), The Cancer Genome Atlas (TCGA), and the Genotype-Tissue Expression (GTEx) project. We identified a total of 101 genes for 51 lead variants after combing the results of a meta-analysis of METABRIC and TCGA, and the results from GTEx at a Benjamini-Hochberg (BH)-adjusted p < 0.05. Using luciferase reporter assays in both estrogen-receptor positive (ER+) and negative (ER-) cell lines, we showed that alternative alleles of potential functional single-nucleotide polymorphisms (SNPs), rs11552449 (DCLRE1B), rs7257932 (SSBP4), rs3747479 (MRPS30), rs2236007 (PAX9), and rs73134739 (ATG10), could significantly change promoter activities of their target genes compared to reference alleles. Furthermore, we performed in vitro assays in breast cancer cell lines, and our results indicated that DCLRE1B, MRPS30, and ATG10 played a vital role in breast tumorigenesis via certain disruption of cell behaviors. Our findings revealed potential target genes for associations of genetic susceptibility risk loci and provided underlying mechanisms for a better understanding of the pathogenesis of breast cancer.

LINC00689 induces gastric cancer progression via modulating the miR-338-3p/HOXA3 axis.

BACKGROUND: LINC00689 acts one critical regulatory role in several tumors. However, the functional, regulatory mechanism and expression of LINC00689 remains unknown in gastric cancer. METHODS: LINC00689 and miR-338-3p levels were determined using a quantitative reverse transcriptase-polymerase chain reaction analysis and an enzyme-linked immunoassay and a cell-counting kit-8 assay were utilized to detect interleukin (IL)-8, IL-6 and IL-1ß expression and cell proliferation, respectively. RESULTS: We found that LINC00689 and HOXA3 are overexpressed and miR-338-3p is decreased in gastric cancer cells. Compared to control specimens, LINC00689 is overexpressed in gastric cancer specimens and the level of LINC00689 was up-regulated in 32 cases (32/40; 80.0%) compared to control samples. LINC00689 increased cell growth, epithelial-mesenchymal transition (EMT) development and secretion of inflammatory factors in gastric cancer. Compared to control specimens, miR-338-3p expression was decreased in gastric cancer specimens and a Pearson's correlation assay revealed that miR-338-3p was negatively correlated with LINC00689 expression in gastric cancer specimens. HOXA3 was identified as one target gene of miR-338-3p and Ectopic expression of LINC00689 suppressed miR-338-3p and enhanced HOXA3 expression in HGC-27 cells. LINC00689 enhanced cell growth, EMT development and secretion of inflammatory factors by promoting HOXA3. CONCLUSIONS: LINC00689 may present a potential future target for gastric cancer treatment.

Effects of a Fermented Beverage of Changbai Mountain Fruit and Vegetables on the Composition of Gut Microbiota in Mice.

Evidence suggests that fermented foods and beverages made from fruits and vegetables benefit human health, including in the prevention of cardiovascular disease, cancer, diabetes and gastrointestinal disorders. However, there are few studies on the effects of fermented fruits and vegetables on intestinal microbiota. In this study, we investigated the changes in the composition of the intestinal microbial community after short-term treatment with a fermented beverage of Changbai Mountain fruit and vegetables (FB). Forty male ICR mice, weighing 17-19 g, were fed diets with different concentrations of the FB or distilled water for 15 days. 16S rDNA gene sequences were used to analyze the gut microbiota with the Illumina sequencing platform and a paired-end method. FB had no effect on weight gain, the adiposity index, or food intake in the treated mice compared with the control group. The cecal index was significantly higher in the FB-administered groups than in the control group. Firmicutes and Bacteroidetes were the dominant phyla in the mice ceca. The Firmicutes/Bacteroidetes ratio was reduced in the FB-administered mice, and proportions of the family Prevotellaceae, Bacteroidales_S24-7_group, family Bacteroidaceae, and genus Bacteroides increased, and these increases were correlated positively with intake of fermented beverage. The FB also altered the diversity of the cecal microbiota in the mice. Graphical Abstract.

Evaluation of potential regulatory function of breast cancer risk locus at 6q25.1.

In a genome-wide association study conducted among Chinese women, we identified the single nucleotide polymorphism (SNP) rs2046210 at 6q25.1 for breast cancer risk. To explore a potential regulatory role for this risk locus, we measured expression levels of nine genes at the locus in breast cancer tissue and adjacent normal tissue samples obtained from 67 patients recruited in the Shanghai Breast Cancer Study. We found that rs2046210 had a statistically significant association with the expression levels of the AKAP12 and ESR1 genes in adjacent normal breast tissues. Women who carry the AA/AG risk genotypes had higher expressions of these two genes compared to those who carry G/G genotypes (P = 0.02 and 0.04 for the AKAP12 and ESR1, respectively). However, no significant differences of SNP rs2046210 with gene expression levels were found in tumor tissues. In The Cancer Genome Atlas samples, the AA/AG risk genotypes of SNP rs2046210 were associated with a significantly higher expression level of the AKAP12 gene and a lower level of the ESR1 gene in tumor tissue. Functional analysis using ENCODE data revealed that SNP rs7763637, which is in strong linkage disequilibrium with SNP rs2046210, is likely a potential functional variant, regulating the AKAP12 gene. Taken together, these results from our study suggest that the association between the 6q25.1 locus and breast cancer risk may be mediated through SNPs that regulate expressions of the AKAP12 gene.

A prospective study of autoantibodies to Ezrin and pancreatic cancer risk.

PURPOSE: No biomarker is available for pancreatic cancer early detection, but a small prospective European study involving 16 cases and 32 controls raised the possibility that anti-Ezrin autoantibodies may be associated with risk of pancreatic cancer. We aimed to validate this finding in a case-control study nested within a prospective study in the USA. METHODS: Levels of anti-Ezrin autoantibodies were examined using ELISA in pre-diagnostic plasma samples of 73 cases and 145 matched controls. Paired t test and paired signed rank tests were used to determine the difference between two groups, and conditional logistic regression was used to evaluate the association between anti-Ezrin autoantibody levels and risk of developing pancreatic cancer. RESULTS: No association was found between levels of anti-Ezrin plasma autoantibodies and subsequent risk of developing pancreatic cancer. CONCLUSION: Anti-Ezrin autoantibodies did not appear to be useful as a plasma biomarker for early detection of pancreatic cancer.

A Sensitive LC-MS/MS Method for the Simultaneous Determination of Skimmin, a Potential Agent for Treating Postpartum Stroke, and Its Metabolite Umbelliferone in Rat Plasma.

BACKGROUND: Skimmin, a potential agent for treating postpartum stroke, is one of the most important coumarins extracted from the leaves of skimmia. OBJECTIVE: In this study, a specific, sensitive, and simple high-performance liquid chromatography with tandem mass spectrometry (HPLC-MS/MS) method for the simultaneous determination of skimmin and its metabolite umbelliferone in rat plasma was established and validated. METHOD: Chromatographic separation was performed by an Inertsil ODS-3 column (50 mm × 4.6 mm, 5 µm) with a mobile phase consisting of 0.1% formic acid in distilled water-acetonitrile at a flow rate of 0.5 mL/min with gradient elution mode. All analytes were detected and quantified in negative multiple reaction monitoring (MRM). RESULTS: All calibration curves showed good linearity (r > 0.995) over the concentration range of 10-10 000 and 2.0-2000 ng/mL for skimmin and umbelliferone, respectively. The selectivity, sensitivity, extraction recovery, matrix effect, and stability met all requirements. CONCLUSIONS: The analysis method was successfully applied to the pharmacokinetic study of skimmin and umbelliferone in rats following oral administration of skimmin at the doses of 10, 30, and 90 mg/kg. With the exception of AUC(0-∞) and Cmax, MRT and Cl/F of skimmin had significant statistical difference with the increasing doses. Skimmin might exhibit nonlinear pharmacokinetic characteristics in rats. HIGHLIGHTS: This was the first study to investigate the pharmacokinetic characteristics of skimmin as a candidate agent for treating postpartum stroke.

Synergistic potential of teriflunomide with fluconazole against resistant Candida albicans in vitro and in vivo.

Introduction: Candida albicans is the primary cause of systemic candidiasis, which is involved in high morbidity and mortality. Drug resistance exacerbates these problems. In addition, there are limited antifungal drugs available. In order to solve these problems, combination therapy has aroused great interest. Teriflunomide is an immunosuppressant. In the present work, we aimed to identify whether teriflunomide can reverse the resistance of Candida albicans in the presence of sub-inhibitory concentrations of fluconazole in vitro and in vivo. Methods: Seven Candida albicans isolates were used in this study. Susceptibility of Candida albicans in vitro to the drugs was determined using a checkerboard microdilution assay in accordance with the recommendations of the Clinical and Laboratory Standards Institute. The effects of drugs on biofilm biomass of Candida albicans were determined by crystal violet staining. The development ability of Candida albicans hyphae was performed using a modified broth microdilution method. Galleria mellonella was used for testing the in vivo efficacy of the combination therapies. Results: We found that the combination of teriflunomide (64 µg/mL) and fluconazole (0.5-1 µg/mL) has a significant synergistic effect in all resistant Candida albicans isolates (n=4). Also, this drug combination could inhibit the immature biofilm biomass and hyphae formation of resistant Candida albicans. Galleria mellonella was used for testing the in vivo efficacy of this combination therapies. As for the Galleria mellonella larvae infected by resistant Candida albicans, teriflunomide (1.6 µg/larvae) combined with fluconazole (1.6 µg/larvae) significantly increased their survival rates, and reduced the fungal burden, as well as damage of tissue in comparison to that in the control group or drug monotherapy group. Conclusion: These results expand our knowledge about the antifungal potential of teriflunomide as an adjuvant of existing antifungal drugs, and also open new perspectives in the treatment of resistant Candida albicans based on repurposing clinically available nonantifungal drugs.

[Corrigendum] FPR2 serves a role in recurrent spontaneous abortion by regulating trophoblast function via the PI3K/AKT signaling pathway.

Subsequently to the publication of the above paper, an interested reader drew to the authors' attention that the '0 h/si-NC + Solvent' and '0 h/si-FPR2 + Solvent' data panels shown in Fig. 4B on p. 7 appeared to contain overlapping sections of data, such that they were potentially derived from the same original source where these panels was intended to show the results from differently performed experiments. After having conducted an independent investigation of the figures in the Editorial Office, it was identified that sections of the 'si-NC + Solvent' and 'si-FPR2 + LY294002' data panels in Fig. 4C also contained overlapping data. After having asked the authors to provide an explanation of these data, they realized that this figure was inadvertently assembled incorrectly. They were, however, able to consult their original data, and the revised version of Fig. 4, containing the correct data panel for the 'si-FPR2 + LY294002' experiment in Fig. 4C and complete data from one of the alternatively performed experiments in Fig. 4B, is shown on the next page. Note that these errors did not adversely affect either the results or the overall conclusions reported in this study. All the authors agree with the publication of this corrigendum, and are grateful to the Editor of Molecular Medicine Reports for allowing them the opportunity to publish this. They also wish to apologize to the readership of the Journal for any inconvenience caused. [Molecular Medicine Reports 24: 838, 2021; DOI: 10.3892/mmr.2021.12478].

Proteomic analysis of amniotic fluid to identify potential targets predicting preterm delivery.

Preterm delivery is a common complication of pregnancy which leads to significant neonatal mortality and morbidity. Identifying predictive markers linked to spontaneous preterm delivery (SPTD) is important for effective treatment and prevention of PTD. To explore potential biomarkers related to SPTD, we performed proteomics analysis in amniotic fluid (AF). In total, we enrolled 30 pregnant women with singleton gestation who underwent clinically indicated amniocentesis at 15-24 weeks of gestation. LC-MS analysis was used to analyze the AF samples of 10 women with SPTD < 34 weeks after cervix cerclage (Preterm group), 10 women with term delivery (TD) ≥ 34 weeks after cervix cerclage (Term group), and 10 women who delivered at term (Normal group). ELISA validation was performed for candidate proteins in a second independent cohort. As a result, we identified 44 differentially expressed proteins (DEPs, P < 0.05) via proteomic analysis, and based on that, 9 primary pathways were also determined in SPTD. Results of the ELISA assay confirmed that the increased concentration of Serpin A1, decreased concentrations of Renin and IGFBP4 were significantly associated with SPTD at ≤34 weeks.

Passive Surveillance of Malaria in Pregnant Women, Non-pregnant Women and Children Under 5 Years of Age in Bannu District, Khyber Pakhtunkhwa Pakistan.

Background: Malaria among pregnant women is one of the major causes of maternal and infant mortality and morbidity, especially in high-risk areas. Therefore, our study identified the burden of malaria for pregnant women, non-pregnant women, and children under 5 years of age, and malaria service health facilities in Bannu district, Khyber Pakhtunkhwa, Pakistan. Methods: A cross-sectional study was conducted. In this survey, 15,650 individuals were surveyed, and 1,283 were malaria-positive detected. The data were collected from 80 different healthcare centers. SPSS version 23 was used for data analysis. ArcGIS version 10.8 was used for study area mapping. Results: Malaria was detected in 23.3% of children under five, 4.4% of pregnant women, and 72.3% of non-pregnant women, respectively. Moreover, P. falciparum, P. vivax, and mixed infection had a prevalence of 2.1, 96.8, and 1.1%. The most often used and effective medications to treat malaria were chloroquine (29.7%) and primaquine (69.4%). Conclusion: This study's findings depict that malaria's prevalence in the non-pregnant women's group was high. Additionally, P. vivax infection was found to be more prevalent than other types of malaria infection. Due to the scarcity of healthcare facilities in this endemic region, special attention should be directed to strengthening the malaria surveillance and eradication programs.

Psoralen, a natural phytoestrogen, improves diaphyseal fracture healing in ovariectomized mice: A preliminary study.

Psoralen is an effective active component extracted from Psoraleacorylifolia, which can promote bone formation in osteoporotic animals. However, to the best of our knowledge, its effect on fracture healing has not yet been examined. In the present study, open femur fractures were created in ovariectomy (OVX)-induced osteoporotic mice. OVX mice were treated with psoralen (psoralen+OVX group) or physiological saline (OVX group) by oral gavage. Radiographic and histological results demonstrated progressed callus consolidation in the psoralen+OVX group compared with the OVX group after 10 and 21 days of treatment. Qualitative histological analysis showed that the number of osteoclasts was significantly reduced in the psoralen+OVX group after treatment. Moreover, reverse transcription-quantitative PCR analysis of callus samples showed increased expression of bone morphogenetic protein-2 (BMP-2) and osteoprotegerin (OPG), and decreased expression of receptor activator of nuclear factor-κB ligand (RANKL) at 10 and 21 days post injury in the psoralen+OVX group compared with the OVX group. Furthermore, western blot analysis showed that psoralen significantly increased the expression of estrogen receptor (ER)-α, but had no effect on ER-ß expression; these results were further confirmed by immunohistochemistry. To conclude, these results indicated that psoralen may promote callus formation and inhibit osteoclast genesis by increasing BMP-2 and ER-α levels, and OPG/RANKL ratio. Consequently, psoralen could be a possible treatment for osteoporotic fracture-related complications.

FPR2 serves a role in recurrent spontaneous abortion by regulating trophoblast function via the PI3K/AKT signaling pathway.

Recurrent spontaneous abortion (RSA) effects both the physical and mental health of women of reproductive age. Trophoblast dysfunction may result in RSA due to shallow placental implantation. The mechanisms underlying formyl peptide receptor 2 (FPR2) on the biological functions of trophoblasts remain to be elucidated. The present study aimed to explore the potential functions of FPR2, a G protein­coupled receptor, in placental trophoblasts. The location and expression levels of FPR2 in the villi tissue of patients with RSA were detected using immunohistochemical staining, reverse transcription­quantitative PCR and western blotting. Following the transfection of small interfering RNA targeting FPR2 in HTR­8/SVneo cells, a Cell Counting Kit­8 assay was used to determine the levels of cell viability. Flow cytometry was used to examine the levels of cell apoptosis and gap closure and Transwell assays were carried out to evaluate the levels of cell migration and invasion. A tube formation assay was performed to detect the levels of capillary­like structure formation. Western blotting was used to detect the expression levels of proteins in the associated signaling pathways. The expression of FPR2 was present in villi trophoblasts and was markedly increased in patients with RSA. The levels of trophoblast invasion, migration and tube formation were markedly increased following FPR2 knockdown, whereas the levels of apoptosis were markedly decreased. In addition, FPR2 knockdown caused an increase in the phosphorylation levels of AKT and PI3K. Thus, FPR2 may be involved in the regulation of trophoblast function via the PI3K/AKT signaling pathway. The results of the present study provided a theoretical basis for the use of FPR2 as a target for the treatment of trophoblast­associated diseases, such as RSA.

Eucommia ulmoides Polysaccharides Attenuate Rabbit Osteoarthritis by Regulating the Function of Macrophages.

Background and purpose: Eucommia ulmoides polysaccharides (EUP) can regulate the immunity of macrophages, but the functional status of macrophages is related to osteoarthritis and synovial inflammation. The purpose of this study is to explore whether EUP has the effect of inhibiting osteoarthritis and its possible mechanism. Methods: MTT test was used to evaluate the appropriate concentration of EUP and real-time quantitative polymerase chain reaction (RT-qPCR) was conducted to detect the effect of EUP on gene expression in RAW 264.7 cells. The osteoarthritis model was constructed by the anterior cruciate ligament transection (ACLT) in the rabbits. These rabbits were divided into three groups, sham operation group, OA group, and EUP group. The changes in articular cartilage were detected by gross observation and histological staining, and Micro-CT tested subchondral bone. Finally, the changes of macrophages in synovial tissue were studied by immunohistochemistry. Results: The results showed that EUP at the concentration of 50ug/mL and 100ug/mL were beneficial to the proliferation of macrophages. The qPCR results indicated that EUP inhibited the expression of inflammation-related genes IL-6, IL-18 and IL-1ß, and promoted the expression of osteogenic and cartilage-related genes BMP-6, Arg-1 and transforming growth factor beta (TGF-ß). The results of in vivo experiments suggested that the degree of destruction of articular cartilage in the EUP group was significantly reduced, and the Osteoarthritis Research Society International (OARSI) score was significantly reduced. Compared with the OA group, the subchondral cancellous bone density of the EUP group increased, the number and thickness of trabecular bone increased, and the separation of trabecular bone decreased. Synovial macrophage immunohistochemistry results manifested that EUP, on the one hand, reduced M1 polarized macrophages, on the other hand, accumulated M2 polarized macrophages. Conclusion: EUP can promote articular cartilage repair and subchondral bone reconstruction. The regulation of the polarization state of macrophages may be one of its mechanisms to delay the progression of osteoarthritis.

LNX (Ligand of Numb-protein X) interacts with RhoC, both of which regulate AP-1-mediated transcriptional activation.

LNX (Ligand of Numb-protein X) was originally isolated as a binding partner to the cell-fate Determinant Numb during development, and then identified to act as a RING finger-type E3 ubiquitin ligase for the ubiquitylation and degradation of Numb. LNX contains 4 PDZ domains which are proved to play a central role in organizing diverse cell signaling assemblies. A yeast two-hybrid screening was used to identify LNX as a potential binding partner for RhoC. RhoC, a member of the Ras family of small GTPases, promotes reorganization of the actin cytoskeleton and regulation of cell shape, attachment, and motility. The interaction between LNX and RhoC in mammalian cells was identified by co-immunoprecipitation assays, and the efficient binding required the first PDZ domain of LNX. LNX and RhoC were further colocalized with each other in mammalian cells, in which RhoC changed its sublocalization from cytoplasm to nucleus when co-transferred with LNX. Furthermore, co-expression of RhoC reduced the transcriptional activity of AP-1, which was up-regulated by over-expression of LNX alone. These results suggest that LNX and RhoC might be part of a larger protein complex that would have important functions in signaling transduction about regulating the transcriptional activities of AP-1.

Improved placental vascular repair in a rat preeclampsia model by implantation of endothelial progenitor cells treated with platelet microparticles.

Objective: To detect the role of endothelial progenitor cells (EPCs) treated with platelet microparticles (PMPs) in preeclampsia. Methods: EPCs treated with/without PMPs were labeled and injected to PE rats. The differentiation of EPCs, the change of endothelial nitric oxide synthase (eNOS), blood pressure and proteinuria were measured. The blood pressure and proteinuria increased in each of PE groups, and were improved by EPCs which was strengthened by PMPs. Transplantation of EPCs increased placental angiogenesis. The trend of change of NO was the same as blood pressure. Conclusion: Transplantation of EPCs treated with PMPs improved blood pressure and proteinuria more effectively.

Mechanism of PM2.5-induced human bronchial epithelial cell toxicity in central China.

Exposure to PM2.5 has been linked to respiratory disorders, yet knowledge of the molecular mechanism is limited. Here, PM2.5 was monitored and collected in central China, and its cytotoxicity mechanism on human bronchial epithelial cells (BEAS-2B) was investigated. With the average concentration of 109 ±â€¯69 µg/m3, PM2.5 was rich in heavy metals and organic pollutants. After exposure to PM2.5, the viability of BEAS-2B cells decreased, where 510 dysregulated genes were predicted to induce necroptosis via inhibiting ATP synthesis through the oxidative phosphorylation signaling pathway. Cellular experiments demonstrated that the content of ATP was downregulated, while the expression of RIP3, a necroptosis indicator, was upregulated. Besides, four enzymes in charge of ATP synthesis were downregulated, including ATP5F, NDUF, COX7A, and UQCR, while two genes of RELA and CAPN1 responsible for necroptosis were upregulated. Furthermore, N-acetylcysteine was applied as an enhancer for ATP synthesis, which reversed the downregulation of ATP5F, NDUF, and COX7A, and consequently alleviated the elevation of RELA, CAPN1, and RIP3. In conclusion, PM2.5 exposure downregulates ATP5F, NDUF, COX7A, and UQCR, and that inhibits ATP synthesis via the oxidative phosphorylation signaling pathway, which subsequently upregulates RELA and CAPN1 and ultimately leads to necroptosis of BEAS-2B cells.

Fractalkine/CX3CR1 Contributes to Endometriosis-Induced Neuropathic Pain and Mechanical Hypersensitivity in Rats.

Pain is the most severe and common symptom of endometriosis. Its underlying pathogenetic mechanism is poorly understood. Nerve sensitization is a particular research challenge, due to the limitations of general endometriosis models and sampling nerve tissue from patients. The chemokine fractalkine (FKN) has been demonstrated to play a key role in various forms of neuropathic pain, while its role in endometriotic pain is unknown. Our study was designed to explore the function of FKN in the development and maintenance of peripheral hyperalgesia and central sensitization in endometriosis using a novel endometriosis animal model developed in our laboratory. After modeling, behavioral tests were carried out and the optimal time for molecular changes was obtained. We extracted ectopic tissues and L4-6 spinal cords to detect peripheral and central roles for FKN, respectively. To assess morphologic characteristics of endometriosis-like lesions-as well as expression and location of FKN/CX3CR1-we performed H&E staining, immunostaining, and western blotting analyses. Furthermore, inhibition of FKN expression in the spinal cord was achieved by intrathecal administration of an FKN-neutralizing antibody to demonstrate its function. Our results showed that implanted autologous uterine tissue around the sciatic nerve induced endometriosis-like lesions and produced mechanical hyperalgesia and allodynia. FKN was highly expressed on macrophages, whereas its receptor CX3CR1 was overexpressed in the myelin sheath of sciatic nerve fibers. Overexpressed FKN was also observed in neurons. CX3CR1/pp38-MAPK was upregulated in activated microglia in the spinal dorsal horn. Intrathecal administration of FKN-neutralizing antibody not only reversed the established mechanical hyperalgesia and allodynia, but also inhibited the expression of CX3CR1/pp38-MAPK in activated microglia, which was essential for the persistence of central sensitization. We concluded that the FKN/CX3CR1 signaling pathway might be one of the mechanisms of peripheral hyperalgesia in endometriosis, which requires further studies. Spinal FKN is important for the development and maintenance of central sensitization in endometriosis, and it may further serve as a novel therapeutic target to relieve persistent pain associated with endometriosis.

Dual specificity phosphotase 18, interacting with SAPK, dephosphorylates SAPK and inhibits SAPK/JNK signal pathway in vivo.

The SAPK/JNKs play important roles in numerous cellular processes, and for this reason they have become putative drug targets. Most dual-specificity protein phosphatases (DSPs) play important roles in the regulation of mitogenic signal transduction and cell cycle control in response to extracellular stimuli. Dual-specificity phosphatase 18 (DUSP18), a newly recognized SAPK/JNK phosphatase, is widely expressed. This expression is modulated in response to extracellular stimuli. By phosphorylation assay, pull down and coimmunoprecipitation experiments, it is shown here that DUSP18 interacts with SAPK/JNK and dephosphorylates it both in vitro and in vivo. DUSP18 does not dephosphorylate p38 or p44ERK1. Furthermore, DUSP18 inhibits SAPK/JNK pathway in vivo. Based on these findings, DUSP18 appears to serve an important role by regulation of SAPK/JNK pathway.