Piper longum L. ameliorates gout through the MAPK/PI3K-AKT pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: Gout, a painful joint disease with a prevalence ranging from 0.86% to 2.2% in China over the past decade. Traditional medicine has long utilized the medicinal and edible Piper longum L. (PL) fruit spikes for treating gout and other joint conditions like rheumatoid arthritis. However, the exact mechanisms behind its effectiveness remain unclear. AIM OF THE STUDY: This study aimed to investigate the potential of alcoholic extracts from PL fruit spikes as a safe and effective treatment for gout. We used a combined network pharmacology and experimental validation approach to evaluate the mechanisms behind the anti-gout properties of PL. MATERIALS AND METHODS: UPLC-Q/TOF-MS analysis determined the major components of PL. Subsequently, network pharmacology analysis predicted potential molecular targets and related signaling pathways for the anti-gout activity of PL. Molecular docking simulations further explored the interactions between PL compounds and proteins and characterized the properties of potential bioactive secondary metabolites. Mouse models of air pouch inflammation and hyperuricemia were further established, and the anti-gout mechanism of PL was confirmed by examining the expression of proteins related to the MAPK and PI3K-AKT pathways in the tissue. RESULTS: Our analysis revealed 220 bioactive secondary metabolites within PL extracts. Network pharmacology and molecular docking results indicated that these metabolites primarily combat gout by modulating the PI3K-AKT and MAPK signaling pathways. In vivo experiments have also proven that PL at a dose of 100 mg/kg can optimally reduce acute inflammation of gout and kidney damage caused by high uric acid. The anti-gout mechanism involves the PI3K-AKT/MAPK signaling pathway and its downstream NF-κB pathway. CONCLUSION: This study provides compelling evidence for PL's therapeutic potential in gout management by modulating key inflammatory pathways. The findings offer a strong foundation for future clinical exploration of PL as a gout treatment option.

Potential anti-gout properties of Wuwei Shexiang pills based on network pharmacology and pharmacological verification.

ETHNOPHARMACOLOGICAL RELEVANCE: Wuwei Shexiang Pills (WWSX), a classic Tibetan medicine, consists of Chebulae Fructus (removed pit), Aucklandiae Radix, Moschus, Aconiti Fiavi Radix, and Acori Calami Rhizoma. It is used clinically in China to treat joint pain, swelling and other symptoms, and has the function of dispelling wind and relieving pain. However, to date, the mechanism of how it works against gout is still unclear. AIMS OF THE STUDY: Using network pharmacology, molecular docking and pharmacological verification to explore the potential anti-gout properties of WWSX. MATERIALS AND METHODS: With the use of UPLC-Q/TOF-MS, the main components of WWSX were obtained and screened for potential anti-inflammatory components by network pharmacology and molecular docking. The anti-inflammatory activity of the components screened from WWSX was also tested by in vitro assays. The anti-gout mechanism of WWSX was predicted by network pharmacology, and the pharmacological validation experiments using gouty arthritis model and mouse air pouch model were used to explore the multifaceted mechanism of WWSX to modify gout. RESULT: Thirty-eight active ingredients were obtained from the UPLC-Q/TOF-MS detection. The network pharmacology and molecular docking analysis showed that 104 co-targets were participated in the treatment of gout, and the main signaling pathways involved were NOD-like receptor pathway, NF-κB pathway and MAPK pathway. Pharmacological evaluation showed that WWSX could significantly improve gout in gouty arthritis models and mouse air pouch models by modulating the above pathways. CONCLUSION: This work has predicted and validated the anti-inflammatory material basis and predicted the anti-gout mechanism of WWSX which was verified by network pharmacology, molecular docking and in vitro cellular studies. The results reveal the mechanism of WWSX in the treatment of gout and provide a theoretical basis for its clinical application.

Integrating network pharmacology and pharmacological validation to explore the effect of Shi Wei Ru Xiang powder on suppressing hyperuricemia.

ETHNOPHARMACOLOGICAL RELEVANCE: Shi Wei Ru Xiang powder (SWR) is a traditional Tibetan medicinal formula with the effect of dispelling dampness and dispersing cold. In clinical practice, SWR is generally used for the treatment of hyperuricemia (HUA). However, its exact pharmacological mechanism remains unclear. AIMS OF THE STUDY: To preliminarily elucidate the regulatory effects and possible mechanisms of SWR on hyperuricemia using network pharmacology and experimental validation. MATERIALS AND METHODS: A mouse model of hyperuricemia was used to evaluate the alleviating effect of SWR on hyperuricemia. The major components of SWR were acquired by UPLC-Q/TOF-MS. The potential molecular targets and associated signaling pathways were predicted through network pharmacology. The mechanism of action of SWR in ameliorating hyperuricemia was further investigated by pharmacological evaluation. RESULTS: Mice with hyperuricemia and renal dysfunction were ameliorated by SWR. The 36 components of SWR included phenolic acids, terpenoids, alkaloids and flavonoids were identified. Network pharmacological analysis showed the involvement of the above compounds, and 115 targets were involved to treat hyperuricemia, involving multiple biological processes and different signaling pathways. Pharmacological experiments validated that SWR ameliorated hyperuricemic nephropathy in mice by modulating the mitogen-activated protein kinase (MAPK) signaling pathway, nuclear factor kappaB (NF-κB) signaling pathway and NOD-like receptor signaling pathway. CONCLUSION: MAPK signaling pathway, NF-κB signaling pathway and NOD-like receptor signaling pathway play important roles in the therapeutic effects of SWR on hyperuricemia.

Dysregulation of iron homeostasis and methamphetamine reward behaviors in Clk1-deficient mice.

Chronic administration of methamphetamine (METH) leads to physical and psychological dependence. It is generally accepted that METH exerts rewarding effects via competitive inhibition of the dopamine transporter (DAT), but the molecular mechanism of METH addiction remains largely unknown. Accumulating evidence shows that mitochondrial function is important in regulation of drug addiction. In this study,  we investigated the role of Clk1, an essential mitochondrial hydroxylase for ubiquinone (UQ), in METH reward effects. We showed that Clk1+/- mutation significantly suppressed METH-induced conditioned place preference (CPP), accompanied by increased expression of DAT in plasma membrane of striatum and hippocampus due to Clk1 deficiency-induced inhibition of DAT degradation without influencing de novo synthesis of DAT. Notably, significantly decreased iron content in striatum and hippocampus was evident in both Clk1+/- mutant mice and PC12 cells with Clk1 knockdown. The decreased iron content was attributed to increased expression of iron exporter ferroportin 1 (FPN1) that was associated with elevated expression of hypoxia-inducible factor-1α (HIF-1α) in response to Clk1 deficiency both in vivo and in vitro. Furthermore, we showed that iron played a critical role in mediating Clk1 deficiency-induced alteration in DAT expression, presumably via upstream HIF-1α. Taken together, these data demonstrated that HIF-1α-mediated changes in iron homostasis are involved in the Clk1 deficiency-altered METH reward behaviors.

Nucleolin-Targeting AS1411-Aptamer-Modified Graft Polymeric Micelle with Dual pH/Redox Sensitivity Designed To Enhance Tumor Therapy through the Codelivery of Doxorubicin/TLR4 siRNA and Suppression of Invasion.

In this article, a novel graft polymeric micelle with targeting function ground on aptamer AS1411 was synthesized. The micelle was based on chitosan-ss-polyethylenimine-urocanic acid (CPU) with dual pH/redox sensitivity and targeting effects. This micelle was produced for codelivering Toll-like receptor 4 siRNA (TLR4-siRNA) and doxorubicin (Dox). In vitro investigation revealed the sustained gene and drug release from Dox-siRNA-loaded micelles under physiological conditions, and this codelivery nanosystem exhibited high dual pH/redox sensitivity, rapid intracellular drug release, and improved cytotoxicity against A549 cells in vitro. Furthermore, the micelles loaded with TLR4-siRNA inhibited the migration and invasion of A549. Excellent tumor penetrating efficacy was also noted in the A549 tumor spheroids and solid tumor slices. In vivo, multiple results demonstrated the excellent tumor-targeting ability of AS1411-chitosan-ss-polyethylenimine-urocanic acid (ACPU) micelle in tumor tissues. The micelles exhibited excellent antitumor efficacy and low toxicity in the systemic circulation in lung-tumor-bearing BALB/c mice. These results conclusively demonstrated the great potential of the new graft copolymer micelle with targeting function for the targeted and efficient codelivery of chemotherapeutic drugs and genes in cancer treatment.

Dihydromyricetin exerts a rapid antidepressant-like effect in association with enhancement of BDNF expression and inhibition of neuroinflammation.

RATIONALE: Major depressive disorder (MDD) is a highly prevalent illness that affects large populations across the world, and increasing evidence suggests that neuroinflammation and levels of brain-derived neurotrophic factor (BDNF) are closely related to depression. Dihydromyricetin (DHM) is a kind of flavonoid natural product that has been reported to display multiple pharmacological effects, including anti-inflammatory and anti-oxidative properties, and these may contribute to ameliorate MDD. OBJECTIVE: This study investigated the effect of DHM on depression-related phenotypes in various experimental animal models. METHODS: The antidepressant-like effect of DHM was validated via depression-related behavioral tests in naïve male C57BL/6 mice, as well as in the acute lipopolysaccharide-induced mouse model of depression. The chronic unpredicted mild stress (CUMS) mouse model of depression was also used to assess the rapid antidepressant-like effect of DHM by tail suspension test (TST), forced swimming test (FST), locomotor activity, and sucrose preference test (SPT). The expression of BDNF and inflammatory factors were determined through Western blotting and enzyme-linked immunosorbent assay, respectively. RESULTS: DHM reduced immobility time in the TST and FST both in mice and the acute LPS-induced mouse model of depression. Seven days of DHM treatment ameliorated depression-related behaviors induced by CUMS, whereas similar treatment with the typical antidepressant venlafaxine did not. DHM activated the ERK1/2-CREB pathway and increased glycogen synthase kinase-3 beta (GSK-3ß) phosphorylation at ser-9, with upregulation of BDNF expression, in both hippocampal tissues and cultured hippocampal cells. CONCLUSION: The present data indicate that DHM exerts a more rapid antidepressant-like effect than does a typical antidepressant, in association with enhancement of BDNF expression and inhibition of neuroinflammation.

Stepwise pH/reduction-responsive polymeric conjugates for enhanced drug delivery to tumor.

In this research, a charge-conversional polymer, poly-l-lysine-lipoic acid (PLL-LA), was prepared by dimethylmaleic anhydride (DA) modification and applied as a carrier with enhanced cell internalization and intracellular pH- and reduction-triggered doxorubicin (Dox) release. The surface charge of dimethylmaleic anhydride-poly-l-lysine-lipoic acid micelles (DA-PLL-LA) was negative at physiological pH and reversed to positive at the extracellular and intracellular pH of cancer cells. At tumor extracellular pH of 6.8, the conjugates underwent a rapid charge-reversible process with almost 80% DA cleavage within 2h, and then endocytosed into the endo/lysosomes more rapidly than at physiological pH of 7.4. The Dox/DA-PLL-LA micelles (Dox-micelles) demonstrated a sustained drug release in vitro under physiological condition, and rapid Dox release was triggered by both extracellular pH and high-concentration reducing glutathione. The Dox-micelles also exhibited enhanced internalization at extracellular pH, rapid intracellular drug release, and improved cytotoxicity against A549 cells in vitro. Excellent tumor-penetrating efficacy was also found in A549 tumor spheroids and solid tumor slices. Moreover, the DA-PLL-LA micelles exhibited excellent tumor-targeting ability in tumor tissues and excellent antitumor efficacy and low systemic toxicity in breast tumor-bearing mice. Therefore, the DA-PLL-LA micelles demonstrated great potential for targeted and efficient drug delivery in cancer treatments.

Binary-copolymer system base on low-density lipoprotein-coupled N-succinyl chitosan lipoic acid micelles for co-delivery MDR1 siRNA and paclitaxel, enhances antitumor effects via reducing drug.

The development of effective and stable carriers of small interfering RNA (siRNA) is important for treating cancer with multidrug resistance (MDR). We developed a new gene and drug co-delivery system and checked its characteristics. Low-density lipoprotein (LDL) was coupled with N-succinyl chitosan (NSC) Lipoic acid (LA) micelles and co-delivered MDR1 siRNA and paclitaxel (PTX-siRNA/LDL-NSC-LA) to enhance antitumor effects by silencing the MDR gene of tumors (Li et al., Adv Mater 2014;26:8217-8224). In our study, we developed a new type of containing paclitaxel-loaded micelles and siRNA-loaded LDL nanoparticle. This "binary polymer" is pH and reduction dual-sensitive core-crosslinked micelles. PTX-siRNA/LDL-NSC-LA had an average particle size of (171.6 ± 6.42) nm, entrapment efficiency of (93.92 ± 1.06) %, and drug-loading amount of (12.35% ± 0.87) %. In vitro, MCF-7 cells, high expressed LDL receptor, were more sensitive to this delivery system than to taxol® and cell activity was inhibited significantly. Fluorescence microscopy showed that PTX-siRNA/LDL-NSC-LA was uptaken very conveniently and played a key role in antitumor activity. PTX-siRNA/LDL-NSC-LA protected the siRNA from degradation by macrophage phagocytosis and evidently down-regulated the level of mdr1 mRNA as well as the expression of P-gp. We tested the target ability of PTX-siRNA/LDL-NSC-LA in vivo in tumor-bearing nude mice. Results showed that this system could directly deliver siRNA and PTX to cancer cells. Thus, new co-delivering siRNA and antitumor drugs should be explored for solving MDR in cancer. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 105B: 1114-1125, 2017.

GSK-3ß Interacts with Dopamine D1 Receptor to Regulate Receptor Function: Implication for Prefrontal Cortical D1 Receptor Dysfunction in Schizophrenia.

INTRODUCTION: Impaired dopamine D1 receptor (D1R) function in prefrontal cortex (PFC) is believed to contribute to the PFC hypofunction that has been hypothesized to be associated with negative symptoms and cognitive deficits in schizophrenia. It is therefore critical to understand the mechanisms for modulation of D1R function. AIMS: To investigate the physical interaction and functional modulation between D1R and GSK-3ß. RESULTS: D1R and GSK-3ß physically interact in cultured cells and native brain tissues. This direct interaction was found to occur at the S(417)PALS(421) motif in the C-terminus of D1R. Inhibition of GSK-3ß impaired D1R activation along with a decrease in D1R-GSK-3ß interaction. GSK-3ß inhibition reduced agonist-stimulated D1R desensitization and endocytosis, the latter associated with the reduction of membrane translocation of ß-arrestin-2. Similarly, inhibition of GSK-3ß in rat PFC also resulted in impaired D1R activation and association with GSK-3ß. Moreover, in a NMDA antagonist animal model of schizophrenia, we detected a decrease in prefrontal GSK-3ß activity and D1R-GSK-3ß association and decreased D1R activation in the PFC. CONCLUSIONS: The present work identified GSK-3ß as a new interacting protein for D1R functional regulation and revealed a novel mechanism for GSK-3ß-regulated D1R function which may underlie D1R dysfunction in schizophrenia.

Clk1 deficiency promotes neuroinflammation and subsequent dopaminergic cell death through regulation of microglial metabolic reprogramming.

Clock (Clk)1/COQ7 is a mitochondrial hydroxylase that is necessary for the biosynthesis of ubiquinone (coenzyme Q or UQ). Here, we investigate the role of Clk1 in neuroinflammation and consequentially dopaminergic (DA) neuron survival. Reduced expression of Clk1 in microglia enhanced the LPS-induced proinflammatory response and promoted aerobic glycolysis. Inhibition of glycolysis abolished Clk1 deficiency-induced hypersensitivity to the inflammatory stimulation. Mechanistic studies demonstrated that mTOR/HIF-1α and ROS/HIF-1α signaling pathways were involved in Clk1 deficiency-induced aerobic glycolysis. The increase in neuronal cell death was observed following treatment with conditioned media from Clk1 deficient microglia. Increased DA neuron loss and microgliosis were observed in Clk1+/- mice after treatment with MPTP, a rodent model of Parkinson's disease (PD). This increase in DA neuron loss was due to an exacerbated microglial inflammatory response, rather than direct susceptibility of Clk1+/- DA cells to MPP+, the active species of MPTP. Exaggerated expressions of proinflammatory genes and loss of DA neurons were also observed in Clk1+/- mice after stereotaxic injection of LPS. Our results suggest that Clk1 regulates microglial metabolic reprogramming that is, in turn, involved in the neuroinflammatory processes and PD.

GSK-3ß inhibitors reverse cocaine-induced synaptic transmission dysfunction in the nucleus accumbens.

Nucleus accumbens receives glutamatergic projection from the prefrontal cortex (PFC) and dopaminergic input from the Ventral tegmental area (VTA). Recent studies have suggested a critical role for serine/threonine kinase glycogen synthase kinase 3ß (GSK3ß) in cocaine-induced hyperactivity; however, the effect of GSK3ß on the modulation of glutamatergic and dopaminergic afferents is unclear. In this study, we found that the GSK3 inhibitors, LiCl (100 mg/kg, i.p.) or SB216763 (2.5 mg/kg, i.p.), blocked the cocaine-induced hyperlocomotor activity in rats. By employing single-unit recordings in vivo, we found that pretreatment with either SB216763 or LiCl for 15 min reversed the cocaine-inhibited firing frequency of medium spiny neuron (MSN) in the nucleus accumbens (NAc). Preperfusion of SB216763 (5 µM) ameliorated the inhibitory effect of cocaine on both the α-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) (up to 99 ± 6.8% inhibition) and N-methyl-D-aspartic acid receptor (NMDAR)-mediate EPSC (up to 73 ± 9.7% inhibition) in the NAc in brain slices. The effect of cocaine on AMPA and NMDA receptor-mediate excitatory postsynaptic current (EPSC) were mimicked by the D1 -like receptor agonist SKF 38393 and blocked by the D1 -like receptor antagonist SCH 23390, whereas D2 -like receptor agonist or antagonist failed to mimic or to block the action of cocaine. Preperfusion of SB216763 for 5 min also ameliorated the inhibitory effect of SKF38393 on both AMPA and NMDA receptor-mediated components of EPSC, indicate the effect of SB216763 on cocaine was via the D1 -like receptor. Moreover, cocaine inhibited the presynaptic release of glutamate in the NAc, and SB216763 reversed this effect. In conclusion, D1 receptor-GSK3ß pathway, which mediates glutamatergic transmission in the NAc core through a presynaptic mechanism, plays an important role in acute cocaine-induced hyperlocomotion.

Dihydromyricetin protects neurons in an MPTP-induced model of Parkinson's disease by suppressing glycogen synthase kinase-3 beta activity.

AIM: It is general believed that mitochondrial dysfunction and oxidative stress play critical roles in the pathology of Parkinson's disease (PD). Dihydromyricetin (DHM), a natural flavonoid extracted from Ampelopsis grossedentata, has recently been found to elicit potent anti-oxidative effects. In the present study, we explored the role of DHM in protecting dopaminergic neurons. METHODS: Male C57BL/6 mice were intraperitoneally injected with 1-methyl4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) for 7 d to induce PD. Additionally, mice were treated with either 5 or 10 mg/kg DHM for a total of 13 d (3 d before the start of MPTP, during MPTP administration (7 d) and 3 d after the end of MPTP). For the saline or DHM alone treatment groups, mice were injected with saline or DHM for 13 d. On d 14, behavioral tests (locomotor activity, the rotarod test and the pole test) were administered. After the behavioral tests, the mice were sacrificed, and brain tissue was collected for immunofluorescence staining and Western blotting. In addition, MES23.5 cells were treated with MPP+ and DHM, and evaluated using cell viability assays, reactive oxygen species (ROS) measurements, apoptosis analysis and Western blotting. RESULTS: DHM significantly attenuated MPTP-induced mouse behavioral impairments and dopaminergic neuron loss. In the MES23.5 cells, DHM attenuated MPP+-induced cell injury and ROS production in a dose-dependent manner. In addition, DHM increased glycogen synthase kinase-3 beta phosphorylation in a dose- and time-dependent manner, which may be associated with DHM-induced dopaminergic neuronal protection. CONCLUSION: The present study demonstrated that DHM is a potent neuroprotective agent for DA neurons by modulating the Akt/GSK-3ß pathway, which suggests that DHM may be a promising therapeutic candidate for PD.

Inhibition of phosphodiesterase10A attenuates morphine-induced conditioned place preference.

BACKGROUND: Phosphodiesterase (PDE) 10A is selectively expressed in medium spiny neurons of the striatum. Nucleus accumbens (NAc) is a key region that mediates drug reward and addiction-related behaviors. To investigate the potential role of PDE10A in the reinforcement properties of morphine, we tested the effect of MP-10, a selective inhibitor of PDE10A, on acquisition, expression, and extinction of morphine-induced conditioned place preference (CPP). RESULTS: The results show that 2.5 mg/kg MP-10, administered subcutaneously, significantly inhibited the acquisition of morphine-induced CPP. The same dose of MP-10 alone did not result in the CPP. Moreover, MP-10 did not alter the expression of morphine-induced CPP, but did accelerate the extinction of morphine-induced CPP. Additionally, chronic treatment with 2.5 mg/kg MP-10 decreased expression of phosphorylated CREB (pCREB), activated cAMP response element binding protein, in dorsomedial striatum, in shell of NAc, and in anterior cingulate cortex (ACC) as well as decreased expression of ΔFosB in the shell of NAc and ACC. CONCLUSION: The results suggest that inhibition of PDE10A may have therapeutic potential in the treatment of opioid addiction.

Pleural fluid prealbumin and C-reactive protein in the differential diagnosis of infectious and malignant pleural effusions.

Clinical history and physical examination are helpful in indicating the potential causes of pleural effusions (PEs). However, the accurate diagnosis and establishment of the causes of PE is an ongoing challenge in daily clinical practice. The primary aim of this study was to distinguish between infectious PE and malignant PE (MPE) by measuring two major acute phase response biomarkers: prealbumin (PA) and C-reactive protein (CRP). The study was a prospective trial involving 151 patients who were diagnosed with infectious PE or MPE. Patients with infectious PE were divided into two subgroups: tuberculous PE (TBPE) and parapneumonic PE (PNPE). A further 58 patients with PEs that showed no evidence of MPE, TBPE or PNPE were classified as the chronic non-specific PE (NSPE) group. Demographic characteristics and pleural fluids of the subjects were collected consecutively. The discriminative properties of pleural fluid routine biochemistries, and PA and CRP were evaluated. PA, CRP and classical fluid parameters were also applied to classify patients with infectious PE and MPE. Receiver operating characteristics (ROC) analysis established the cutoffs of PA and CRP for discriminating between groups. Pleural fluid PA levels were significantly higher in the MPE group (n=47) than in the infectious PE group (n=104). Pleural fluid CRP levels were significantly higher in the infectious PE group than in the MPE group. Pleural fluid PA levels were identified to be moderately negatively correlated with CRP levels in the MPE group, with a statistically significant correlation coefficient of -0.352. The ROC curve showed that the sensitivity and specificity of PA for the diagnosis of MPE were 0.851 and 0.548, respectively, at the cutoff of 28.3 mg/l. The area under the curve (AUC) was 0.784 (95% CI, 0.707-0.861). Using CRP as a diagnostic parameter resulted in an comparable AUC of 0.810 (95% CI, 0.736-0.885), at the cutoff of 35.2 mg/l. Combinations of PA and CRP resulted in incrementally discriminating values for MPE, with a sensitivity of 0.617 and a specificity of 0.903. The measurement of PA and CRP levels in pleural fluid may be a useful adjunctive test in PE, as a potential differentiator between infectious PE and MPE.

Serum angiopoietin-2 and ß-hCG as predictors of prolonged uterine bleeding after medical abortion in the first trimester.

OBJECTIVE: The combination of mifepristone and misoprostol is an established method for induction of early first trimester abortion, but there is no consensus about the best evaluation of treatment outcome. We evaluate serum Angiopoietin-2 (Ang-2) and ß human chorionic gonadotropin (ß-hCG) in women who had undergone a medical abortion as markers of prolonged uterine bleeding (PUB). METHODS: Prospective trial involving 2843 women attending an gynecology outpatient clinic who following a medical abortion with mifepristone and misoprostol, the study cohort was divided into women with duration of uterine bleeding >14 days (PUB) and women with duration of uterine bleeding ≤14 days (normal uterine bleeding, NUB). Serum determinations of Ang-2 levels by ELISA and ß-hCG levels by electrochemiluminiscence immunoassay. Receiver Operating Characteristics (ROC) analyses were calculated and plotted for the diagnostic accuracy of serum ß-hCG and Ang-2 concentration to discriminate PUB and NUB. RESULTS: Baseline characteristics for both groups were similar, Only duration of bleeding showed a significant difference between the PUB group and NUB group. Ang-2 serum levels moderately correlated with serum ß-hCG levels with statistically significant correlation coefficients of 0.536. Serum ß-hCG and Ang-2 levels on day 7 and on day 14 after medical abortion were signifcantly higher in PUB group than in NUB group. Plotted as ROC curves, ß-hCG area under curve (AUC) was 0.65 (95% CI, 0.53-0.76) on day 7, rising to AUC = 0.83 (95% CI, 0.75-0.92) on day 14. Using Ang-2 on day 7 and day 14 as predictive parameter resulted in an analogous AUC (AUC = 0.61 on day 7, AUC = 0.78 on day 14). CONCLUSIONS: Both parameters are clinically useful as a diagnostic test in predicting PUB after medical abortion, and can be helpful in uncertain clinical situations, but should be considered as supplementary to a general clinical evaluation.