Identification of faecal extracellular vesicles as novel biomarkers for the non-invasive diagnosis and prognosis of colorectal cancer.

Colorectal cancer (CRC) is one of the most common malignancies that is usually detected late in the clinic. The currently available diagnostic tools for CRC are either invasive or insensitive to early lesions due to the dearth of reliable biomarkers. In this study, we discovered that the extracellular vesicles (EVs) in the faeces of CRC patients can act as a potent biomarker for the non-invasive diagnosis and prognosis of CRC. This finding is based on the identification of two transmembrane proteins-CD147 and A33-on faeces-derived EVs (fEVs) that are intrinsically associated with CRC. The detection results show that the levels of CD147 and A33 on fEVs were upregulated in the CRC patients (n = 48), dramatically distinguishing them from the healthy donors (n = 16). The CD147/A33-enriched EVs offer a clinical sensitivity of 89%, much higher than that (40%) of carcinoembryonic antigen (CEA), a clinically-established serum biomarker for CRC diagnosis. In addition, the analysis of longitudinal faeces samples (n = 29) demonstrated that the CD147/A33-enriched fEVs can be utilized to track the prognosis of CRC. Due to the high compliance of faeces-based detection, the CD147/A33-enriched fEVs could serve as new-generation CRC biomarkers for large-scale, non-invasive CRC screening as well as real-time monitoring of patient outcomes during clinical interventions.

HM-3-HSA exhibits potent anti-angiogenesis and antitumor activity in hepatocellular carcinoma.

HM-3-HSA is an antitumor fusion protein which improved the pharmacokinetics of HM-3. Previous studies reported that HM-3-HSA enhanced antitumor activity of HM-3 in melanoma cells. However, the efficacy and the mechanism of HM-3-HSA in hepatocellular carcinoma, especially its effect on tumor angiogenesis, have not been elucidated. Herein, we showed that HM-3-HSA significantly inhibited the H22 and SMMC-7721 tumor xenografts growth and tumor angiogenesis in vivo, indicating the antitumor activity exerted by HM-3-HSA was closely corrected with its potency on tumor angiogenesis. To investigate the anti-angiogenic mechanism, we evaluated the efficacy of HM-3-HSA in HUVECs in vitro. The results showed that multiple steps of tumor angiogenesis, including endothelial cell proliferation, migration, invasion and tube formation, were substantially inhibited by HM-3-HSA. Mechanism investigations revealed that HM-3-HSA could bind HUVECs via integrin αvß3 and α5ß1 and inhibited phosphorylation of the downstream protein kinases including FAK, Src and PI3 K. Our study was the first to report the activity of HM-3-HSA against hepatocellular carcinoma and tumor angiogenesis as well as the underlying mechanism by which HM-3-HSA to exert its anti-angiogenic activity.

A novel realgar-indigo naturalis formula more effectively induces apoptosis in NB4 cells.

Realgar as a kind of arsenic agent is currently used to treat APL in China. The effectiveness and low toxicity of realgar have been verified, lower than arsenic trioxide. Although the therapeutic efficacy of realgar is blocked severely by its poor insolubility in water. In our lab, we addressed this problem by obtaining realgar bioleaching solution (RBS) from microbiological leaching technique. To develop a tradition Chinese medicinal formula (TCMF) for clinical application realgar is usually used with other herbs. However, treated realgar with RBS has not been evaluated in TCMF contain realgar. In the present study we used NB4 to investigate the effects of novel Realgar-Indigo naturalis formula (FRBS) on cell proliferation and apoptosis. We used MTT assay to measure anti proliferative activity of FRBS. We further study the effects of FRBS on cell growth and apoptosis according flow cytometry, DNA fragmentation assay and Fluorescence microscopy and Western blot. The results revealed that FRBS significantly inhibited growth in a dose-dependent manner, and induced apoptosis in NB4 cells. NB4 cell inhibitory response to FRBS at 2µg ml-1 of arsenic concentration was twofold higher, dissimilar to RIF, and induced apoptosis more effectively. Further, a higher expression of caspase-3, caspase-9 and cytochrome C from increased from FRBS. RBS can substitute the traditional realgar powder in RIF in order to provide a novel and promising Realgar-Indigo naturalis formula to treat acute promyelocytic leukemia.

Realgar transforming solution as a novel arsenic agent with a lower risk of cardiotoxicity.

QTc prolongation has been observed during arsenic trioxide and realgar's clinical use, and become a huge obstacle for the application. Our lab has obtained the soluble arsenic from realgar named realgar transforming solution or RTS. In this study, we first evaluated the cytotoxicity on NB4 cell and found that RTS could remarkably inhibit proliferation of NB4 than arsenic trioxide. Then we figured out the QTc prolongation of RTS treatment contrasted with arsenic trioxide; results revealed that arsenic trioxide prolonged corrected QTc of mice by 20.1% and showed 1.9-fold higher cytotoxicity on H9c2 cell than RTS. On the contrary, there could not find any QTc prolongation of mice in RTS treatment. Also, arsenic trioxide elevated the intercellular calcium accumulation of H9c2 cell by 2.02-fold v.s control and RTS. HE staining and Masson's trichrome staining had shown that there was no injured section after RTS treatments. IK1 currents of rat ventricular cardiomyocytes were diminished by 45.0% after treating with arsenic trioxide while RTS showed no significance than the control group. The results above indicated that RTS could serve as an alternative arsenic agent on leukemia and had a lower risk of cardiotoxicity.

Realgar transforming solution-induced differentiation of NB4 cell by the degradation of PML/RARα partially through the ubiquitin-proteasome pathway.

PML/retinoic acid receptor alpha (RARα), as a hallmark of acute promyeloid leukemia (APL), is directly related to the outcome of clinical APL remedy. It is reported that arsenicals can effectively degrade PML/RARα, such as arsenic trioxide and realgar. However, the high toxicity or insolubility have hampered their clinical applications. Realgar transforming solution (RTS) was produced from realgar by bioleaching process in our lab. Previous studies demonstrated that RTS had a significant anti-cancer ability on chronic myeloid leukemia through oncoprotein degradation. The capacity of RTS on treating APL is what is focused on in this study. The results showed that RTS had a noticeable sensitivity in NB4 cell, and RTS remarkably down-regulated PML/RARα expression and induced cell differentiation. Further, RTS could accumulate PML/RARα into the nuclear bodies and then execute degradation, which could be reversed by proteasome inhibitor MG132. The results also exhibited that the reduction of RTS-induced PML/RARα expression accompanied by the elevation of ubiquitin and SUMO-1 protein expression. Finally, PML and SUMO-1 had been demonstrated to be co-localized after RTS treatment by immunofluorescence co-localization assay and immunoprecipitation assay. In conclusion, these results suggested that RTS-induced cell differentiation may attribute to the PML/RARα degradation partially through the ubiquitin-proteasome pathway.

Arsenic trioxide combined with transarterial chemoembolization for unresectable primary hepatic carcinoma: A systematic review and meta-analysis.

BACKGROUND: Primary hepatic carcinoma (PHC) is the third commonest leading to cancer death around the world, and transarterial chemoembolization (TACE) has been proposed as the first-line therapeutic treatment for patients with unresectable PHC. This study aims to determine whether the combination of As2O3 and TACE is superior to alone TACE for achieving more clinical therapeutic efficacy, survival time, life quality and safety in patients with unresectable PHC. METHODS: A comprehensive literature search was conducted on the clinical controlled trials comparing therapeutic effects of As2O3 & TACE versus alone TACE for unresectable PHC through English databases (including PubMed, Embase, and the Cochrane Library) and Chinese databases (including China Knowledge Resource Integrated Database, Wanfang Database, Weipu Database, and Chinese Biomedical Database). The last search was in 30 August 2017. A recursive search was performed with bibliographies of relevant studies. There were no language restrictions. Primary outcomes, defined a priori, were therapeutic responses (clinical effective rate and clinical benefit rate), survival time, life quality, and adverse events of As2O3 & TACE compared with alone TACE expressed as relative risk (RR) with 95% confidence intervals (CI). RESULTS: 25 clinical controlled trials involving 1886 participants were included. We found that there were significant superiority associated with As2O3 & TACE compared with alone TACE in clinical benefit rate (RR: 1.24, 95% CI: 1.12-1.37), clinical effective rate (RR: 1.35, 95% CI: 1.17-1.55), 2-year survival rate (RR: 1.45, 95% CI: 1.20-1.75), and improving of KPS (RR: 1.31, 95% CI: 1.14-1.50). These associations were also observed in subgroups by intervened methods of As2O3 and pulmonary metastasis. Notably, the pooled relative risk of retention of sodium and water was obviously raised in patients with As2O3 & TACE therapy (RR: 16.616, 95% CI: 8.01 - 34.486). CONCLUSION: The superiority of adjuvant As2O3 therapy combined with TACE in PHC individuals will outweigh alone TACE therapy, especially in PHC populations with pulmonary metastasis.

Realgar transforming solution suppresses angiogenesis and tumor growth by inhibiting VEGF receptor 2 signaling in vein endothelial cells.

Realgar (As4S4), as an arsenic sulfide mineral drug, has a good therapeutic reputation for anticancer in Traditional Chinese Medicine, and has recently been reported to inhibit angiogenesis in tumor growth. However, considering the poor solubility and low bioavailability of realgar, large dose of realgar and long period of treatment are necessary for achieving the effective blood medicine concentration. In present study, we resolved the crucial problem of poor solubility of realgar by using intrinsic biotransformation in microorganism, and investigated underlying mechanisms of realgar transforming solution (RTS) for antiangiogenesis. Our results demonstrated that RTS had a strong activity to inhibit HUVECs proliferation, migration, invasion, and tube formation. Moreover, RTS inhibited VEGF/bFGF-induced phosphorylation of VEGFR2 and the downstream protein kinases including ERK, FAK, and Src. In vivo zebrafish and chicken chorioallantoic membrane model experiments showed that RTS remarkably blocked angiogenesis. Finally, compared with the control, administration of 2.50 mg/kg RTS reached more than 50% inhibition against H22 tumor allografts in KM mice, but caused few toxic effects in the host. The antiangiogenic effect was indicated by CD31 immunohistochemical staining and alginate-encapsulated tumor cell assay. In summary, our findings suggest that RTS inhibits angiogenesis and may be a potential drug candidate in anticancer therapy.

Autophagy enhanced antitumor effect in K562 and K562/ADM cells using realgar transforming solution.

Realgar transforming solution (RTS) can be produced from a biotransformation process by using microorganisms cultured with realgar in our lab. RTS has been demonstrated as a novel arsenic anti-leukemia agent in K562 and K562/ADM. However, its underlying mechanism is unclear. In this study, we showed that RTS could strongly induce apoptosis in K562 and K562/ADM cells. After the cells were treated by RTS, apoptotic population were increased compared to control and clearly distinguishable by DAPI nuclei staining. With increasing the dose of RTS, more cells arrested in S phase and G2/M phase. Secondly, we also showed that RTS could induce autophagy via up-regulation of LC3, p62/SQSTM1 and inhibition of mTOR in a much lower arsenic dosage in contrast to ATO and realgar. In addition, autophagy induced by RTS partially due to the degradation of fusion oncoprotein Bcr-Abl, which is associated with multidrug resistant in (MDR)-CML. Our results also showed that the apoptotic rate decreased when autophagic flux was attenuated by CQ via inhibiting cleaved-caspase-3 and alleviating Bcl-2 level. These suggested that RTS triggered autophagy is a pro-death process in CML and MDR-CML cells. In conclusion, our findings demonstrated that RTS could serve as a promising arsenic candidate for anti-CML/MDR-CML by inducing apoptosis and autophagy and is more potent than ATO and realgar.

Properties of realgar bioleaching using an extremely acidophilic bacterium and its antitumor mechanism as an anticancer agent.

Realgar is a naturally occurring arsenic sulfide (or Xionghuang, in Chinese). It contains over 90% tetra-arsenic tetra-sulfide (As4S4). Currently, realgar has been confirmed the antitumor activities, both in vitro and in vivo, of realgar extracted using Acidithiobacillus ferrooxidans (A. ferrooxidans). Bioleaching, a new technology to greatly improve the use rate of arsenic extraction from realgar using bacteria, is a novel methodology that addressed a limitation of the traditional method for realgar preparation. The present systematic review reports on the research progress in realgar bioleaching and its antitumor mechanism as an anticancer agent. A total of 93 research articles that report on the biological activity of extracts from realgar using bacteria and its preparation were presented in this review. The realgar bioleaching solution (RBS) works by inducing apoptosis when it is used to treat tumor cells in vitro and in vivo. When it is used to treat animal model organisms in vivo, such as mice and Caenorhabditis elegans, tumor tissues grew more slowly, with mass necrosis. Meanwhile, the agent also showed obvious inhibition of tumor cell growth. Bioleaching technology greatly improves the utilization of realgar and is a novel methodology to improve the traditional method.

Sporosarcina terrae sp. nov., isolated from orchard soil.

A Gram-stain-positive, motile and rod-shaped bacterium, designated strain LZ2T, was isolated from a sample of orchard soil from Laizhou city, Shandong province, PR China. On the basis of 16S rRNA gene sequence analysis, strain LZ2T was closely related to members of the genus Sporosarcina, sharing highest levels of sequence similarity with Sporosarcina pasteurii NCIMB 8841T (98.8 %), Sporosarcina soli I80T (95.9 %). The value for the DNA-DNA relatedness between strain LZ2T and Sporosarcina pasteurii NCIMB 8841T was 39.8±1.7 %. Growth occurred at 10-44 °C (optimum, 30-35 °C), pH 5.0-11.0 (optimum pH 9.0-10.0); NaCl concentrations of up to 7.0 % (w/v) were tolerated. The dominant respiratory quinone was MK-7 and the G+C content was 39.2 mol%. The major fatty acids were anteiso-C15 : 0 and iso-C15 : 0. The major polar lipids of strain LZ2T were diphosphatidylglycerol, phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol and an unidentified phospholipid. Based on phenotypic and chemotaxonomic characteristics, and phylogenetic data strain LZ2T represents a novel species of the genus Sporosarcina, for which the name Sporosarcina terrae sp. nov. (type strain LZ2T=KACC 18822T=MCCC 1K03174T) is proposed.

Synthesis of hydroxycinnamic acid derivatives as mitochondria-targeted antioxidants and cytotoxic agents.

In order to develop agents with superior chemopreventive and chemotherapeutic properties against hepatocellular carcinomas, mitochondria-targeted hydroxycinnamic acids (MitoHCAs) were synthesized by conjugation with a triphenylphosphonium cation. These synthetic compounds were evaluated for their antioxidant activities in hepatic mitochondria, including against OH∙- and ROO∙- induced lipid peroxidation. H2O2 production was decreased significantly by increasing glutathione peroxidase and catalase activities. In addition, cell proliferation data from three cell lines (HepG2, L02 and WI38) indicated that the MitoHCAs were selective for cancer cells. Interestingly, the MitoHCAs both with or without Ca2+ triggered mitochondrial dysfunction by inducing mitochondrial swelling, collapsing the mitochondrial membrane potential and causing cytochrome c release. In particular, an inhibitor of the mitochondrial permeability transition pore (mPTP), cyclosporin A, attenuated mitochondrial damage and cell apoptosis, indicating that mPTP may be involved in the antiproliferative activity of MitoHCAs. Further studies focused on structural optimization of these compounds are onging.

Properties of realgar bioleaching using an extremely acidophilic bacterium and its antitumor mechanism as an anticancer agent

Realgar is a naturally occurring arsenic sulfide (or Xionghuang, in Chinese). It contains over 90% tetra-arsenic tetrasulfide (As4S4). Currently, realgar has been confirmed the antitumor activities, both in vitro and in vivo, of realgar extracted using Acidithiobacillus ferrooxidans (A. ferrooxidans). Bioleaching, a new technology to greatly improve the use rate of arsenic extraction from realgar using bacteria, is a novel methodology that addressed a limitation of the traditional method for realgar preparation. The present systematic review reports on the research progress in realgar bioleaching and its antitumor mechanism as an anticancer agent. A total of 93 research articles that report on the biological activity of extracts from realgar using bacteria and its preparation were presented in this review. The realgar bioleaching solution (RBS) works by inducing apoptosis when it is used to treat tumor cells in vitro and in vivo. When it is used to treat animal model organisms in vivo, such as mice and Caenorhabditis elegans, tumor tissues grew more slowly, with mass necrosis. Meanwhile, the agent also showed obvious inhibition of tumor cell growth. Bioleaching technology greatly improves the utilization of realgar and is a novel methodology to improve the traditional method.

Paenibacillus ripae sp. nov., isolated from bank side soil.

A Gram-stain-variable, rod-shaped, non-motile and endospore-forming bacterium, designated strain HZ1T, was isolated from a sample of bank side soil from Hangzhou city, Zhejiang province, PR China. On the basis of 16S rRNA gene sequence analysis, strain HZ1T was closely related to members of the genus Paenibacillus, sharing the highest levels of sequence similarity with Paenibacillus agarexedens DSM 1327T (94.4 %), Paenibacillus sputi KIT00200-70066-1T (94.4 %). Growth occurred at 15-42 °C (optimum 30-37 °C), pH 5.0-9.5 (optimum pH 7.0-8.0) and NaCl concentrations of up to 6.0 % (w/v) were tolerated (optimum 0.5 %). The dominant respiratory quinone was MK-7 and the DNA G+C content was 40.1 mol%. The major fatty acids were anteiso-C15 : 0 and iso-C16 : 0. The major polar lipids of strain HZ1T were diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine and several unknown lipids. The diagnostic diamino acid found in the cell-wall peptidoglycan was meso-diaminopimelic acid. Based on its phenotypic and chemotaxonomic characteristics and phylogenetic data, strain HZ1T represents a novel species of the genus Paenibacillus, for which the name Paenibacillus ripae sp. nov. (type strain HZ1T = CCTCC AB 2014276T = LMG 28639T) is proposed.

Effects of pyrite bioleaching solution of Acidithiobacillus ferrooxidans on viability, differentiation and mineralization potentials of rat osteoblasts.

Iron pyrite, an important component of traditional Chinese medicine, has a poor solubility, bioavailability, and patient compliance due to a high dose required and associated side effects, all of which have limited its clinical applications and experimental studies on its action mechanisms in improving fracture healing. This study investigated Acidithiobacillus ferrooxidans (A.f)-bioleaching of two kinds of pyrites and examined bioactivities of the derived solutions in viability and osteogenic differentiation in rat calvarial osteoblasts. A.f bioleaching improved element contents (Fe, Mn, Zn, Cu, and Se) in the derived solutions and the solutions concentration-dependently affected osteoblast viability and differentiation. While the solutions had no effects at low concentrations and inhibited the osteoblast alkaline phosphatase (ALP) activity at high concentrations, they improved ALP activity at their optimal concentrations. The improved osteoblast differentiation and osteogenic function at optimal concentrations were also revealed by levels of ALP cytochemical staining, calcium deposition, numbers and areas of mineralized nodules formed, mRNA and protein expression levels of osteogenesis-related genes (osteocalcin, Bmp-2, Runx-2, and IGF-1), and Runx-2 nuclear translocation. Data from this study will be useful in offering new strategies for improving pyrite bioavailability and providing a mechanistic explanation for the beneficial effects of pyrite in improving bone healing.

[Chemical Constituents from Young Fruits of Citrus maxima cv. Shatian].

OBJECTIVE: To investigate the chemical constituents from the young fruits of Citrus maxima cv. Shatian. METHODS: The chemical constituents were isolated and purified by silica gel column chromatography and recrystallization, and their structures were identified on the basis of physicochemical properties and spectral analysis. RESULTS: Seven compounds were isolated and identified as naringenin (I), marmin (II), naringin (III), ß-sitosterol (IV) 5,7-dihydroxylcoumarin (V) 1, 3,5-trihydroxyhenzene (VI) and xanthotoxol (VII). CONCLUSION: Except compound III, all compounds are isolated from the young fruits of Citrus maxima cv. Shatian for the first time.

Anti-tumor effects and apoptosis induction by Realgar bioleaching solution in Sarcoma-180 cells in vitro and transplanted tumors in mice in vivo.

BACKGROUND: Realgar which contains arsenic components has been used in traditional Chinese medicine (TCM) as an anticancer drug. However, neither Realgar nor its formula are soluble in water. As a result, high dose of Realgar has to be administered to achieve an effective blood medicine concentration, and this is associated with adverse side effects. The objective of the present study was to increase the solubility of a formula using hydrometallurgy technology as well as investigating its effects on in vitro and in vivo cell proliferation and apoptosis in Sarcoma-180 cell line. MATERIALS AND METHODS: Antiproliferative activity of Realgar Bioleaching Solution (RBS) was evaluated by MTT assay. Further, effects of RBS on cell proliferation and apoptosis were studied using flow cytometry and transmission electron microscopy. Kunming mice were administered RBS in vivo, where arsenic specifically targeted solid tumors. RESULTS: The results indicated that RBS extract potently inhibited the tumor growth of Sarcoma-180 cell line in a dose-dependent manner. Flow cytometry and transmission electron microscopy further indicated that RBS significantly induced cell apoptosis through the inhibition of cell cycle pathway in a dose-dependent manner. Further, on RBS administration to mice, arsenic was specifically targeted to solid tumors CONCLUSIONS: RBS could substitute for traditional Realgar or its formula to work as a potent tool in cancer treatment.

[Study on the chemical constituents of Buddleja davidii].

OBJECTIVE: To study the chemical constituents of Buddleja davidii. METHODS: The constituents were isolated and purified by silica gel column chromatography, polyamide column chromatography and macroporous adsorption resin and their structures were identified by spectroscopic analysis. RESULTS: Eight compounds were elucidated as : Cranioside A (1), Eutigoside A (2), 1-O-4-Dimethoxyphenylethyl-4-O-3,4-dimethoxyphenylethy-beta-D-glucopyranoside (3), Isomartynoside (4'), 4"-O-Acetylmartynoside (5), Stigmasterol glueoside (6), beta-Sitosterol (7), Daucosterol (8). CONCLUSION: All these compounds are obtained from this plant for the first time.

Biological extraction of realgar by Acidithiobacillus ferrooxidans and its in vitro and in vivo antitumor activities.

Acidithiobacillus ferrooxidans is a Gram-negative, chemolithoautotrophic bacterium involved in metal bioleaching. It is used for the extraction of coarse medical realgar, which is converted into an aqueous solution. To prove its feasibility as an anticancer drug candidate, extracted realgar (ER/Af) was evaluated for its antitumor activities both in vitro and in vivo. In cytotoxicity tests, ER/Af displayed significant inhibition on cell proliferation of HepG2, SMMC7721, and H22 cells in a time and dose dependent manner. Remarkable tumor growth inhibition and survival time prolongation effects, along with no obvious toxicity, were observed in antitumor experiments against H22 cell-bearing mice. Apoptosis induction was also confirmed as one of the mechanisms involved in the efficacy of ER/Af both in vitro and in vivo. The most important observation is that ER/Af showed high selective affinity to tumor tissues with about eight-fold higher arsenic accumulations at the tumor site of mice than those of the arsenic trioxide (ATO)-treated group at the same dose (57.8 +/- 3.34 microg/g dry tissue vs. 7.6 +/- 0.88 microg/g dry tissue). In conclusion, A. ferrooxidans could be successfully used for the extraction of realgar and ER/Af was proved to be a promising anticancer drug candidate, which is valuable for further study and clinical trials.

[The inhibitory effects of Realgar bioleaching solution on S180 ascites-tumor and its toxicity].

OBJECTIVE: To investigate the inhibitory effect of Realgar bioleaching solution (RBS) on tumor S180 cells line and estimate its toxicity, to provide experimental evidence for the further exploit of Realgar. METHODS: 24 hours after the models of ascites-tumor bearing mice were established, the mice were injected RBS once a day. The survival rates of S180 ascites-tumor bearing mice injected in RBS was studied, and the RBS acute toxicity of mice produced by oral, intraperitoneal or intravenous was evaluated by Drug Median Lethal Dose (LD50). RESULTS: The inhibitory effect of S180 cells in vivo had a dose-dependent manner. The survival rates of mice were 10% - 60% in the different dose in 15 days. The LD50 values of RBS by oral, intraperitoneal or intravenous were 5.27 mg/kg, 3.63 mg/kg and 2.68 mg/kg, respectively. CONCLUSION: RBS has potent antitumor effect, LD50 value of RBS is lower than that of traditional medicine.

Peripheral inflammation increased the synaptic expression of NMDA receptors in spinal dorsal horn.

Considerable evidence has indicated that the aberrant, sustained enhancement of spinal NMDA receptors (NMDARs) function is closely associated with behavioral sensitization during inflammatory pain. However, the molecular mechanisms underlying inflammation-induced NMDARs hyperfunction remain poorly understood. The present study performed immunoblotting analysis to evaluate the possible changes in the protein expression of spinal NMDARs after injection of complete Freund's adjuvant (CFA) in mice. We found that CFA did not affect the total protein level of NMDARs subunit NR1 in spinal dorsal horn. However, NR1 immunoreactivity at synapses significantly increased after CFA injection, which was correlated in the time course with the development of mechanical allodynia. Inhibition of spinal NMDARs with D-APV completely eliminated the CFA-induced increase in NR1 immunoreactive density at synapses, and direct application of NMDA onto the spinal cord of naïve mice mimicked the effects of CFA, suggesting the importance of NMDARs activity in regulating the synaptic content of NR1 during inflammatory pain. Moreover, cAMP-dependent protein kinase (PKA) downstream to NMDARs was also required for NR1 synaptic expression because inhibition of PKA activity abolished the enhancement of synaptic NR1 immunoreactivity evoked by either CFA or NMDA. Thus, our data suggested that NMDARs- and PKA-dependent increase in NR1 synaptic expression represented an important mechanism for the hyperfunction of spinal NMDARs following peripheral inflammation.