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Research question Whether extracellular vesicle-derived lncRNAs could serve as promising circulating biomarkers for endometriosis? Design To obtain novel diagnostic markers, we enrolled 85 patients with endometriosis as the endometriosis group and 86 unaffected participants as the control group. RNA sequencing was performed to identify exosomal long non-coding RNAs (lncRNAs) that are differentially expressed between women with endometriosis (n=5) and unaffected participants (n=6). Messenger RNA and lncRNA sequences of the plasma exosomes were analyzed using Gene Ontology and Kyoto Encyclopedia of Genes and Genomes analyses. LncRNA expression levels were further validated using quantitative reverse transcriptase polymerase chain reaction. Moreover, receiver operating characteristic curve analysis was performed to determine the diagnostic value of candidate lncRNAs. Clinical features were correlated to the expression levels of candidate lncRNAs. Results We found that 210 lncRNAs were significantly dysregulated; among these, expression of LINC01569, RP3-399L15.2, FAM138B, and CH507-513H4.6 was significantly decreased, whereas expression of RP11-326N17.2, KLHL7-AS1, and MIR548XHG was increased, in the plasma of patients with endometriosis. Combined expression level of RP3-399L15.2 and CH507-513H4.6 was used to distinguish patients with endometriosis from control participants; the results revealed a sensitivity of 80.00% and specificity of 85.45% at the cutoff point, and an area under the ROC curve of 0.9045. Our findings demonstrated the potential of these two lncRNAs as diagnostic biomarkers for endometriosis. Moreover, CH507-513H4.6 alone may be useful to detect early-stage endometriosis lesions. Conclusions The combination of RP3-399L15.2 and CH507-513H4.6 can be potential candidates for endometriosis biomarkers.

Diffuse large B cell lymphoma (DLBCL) exhibits a tightly complexity immune landscape. In this study, we intended to identify different immune phenotype and to examine the immune related mRNA signature for clinical characteristic, therapeutic responsiveness as well as risk stratification and survival prediction in DLBCL. We separated 738 DLCBL patients into two robust subtypes with distinct immune features based on 28 immune cell types. GO analysis uncovered the concordant classification of two robust significant subtypes of DLBCL. Immunity-L group, associated with poorer outcomes, was characterized by significantly lower immune cell infiltration. Immunity-H group, associated with better outcomes, was characterized by significantly higher immune cell infiltration. Further, a prognostic gene signature related immune infiltration was established by a least absolute shrinkage and selection operator (LASSO)-Cox regression model. The comprehensive results showed that the high-risk group was correlated with lower immune infiltration, more aggressive phenotypes, lower overall survival and more sensitive to lenalidomide. In contrast, a low-risk group score was associated with higher immune infiltration, less aggressive phenotypes, better overall survival and more likely to benefit from PD-1/PD-L1 inhibitors. Finally, a nomogram comprised of the immune risk score and IPI score was verified to more accurately predict the overall survival of DLBCL than traditional clinical prediction models. Altogether, our data demonstrate the heterogeneity of immune patterns within DLBCL and deepen our molecular understanding of this tumor entity.

Diabetes mellitus (DM) is considered the primary cause of neuropathic pain. Osthole (7-methoxy-8[3-methylpent 2-enyl]coumarin) is a component extracted from Cnidium monnieri (L.) cusson plant seeds and has anti-inflammatory and anti-oxidative properties. The aim of the present study was to investigate the effects of osthole on diabetic neuropathic pain (DNP) involving the P2X4 receptor on satellite glial cells (SGCs) in the dorsal root ganglia (DRG) of type 2 diabetic rats. These data showed that the mechanical withdrawal threshold (MWT) and thermal withdrawal latency (TWL) in DM rats were lower than those in control rats. MWT and TWL in DM rats treated with osthole were higher compared with those in untreated DM rats. The expression levels of P2X4 mRNA and protein in the DRG of DM rats were higher compared with those in the control rats, while those in DM rats treated with osthole were significantly lower compared with those in the untreated DM rats. Osthole treatment decreased the co-expression levels of P2X4 and glial fibrillary acidic protein (GFAP) and reduced the up-regulated expression of interleukin-1 beta (IL-1β), tumour necrosis factor-α (TNF-α), brain-derived neurotrophic factor (BDNF) and phosphorylated-p38MAPK and enhanced the down-regulation of IL-10 in DM rats. Thus, osthole treatment may act on the P2X4 receptor to alleviate the mechanical and thermal hyperalgesia in DM rats.

Brain-derived neurotrophic factor (BDNF) is a biomarker of depression. Recent studies have found adenosine deaminase acting on RNA1 (ADAR1) is a novel target being sensitive to stress at epigenetic level. The epigenetic regulation mechanism of stress-related depression is still unclear so far. To explore the potential regulating mechanism of ADAR1 on BDNF, over and low expression of ADAR1 in PC12 and SH-SY5Y cell lines are prepared. In the meanwhile, chronic unpredictable stress (CUS) mice are treated with ADAR1 inducer (interferon-γ, IFN-γ). ADAR1 regulates BDNF expression, which is proven by that over and low expressions of ADAR1 increase and decrease BDNF mRNA and protein respectively in vitro. Additionally, ADAR1 inducer alleviates the depressive-like behavior of CUS mice by recovering the decreased BDNF protein in brain and serum. Moreover, over and low expressions of ADAR1 reduce and enhance microRNA-432 (miR-432) expression respectively in vitro. Furtherly, over and low miR-432 expressions lead to decreased and increased BDNF and ADAR1 mRNA, protein and immunoreactivity respectively in vitro. The above results demonstrate that ADAR1 is involved in antidepressant action by regulating BDNF via miR-432. Those novel findings can provide a new idea for the study of epigenetic regulation mechanism, early diagnosis, and effective treatment of stress-related depression.

There is a need to investigate the role of nuclear factor kappa B in the regulation of cyclooxygenase-2 expression in the epileptic rat brain and cultured hippocampal neurons. Immunofluorescence and polymerase chain reaction was used to detect the expression of nuclear factor kappa B and cyclooxygenase-2. In cultured hippocampal neurons and rat brain: the control group compared with the normal group, nuclear factor kappa B expression in the hippocampal dentate gyrus, cerebral cortex, the piriform cortex brain regions were significantly increased (P < 0.01). This is accompanied by a significant increase in cyclooxygenase-2 protein and mRNA expressions in the hippocampus (P < 0.01). In the experimental group compared to the control group, the nuclear factor-kappa B expression in the hippocampal dentate gyrus, cerebral cortex, piriform cortex, and other brain regions was significantly lower (P < 0.01), with the accompanying decrease in cyclooxygenase-2 protein and mRNA expression (P < 0.01) in the hippocampus. In conclusion, κB-decoy can inhibit nuclear factor kappa B activation in epileptic rat brain and cyclooxygenase-2 overexpression.

Peroxisome proliferator-activated receptor α (PPARα) activation has been reported to exert protective effects on podocytes, whereas angiopoietin-like 3 (ANGPTL3) has been shown to exert significant pathogenic effects on these cells. This study aimed to investigate the link between the protective effects of PPARα activation and the pathogenic effects of ANGPTL3 in podocytes. Both PPARα and ANGPTL3 were expressed in cultured podocytes. PPARα mRNA and protein levels decreased whereas ANGPTL3 mRNA and protein levels increased in a time-dependent manner in podocytes treated with puromycin aminonucleoside (PAN). Gemfibrozil, a pharmacological agonist of PPARα, increased PPARα levels and activity in podocytes. The drug also decreased ANGPTL3 levels by potentially weakening ANGPTL3 promoter activity in both normal and PAN-treated podocytes. Furthermore, gemfibrozil significantly decreased PAN-induced apoptosis and F-actin rearrangement. Primary podocytes from Angptl3-knockout mice were cultured. There was no significant difference between Angptl3-/- podocytes treated with or without gemfibrozil in the lamellipodia numbers after PAN treatment. The results suggested that the protective effects of gemfibrozil on podocytes were not exerted following knockout of the Angptl3 gene. This study identified a novel mechanism of the PPARα agonist gemfibrozil that exerts its protective effects by inhibiting PAN-induced apoptosis and cytoskeleton rearrangements through inhibition of ANGPTL3 expression.

This study aimed to observe changes in the hydrogen sulfide (H2S) system in the blood and liver tissue of rats with hepatic cirrhosis at different stages by studying the effect of H2S on the course of hyperdynamic circulation in rats with hepatic cirrhosis. H2S concentration in the blood from the portal vein and inferior vena cava of hepatic cirrhosis rat model induced with carbon tetrachloride was detected on the 15th, 30th, and 52nd day. The expression of cystathionine β-synthase (CBS) and cystathionine γ-lyase (CSE) protein, and CBS and CSE mRNA in the liver was detected by immunohistochemistry and reverse transcriptase polymerase chain reaction (RT-PCR), respectively. The results indicated that H2S concentration in the blood from the portal vein and inferior vena cava of rats with hepatic cirrhosis was significantly lower than that in the control group. H2S was gradually decreased with the development of the disease and significantly lower in the blood from portal vein than in the blood of inferior vena cava at the mid-stage and the late stage groups. The expression levels of CBS and CSE protein, and CBS and CSE mRNA in the livers with hepatic cirrhosis at different stages were all higher than those in the control group, and the expression gradually increased with the development of the disease. The expression of CBS was lower than CSE in the same stages. The results indicated that the CSE mRNA was expressed predominantly in the cirrhosis groups as compared with CBS mRNA. Among experimental rats, the H2S system has an important effect on the occurrence and development of hyperdynamic circulation in rats with hepatic cirrhosis. This finding adds to the literature by demonstrating that H2S protects vascular remodelling in the liver, and that CSE is indispensable in this process.

HIV-1 envelope glycoprotein (Glycoprotein 120, gp120) can directly stimulate primary sensory afferent neurons and cause chronic neuropathic pain. The P2X3 receptor in the dorsal root ganglia (DRG) is associated with the transmission of neuropathic pain. Curcumin isolated from the herb Curcuma rhizome has anti-inflammatory and anti-tumor effects. The water solubility, targeting and bioavailability of curcumin can be improved by nanoparticle encapsulation. In this study, we sought to explore the effects of nanoparticle-encapsulated curcumin (nano curcumin) on HIV-gp120-induced neuropathic pain mediated by the P2X3 receptor in DRG neurons. The results showed that mechanical and thermal hyperalgesia in rats treated with gp120 were increased compared to those in the control group. The expression levels of P2X3 mRNA and protein in rats treated with gp120 were higher than those in the control group. Nano curcumin treatment decreased mechanical hyperalgesia and thermal hyperalgesia and upregulated the expression levels of P2X3 mRNA and protein in rats treated with gp120. Nano curcumin treatment also reduced the ERK1/2 phosphorylation levels in gp120-treated rat DRG. In addition, P2X3 agonist α,β-methylene ATP (α,β-meATP)-induced currents in DRG neurons cultured with gp120 significantly decreased after co-treatment with nano curcumin. Therefore, nano curcumin treatment may inhibit P2X3 activation, decrease the sensitizing DRG primary afferents and relieve mechanical hyperalgesia and thermal hyperalgesia in gp120-treated rats.

Aflatoxin B1 (AFB1) develops various toxic effects in the liver by impairing mitochondrial function, inducing cell apoptosis. However, little is focused on its toxicity to broiler cardiomyocytes (BCMs). Here, the mitochondrial membrane potential (MMP), reactive oxygen species (ROS) generation, cardiac troponin T (cTnT) location, apoptosis induced by AFB1, and antioxidative genes were investigated in BCMs. It was found that AFB1 evoked intracellular ROS generation, and induced apoptosis in BCMs. AFB1 treatment resulted in increased percentage of apoptotic cells, increased location range of cTnT in cytoplasm, upregulated messenger RNA (mRNA) expression of nuclear factor erythroid 2-related factor 2 (Nrf2) and downregulated mRNA expressions of Mn-superoxide dismutase in BCMs. These findings suggested AFB1 treatment caused significant cardiomyocyte damage and cardiotoxicity, impairment of mitochondrial functions, activated ROS generation, and induced apoptosis, and probably was involved in the Nrf2 signal pathway in BCMs.

Cardiac autonomic neuropathy in Type 2 diabetes (T2D) is often a devastating complication. Long non-coding RNAs (lncRNAs) have important effects on both normal development and disease pathogenesis. In this study, we explored the expression profiles of some lncRNAs involved in inflammation which may be co-expressed with messenger RNA (mRNA) in superior cervical and stellate ganglia after type 2 diabetic injuries. Total RNA isolated from 10 pairs of superior cervical and stellate ganglia in diabetic and normal male rats was hybridized to lncRNA arrays for detections. Pathway analysis indicated that the most significant gene ontology (GO) processes that were upregulated in diabetes were associated with immune response, cell migration, defense response, taxis, and chemotaxis. The Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway revealed that most of the target genes of the lncRNAs were located in cytokine-cytokine receptor interactions, the chemokine signaling pathway and cell adhesion molecules, which were involved in T2D. Gene co-expression network construction showed that the co-expression network in the experimental rats consisted of 268 regulation edges among 105 lncRNAs and 11 mRNAs. Our studies demonstrated the co-expression profile of lncRNAs and mRNAs in diabetic cardiac autonomic ganglia, suggesting possible roles for multiple lncRNAs as potential targets for the development of therapeutic strategies or biomarkers for diabetic cardiac autonomic neuropathy. © 2016 Wiley Periodicals, Inc.

Schistosomula antigens play an important role in the growth and development of Schistosoma japonicum. We investigated the role of S. japonicum adenylate kinase 1 (SjAK1) in the growth and development of schistosomula. Quantitative real-time PCR showed that SjAK1 mRNA was expressed in all schistosomula stages, but increased gradually with the development of S. japonicum schistosomula. Using immunohistochemical techniques, the AK1 protein was found to be mainly distributed in the tegument and in some parenchymal tissues of the schistosomula. Double-stranded RNA-mediated knockdown of AK1 reduced AK1 mRNA transcripts by more than 90%; western blot analysis demonstrated that AK1 protein expression decreased by 66%. Scanning electron microscopy following RNA-mediated AK1 knockdown demonstrated that the sensory papillae degenerated significantly. Transmission electron microscopy demonstrated that the mean thickness of the tegument in the SjAK1 interference group was lower than that in the negative control group. Terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) suggested that, compared with the negative control group, apoptosis increased in the interference group. These results show that AK1 may be involved in the growth and development of S. japonicum schistosomula, and thus may be a target when developing treatments for schistosomiasis.

Background The aim of this study is to explore the molecular mechanism of the LIM protein Ajuba and the transcription factor SP1 in the pathogenesis and progression of PDAC. Ajuba is a newly defined transcriptional co-regulator and plays important role in various cancer development, while SP1 is a classic transcription factor, and is closely related with a variety of gene expression and cancer development including PDAC. Methods The expression of Ajuba and SP1 in PDAC tissues was detected by immunohistochemistry (IHC), and the correlation between expression level and clinical prognosis of Ajuba and SP1 was extensively analyzed using online tools. The interaction between Ajuba and SP1 was examined by co-immunoprecipitation (co-IP) and GST-pulldown assays. Stable cell lines were established via lentiviral infection, and was examined by qRT-PCR and western blot assays. The effects of Ajuba/SP1 on PDAC cell proliferation were examined using CCK8 and colony formation assays. Luciferase reporter and chromatin immunoprecipitation (ChIP) assays were employed to examine the transcription activity. Results The expression level (protein and mRNA) of Ajuba and SP1 was elevated in PDAC tissues and was positively correlated; patients with high Ajuba and SP1 expression had a poor prognosis. Mechanistically, Ajuba binds to the C-terminus of SP1 and functions as a co-activator to enhance SP1 gene expression and promote cell proliferation; the promoter of Ajuba contains functional SP1 responsive elements and Ajuba itself is a target gene of SP1. Conclusion Ajuba functions as a co-activator of SP1 to induce its target gene, and that Ajuba itself is a target genes of SP1. Ajuba/SP1 complex could form a feed forward loop to drive SP1 target gene transcription and promote cell proliferation of pancreatic cancer cells. Ajuba and SP1 might be biomarkers for PDAC diagnostics, prognosis and targets for new therapeutics.

Long noncoding RNAs (lncRNAs) participate in physiological and pathophysiological processes. Type 2 diabetes mellitus (T2DM) accounts for more than 90 % of all cases of diabetes mellitus (DM). Diabetic neuropathic pain (DNP) is a common complication of T2DM. The aim of this study was to investigate the effects of lncRNA NONRATT021972 small interference RNA (siRNA) on DNP mediated by the P2X3 receptor in dorsal root ganglia (DRG). These experiments showed that the expression levels of NONRATT021972 in DRG were increased in the T2DM rat model (intraperitoneal injection of STZ with 30 mg/kg). The concentration of NONRATT021972 in T2DM patient serum was higher compared to control healthy subjects. The mechanical withdrawal threshold (MWT) and thermal withdrawal latency (TWL) in T2DM rats were lower compared to control rats. MWT and TWL in T2DM rats treated with NONRATT021972 siRNA were higher compared with those in T2DM rats. The expression levels of the P2X3 protein and messenger RNA (mRNA) of T2DM rat DRG were higher compared to the control, while those in T2DM rats treated with NONRATT021972 siRNA were significantly lower compared to T2DM rats. The level of tumor necrosis factor-α (TNF-α) in the serum of T2DM rats treated with NONRATT021972 siRNA was significantly decreased compared with T2DM rats. NONRATT021972 siRNA inhibited the phosphorylation and activation of ERK1/2 in T2DM DRG. Thus, NONRATT021972 siRNA treatment may suppress the upregulated expression and activation of the P2X3 receptor and reduce the hyperalgesia potentiated by the pro-inflammatory cytokine TNF-α in T2DM rats.

Adenosine triphosphate (ATP) acts on P2X receptors to initiate signal transmission. P2X7 receptors play a role in the pathophysiological process of myocardial ischemic injury. Long noncoding RNAs (lncRNAs) participate in numerous biological functions independent of protein translation. LncRNAs are implicated in nervous system diseases. This study investigated the effects of NONRATT021972 small interference RNA (siRNA) on the pathophysiologic processes mediated by P2X7 receptors in stellate ganglia (SG) after myocardial ischemic injury. Our results demonstrated that the expression of NONRATT021972 in SG was significantly higher in the myocardial ischemic (MI) group than in the control group. Treatment of MI rats with NONRATT021972 siRNA, the P2X7 antagonist brilliant blue G (BBG), or P2X7 siRNA improved the histology of injured ischemic cardiac tissues and decreased the elevated concentrations of serum myocardial enzymes, creatine kinase (CK), CK isoform MB (CK-MB), lactate dehydrogenase (LDH) and aspartate aminotransferase (AST) compared to the MI rats. NONRATT021972 siRNA, BBG, or P2X7 siRNA treatment in MI rats decreased the expression levels of P2X7 immunoreactivity, P2X7 messenger RNA (mRNA), and P2X7 protein, interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), and phosphorylated p38 mitogen-activated protein kinase (p38 MAPK) in the SG compared to MI rats. NONRATT021972 siRNA treatment prevented the pathophysiologic processes mediated by P2X7 receptors in the SG after myocardial ischemic injury.

Adenosine triphosphate (ATP) plays an important role in signal transmission via acting on P2X receptors. P2X 7 receptor is involved in pathophysiological changes of ischemic diseases. The PC12 cell line is a popular model system to study sympathetic neuronal function. In this study, the effects of P2X 7 on the viability or [Ca 2 + ] i in PC12 cells after exposure to oxygen–glucose deprivation (OGD) were investigated. The results showed that the viability of PC12 cells was decreased under the condition of OGD. BzATP, a P2X 7 agonist, decreased the viability, while P2X 7 antagonist oxATP or P2X 7 siRNA reversed the viability of PC12 cells under the condition of OGD. The expression levels of P2X 7 mRNA and protein in PC12 cells were up-regulated under the condition of OGD or BzATP treatment. The expression levels of P2X 7 mRNA and protein were significantly decreased in OGD PC12 cells, which were pretreated with oxATP or P2X 7 siRNA. It was also found that oxATP or P2X 7 siRNA effectively suppressed the increase of [Ca 2 + ] i induced by OGD. P2X 7 agonist ATP or BzATP enhanced the [Ca 2 + ] i rise induced by OGD in PC12 cells. The [Ca 2 + ] i peak induced by ATP or BzATP in OGD group was decreased by ERK inhibitor U0126. Therefore, P2X 7 antagonists or P2X 7 siRNA could depress the sympathetic neuronal damage induced by ischemia.

Sperm motility is the most important indicator in evaluating roosters’ fecundity. However, the genetic mechanisms underlying chicken sperm motility is not yet clear. Long non-coding RNA (lncRNA) play epigenetic roles in reproduction. In this study, RNA sequencing was employed to profile the testis transcriptome (lncRNA and mRNA) of six Beijing-you cocks divergent in sperm motility. In total, 2,597 lncRNAs were identified in the chicken testis, including 1,267 lincRNAs, 975 anti-sense lncRNAs, and 355 intronic lncRNAs. They shared similar features with previous studies. Of these lncRNAs, 124 were differentially expressed. Among 17,690 mRNAs detected in this study, 544 were differentially expressed, including a bunch of genes with known functions on sperm motility. GO annotation analysis revealed these genes were involved in ATP binding, cilium assembly, and oxidation-reduction processes. Integrating analysis of lncRNA and mRNA profiles predicted 10 lncRNA-gene pairs, including 8 co-regulated and 2 inversely-regulated pairs. To the best of our knowledge, this is the first genome-wide investigation of the lncRNAs in the chicken testis associated with sperm motility. Our results provided a catalog of chicken testis lncRNAs and genes worthy of further studies to understand their roles in cocks’ reproductive performance regulation.

Diabetic peripheral neuropathy (DPN) is the most common complication of diabetes mellitus (DM). More than 90% of all cases of DM belong to type 2 diabetes mellitus (T2DM). Emodin is the main active component of Radix et rhizoma rhei and has anti-bacterial, anti-viral, anti-ulcerogenic, anti-inflammatory, and anti-cancer effects. Nanoparticle encapsulation of drugs is beneficial for drug targeting and bioavailability as well as for lowering drug toxicity side effects. The aim of this study was to investigate the effects of nanoparticle-encapsulated emodin (nano emodin) on diabetic neuropathic pain (DNP) mediated by the Purin 2X3 (P2X3) receptor in the dorsal root ganglia (DRG). Mechanical withdrawal threshold (MWT) and thermal withdrawal latency (TWL) values in T2DM rats were lower than those of control rats. MWT and TWL in T2DM rats treated with nano emodin were higher compared with those in T2DM rats. Expression levels of P2X3 protein and messenger RNA (mRNA) in the DRG of T2DM rats were higher than those of controls, while levels in T2DM rats treated with nano emodin were significantly lower than those of the T2DM rats. Phosphorylation and activation of ERK1/2 in the T2DM DRG were decreased by nano emodin treatment. Nano emodin significantly inhibited currents activated by the P2X3 agonist α,β-meATP in HEK293 cells transfected with the P2X3 receptor. Therefore, nano emodin treatment may relieve DNP by decreasing excitatory transmission mediated by the DRG P2X3 receptor in T2DM rats.

P2X₃ receptors in stellate ganglia (SG) and cervical dorsal root ganglia (DRG) neurons are involved in sympathoexcitatory reflex induced by myocardial ischemic damage. Puerarin, a major active ingredient extracted from the traditional Chinese plant medicine Ge-gen, has been widely used in treatment of myocardial and cerebral ischemia. The present study is aimed to observe the effects of puerarin on the signaling transmission mediated by P2X₃ receptor in SG and DRG after myocardial ischemic damage. Our results showed that systolic blood pressure and heart rate increased, and the expression levels of P2X₃ mRNA and protein in SG and DRG were up-regulated after myocardial ischemic damage. Puerarin reduced systolic blood pressure and heart rate, relieved pain and decreased up-regulated expression of P2X₃ mRNA and protein in SG and DRG after myocardial ischemia. Puerarin inhibited the up-regulated ATP-activated currents in DRG neurons after myocardial ischemia. Thus, puerarin can relieve myocardial ischemic damage through blocking the P2X₃ signaling transmission and then depressed the aggravated sympathoexcitatory reflex.

The effects of puerarin on acute nociception mediated by the P2X3 receptor in rat dorsal root ganglia (DRG) were studied. Intrathecal injection of adenosine triphosphate (ATP) or α,β-methylene-ATP (α,β-meATP) could potentiate acute nociceptive responses induced by intraplantar injection of ATP or α,β-meATP in rat hindpaw. Intraplantar injection of formalin increased the expression of P2X3 in rat DRG and induced acute nociceptive responses. These induced nociceptive responses were inhibited by intrathecal injection of puerarin, which also decreased the up-regulated expression of P2X3 mRNA and protein in DRG induced by intraplantar injection of formalin. Molecular docking studies revealed that puerarin could interact perfectly with homology-modeled rat P2X3 receptor (rP2X3). rP2X3 can be restricted to binding ATP because of its combination with puerarin (increasing the concentration of free ATP), therefore blocking the rP2X3 channel. Whole cell patch clamp recording showed that puerarin inhibited the potentiation of P2X3 receptor-mediated currents induced by lipopolysaccharide. These results demonstrated that puerarin inhibited nociceptive responses induced by ATP, α,β-meATP, or formalin and its inhibitory effect was mediated by reduction of inflammatory pain-induced up-regulation of P2X3 receptor expression and blockade of ATP binding sites on the P2X3 receptor. Our results suggest that puerarin could decrease acute pain mediated by the P2X3 receptor in the DRG. Key words: Puerarin, acute nociceptive response, P2X3 receptor, dorsal root ganglia, whole cell patch clamp.

Background Long non-protein-coding RNAs (lncRNAs) are involved in the pathological processes of nervous system diseases. NONRATT021972 is an lncRNA. This study explores the effects of lncRNA NONRATT021972 small interference RNA (siRNA) on diabetic neuropathic pain (DNP) mediated by the P2X7 receptor in the rat dorsal root ganglia (DRG). Results Our results show that NONRATT021972 expression was significantly higher in the DRG of diabetes mellitus (DM) group compared with control group. NONRATT021972 expression in the DRG was reduced when DM rats were treated with NONRATT021972 siRNA. NONRATT021972 siRNA treatment in type 2 DM rats increased the mechanical withdrawal threshold (MWT), the thermal withdrawal latency (TWL) and the sensory nerve conduction velocity (SNCV) of rat tail nerves. After intravenous injection with NONRATT021972 siRNA in DM rats, the P2X7, GFAP and TNF-ɑ expression levels in DRG were decreased. An interaction between the RNA (NONRATT021972) and protein (P2X7) was predicted by the application of bioinformatics technology. The BzATP-activated currents in DRG non-neurons (satellite glial cells) of DM rats were significantly increased compared to control rats. NONRATT021972 siRNA treatment inhibited the ATP-activated currents in HEK293 cells transfected with pEGFP-P2X7. Conclusions NONRATT021972 siRNA treatment can decrease the expression levels of P2X7 mRNA and protein and inhibit the activation of satellite glial cells (SGCs) in the DRG of type 2 DM rats. Moreover, NONRATT021972 siRNA treatment reduced the release of inflammatory factors (TNF-α), thereby inhibiting the excitability of DRG neurons and reducing mechanical and thermal hyperalgesia in type 2 DM rats.

Fascin actin-bundling protein 1 (FSCN1) plays significant roles in biological processes such as tumor cell invasion and metastasis. However, little is known about prognostic value of FSCN1 in non-small cell lung cancer (NSCLC). FSCN1 mRNA and protein expression were detected by real-time PCR and Western blot in NSCLC tissues and paired adjacent normal lung tissues. Furthermore, the association of FSCN1 protein expression with clinicopathological characteristics including the survival was explored in 156 NSCLC patients. In our results, FSCN1 mRNA and protein expression were obviously higher in NSCLC tissues than in normal lung tissues. High levels of FSCN1 protein were positively correlated with status of differentiated degree, clinical stage, N classification, and M classification in NSCLC. Meanwhile, higher FSCN1 protein expression was significantly associated with poor overall survival in patients with NSCLC. Multivariate analyses showed that increased FSCN1 protein expression was a poor independent prognostic biomarker for NSCLC patients. In conclusions, FSCN1 plays an important role on NSCLC progression and prognosis and may serve as a convictive prognostic biomarker for NSCLC patients.

We investigated the effects of dietary D-penicillamine on copper and zinc contents in the liver, serum, and urine of Long-Evans Cinnamon (LEC) rats, as well as the gene expression of hepatic metallothionein-1 and hepatic metallothionein protein level in these animals. Young male LEC and Fischer rats were fed a basal or D-penicillamine (5 g D-penicillamine/kg diet) diet for 16 days. The urinary output of copper and zinc was markedly increased by the D-penicillamine diet, and the copper and zinc contents in the liver were significantly decreased in LEC rats. Excess copper accumulation in the liver induced hepatic metallothionein in LEC rats, but the D-penicillamine diet depressed the level of hepatic metallothionein-1 mRNA as well as the hepatic metallothionein protein level. When fed the basal diet, serum ornithine carbamoyltransferase activity was higher in LEC rats than in Fischer rats, but a significant decrease in serum ornithine carbamoyltransferase activity rats was observed in LEC rats on feeding the D-penicillamine diet. These results indicated that dietary D-penicillamine significantly reduced excess copper and zinc in the livers of LEC rats through urinary excretion, and the decrease in hepatic copper and zinc contents caused a decrease in the levels of metallothionein-1 mRNA and metallothionein protein in LEC rats.

Vesicoureteral reflux (VUR) is the retrograde passage of urine from the bladder to the urinary tract; this leads to renal scarring and end-stage renal disease in children. Pax2 is a nuclear transcription factor that is involved in urinary system development. We measured the expression of Pax2 in the ureters of 47 patients with VUR. The messenger RNA expression and the protein level of Pax2 were significantly increased in patients with VUR, suggesting a correlation with VUR. Further studies demonstrated that Pax2 was hypomethylated, and Dnmt3a messenger RNA expression was significantly lower than in the control group. We speculate that the low level of Dnmt2a might decrease PAX2 gene methylation and up-regulate the Pax2 protein. The high level of Pax2 might be related to cellular apoptosis and functional lesions in ureters. In conclusion, our results revealed that the level of Pax2 is correlated with VUR; thus, Pax2 represents a possible target for VUR therapy.

To investigate the mRNA and protein expression of FK506-binding protein 52 (FKBP52) in the chorionic villi of patients with recurrent spontaneous abortion (RSA) and normal women during early pregnancy.Fresh chorionic villus tissues were collected from 60 subjects. A total of 30 patients with a history of RSA were enrolled into the RSA group and 30 normal pregnant women were enrolled into the control group. The FKBP52 mRNA expression levels in chorionic villi of the RSA patients and healthy controls were measured via semiquantitative RT-PCR. The protein distribution and expression levels of FKBP52 in chorionic villi were analyzed through immunohistochemistry (IHC). The correlation between FKBP52 expression and RSA was analyzed.We demonstrated that FKBP52 mRNA is expressed in chorionic villi samples of normal pregnancy and RSA. RSA patients exhibited significantly lower FKBP52 gene expression levels compared with those in normal pregnancies (p0.05). FKBP52 immunoreactivity in chorionic villi was mainly observed in trophoblast cell cytoplasm. The FKBP52 protein expression levels in the chorionic villi of RSA patients was significantly lower than in normal women during pregnancy (p0.05).FKBP52 protein levels were decreased in the chorionic villi of RSA patients, which indicate that the decrease in FKBP52 may be associated with RSA. The low FKBP52 mRNA expression level, which is consistent with the IHC result, may affect embryonic development and even lead to abortion. FKBP52 may be involved in the pathogenesis of RSA and new therapies that increase the FKBP52 expression may help treat RSA.

Previous studies have suggested anti-tumor effects of asiatic acid in some human cancer cell lines. This agent is reported to increase the levels of p21 WAF1/CIP1 in human breast cancer cell lines. However, the molecular mechanisms have not been established. Here we report that asiatic acid up-regulates p21 WAF1/CIP1 protein expression but not the level of p21 WAF1/CIP1 mRNA in HepG2 human hepatoma cells. Furthermore, we found that the asiatic acid induced increase of p21 WAF1/CIP1 protein was associated with decreased phosphorylation (ser-146) of p21 WAF1/CIP1 . Knockdown of NDR1/2 kinase, which directly phosphorylates p21 WAF1/CIP1 protein at ser-146 and enhances its proteasomal degradation, increased the levels of p21 WAF1/CIP1 protein and eliminated the regulation of p21 WAF1/ CIP1 stability by asiatic acid. At the same time, the expression of NDR1/2 kinase decreased during treatment with asiatic acid in HepG2 cells. Moreover, asiatic acid inhibited the proliferation of HepG2 cells, this being attenuated by knockdown of p21 WAF1/CIP1 . In conclusion, we propose that asiatic acid inhibits the expression NDR1/2 kinase and promotes the stability of p21 WAF1/CIP1 protein through attenuating NDR1/2 dependent phosphorylation of p21 WAF1/CIP1 in HepG2 cells.

We investigated the expression of the cognate chemokine receptor 1 (CCR1) messenger ribonucleic acid, a G-protein-coupled cognate chemokine receptor with high affinity for RANTES (Regulated upon Activation, Normal T cells Expressed and Secreted), in peripheral blood leukocytes of women with and without endometriosis, and its potential use as a diagnostic test for endometriosis. Because patients with an earlier diagnosis of this disease have a better treatment outcome and a reduced recurrence rate, CCR1 mRNA measurement in the peripheral blood of patients with suspected endometriosis might give us a new perspective in diagnosing and treating this disease earlier and better.

There is a growing evidence that regucalcin (RGN) plays a multifunctional role in liver cancer cells. Previous reports showed that the presence of phorbol 12-myristate 13-acetate (PMA) caused a significant increase in RGN mRNA expression and promoter activity in rat hepatoma cells. In this study, we confirmed that human RGN is also up-regulated by PMA treatment independent of translation, and we identified the mechanism by which PMA up-regulates the expression of human RGN via driving SP1 away from a SP1 motif located within −188/−180 of the promoter in HepG2 cells. Overexpression of SP1 dramatically reduces PMA-induced up-regulation of both internal expression of mRNA and promoter activity, whereas knockdown of SP1 has the opposite effect. Therefore, the present study delineates the fundamental elements in the promoter which will be helpful in the future studies on the regulation of RGN expression in liver cancer.

Adhalin, or α-sarcoglycan, is a 50-kDa glycoprotein that was originally characterized as a muscle membrane protein. The importance of adhalin is suggested by the diseases associated with its absence, notably the limb-girdle muscular dystrophies. However, the function of adhalin is unknown. To analyze the biological roles of adhalin, we cloned the mouse adhalin cDNA, raised peptide-specific antibodies to its cytoplasmic domain, and examined its expression and localization in vivo and in vitro. The mouse adhalin sequence was 80% identical to that of human, rabbit, and hamster. Adhalin was specifically expressed in striated muscle cells and their immediate precursors, and absent in many other cell types. Adhalin expression in embryonic mouse muscle was coincident with primary myogenesis. Its expression was found to be up-regulated at mRNA and protein levels during myogenic differentiation in vitro. The proper localization of adhalin to the muscle cell membrane was observed only in late stages of myotube maturation, coincident with the re-distribution of caveolin-3 and dystrophin. These data suggest that adhalin is highly specific for striated muscle and that it is linked with the formation of a fully functional muscle fiber.