Transcranial direct current stimulation relieves visceral hypersensitivity via normalizing GluN2B expression and neural activity in anterior cingulate cortex.

Irritable bowel syndrome (IBS) is one of the most common challenging diseases for clinical treatment. The aim of this study is to investigate whether transcranial direct current stimulation (tDCS) has analgesic effect on visceral hypersensitivity (VH) in an animal model of IBS as well as the underlying mechanism. As the activation of GluN2B in anterior cingulate cortex (ACC) takes part in VH, we examined whether and how GluN2B in ACC takes part in the effect of tDCS. Neonatal maternal deprivation (NMD), a valuable experimental model to study the IBS pathophysiology, was used to induce visceral hypersensitivity of rats. We quantified VH as colorectal distention threshold and performed patch-clamp recordings of ACC neurons. The expression of GluN2B were determined by RT-qPCR and Western blotting. The GluN2B antagonist Ro 25-6981 was microinjected into the rostral and caudal ACC. tDCS was performed for 7 consecutive days. It was found that NMD decreased expression of GluN2B, which could be obviously reversed by tDCS. Injection of Ro 25-6981 into rostral and caudal ACC of normal rats induced VH and also reversed the analgesic effect of tDCS. Our data sheds light on the nonpharmacological therapy for chronic VH in pathological states such as IBS.NEW & NOTEWORTHY Irritable bowel syndrome (IBS) is a gastrointestinal disease characterized by visceral hypersensitivity. This study showed a decrease of GluN2B expression and neural activity in ACC of IBS-model rats, which could be obviously reversed by tDCS. In addition, blockade of GluN2B in rostral and caudal ACC induced VH of normal rats. Furthermore, analgesic effect of tDCS on NMD rats was reversed by GluN2B antagonist.

Causes and Counter Measure Analysis of Inpatients Unqualified Clinical Specimens from 2015 to 2019.

BACKGROUND: To improve the quality of pre-analytical phase and provide targeted suggestions, this study analyzed factors causing unqualified clinical specimens in patients of the Department of Clinical Laboratory of Renmin Hospital of WuHan University from 2015 to 2019. METHODS: Inpatient specimens from January 2015 to December 2019 were retrospectively analyzed. Unqualified specimens were identified by referring to the general principle of rejection. The analytical indicators included incidence rate of unqualified specimens and constituent ratio of reasons of unqualified specimens. These two indicators were analyzed according to the inpatient wards and types of specimens. RESULTS: From 2015 to 2019, 21,674 inpatient unqualified specimens were collected, the incidence rate of unqualified specimens was 0.22% (21,674/9,700,869), the number and rate of unqualified specimens decreased year by year. The main reasons of unqualified specimens were insufficient volume (29.67%, 6,430/21,674) and clotting (26.31%, 5,703/21,674). The number of unqualified specimens in the departments of cardiovascular, pediatrics, neurology, oncology, urinary surgery, and intensive care unit ranked the top each year. Clotting (39.29%, 5,682/14,462) was the main reason of unqualified blood specimens while insufficient volume (71.18%, 3,365/4,727) was for urine specimens. Wrong identification caused unqualified feces (62.65%, 728/1,162) and body fluid (40.74%, 539/1,323) specimens. CONCLUSIONS: Clinical laboratory could make effective measures to improve pre-analytical quality by retrospectively analyzing data of unqualified specimens.

Downregulation of GRK6 in arcuate nucleus promotes chronic visceral hypersensitivity via NF-κB upregulation in adult rats with neonatal maternal deprivation.

AIMS: The arcuate nucleus is a vital brain region for coursing of pain command. G protein-coupled kinase 6 (GRK6) accommodates signaling through G protein-coupled receptors. Studies have demonstrated that GRK6 is involved in inflammatory pain and neuropathic pain. The present study was designed to explore the role and the underlying mechanism of GRK6 in arcuate nucleus of chronic visceral pain. METHODS: Chronic visceral pain of rats was induced by neonatal maternal deprivation and evaluated by monitoring the threshold of colorectal distension. Western blotting, immunofluorescence, real-time quantitative polymerase chain reaction techniques, and Nissl staining were employed to determine the expression and mutual effect of GRK6 with nuclear factor κB (NF-κB). RESULTS: Expression of GRK6 in arcuate nucleus was significantly reduced in neonatal maternal deprivation rats when compared with control rats. GRK6 was mainly expressed in arcuate nucleus neurons, but not in astrocytes, and a little in microglial cells. Neonatal maternal deprivation reduced the percentage of GRK6-positive neurons of arcuate nucleus. Overexpression of GRK6 by Lentiviral injection into arcuate nucleus reversed chronic visceral pain in neonatal maternal deprivation rats. Furthermore, the expression of NF-κB in arcuate nucleus was markedly upregulated in neonatal maternal deprivation rats. NF-κB selective inhibitor pyrrolidine dithiocarbamate suppressed chronic visceral pain in neonatal maternal deprivation rats. GRK6 and NF-κB were expressed in the arcuate nucleus neurons. Importantly, overexpression of GRK6 reversed NF-κB expression at the protein level. In contrast, injection of pyrrolidine dithiocarbamate once daily for seven consecutive days did not alter GRK6 expression in arcuate nucleus of neonatal maternal deprivation rats. CONCLUSIONS: Present data suggest that GRK6 might be a pivotal molecule participated in the central mechanisms of chronic visceral pain, which might be mediated by inhibiting NF-κB signal pathway. Overexpression of GRK6 possibly represents a potential strategy for therapy of chronic visceral pain.

Comparison of Application Biological Variation and Patient Data for Selecting Delta Check Limits.

BACKGROUND: This study aimed to evaluate delta check limits set by reference change value (RCV) and patient data. METHODS: Patient data of 11 clinical chemistry analytes from June 2018 to May 2019 were collected. RCV with 95% or 99% levels of probability were calculated based on biological variation. The corresponding delta check limits for outpatients and inpatients were calculated by 95% or 99% central range of delta% which was the difference of two consecutive results within thirty days of the same patient for each analyte. Patient data in June 2019 were used to analyze the utility of delta check limits. RESULTS: In total, 434,927 paired results for these 11 analytes were included. The delta check limits were different between outpatients and inpatients, but were wider than those established by RCV. The difference between Glu's outpatient and inpatient boundaries was the largest, 95% central range from the outpatient (inpatients) was from -32.29% (-56.97%) to 38.78% (106.00%) while 99% central range from the outpatient (inpatients) was from -56.86% (-90.56%) to 89.96% (262.54%). The RCV is mainly determined by within-individual biological variation so that the RCV of each analyte varied from each other. As for RCV, Na had the lowest value and BUN had the highest one. In addition, the main reason for delta% exceeding delta check limits was a clinically significant change. CONCLUSIONS: Laboratories could use delta check procedure to find out errors in sample collection and monitor clinical significance. When delta% of patients exceed corresponding delta check limits in a short time, clinicians and personnel of clinical laboratory should pay more attention. Delta check limits should be reviewed regularly to check the utility of procedure.

Downregulation of glucose-6-phosphate dehydrogenase contributes to diabetic neuropathic pain through upregulation of toll-like receptor 4 in rats.

BACKGROUND AND AIM: Diabetic neuropathic pain is a refractory and disabling complication of diabetes mellitus. The pathogenesis of the diabetic neuropathic pain is still unclear, and treatment is insufficient. The aim of this study is to investigate the roles of glucose-6-phosphate dehydrogenase (G6PD) and toll-like receptor 4 (TLR4) in neuropathic pain in rats with diabetes. METHODS: Type 1 diabetes model was induced by intraperitoneal injection of streptozotocin (STZ, 75 mg/kg) in adult female Sprague-Dawley rats. Paw withdrawal threshold and paw withdrawal latency of rats were measured by von Frey filaments and thermal radiation, respectively. The expressions of G6PD and TLR4 in L4-L6 dorsal root ganglions (DRGs) were measured by western blotting and quantitative real-time polymerase chain reaction analysis. Fluorescent immunohistochemistry was employed to detect expressions of G6PD and TLR4 and co-location of G6PD with TLR4. RESULTS: The mRNA and protein expression levels of G6PD in DRGs were significantly decreased in diabetic rats when compared with age-matched control rats. Upregulation of G6PD by intrathecal injection of G6PD overexpression adenovirus markedly attenuated hindpaw pain hypersensitivity of diabetic rats. The mRNA and protein expression levels of TLR4 in DRGs of diabetic rats were significantly increased when compared with control rats. Intrathecal injection of TLR4-selective inhibitor CLI-095 attenuated diabetic pain in dose- and time-dependent manners. Furthermore, G6PD and TLR4 were co-localized in DRG neurons. Intrathecal injection of G6PD overexpression adenovirus greatly reduced TLR4 expression, while intrathecal injection of CLI-095 had no significant effect on G6PD expression in diabetic rats. CONCLUSIONS: Our results suggest that decrease in G6PD expression was involved in diabetic peripheral neuropathic pain, which was most likely through upregulation of TLR4 expression in the DRGs of rats.

Relationship Between Diabetes Mellitus and Cirrhosis Risk in Chronic Hepatitis B Patients in Wuhan, China.

BACKGROUND The objective of our research was to assess the possible link between diabetes mellitus (DM) and liver cirrhosis in chronic hepatitis B (CHB) patients in Wuhan, China. MATERIAL AND METHODS Individuals with a diagnosis of both liver cirrhosis and chronic HBV infection (n=257), and CHB-only patients (n=514) were matched 1: 2 by age and sex. Demographic, lifestyle, laboratory, and clinical characteristics were reviewed. Univariate and the multiple logistic regression analysis were conducted to investigate the association between DM and HBV-related liver cirrhosis. RESULTS The prevalence of DM was higher among CHB patients with liver cirrhosis than in those without liver cirrhosis (22.2% vs. 12.8%, P=0.001), yielding an adjusted odds ratio (AOR) of 2.317 and a 95% confidence interval (CI) of 1.528-3.513. Among them, 87.7% of liver cirrhosis patients were diagnosed with DM before liver cirrhosis diagnosis, yielding an AOR (95% CI) of 2.386 (1.533-3.714). In comparison to patients with a DM duration of 2-5 years, the AOR (95% CI) for those with a DM duration >5 years was 2.073 (0.701-6.132). In DM treatment, the AOR (95% CI) for those treated with insulin was 4.746 (1.329-16.949). CONCLUSIONS DM was associated with cirrhosis risk in CHB patients in Wuhan, China.

Sensitization of P2X3 receptors in insular cortex contributes to visceral pain of adult rats with neonatal maternal deprivation.

Aims Insular cortex is a brain region critical for processing of the sensation. Purinergic receptors are involved in the formation of chronic pain. The aim of the present study was to explore the role and mechanism of P2X3 receptors (P2X3Rs) in insular cortex in chronic visceral pain. Methods Chronic visceral pain in adult rats was induced by neonatal maternal deprivation and measured by detecting the threshold of colorectal distension. Western blotting, immunofluorescence, and real-time quantitative polymerase chain reaction techniques were used to detect the expression and distribution of P2X3Rs. Synaptic transmission in insular cortex was recorded in brain slices by patch clamp techniques. Results Expression of P2X3Rs both at mRNA and protein levels in right hemisphere of insular cortex was significantly increased in neonatal maternal deprivation rats. In addition, P2X3Rs were expressed with NeuN or synaptophysin but not with glial fibrillary acidic protein and CD11b. The co-localization of P2X3Rs with NeuN or synaptophysin was greatly enhanced in right hemisphere of insular cortex in neonatal maternal deprivation rats. Furthermore, neonatal maternal deprivation markedly increased both the frequency and amplitude of miniature excitatory postsynaptic current in right hemisphere of insular cortex. Incubation of A347091 significantly decreased the frequency of spontaneous excitatory postsynaptic current and miniature excitatory postsynaptic current of insular cortex neurons of neonatal maternal deprivation rats. Incubation of P2X3Rs agonists α,ß-mATP remarkably increased the frequency of spontaneous excitatory postsynaptic current and miniature excitatory postsynaptic current of the right hemisphere of insular cortex neurons of healthy control rats. Importantly, injection of A317491 significantly enhanced the colorectal distension threshold of neonatal maternal deprivation rats, while injection of α,ß-mATP into right but not left insular cortex markedly decreased the colorectal distension threshold in healthy control rats. Conclusions Overall, our data provide integrated pharmacological, biochemical, and functional evidence demonstrating that P2X3Rs are physically and functionally interconnected at the presynaptic level to control synaptic activities in the right insular cortex, thus contributing to visceral pain of neonatal maternal deprivation rats.

Prenatal maternal stress induces visceral hypersensitivity of adult rat offspring through activation of cystathionine-ß-synthase signaling in primary sensory neurons.

Irritable bowel syndrome is a disorder of unknown etiology characterized by widespread, chronic abdominal pain associated with altered bowel movements. Increasing amounts of evidence indicate that stressors presented during gestational periods could have long-term effects on the offspring's tissue structure and function, which may predispose to gastrointestinal diseases. The aim of the present study is to determine whether prenatal maternal stressis a adverse factor affecting gastrointestinal sensitivity and to investigate possible mechanisms underlying prenatal maternal stress-induced visceral hypersensitivity in adult offspring. Prenatal maternal stress was induced in pregnant Sprague-Dawley rats by exposure to heterotypic intermitent stress from gestational day 7 to delivery. Prenatal maternal stress significantly increased visceromotor response to colorectal distention in adult offspring from the age of 6 weeks to 10 weeks. Prenatal maternal stress also enhanced neuronal excitability including depolarization of resting membrane potentials, reduction in rheobase, and an increase in the number of action potentials evoked by 2× and 3× rheobase current stimultion of colon-specific dorsal root ganglion neurons. Prenatal maternal stress remarkably enhanced expression of cystathionine-ß-synthase and Nav1.7 in T13-L2 thoracolumbar dorsal root ganglions both at protein and mRNA levels. Intraperitoneal injection of aminooxyacetic acid, an inhibitor of cystathionine-ß-synthase, attenuated prenatal maternal stress-induced visceral hypersensitivity in a dose-dependent manner. A consecutive seven-day administration of aminooxyacetic acid reversed the hyperexcitability of colon-specific dorsal root ganglion neurons and markedly reduced Nav1.7 expression. These results indicate that the presence of multiple psychophysical stressors during pregnancy is associated with visceral hypersensitivity in offspring, which is likely mediated by an upregualtion of cystathionine-ß-synthase and Nav1.7 expression. Prenatal maternal stress might be a significant contributor to irritable bowel syndrome, and cystathionine-ß-synthase might be a potential target for treatment for chronic visceral hypersensitivity in patients with irritable bowel syndrome.

Association of plasma interleukin-18 levels and polymorphisms in interleukin-18 gene with outcomes of hepatitis C virus infections: a meta-analysis.

Several polymorphisms in the interleukin-18 (IL-18) gene and plasma IL-18 levels have been reported to influence hepatitis C virus (HCV) infection. However, the published findings have been conflicting. We conducted meta-analyses of randomized, controlled trials to address the association of IL-18 polymorphisms and plasma IL-18 levels and the outcomes of HCV infection. The results showed that there was no evidence for an association between the HCV infections and the polymorphisms genotypes of IL-18. However, plasma IL-18 levels were found higher in HCV infection patients than in healthy controls. The pooled SMD was 2.911(95% CI 2.556-3.265, z = 16.09, P < 0.001).

Analysis of Key Genes and miRNA-mRNA Networks Associated with Glucocorticoids Treatment in Chronic Obstructive Pulmonary Disease.

Background: Some patients with chronic obstructive pulmonary disease (COPD) benefit from glucocorticoid (GC) treatment, but its mechanism is unclear. Objective: With the help of the Gene Expression Omnibus (GEO) database, the key genes and miRNA-mRNA related to the treatment of COPD by GCs were discussed, and the potential mechanism was explained. Methods: The miRNA microarray dataset (GSE76774) and mRNA microarray dataset (GSE36221) were downloaded, and differential expression analysis were performed. Gene Ontology (GO) function and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses were performed on the differentially expressed genes (DEGs). The protein interaction network of the DEGs in the regulatory network was constructed with the STRING database, and the key genes were screened through Cytoscape. Potential downstream target genes regulated by differentially expressed miRNAs (DEMs) were predicted by the miRWalk3.0 database, and miRNA-mRNA regulatory networks were constructed. Finally, some research results were validated. Results: ① Four DEMs and 83 DEGs were screened; ② GO and KEGG enrichment analysis mainly focused on the PI3K/Akt signalling pathway, ECM receptor interaction, etc.; ③ CD2, SLAMF7, etc. may be the key targets of GC in the treatment of COPD; ④ 18 intersection genes were predicted by the mirwalk 3.0 database, and 9 pairs of miRNA-mRNA regulatory networks were identified; ⑤ The expression of miR-320d-2 and TFCP2L1 were upregulated by dexamethasone in the COPD cell model, while the expression of miR-181a-2-3p and SLAMF7 were downregulated. Conclusion: In COPD, GC may mediate the expression of the PI3K/Akt signalling pathway through miR-181a-2-3p, miR-320d-2, miR-650, and miR-155-5p, targeting its downstream signal factors. The research results provide new ideas for RNA therapy strategies of COPD, and also lay a foundation for further research.

DMSO-soluble cigarette smoke particles alter the expression of endothelin B receptor in rat coronary artery.

In coronary artery diseases, cigarette smoking is a risk factor and the endothelin system plays a key role in the pathogenesis. This study was to examine if dimethylsulfoxide-soluble smoke particles (DSP) upregulate endothelin type-B (ETB) receptors in the coronary artery and investigate the mechanism. The isolated rat coronary arteries were organ-cultured for 24 h. The contractile response of the coronary artery was recorded by myograph. The mRNA and protein expression of the ETB receptors was studied using quantitative real-time PCR and immunohistochemistry. Results showed that the ETB receptor agonist, sarafotoxin 6c, induced a weak contraction in the fresh coronary artery. After culture, the contraction curve mediated by ETB receptor was shifted towards the left with an increased Emax of 152 ± 12%. DSP of 0.2 and 0.4 µl/ml shifted the concentration-contractile curves towards the left with further increased Emax of 270 ± 26 and 280 ± 29%, respectively. The culture increased ETB receptor mRNA and protein levels from fresh arteries, which was further enhanced by DSP. PD98059 (ERK1/2 inhibitor), wedelolactone (NF-κB inhibitor), actinomycin D or cycloheximide significantly inhibited the DSP-enhanced contraction and expression of mRNA and protein of the ETB receptor. However, SB203580 (p38 inhibitor) further increased DSP-enhanced contraction and protein expression of the ETB receptor. The results indicate that DSP upregulates ETB receptors in rat coronary artery via ERK1/2 and the NF-κB pathway.

ZnO Nanoparticles Modified by Carbon Quantum Dots for the Photocatalytic Removal of Synthetic Pigment Pollutants.

Synthetic pigment pollutants caused by the rapid development of the modern food industry have become a serious threat to people's health and quality of life. Environmentally friendly ZnO-based photocatalytic degradation exhibits satisfactory efficiency, but some shortcomings of large band gap and rapid charge recombination reduce the removal of synthetic pigment pollutants. Here, carbon quantum dots (CQDs) with unique up-conversion luminescence were applied to decorate ZnO nanoparticles to effectively construct the CQDs/ZnO composites via a facile and efficient route. The ZnO nanoparticles with a spherical-like shape obtained from a zinc-based metal organic framework (zeolitic imidazolate framework-8, ZIF-8) were coated by uniformly dispersive quantum dots. Compared with single ZnO particles, the obtained CQDs/ZnO composites exhibit enhanced light absorption capacity, decreased photoluminescence (PL) intensity, and improved visible-light degradation for rhodamine B (RhB) with the large apparent rate constant (k app). The largest k app value in the CQDs/ZnO composite obtained from 75 mg of ZnO nanoparticles and 12.5 mL of the CQDs solution (∼1 mg·mL-1) was 2.6 times that in ZnO nanoparticles. This phenomenon may be attributed to the introduction of CQDs, leading to the narrowed band gap, an extended lifetime, and the charge separation. This work provides an economical and clean strategy to design visible-light-responsive ZnO-based photocatalysts, which is expected to be used for the removal of synthetic pigment pollutants in food industry.

miR-1306-3p directly activates P2X3 receptors in primary sensory neurons to induce visceral pain in rats.

ABSTRACT: Mounting evidence indicates that microRNAs (miRNAs) play critical roles in various pathophysiological conditions and diseases, but the physiological roles of extracellular miRNAs on the disease-related ion channels remain largely unknown. Here, we showed that miR-1306-3p evoked action potentials and induced inward currents of the acutely isolated rat dorsal root ganglion (DRG) neurons. The miR-1306-3p-induced effects were significantly inhibited by A317491, a potent inhibitor of the P2X3 receptor (P2X3R), or disappeared after the knockdown of P2X3Rs in DRG neurons. We further identified R180, K315, and R52 as the miR-1306-3p interaction sites on the extracellular domain of P2X3Rs, which were distinct from the orthosteric ATP-binding sites. Intrathecal injection of miR-1306-3p produced visceral pain but not somatic pain in normal control rats. Conversely, intrathecal application of a miR-1306-3p antagomir and A317491 significantly alleviated visceral pain in a rat model of chronic visceral pain. Together, our findings suggest that miR-1306-3p might function as an endogenous ligand to activate P2X3Rs, eventually leading to chronic visceral pain.

Fu-Huang ointment ameliorates impaired wound healing associated with diabetes through PI3K-AKT signalling pathway activation.

A diabetic ulcer (DU) is a dreaded and resistant complication of diabetes mellitus with high morbidity. Fu-Huang ointment (FH ointment) is a proven recipe for treating chronic refractory wounds; however, its molecular mechanisms of action are unclear. In this study, we identified 154 bioactive ingredients and their 1127 target genes in FH ointment through the public database. The intersection of these target genes with 151 disease-related targets in DUs resulted in 64 overlapping genes. Overlapping genes were identified in the PPI network and enrichment analyses. The PPI network identified 12 core target genes, whereas Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis indicated that upregulation of the PI3K/Akt signalling pathway was involved in the role of FH ointment in treating diabetic wounds. Molecular docking showed that 22 active compounds in FH ointment could enter the active pocket of PIK3CA. Molecular dynamics was used to prove the binding stability of the active ingredients and protein targets. We found that PIK3CA/Isobutyryl shikonin and PIK3CA/Isovaleryl shikonin combinations had strong binding energies. An in vivo experiment was conducted on PIK3CA, which was the most significant gene.This study comprehensively elucidated the active compounds, potential targets, and molecular mechanism of FH ointment application in treating DUs, and believed that PIK3CA is a promising target for accelerated healing.

The Difference in All-Cause Mortality Between Allergic Bronchopulmonary Aspergillosis with and without Chronic Obstructive Pulmonary Disease.

Background: Allergic bronchopulmonary aspergillosis (ABPA) primarily complicates the course of asthma, cystic fibrosis, and chronic obstructive pulmonary disease (COPD). Mortality data of ABPA and the difference in all-cause mortality between ABPA with and without COPD are not available. Objective: We investigated the difference in all-cause mortality between ABPA with and without COPD. Methods: A retrospective review was performed among patients with the diagnosis of ABPA at Peking University People's Hospital between January 2010 and March 2022. Logrank test was performed to investigate the difference between all-cause mortality for ABPA with and without COPD and Cox regression analysis was performed to investigate the independent risk factors for all-cause mortality in patients with ABPA. Results: Sixty-one patients with ABPA were enrolled in this study. The follow-up duration was 50.38 months (3-143 months). In the COPD group, 7 patients died (7/10), while in the non-COPD group, 4 patients died (4/51). The 1-year survival rates of ABPA with and without COPD were 60% and 97.8%, respectively. The 5-year survival rates of ABPA with and without COPD were 40% and 94%, respectively. The Cox regression analysis showed that higher C-reactive protein (CRP) (HR = 1.017, 95% CI 1.004-1.031, P = 0.013) and complicating COPD (HR = 8.525, 95% CI 1.827-39.773, P = 0.006) were independent risk factors associated with mortality in patients with ABPA. Conclusion: The all-cause mortality for ABPA with COPD is higher than that for ABPA without COPD. Higher CRP and complicating COPD are independent risk factor for mortality in patients with ABPA.

Overexpression of GRK6 alleviates chronic visceral hypersensitivity through downregulation of P2Y6 receptors in anterior cingulate cortex of rats with prenatal maternal stress.

AIMS: Visceral hypersensitivity is a major clinic symptom in patients with irritable bowel syndrome (IBS). Anterior cingulate cortex (ACC) is involved in processing the information of pain. Both G protein-coupled receptor kinase 6 (GRK6) and P2Y purinoceptor 6 (P2Y6) are associated with neuroinflammation and pathological pain. The aim of this study was to investigate the interaction between GRK6 and P2Y6 in ACC in the development of visceral hypersensitivity of adult offspring rats with prenatal maternal stress (PMS). METHODS: Visceral hypersensitivity was quantified by abdominal withdrawal reflex threshold to colorectal distension (CRD). The expression and cellular distribution of GRK6 and P2Y6 were determined by Western blotting, qPCR, and fluorescence immunohistochemistry. Co-immunoprecipitation was used to evaluate the interaction between GRK6 and P2Y6. RESULTS: The mRNA and protein levels of GRK6 were significantly decreased in ACC of PMS rats. The injection of GRK6 overexpression virus significantly attenuated visceral hypersensitivity of PMS rats. P2Y6's mRNA level, protein level, and ratio of membrane protein over total protein expression was markedly increased in PMS rats. P2Y6 antagonist MRS2578 microinjection reversed visceral hypersensitivity of PMS rats. GRK6 overexpression significantly reduced P2Y6's expression in membrane proteins and P2Y6's ratio of membrane protein over total protein expression. CONCLUSIONS: These results indicate that decreased GRK6 leads to the accumulation of P2Y6 at neuron membrane in ACC, thereby contributing to visceral hypersensitivity of PMS rats.

Targeting GATA1 and p2x7r Locus Binding in Spinal Astrocytes Suppresses Chronic Visceral Pain by Promoting DNA Demethylation.

Irritable bowel syndrome is a gastrointestinal disorder of unknown etiology characterized by widespread, chronic abdominal pain associated with altered bowel movements. Increasing amounts of evidence indicate that injury and inflammation during the neonatal period have long-term effects on tissue structure and function in the adult that may predispose to gastrointestinal diseases. In this study we aimed to investigate how the epigenetic regulation of DNA demethylation of the p2x7r locus guided by the transcription factor GATA binding protein 1 (GATA1) in spinal astrocytes affects chronic visceral pain in adult rats with neonatal colonic inflammation (NCI). The spinal GATA1 targeting to DNA demethylation of p2x7r locus in these rats was assessed by assessing GATA1 function with luciferase assay, chromatin immunoprecipitation, patch clamp, and interference in vitro and in vivo. In addition, a decoy oligodeoxynucleotide was designed and applied to determine the influence of GATA1 on the DNA methylation of a p2x7r CpG island. We showed that NCI caused the induction of GATA1, Ten-eleven translocation 3 (TET3), and purinergic receptors (P2X7Rs) in astrocytes of the spinal dorsal horn, and demonstrated that inhibiting these molecules markedly increased the pain threshold, inhibited the activation of astrocytes, and decreased the spinal sEPSC frequency. NCI also markedly demethylated the p2x7r locus in a manner dependent on the enhancement of both a GATA1-TET3 physical interaction and GATA1 binding at the p2x7r promoter. Importantly, we showed that demethylation of the p2x7r locus (and the attendant increase in P2X7R expression) was reversed upon knockdown of GATA1 or TET3 expression, and demonstrated that a decoy oligodeoxynucleotide that selectively blocked the GATA1 binding site increased the methylation of a CpG island in the p2x7r promoter. These results demonstrate that chronic visceral pain is mediated synergistically by GATA1 and TET3 via a DNA-demethylation mechanism that controls p2x7r transcription in spinal dorsal horn astrocytes, and provide a potential therapeutic strategy by targeting GATA1 and p2x7r locus binding.

Metformin attenuates diabetic neuropathic pain via AMPK/NF-κB signaling pathway in dorsal root ganglion of diabetic rats.

Neuropathic pain is a common complication of diabetes mellitus with poorly relieved by conventional analgesics. Metformin, a first-line drug for type 2 diabetes, reduces blood glucose by activating adenosine monophosphate protein kinase (AMPK) signalling system. However, the effect of Metformin on diabetic neuropathic pain is still unknown. In the present study, we showed that Metformin was capable of attenuating diabetes induced mechanical allodynia, and the analgesia effect could be blocked by Compound C (an AMPK inhibitor). Importantly, Metformin enhanced the phosphorylation level of AMPK in L4-6 DRGs of diabetic rats but not affect the expression of total AMPK. Intrathecal injection of AICAR (an AMPK agonist) could activate AMPK and alleviate the mechanical allodynia of diabetic rats. Additionally, phosphorylated AMPK and NF-κB was co-localized in small and medium neurons of L4-6 DRGs. Interestingly, the regulation of NF-κB in diabetic rats was obviously reduced when AMPK was activated by AICAR. Notably, Metformin could decrease NF-κB expression in L4-6 DRGs of diabetic rats, but the decrease was blocked by Compound C. In conclusion, Metformin alleviates diabetic mechanical allodynia via activation of AMPK signaling pathway in L4-6 DRGs of diabetic rats, which might be mediated by the downregulation of NF-κB, and this providing certain basis for Metformin to become a potential drug in the clinical treatment of diabetic neuropathic pain.

Metformin Attenuates Bone Cancer Pain by Reducing TRPV1 and ASIC3 Expression.

Bone cancer pain (BCP) is a common pathologic pain associated with destruction of bone and pathological reconstruction of nervous system. Current treatment strategies in clinical is inadequate and have unacceptable side effects due to the unclear pathology mechanism. In the present study, we showed that transplantation of Walker 256 cells aggravated mechanical allodynia of BCP rats (**p < 0.01 vs. Sham), and the expression of ASIC3 (Acid-sensitive ion channel 3) and TRPV1 was obviously enhanced in L4-6 dorsal root ganglions (DRGs) of BCP rats (**p < 0.01 vs. Sham). ASIC3 and TRPV1 was mainly expressed in CGRP and IB4 positive neurons of L4-6 DRGs. While, TRPV1 but not ASIC3 was markedly upregulated in L4-6 spinal dorsal horn (SDH) of BCP rats (**p < 0.01 vs. Sham). Importantly, intrathecal injection of CPZ (a TRPV1 inhibitor) or Amiloride (an ASICs antagonist) markedly increased the paw withdraw threshold (PWT) of BCP rats response to Von Frey filaments (**p < 0.01 vs. BCP + NS). What's more, intraperitoneally injection of Metformin or Vinorelbine markedly elevated the PWT of BCP rats, but reduced the expression of TRPV1 and ASIC3 in L4-6 DRGs and decreased the TRPV1 expression in SDH (*p < 0.05, **p < 0.01 vs. BCP + NS). Collectively, these results suggest an effective analgesic effect of Metformin on mechanical allodynia of BCP rats, which may be mediated by the downregulation of ASIC3 and TRPV1.