miR-873a-5p Targets A20 to Facilitate Morphine Tolerance in Mice

Long-term morphine administration leads to tolerance and a gradual reduction in analgesic potency. Noncoding microRNAs (miRNAs) modulate gene expression in a posttranscriptional manner, and their dysregulation causes various diseases. Emerging evidence suggests that miRNAs play a regulatory role in the development of morphine tolerance. In the present study, we hypothesized that miR-873a-5p is a key functional small RNA that participates in the development and maintenance of morphine tolerance through the regulation of A20 (tumor necrosis factor α-induced protein 3, TNFAIP3) in mice. We measured the percentage of maximum possible effect (MPE %) to evaluate the analgesic effect of morphine. The expression of miR-873a-5p and its target gene A20 were determined after the morphine-tolerant model was successfully established. Intrathecal injection with lentivirus to intervene in the expression of A20 and the miR-873a-5p antagomir was used to explore the role of miR-873a-5p in the development of morphine tolerance. Chronic morphine administration significantly increased the expression of miR-873a-5p, which was inversely correlated with decreased A20 expression in the spinal cord of morphine-tolerant mice. Downregulation of miR-873a-5p in the spinal cord attenuated and partly reversed the development of morphine tolerance accompanied by overexpression of A20. Similarly, A20 was upregulated by a recombinant lentivirus vector, which attenuated and reversed the pathology of morphine tolerance by inhibiting the activation of nuclear factor (NF)-κB. Collectively, our results indicated that miR-873a-5p targets A20 in the spinal cord to facilitate the development of morphine tolerance in mice. Downregulating the expression of miR-873a-5p may be a potential strategy to ameliorate morphine tolerance

Overexpression of GDNF in Spinal Cord Attenuates Morphine Analgesic Tolerance in Rats with Bone Cancer Pain

Bone cancer pain (BCP) is one of the typical and distressing symptoms in cancer patients. Morphine is a widely used analgesic drug for BCP; however, long-term morphine administration will lead to analgesic tolerance. Our previous study indicated that spinal glial cell line-derived neurotrophic factor (GDNF) exerts analgesic effects in rats with BCP. In this study, BCP was established by inoculated Walker 256 carcinoma cells into rat tibias, while morphine tolerance (MT) was induced by intrathecally injecting morphine twice daily from the 9th to 15th postoperative day (POD) in BCP rats. The BCP rats developed mechanical and thermal hyperalgesia on POD 5 and it lasted to POD 15. The analgesic effect of morphine was decreased after repeat administration. Western blots and immunochemistry tests showed that GDNF was gradually decreased in the spinal cord after the development of MT in rats with BCP, and GDNF was colocalized with the μ opioid receptor (MOR) in the superficial laminate of the spinal cords. The overexpression of GDNF by lentivirus significantly attenuated MT, and restored the expression of MOR in the spinal cord. In summary, our results suggest that the reduction of GDNF expression participated in the development of MT in rats with BCP and could be a promising therapeutic option for BCP

Identification of lncRNA expression profiles and ceRNA analysis in the spinal cord of morphine-tolerant rats

Abstract Morphine tolerance is a challenging clinical problem that limits the use of morphine in pain treatment, but the mechanisms of morphine tolerance remain unclear. Recent research indicates that long noncoding RNAs (lncRNAs) might be a novel and promising target in the pathogeneses of diseases. Therefore, we hypothesized that lncRNAs might play a role in the development of morphine tolerance. Male Sprague-Dawley rats were intrathecally injected with 10 μg morphine twice daily for 7 consecutive days. The animals were then sacrificed for lncRNA microarray tests, and the results were validated by RT-qPCR. Next, functional predictions for the differentially expressed mRNAs (DEmRNAs) were made with the Gene Ontology/Kyoto Encyclopedia of Genes and Genomes (GO/KEGG), and predictions for the differentially expressed lncRNAs (DElncRNAs) were made based on competitive endogenous RNA (ceRNA) analyses. The rats successfully developed morphine tolerance. LncRNA microarray analysis revealed that, according to the criteria of a log2 (fold change) > 1.5 and a P-value < 0.05, 136 lncRNAs and 278 mRNAs were differentially expressed in the morphine tolerance group (MT) compared with the normal saline group (NS). The functions of the DEmRNAs likely involve in the processes of the ion channel transport, pain transmission and immune response. The ceRNA analysis indicated that several possible interacting networks existed, including (MRAK150340, MRAK161211)/miR-219b/Tollip.Further annotations of the potential target mRNAs of the miRNAs according to the gene database suggested that the possible functions of these mRNAs primarily involved the regulation of ubiquitylation, G protein-linked receptors, and Toll-like receptors, which play roles in the development of morphine tolerance. Our findings revealed the profiles of differentially expressed lncRNAs in morphine tolerance conditions, and among these lncRNAs, some DElncRNAs might be new therapeutic targets for morphine tolerance

Neonatal isoflurane exposure induces neurocognitive impairment and abnormal hippocampal histone acetylation in mice.

Neonatal exposure to isoflurane may induce long-term memory impairment in mice. Histone acetylation is an important form of chromatin modification that regulates the transcription of genes required for memory formation. This study investigated whether neonatal isoflurane exposure-induced neurocognitive impairment is related to dysregulated histone acetylation in the hippocampus and whether it can be attenuated by the histone deacetylase (HDAC) inhibitor trichostatin A (TSA).C57BL/6 mice were exposed to 0.75% isoflurane three times (each for 4 h) at postnatal days 7, 8, and 9. Contextual fear conditioning (CFC) was tested at 3 months after anesthesia administration. TSA was intraperitoneally injected 2 h before CFC training. Hippocampal histone acetylation levels were analyzed following CFC training. Levels of the neuronal activation and synaptic plasticity marker c-Fos were investigated at the same time point.Mice that were neonatally exposed to isoflurane showed significant memory impairment on CFC testing. These mice also exhibited dysregulated hippocampal H4K12 acetylation and decreased c-Fos expression following CFC training. TSA attenuated isoflurane-induced memory impairment and simultaneously increased histone acetylation and c-Fos levels in the hippocampal cornu ammonis (CA)1 area 1 h after CFC training.Memory impairment induced by repeated neonatal exposure to isoflurane is associated with dysregulated histone H4K12 acetylation in the hippocampus, which probably affects downstream c-Fos gene expression following CFC training. The HDAC inhibitor TSA successfully rescued impaired contextual fear memory, presumably by promoting histone acetylation and histone acetylation-mediated gene expression

Table_3_Predicting the prognosis in patients with sepsis by a pyroptosis-related gene signature.xlsx

BackgroundSepsis remains a life-threatening disease with a high mortality rate that causes millions of deaths worldwide every year. Many studies have suggested that pyroptosis plays an important role in the development and progression of sepsis. However, the potential prognostic and diagnostic value of pyroptosis-related genes in sepsis remains unknown.MethodsThe GSE65682 and GSE95233 datasets were obtained from Gene Expression Omnibus (GEO) database and pyroptosis-related genes were obtained from previous literature and Molecular Signature Database. Univariate cox analysis and least absolute shrinkage and selection operator (LASSO) cox regression analysis were used to select prognostic differentially expressed pyroptosis-related genes and constructed a prognostic risk score. Functional analysis and immune infiltration analysis were used to investigate the biological characteristics and immune cell enrichment in sepsis patients who were classified as low- or high-risk based on their risk score. Then the correlation between pyroptosis-related genes and immune cells was analyzed and the diagnostic value of the selected genes was assessed using the receiver operating characteristic curve.ResultsA total of 16 pyroptosis-related differentially expressed genes were identified between sepsis patients and healthy individuals. A six-gene-based (GZMB, CHMP7, NLRP1, MYD88, ELANE, and AIM2) prognostic risk score was developed. Based on the risk score, sepsis patients were divided into low- and high-risk groups, and patients in the low-risk group had a better prognosis. Functional enrichment analysis found that NOD-like receptor signaling pathway, hematopoietic cell lineage, and other immune-related pathways were enriched. Immune infiltration analysis showed that some innate and adaptive immune cells were significantly different between low- and high-risk groups, and correlation analysis revealed that all six genes were significantly correlated with neutrophils. Four out of six genes (GZMB, CHMP7, NLRP1, and AIM2) also have potential diagnostic value in sepsis diagnosis.ConclusionWe developed and validated a novel prognostic predictive risk score for sepsis based on six pyroptosis-related genes. Four out of the six genes also have potential diagnostic value in sepsis diagnosis.</p

Table_2_Predicting the prognosis in patients with sepsis by a pyroptosis-related gene signature.xlsx

BackgroundSepsis remains a life-threatening disease with a high mortality rate that causes millions of deaths worldwide every year. Many studies have suggested that pyroptosis plays an important role in the development and progression of sepsis. However, the potential prognostic and diagnostic value of pyroptosis-related genes in sepsis remains unknown.MethodsThe GSE65682 and GSE95233 datasets were obtained from Gene Expression Omnibus (GEO) database and pyroptosis-related genes were obtained from previous literature and Molecular Signature Database. Univariate cox analysis and least absolute shrinkage and selection operator (LASSO) cox regression analysis were used to select prognostic differentially expressed pyroptosis-related genes and constructed a prognostic risk score. Functional analysis and immune infiltration analysis were used to investigate the biological characteristics and immune cell enrichment in sepsis patients who were classified as low- or high-risk based on their risk score. Then the correlation between pyroptosis-related genes and immune cells was analyzed and the diagnostic value of the selected genes was assessed using the receiver operating characteristic curve.ResultsA total of 16 pyroptosis-related differentially expressed genes were identified between sepsis patients and healthy individuals. A six-gene-based (GZMB, CHMP7, NLRP1, MYD88, ELANE, and AIM2) prognostic risk score was developed. Based on the risk score, sepsis patients were divided into low- and high-risk groups, and patients in the low-risk group had a better prognosis. Functional enrichment analysis found that NOD-like receptor signaling pathway, hematopoietic cell lineage, and other immune-related pathways were enriched. Immune infiltration analysis showed that some innate and adaptive immune cells were significantly different between low- and high-risk groups, and correlation analysis revealed that all six genes were significantly correlated with neutrophils. Four out of six genes (GZMB, CHMP7, NLRP1, and AIM2) also have potential diagnostic value in sepsis diagnosis.ConclusionWe developed and validated a novel prognostic predictive risk score for sepsis based on six pyroptosis-related genes. Four out of the six genes also have potential diagnostic value in sepsis diagnosis.</p

Proteomic Analysis of PKCγ-Related Proteins in the Spinal Cord of Morphine-Tolerant Rats

<div><h3>Background</h3><p>Morphine tolerance is a common drawback of chronic morphine exposure, hindering use of this drug. Studies have shown that PKCã may play a key role in the development of morphine tolerance, although the mechanisms are not fully known.</p> <h3>Methodology/Principal Findings</h3><p>In a rat model of morphine tolerance, PKCã knockdown in the spinal cord was successfully carried out using RNA interference (RNAi) with lentiviral vector-mediated short hairpin RNA of PKCã (LV-shPKCã). Spinal cords (L4-L5) were obtained surgically from morphine-tolerant (MT) rats with and without PKCã knockdown, for comparative proteomic analysis. Total proteins from the spinal cords (L4-L5) were extracted and separated using two-dimensional gel electrophoresis (2DGE); 2D gel images were analyzed with PDQuest software. Seven differential gel-spots were observed with increased spot volume, and 18 spots observed with decreased spot volume. Among these, 13 differentially expressed proteins (DEPs) were identified with matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS), comparing between MT rats with and without PKCã knockdown. The DEPs identified have roles in the cytoskeleton, as neurotrophic factors, in oxidative stress, in ion metabolism, in cell signaling, and as chaperones. Three DEPs (GFAP, FSCN and GDNF) were validated with Western blot analysis, confirming the DEP data. Furthermore, using immunohistochemical analysis, we reveal for the first time that FSCN is involved in the development of morphine tolerance.</p> <h3>Conclusions/Significance</h3><p>These data cast light on the proteins associated with the PKCã activity during morphine tolerance, and hence may contribute to clarification of the mechanisms by which PKCã influences MT.</p> </div

Table_1_Predicting the prognosis in patients with sepsis by a pyroptosis-related gene signature.docx

BackgroundSepsis remains a life-threatening disease with a high mortality rate that causes millions of deaths worldwide every year. Many studies have suggested that pyroptosis plays an important role in the development and progression of sepsis. However, the potential prognostic and diagnostic value of pyroptosis-related genes in sepsis remains unknown.MethodsThe GSE65682 and GSE95233 datasets were obtained from Gene Expression Omnibus (GEO) database and pyroptosis-related genes were obtained from previous literature and Molecular Signature Database. Univariate cox analysis and least absolute shrinkage and selection operator (LASSO) cox regression analysis were used to select prognostic differentially expressed pyroptosis-related genes and constructed a prognostic risk score. Functional analysis and immune infiltration analysis were used to investigate the biological characteristics and immune cell enrichment in sepsis patients who were classified as low- or high-risk based on their risk score. Then the correlation between pyroptosis-related genes and immune cells was analyzed and the diagnostic value of the selected genes was assessed using the receiver operating characteristic curve.ResultsA total of 16 pyroptosis-related differentially expressed genes were identified between sepsis patients and healthy individuals. A six-gene-based (GZMB, CHMP7, NLRP1, MYD88, ELANE, and AIM2) prognostic risk score was developed. Based on the risk score, sepsis patients were divided into low- and high-risk groups, and patients in the low-risk group had a better prognosis. Functional enrichment analysis found that NOD-like receptor signaling pathway, hematopoietic cell lineage, and other immune-related pathways were enriched. Immune infiltration analysis showed that some innate and adaptive immune cells were significantly different between low- and high-risk groups, and correlation analysis revealed that all six genes were significantly correlated with neutrophils. Four out of six genes (GZMB, CHMP7, NLRP1, and AIM2) also have potential diagnostic value in sepsis diagnosis.ConclusionWe developed and validated a novel prognostic predictive risk score for sepsis based on six pyroptosis-related genes. Four out of the six genes also have potential diagnostic value in sepsis diagnosis.</p