ß-Lapachone, an NQO1 activator, alleviates diabetic cardiomyopathy by regulating antioxidant ability and mitochondrial function.

BACKGROUND: Diabetic cardiomyopathy (DC) is one of the major lethal complications in patients with diabetes mellitus (DM); however, no specific strategy for preventing or treating DC has been identified. PURPOSE: This study aimed to investigate the effects of ß-lapachone (Lap), a natural compound that increases antioxidant activity in various tissues, on DC and explore the underlying mechanisms. STUDY DESIGN AND METHODS: As an in vivo model, C57BL/6 mice were fed with the high-fat diet (HF) for 10 weeks to induce type 2 DM. Mice were fed Lap with the HF or after 5 weeks of HF treatment to investigate the protective effects of Lap against DC. RESULTS: In the two in vivo models, Lap decreased heart weight, increased heart function, reduced oxidative stress, and elevated mitochondrial content under the HF. In the in vitro model, palmitic acid (PA) was used to mimic the effects of an HF on the differentiated-cardiomyoblast cell line H9c2. The results demonstrated that Lap reduced PA-induced ROS production by increasing the expression of antioxidant regulators and enzymes, inhibiting inflammation, increasing mitochondrial activity, and thus reducing cell damage. Via the use of specific inhibitors and siRNA, the protective effects of Lap were determined to be mediated mainly by NQO1, Sirt1 and mitochondrial activity. CONCLUSION: Heart damage in DM is usually caused by excessive oxidative stress. This study showed that Lap can protect the heart from DC by upregulating antioxidant ability and mitochondrial activity in cardiomyocytes. Lap has the potential to serve as a novel therapeutic agent for both the prevention and treatment of DC.

Mitochondrial activity is the key to the protective effect of ß-Lapachone, a NAD+ booster, in healthy cells against cisplatin cytotoxicity.

BACKGROUND: Cisplatin (CDDP) is a first-line chemotherapeutic drug for treating various cancers. However, CDDP also damages normal cells and causes many side effects. Recently, CDDP has been demonstrated to kill cancer cells by targeting mitochondria. Protecting mitochondria might be a potential therapeutic strategy for CDDP-induced side effects. ß-Lapachone (ß-lap), a recognized NAD+ booster, has been reported to regulate mitochondrial activity. However, it remains unclear whether maintaining mitochondrial activity is the key factor in the protective effects of ß-lap in CDDP-treated normal cells. PURPOSE: In this study, the protective effects of ß-lap on mitochondria against CDDP cytotoxicity in normal cells were evaluated. STUDY DESIGN: In vitro cell models were used in this study, including 3T3 fibroblasts, human dermal fibroblasts, MCF-7 breast cancer cells, and MDA-MB-231 breast cancer cells. METHODS: Cells were treated with CDDP and ß-lap, and cell survival, NAD+, mitochondrial activity, autophagy, and ATP production were measured. Various inhibitors and siRNAs were used to confirm the key signal underlying the protective effects of ß-lap. RESULTS: The results demonstrated that ß-lap significantly decreased CDDP cytotoxicity in normal fibroblasts. With various inhibitors and siRNAs, ß-lap reduced CDDP-induced damage to normal fibroblasts by maintaining mitochondrial activity and increasing autophagy through the NQO1/NAD+/SIRT1 axis. Most importantly, the protective effects of ß-lap in fibroblasts did not affect the therapeutic effects of CDDP in cancer cells. This study indicated that mitochondrial activity, energy production, and NQO1 levels might be crucial responses separating normal cells from cancer cells under exposure to CDDP and ß-lap. CONCLUSION: ß-lap could be a good synergistic drug for reducing the side effects of CDDP without affecting the anticancer drug effect.

Hepatoma upregulated protein and Ki-67 expression in resectable hepatocellular carcinoma.

BACKGROUND: Hepatoma upregulated protein (HURP) and Ki-67 have been identified as cancer-related genes involved in cell growth and proliferation. Previous experimental studies have suggested an essential role for HURP expression in liver carcinogenesis. However, data regarding HURP expression in hepatocellular carcinoma (HCC) and its correlation with patient outcomes are limited. In this study, we examined the clinicopathologic features associated with HURP expression in HCC, and compared them to the results of the Ki-67 study. METHODS: Eighty-seven resected HCC at tumor, node, metastasis (TNM) stages I (n = 28), II (n = 29), and III (n = 30) were evaluated. HURP and Ki-67 expression were assessed by immunohistochemistry. Multivariate analysis was used to examine the prognostic significance of HURP and Ki-67 expression. RESULTS: HURP expression in HCC tissue was observed in 59% of patients and associated with female sex, low white blood cell count, and low platelet count. Ki-67 expression was observed in 67% of patients and associated with younger age, higher serum α-fetoprotein (AFP) levels, and frequent microvascular invasion. Univariate analysis showed that factors related to overall survival were: age >55 years, AFP >20 ng/mL, indocyanine green retention rate at 15 minutes (ICG-15) >15%, tumor size >5 cm, multiple tumors, macrovascular invasion, microvascular invasion, Ki-67 expression, and serum vascular endothelial growth factor >170 pg/mL. HURP expression was not associated with postresection survival. Multivariate analysis indicated that macrovascular invasion, multiple tumors, ICG-15 >15%, and Ki-67 expression were independent factors for overall survival. Multiple tumors and Ki-67 expression were independent factors related to recurrence-free survival. CONCLUSION: In our study, HURP expression in HCC tissue was not associated with post-resection survival. Ki-67 expression was an independent prognostic factor for survival. Our results suggest that the effect of HURP activity on growth, invasion, and postresection outcome of HCC in actual patients is less than previously demonstrated in experimental studies.

Clusterin expression in nontumor tissue in patients with resectable hepatocellular carcinoma related with postresectional survival.

BACKGROUND: Surgical resection offers an effective treatment for patients with hepatocellular carcinoma (HCC); however, it has high tumor recurrence rate. Clusterin is a highly conserved glycoprotein that enhances cell aggregation in vitro. It is upregulated in several types of cancers such as breast, ovarian, colon, prostate and kidney cancers, and HCC. Clusterin overexpression is correlated with tumor metastasis. We evaluated the significance of clusterin expression levels in serum and resected tissues of patients with HCC. METHODS: Serum, resected tumor tissue, and nontumor tissue were collected from 140 patients with HCC undergoing hepatic resection. Serum clusterin levels were determined by enzyme-linked immunosorbent assay. Clusterin expression in resected tissue was evaluated by immunohistochemistry. Median follow-up time was 57.8 months. RESULTS: Mean serum clusterin levels were found to be 130.0 ± 58.7 µg/mL (range, 10.1-366.6 µg/mL). Serum clusterin levels were independent of tumor stage and deterioration of liver function in patients. No significant difference was observed in the survival of patients with high (>130.0 ± 58.7 µg/mL) or low (≤130.0 ± 58.7 µg/mL) serum clusterin level. Clusterin was expressed in HCC tissues of 76 patients (54.3%) and nontumor liver tissues of 53 patients (37.9%). No significant difference was observed in the survival of patients with positive or negative clusterin expression in HCC tissues. In nontumor tissues, patients with positive clusterin expression were observed to have low postoperative disease-free survival rate (p = 0.001) compared to patients with negative clusterin expression. Multivariate analysis showed that tumor with macrovascular/microvascular invasion and clusterin expression in nontumor tissues are independent prognostic factors following hepatic resection. CONCLUSION: In HCC, clusterin expression in nontumor tissue shows worse prognosis after hepatic resection. Clusterin can be a prognostic marker for patients with postresection HCC.

Mitochondrial­DNA­associated TLR9 signalling is a potential serological biomarker for non­small cell lung cancer.

Mitochondrial (mt) DNA has been long suggested to contribute to carcinogenesis, and a variety of mutations in mtDNA have been confirmed to be related to various early stages of cancers; these data revealed that the detection of mtDNA in clinical samples may be a promising approach for cancer diagnosis. In the present study, the serum mtDNA in healthy donors and groups of patients with cancer was detected. It was revealed that patients with lung cancer without metastasis had more mtDNA in serum compared to patients with metastasis. Moreover, TLR9­associated signalling was activated in vitro after treatment with a synthetic CpG oligodeoxyribonucleotide (ODN) called ODN­M362. In addition, our data revealed that TLR9 and its adaptor protein, MyD88, were induced by ODN­M362 in a dose­dependent manner. A human cytokine array to evaluate stimulation of cytokine secretion by ODN­M362 was also used. Our findings may identify the role that TLR9 and mtDNA play in lung cancer progression and metastasis.

GluN2B/CaMKII mediates CFA-induced hyperalgesia via HDAC4-modified spinal COX2 transcription.

Histone deacetylase 4 (HDAC4), which actively shuttles between the nucleus and cytoplasm, is an attractive candidate for a repressor mechanism in epigenetic modification. However, the potential role of HDAC4-dependent epigenetics in the neural plasticity underlying the development of inflammatory pain has not been well established. By injecting complete Freund's adjuvant (CFA) into the hind-paw of Sprague-Dawley rats (200-250 g), we found animals displayed behavioral hyperalgesia was accompanied with HDAC4 phosphorylation and cytoplasmic redistribution in the dorsal horn neurons. Cytoplasmic HDAC4 retention led to its uncoupling with the COX2 promoter, hence prompting spinal COX2 transcription and expression in the dorsal horn. Moreover, the GluN2B-bearing N-methyl-d-aspartate receptor (GluN2B-NMDAR)/calmodulin-dependent protein kinase II (CaMKII) acted as an upstream cascade to facilitate HDAC4 phosphorylation/redistribution-associated spinal COX2 expression after inflammatory insults. The results of this pilot study demonstrated that the development and/or maintenance of inflammatory pain involved the spinal HDAC4-dependent epigenetic mechanisms. Our findings open up a new avenue for the development of a novel medical strategy for the relief of inflammatory pain.

Periaqueductal Gray Glutamatergic Transmission Governs Chronic Stress-Induced Depression.

The mechanisms underlying chronic stress-induced dysfunction of glutamatergic transmission that contribute to helplessness-associated depressive disorder are unknown. We investigated the relationship of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors and stress, and the neuroplastic changes of stress-induced depression-like behavior in the ventrolateral periaqueductal gray (vlPAG). We conducted whole-cell patch-clamp electrophysiological recordings in the vlPAG neurons. Depression-like behavior was assayed using tail suspension test and sucrose preference test. Surface and cytosolic glutamate receptor 1 (GluR1) AMPA receptor expression was analyzed using western blotting. Phosphorylated GluR1 expression was quantified using western blotting and immunohistochemical analysis. Unpredictable inescapable foot shock stress caused reduction in glutamatergic transmission originating from both presynaptic and postsynaptic loci in the vlPAG that was associated with behavioral despair and anhedonia in chronic stress-induced depression. Pharmacological inhibition of GluR1 function in the vlPAG caused depression-like behavior. Diminished glutamatergic transmission was due to reduced glutamate release presynaptically and enhanced GluR1-endocytosis from the cell surface postsynaptically. Chronic stress-induced neuroplastic changes and maladaptive behavior were reversed and mimicked by administration of glucocorticoid receptor (GR) antagonist and agonist, respectively. However, chronic stress did not affect γ-aminobutyric acid (GABA)-mediated inhibitory synaptic transmission in the vlPAG. These results demonstrate that depression-like behavior is associated with remarkable reduction in glutamatergic, but not GABAergic, transmission in the vlPAG. These neuroplastic changes and maladaptive behavior are attributed to GR-dependent mechanisms. As reduced GluR1-associated responses in the vlPAG contribute to chronic stress-induced neuroplastic changes, this cellular mechanism may be a critical component in the pathogenesis of stress-associated neuropsychiatric disorders.

Bromodomain-containing Protein 4 Activates Voltage-gated Sodium Channel 1.7 Transcription in Dorsal Root Ganglia Neurons to Mediate Thermal Hyperalgesia in Rats.

BACKGROUND: Bromodomain-containing protein 4 binds acetylated promoter histones and promotes transcription; however, the role of bromodomain-containing protein 4 in inflammatory hyperalgesia remains unclear. METHODS: Male Sprague-Dawley rats received hind paw injections of complete Freund's adjuvant to induce hyperalgesia. The dorsal root ganglia were examined to detect changes in bromodomain-containing protein 4 expression and the activation of genes involved in the expression of voltage-gated sodium channel 1.7, which is a key pain-related ion channel. RESULTS: The intraplantar complete Freund's adjuvant injections resulted in thermal hyperalgesia (4.0 ± 1.5 s; n = 7). The immunohistochemistry and immunoblotting results demonstrated an increase in the bromodomain-containing protein 4-expressing dorsal root ganglia neurons (3.78 ± 0.38 fold; n = 7) and bromodomain-containing protein 4 protein levels (2.62 ± 0.39 fold; n = 6). After the complete Freund's adjuvant injection, histone H3 protein acetylation was enhanced in the voltage-gated sodium channel 1.7 promoter, and cyclin-dependent kinase 9 and phosphorylation of RNA polymerase II were recruited to this area. Furthermore, the voltage-gated sodium channel 1.7-mediated currents were enhanced in neurons of the complete Freund's adjuvant rats (55 ± 11 vs. 19 ± 9 pA/pF; n = 4 to 6 neurons). Using bromodomain-containing protein 4-targeted antisense small interfering RNA to the complete Freund's adjuvant-treated rats, the authors demonstrated a reduction in the expression of bromodomain-containing protein 4 (0.68 ± 0.16 fold; n = 7), a reduction in thermal hyperalgesia (7.5 ± 1.5 s; n = 7), and a reduction in the increased voltage-gated sodium channel 1.7 currents (21 ± 4 pA/pF; n = 4 to 6 neurons). CONCLUSIONS: Complete Freund's adjuvant triggers enhanced bromodomain-containing protein 4 expression, ultimately leading to the enhanced excitability of nociceptive neurons and thermal hyperalgesia. This effect is likely mediated by the enhanced expression of voltage-gated sodium channel 1.7.

Targeting cannabinoid signaling for peritoneal dialysis-induced oxidative stress and fibrosis.

Long-term exposure to bioincompatible peritoneal dialysis (PD) solutions frequently results in peritoneal fibrosis and ultrafiltration failure, which limits the life-long use of and leads to the cessation of PD therapy. Therefore, it is important to elucidate the pathogenesis of peritoneal fibrosis in order to design therapeutic strategies to prevent its occurrence. Peritoneal fibrosis is associated with a chronic inflammatory status as well as an elevated oxidative stress (OS) status. Beyond uremia per se, OS also results from chronic exposure to high glucose load, glucose degradation products, advanced glycation end products, and hypertonic stress. Therapy targeting the cannabinoid (CB) signaling pathway has been reported in several chronic inflammatory diseases with elevated OS. We recently reported that the intra-peritoneal administration of CB receptor ligands, including CB1 receptor antagonists and CB2 receptor agonists, ameliorated dialysis-related peritoneal fibrosis. As targeting the CB signaling pathway has been reported to be beneficial in attenuating the processes of several chronic inflammatory diseases, we reviewed the interaction among the cannabinoid system, inflammation, and OS, through which clinicians ultimately aim to prolong the peritoneal survival of PD patients.

Growth Arrest and DNA-damage-inducible Protein 45ß-mediated DNA Demethylation of Voltage-dependent T-type Calcium Channel 3.2 Subunit Enhances Neuropathic Allodynia after Nerve Injury in Rats.

BACKGROUND: Growth arrest and DNA-damage-inducible protein 45ß reactivates methylation-silenced neural plasticity-associated genes through DNA demethylation. However, growth arrest and DNA-damage-inducible protein 45ß-dependent demethylation contributes to neuropathic allodynia-associated spinal plasticity remains unclear. METHODS: Adult male Sprague-Dawley rats (654 out of 659) received a spinal nerve ligation or a sham operation with or without intrathecal application of one of the following: growth arrest and DNA-damage-inducible protein 45ß messenger RNA-targeted small interfering RNA, lentiviral vector expressing growth arrest and DNA-damage-inducible protein 45ß, Ro 25-6981 (an NR2B-bearing N-methyl-D-aspartate receptor antagonist), or KN-93 (a calmodulin-dependent protein kinase II antagonist) were used for behavioral measurements, Western blotting, immunofluorescence, dot blots, detection of unmodified cytosine enrichment at cytosine-phosphate-guanine site, chromatin immunoprecipitation quantitative polymerase chain reaction analysis, and slice recordings. RESULTS: Nerve ligation-enhanced growth arrest and DNA-damage-inducible protein 45ß expression (n = 6) in ipsilateral dorsal horn neurons accompanied with behavioral allodynia (n = 7). Focal knockdown of growth arrest and DNA-damage-inducible protein 45ß expression attenuated ligation-induced allodynia (n = 7) by reducing the binding of growth arrest and DNA-damage-inducible protein 45ß to the voltage-dependent T-type calcium channel 3.2 subunit promoter (n = 6) that decreased expression of and current mediated by the voltage-dependent T-type calcium channel 3.2 subunit (both n = 6). In addition, NR2B-bearing N-methyl-D-aspartate receptors and calmodulin-dependent protein kinase II act in an upstream cascade to increase growth arrest and DNA-damage-inducible protein 45ß expression, hence enhancing demethylation at the voltage-dependent T-type calcium channel 3.2 subunit promoter and up-regulating voltage-dependent T-type calcium channel 3.2 subunit expression. Intrathecal administration of Ro 25-6981, KN-93, or a growth arrest and DNA-damage-inducible protein 45ß-targeting small interfering RNA (n = 6) reversed the ligation-induced enrichment of unmodified cytosine at the voltage-dependent T-type calcium channel 3.2 subunit promoter by increasing the associated 5-formylcytosine and 5-carboxylcytosine levels. CONCLUSIONS: By converting 5-formylcytosine or 5-carboxylcytosine to unmodified cytosine, the NR2B-bearing N-methyl-D-aspartate receptor, calmodulin-dependent protein kinase II, or growth arrest and DNA-damage-inducible protein 45ß pathway facilitates voltage-dependent T-type calcium channel 3.2 subunit gene demethylation to mediate neuropathic allodynia.

Tet1-dependent epigenetic modification of BDNF expression in dorsal horn neurons mediates neuropathic pain in rats.

Ten-eleven translocation methylcytosine dioxygenase 1 (Tet1) mediates the conversion of 5-methylcytosine (5 mC) to 5-hydroxymethylcytosine (5 hmC), hence promoting DNA demethylation. Although recent studies have linked the DNA demethylation of specific genes to pain hypersensitivity, the role of spinal Tet1-dependent DNA demethylation in nociception hypersensitivity development remains elusive. Here, we report correlated with behavioral allodynia, spinal nerve ligation (SNL) upregulated Tet1 expression in dorsal horn neurons that hydroxylate 5 mC to 5 hmC at CpG dinucleotides in the bdnf promoter to promote spinal BDNF expression at day 7 after operation. Focal knockdown of spinal Tet1 expression decreased Tet1 binding and 5 hmC enrichment, further increased 5 mC enrichment at CpG sites in the bdnf promoter and decreased spinal BDNF expression accompanied by the alleviation of the developed allodynia. Moreover, at day 7 after operation, SNL-enhanced Tet1 expression also inhibited the binding of DNA methyltransferases (DNMTs, i.e., DNMT1, DNMT3a, and DNMT3b) to the bdnf promoter, a requirement for transcriptional silencing by catalysing 5-cytosine (5C) to 5 mC. Together, these data suggest at CpG sites of the bdnf promoter, SNL-enhanced Tet1 expression promotes DNA demethylation both by converting 5 mC to 5 hmC and inhibiting DNMT binding to regulate spinal BDNF expression, hence contributing to behavioral allodynia development.

Spinal Fbxo3-Dependent Fbxl2 Ubiquitination of Active Zone Protein RIM1α Mediates Neuropathic Allodynia through CaV2.2 Activation.

UNLABELLED: Spinal plasticity, a key process mediating neuropathic pain development, requires ubiquitination-dependent protein turnover. Presynaptic active zone proteins have a crucial role in regulating vesicle exocytosis, which is essential for synaptic plasticity. Nevertheless, the mechanism for ubiquitination-regulated turnover of presynaptic active zone proteins in the progression of spinal plasticity-associated neuropathic pain remains unclear. Here, after research involving Sprague Dawley rats, we reported that spinal nerve ligation (SNL), in addition to causing allodynia, enhances the Rab3-interactive molecule-1α (RIM1α), a major active zone protein presumed to regulate neural plasticity, specifically in the synaptic plasma membranes (SPMs) of the ipsilateral dorsal horn. Spinal RIM1α-associated allodynia was mediated by Fbxo3, which abates Fbxl2-dependent RIM1α ubiquitination. Subsequently, following deubiquitination, enhanced RIM1α directly binds to CaV2.2, resulting in increased CaV2.2 expression in the SPMs of the dorsal horn. While exhibiting no effect on Fbxo3/Fbxl2 signaling, the focal knockdown of spinal RIM1α expression reversed the SNL-induced allodynia and increased spontaneous EPSC (sEPSC) frequency by suppressing RIM1α-facilitated CaV2.2 expression in the dorsal horn. Intrathecal applications of BC-1215 (a Fbxo3 activity inhibitor), Fbxl2 mRNA-targeting small-interfering RNA, and ω-conotoxin GVIA (a CaV2.2 blocker) attenuated RIM1α upregulation, enhanced RIM1α expression, and exhibited no effect on RIM1α expression, respectively. These results confirm the prediction that spinal presynaptic Fbxo3-dependent Fbxl2 ubiquitination promotes the subsequent RIM1α/CaV2.2 cascade in SNL-induced neuropathic pain. Our findings identify a role of the presynaptic active zone protein in pain-associated plasticity. That is, RIM1α-facilitated CaV2.2 expression plays a role in the downstream signaling of Fbxo3-dependent Fbxl2 ubiquitination/degradation to promote spinal plasticity underlying the progression of nociceptive hypersensitivity following neuropathic injury. SIGNIFICANCE STATEMENT: Ubiquitination is a well known process required for protein degradation. Studies investigating pain pathology have demonstrated that ubiquitination contributes to chronic pain by regulating the turnover of synaptic proteins. Here, we found that the spinal presynaptic active zone protein Rab3-interactive molecule-1α (RIM1α) participates in neuropathic pain development by binding to and upregulating the expression of CaV2.2. In addition, Fbxo3 modifies this pathway by inhibiting Fbxl2-mediated RIM1α ubiquitination, suggesting that presynaptic protein ubiquitination makes a crucial contribution to the development of neuropathic pain. Research in this area, now in its infancy, could potentially provide a novel therapeutic strategy for pain relief.

Melatonin relieves neuropathic allodynia through spinal MT2-enhanced PP2Ac and downstream HDAC4 shuttling-dependent epigenetic modification of hmgb1 transcription.

Melatonin (MLT; N-acetyl-5-methoxytryptamine) exhibits analgesic properties in chronic pain conditions. While researches linking MLT to epigenetic mechanisms have grown exponentially over recent years, very few studies have investigated the contribution of MLT-associated epigenetic modification to pain states. Here, we report that together with behavioral allodynia, spinal nerve ligation (SNL) induced a decrease in the expression of catalytic subunit of phosphatase 2A (PP2Ac) and enhanced histone deacetylase 4 (HDAC4) phosphorylation and cytoplasmic accumulation, which epigenetically alleviated HDAC4-suppressed hmgb1 gene transcription, resulting in increased high-mobility group protein B1 (HMGB1) expression selectively in the ipsilateral dorsal horn of rats. Focal knock-down of spinal PP2Ac expression also resulted in behavioral allodynia in association with similar protein expression as observed with SNL. Notably, intrathecal administration with MLT increased PP2Ac expression, HDAC4 dephosphorylation and nuclear accumulation, restored HDAC4-mediated hmgb1 suppression and relieved SNL-sensitized behavioral pain; these effects were all inhibited by spinal injection of 4P-PDOT (a MT2 receptor antagonist, 30 minutes before MLT) and okadaic acid (OA, a PP2A inhibitor, 3 hr after MLT). Our findings demonstrate a novel mechanism by which MLT ameliorates neuropathic allodynia via epigenetic modification. This MLT-exhibited anti-allodynia is mediated by MT2-enhanced PP2Ac expression that couples PP2Ac with HDAC4 to induce HDAC4 dephosphorylation and nuclear import, herein increases HDAC4 binding to the promoter of hmgb1 gene and upregulates HMGB1 expression in dorsal horn neurons.

Arecoline Alters Taste Bud Cell Morphology, Reduces Body Weight, and Induces Behavioral Preference Changes in Gustatory Discrimination in C57BL/6 Mice.

Arecoline, a major alkaloid in areca nuts, is involved in the pathogenesis of oral diseases. Mammalian taste buds are the structural unit for detecting taste stimuli in the oral cavity. The effects of arecoline on taste bud morphology are poorly understood. Arecoline was injected intraperitoneally (IP) into C57BL/6 mice twice daily for 1-4 weeks. After arecoline treatment, the vallate papillae were processed for electron microscopy and immunohistochemistry analysis of taste receptor proteins (T1R2, T1R3, T1R1, and T2R) and taste associated proteins (α-gustducin, PLCß2, and SNAP25). Body weight, food intake and water consumption were recorded. A 2-bottle preference test was also performed. The results demonstrated that 1) arecoline treatment didn't change the number and size of the taste buds or taste bud cells, 2) electron microscopy revealed the change of organelles and the accumulation of autophagosomes in type II cells, 3) immunohistochemistry demonstrated a decrease of taste receptor T1R2- and T1R3-expressing cells, 4) the body weight and food intake were markedly reduced, and 5) the sweet preference behavior was reduced. We concluded that the long-term injection of arecoline alters the morphology of type II taste bud cells, retards the growth of mice, and affects discrimination competencies for sweet tastants.

Fbxo3-Dependent Fbxl2 Ubiquitination Mediates Neuropathic Allodynia through the TRAF2/TNIK/GluR1 Cascade.

Emerging evidence has indicated that the pathogenesis of neuropathic pain is mediated by spinal neural plasticity in the dorsal horn, which provides insight for analgesic therapy. Here, we report that the abundance of tumor necrosis factor receptor-associated factor 2 and NcK-interacting kinase (TNIK), a kinase that is presumed to regulate neural plasticity, was specifically enhanced in ipsilateral dorsal horn neurons after spinal nerve ligation (SNL; left L5 and L6). Spinal TNIK-associated allodynia is mediated by downstream TNIK-GluR1 coupling and the subsequent phosphorylation-dependent trafficking of GluR1 toward the plasma membrane in dorsal horn neurons. Tumor necrosis factor receptor-associated factor 2 (TRAF2), which is regulated by spinal F-box protein 3 (Fbxo3)-dependent F-box and leucine-rich repeat protein 2 (Fbxl2) ubiquitination, contributes to SNL-induced allodynia by modifying TNIK/GluR1 phosphorylation-associated GluR1 trafficking. Although exhibiting no effect on Fbxo3/Fbxl2/TRAF2 signaling, focal knockdown of spinal TNIK expression prevented SNL-induced allodynia by attenuating TNIK/GluR1 phosphorylation-dependent subcellular GluR1 redistribution. In contrast, intrathecal administration of BC-1215 (N1,N2-Bis[[4-(2-pyridinyl)phenyl]methyl]-1,2-ethanediamine) (a novel Fbxo3 inhibitor) prevented SNL-induced Fbxl2 ubiquitination and subsequent TFAF2 de-ubiquitination to ameliorate behavioral allodynia via antagonizing TRAF2/TNIK/GluR1 signaling. By targeting spinal Fbxo3-dependent Fbxl2 ubiquitination and the subsequent TRAF2/TNIK/GluR1 cascade, spinal application of a TNF-α-neutralizing antibody ameliorated SNL-induced allodynia, and, conversely, intrathecal TNF-α injection into naive rats induced allodynia via a spinal Fbxo3/Fbxl2-dependent modification of the TRAF2/TNIK/GluR1 cascade. Together, our results suggest that spinal TNF-α contributes to the development of neuropathic pain by upregulating TRAF2/TNIK/GluR1 signaling via Fbxo3-dependent Fbxl2 ubiquitination and degradation. Thus, we propose a potential medical treatment strategy for neuropathic pain by targeting the F-box protein or TNIK. SIGNIFICANCE STATEMENT: TNF-α participates in neuropathic pain development by facilitating the spinal TRAF2-dependent TNIK-GluR1 association, which drives GluR1-containing AMPA receptor trafficking toward the plasma membrane. In addition, F-box protein 3 modifies this pathway by inhibiting F-box and leucine-rich repeat protein 2-mediated TRAF2 ubiquitination, suggesting that protein ubiquitination contributes crucially to the development of neuropathic pain. These results provide a novel therapeutic strategy for pain relief.

VPS26A-SNX27 Interaction-Dependent mGluR5 Recycling in Dorsal Horn Neurons Mediates Neuropathic Pain in Rats.

Retromer, which crucially contributes to endosomal sorting machinery through the retrieval and recycling of signaling receptors away from degradation, has been identified as a critical element for glutamatergic-receptor-dependent neural plasticity at excitatory synapses. We observed it accompanied by behavioral allodynia; neuropathic injury time-dependently enhanced VPS26A and SNX27 expression; VPS26A-SNX27 coprecipitation; and VPS26A-positive, SNX27-positive, and VPS26A-SNX27 double-labeled immunoreactivity in the dorsal horn of Sprague Dawley rats that were all sufficiently ameliorated through the focal knock-down of spinal VPS26A expression. Although the knock-down of spinal SNX27 expression exhibited similar effects, spinal nerve ligation (SNL)-enhanced VPS26A expression remained unaffected. Moreover, SNL also increased membrane-bound and total mGluR5 abundance, VPS26A-bound SNX27 and mGluR5 and mGluR5-bound VPS26A and SNX27 coprecipitation, and mGluR5-positive and VPS26A/SNX27/mGluR5 triple-labeled immunoreactivity in the dorsal horn, and these effects were all attenuated through the focal knock-down of spinal VPS26A and SNX27 expression. Although administration with MPEP adequately ameliorated SNL-associated allodynia, mGluR5 expression, and membrane insertion, SNL-enhanced VPS26A and SNX27 expression were unaffected. Together, these results suggested a role of spinal VPS26A-SNX27-dependent mGluR5 recycling in the development of neuropathic pain. This is the first study that links retromer-associated sorting machinery with the spinal plasticity underlying pain hypersensitivity and proposes the possible pathophysiological relevance of endocytic recycling in pain pathophysiology through the modification of glutamatergic mGluR5 recycling. SIGNIFICANCE STATEMENT: VPS26A-SNX27-dependent mGluR5 recycling plays a role in the development of neuropathic pain. The regulation of the VPS26A-SNX27 interaction that modifies mGluR5 trafficking and expression in the dorsal horn provides a novel therapeutic strategy for pain relief.

Neuropathic Allodynia Involves Spinal Neurexin-1ß-dependent Neuroligin-1/Postsynaptic Density-95/NR2B Cascade in Rats.

BACKGROUND: Neuroligin-1 (NL1) forms a complex with the presynaptic neurexin-1ß (Nrx1b), regulating clustering of N-methyl-D-aspartate receptors with postsynaptic density-95 (PSD-95) to underlie learning-/memory-associated plasticity. Pain-related spinal neuroplasticity shares several common features with learning-/memory-associated plasticity. The authors thereby investigated the potential involvement of NL1-related mechanism in spinal nerve ligation (SNL)-associated allodynia. METHODS: In 626 adult male Sprague-Dawley rats, the withdrawal threshold and NL1, PSD-95, phosphorylated NR2B (pNR2B) expressions, interactions, and locations in dorsal horn (L4 to L5) were compared between the sham operation and SNL groups. A recombinant Nrx1b Fc chimera (Nrx1b Fc, 10 µg, 10 µl, i.t., bolus), antisense small-interfering RNA targeting to NL1 (10 µg, 10 µl, i.t., daily for 4 days), or NR2B antagonist (Ro 25-6981; 1 µM, 10 µl, i.t., bolus) were administered to SNL animals to elucidate possible cascades involved. RESULTS: SNL-induced allodynia failed to affect NL1 or PSD-95 expression. However, pNR2B expression (mean ± SD from 13.1 ± 2.87 to 23.1 ± 2.52, n = 6) and coexpression of NL1-PSD-95, pNR2B-PSD-95, and NL1-total NR2B were enhanced by SNL (from 10.7 ± 2.27 to 22.2 ± 3.94, 11.5 ± 2.15 to 23.8 ± 3.32, and 8.9 ± 1.83 to 14.9 ± 2.27 at day 7, n = 6). Furthermore, neuron-localized pNR2B PSD-95-pNR2B double-labeled and NL1/PSD-95/pNR2B triple-labeled immunofluorescence in the ipsilateral dorsal horn was all prevented by Nrx1b Fc and NL1-targeted small-interfering RNA designed to block and prevent NL1 expression. Without affecting NL1-PSD-95 coupling, Ro 25-6981 decreased the SNL-induced PSD-95-pNR2B coprecipitation (from 18.7 ± 1.80 to 14.7 ± 2.36 at day 7, n = 6). CONCLUSION: SNL-induced allodynia, which is mediated by the spinal NL1/PSD-95/pNR2B cascade, can be prevented by blockade of transsynaptic Nrx1b-NL1 interactions.

Uremia Induces Dental Pulp Ossification but Reciprocally Inhibits Adjacent Alveolar Bone Osteogenesis.

Uremic patients are predisposed to atrophy of the alveolar bone and narrowing of the dental pulp chamber. Such pulp chamber changes have only been diagnosed radiologically; however, this has not been supported by any pathological evidence. We used a uremic rat model with secondary hyperparathyroidism induced by 5/6 nephrectomy surgery and high-phosphate diet to examine the dental pulp and adjacent alveolar bone pathology. In addition, we collected pulp tissues for real-time PCR. We found an opposite histopathological presentation of the ossified dental pulp and the osteomalacic adjacent alveolar bone. Furthermore, pulp cells with positive staining for Thy-1, a surrogate stem cell marker, were significantly reduced in the pulp of uremic rats compared to the controls, indicating a paucity of stem cells. This was further evidenced by the reduced pulp expression of dickkopf-1 (Dkk-1), a Wnt/ß-catenin signaling inhibitor produced by mesenchymal stem cells. In contrast, expressions of receptor activator of nuclear factor κB ligand (RANKL) and RANK in uremic pulp were up-regulated, probably to counteract the ossifying process of uremic pulp. In conclusion, uremic pulp ossifications were associated with a paucity of stem cells and dysregulated Dkk-1 and RANKL signaling systems, further shifting the imbalance toward osteogenesis. Strategies to counteract such an imbalance may offer a potential therapeutic target to improve dental health in uremic patients, which warrants further interventional studies.

Modulation of Nerve Injury-induced HDAC4 Cytoplasmic Retention Contributes to Neuropathic Pain in Rats.

BACKGROUND: The histone deacetylases (HDACs) have been implicated in pain hypersensitivity. This study investigated the potential involvement of an HDAC4-related mechanism in the spinal nerve ligation (SNL)-induced nociceptive hypersensitivity. METHODS: The left L5 to L6 spinal nerves of 627 adult male Sprague-Dawley rats were surgically ligated. The withdrawal threshold of hind paws and the abundances, cellular location, and interactions of proteins in the dorsal horn were assayed before and after surgery. The 14-3-3ß-targeting small-interfering RNA, a serum- and glucocorticoid-inducible kinase 1 (SGK1) antagonist, or an HDAC inhibitor was spinally injected to elucidate the role of 14-3-3ß, SGK1, and HDAC4. RESULTS: Without affecting the HDAC4 level, SNL provoked SGK1 phosphorylation (mean ± SEM from 0.24 ± 0.02 to 0.78 ± 0.06 at day 7, n = 6), HDAC4 phosphorylation (from 0.38 ± 0.03 to 0.72 ± 0.06 at day 7, n = 6), 14-3-3ß expression (from 0.53 ± 0.09 to 0.88 ± 0.09 at day 7, n = 6), cytoplasmic HDAC4 retention (from 1.18 ± 0.16 to 1.92 ± 0.11 at day 7, n = 6), and HDAC4-14-3-3ß coupling (approximately 2.4-fold) in the ipsilateral dorsal horn in association with behavioral allodynia. Knockdown of spinal 14-3-3ß expression prevented the SNL-provoked HDAC4 retention (from 1.89 ± 0.15 to 1.32 ± 0.08 at day 7, n = 6), HDAC4-14-3-3ß coupling (approximately 0.6-fold above SNL 7D), and behavioral allodynia (from 0.16 ± 0.3 to 6 ± 1.78 at day 7, n = 7), but not SGK1 (from 0.78 ± 0.06 to 0.71 ± 0.04 at day 7, n = 6) or HDAC4 (from 0.75 ± 0.15 to 0.68 ± 0.11 at day 7, n = 6) phosphorylation. CONCLUSION: Neuropathic pain maintenance involves the spinal SGK1 activation-dependent HDAC4 phosphorylation and its subsequent association with 14-3-3ß that promotes cytoplasmic HDAC4 retention in dorsal horn neurons.

Circulating Wnt/ß-catenin signalling inhibitors and uraemic vascular calcifications.

BACKGROUND: The process of vascular calcification has been associated with the canonical Wnt/ß-catenin signalling pathway in cell cultures and animal studies. The relationship between circulating Wnt/ß-catenin inhibitors and vascular calcification in dialysis patients is unknown. The aim of this study was to investigate the associations between serum dickkopf-1 (Dkk-1) and sclerostin, two circulating inhibitors of the Wnt/ß-catenin signalling pathway, and the severity of aortic calcification (AoC) and cardiovascular outcomes in dialysis patients. METHODS: This was a prospective observational cohort study. One hundred and twenty-five patients on maintenance haemodialysis participated in the study. Serum levels of Dkk-1 and sclerostin were measured. AoC scores were calculated from plain films of both posterior-anterior and lateral views. The patients were followed up for 2 years or until death or withdrawal. RESULTS: The circulating sclerostin level was inversely associated with the severity of AoC (P = 0.035) and indicators of the bone turnover rate including serum alkaline phosphatase (ALP) (r = -0.235, P = 0.008) and intact parathyroid hormone (r = -0.523, P < 0.001). Furthermore, Cox regression analysis indicated that the patients with high circulating sclerostin levels were less likely to experience future cardiovascular events [1 pmol/L sclerostin increase, hazard ratio 0.982 (95% CI, 0.967-0.996), P = 0.015] after adjusting for a propensity score. In contrast, serum Dkk-1 was not associated with AoC and clinical outcomes. CONCLUSIONS: In long-term haemodialysis patients, circulating sclerostin but not Dkk-1 is inversely associated with AoCs and future cardiovascular events. Our findings suggest that sclerostin, as a bone-related protein, might act as a communicator between uraemic bone and vasculature.