Resolving neuroinflammatory and social deficits in ASD model mice: Dexmedetomidine downregulates NF-κB/IL-6 pathway via α2AR.

Autism spectrum disorder (ASD) is a neurodevelopmental disorder that severely affects individuals' daily life and social development. Unfortunately, there are currently no effective treatments for ASD. Dexmedetomidine (DEX) is a selective agonist of α2 adrenergic receptor (α2AR) and is widely used as a first-line medication for sedation and hypnosis in clinical practice. In recent years, there have been reports suggesting its potential positive effects on improving emotional and cognitive functions. However, whether dexmedetomidine has therapeutic effects on the core symptoms of ASD, namely social deficits and repetitive behaviors, remains to be investigated. In the present study, we employed various behavioral tests to assess the phenotypes of animals, including the three-chamber, self-grooming, marble burying, open field, and elevated plus maze. Additionally, electrophysiological recordings, western blotting, qPCR were mainly used to investigate and validate the potential mechanisms underlying the role of dexmedetomidine. We found that intraperitoneal injection of dexmedetomidine in ASD model mice-BTBR T+ Itpr3tf/J (BTBR) mice could adaptively improve their social deficits. Further, we observed a significant reduction in c-Fos positive signals and interleukin-6 (IL-6) expression level in the prelimbic cortex (PrL) of the BTBR mice treated with dexmedetomidine. Enhancing or inhibiting the action of IL-6 directly affects the social behavior of BTBR mice. Mechanistically, we have found that NF-κB p65 is a key pathway regulating IL-6 expression in the PrL region. In addition, we have confirmed that the α2AR acts as a receptor switch mediating the beneficial effects of dexmedetomidine in improving social deficits. This study provides the first evidence of the beneficial effects of dexmedetomidine on core symptoms of ASD and offers a theoretical basis and potential therapeutic approach for the clinical treatment of ASD.

MicroRNA-Based Discovery of Biomarkers, Therapeutic Targets, and Repositioning Drugs for Breast Cancer.

Breast cancer treatment can be improved with biomarkers for early detection and individualized therapy. A set of 86 microRNAs (miRNAs) were identified to separate breast cancer tumors from normal breast tissues (n = 52) with an overall accuracy of 90.4%. Six miRNAs had concordant expression in both tumors and breast cancer patient blood samples compared with the normal control samples. Twelve miRNAs showed concordant expression in tumors vs. normal breast tissues and patient survival (n = 1093), with seven as potential tumor suppressors and five as potential oncomiRs. From experimentally validated target genes of these 86 miRNAs, pan-sensitive and pan-resistant genes with concordant mRNA and protein expression associated with in-vitro drug response to 19 NCCN-recommended breast cancer drugs were selected. Combined with in-vitro proliferation assays using CRISPR-Cas9/RNAi and patient survival analysis, MEK inhibitors PD19830 and BRD-K12244279, pilocarpine, and tremorine were discovered as potential new drug options for treating breast cancer. Multi-omics biomarkers of response to the discovered drugs were identified using human breast cancer cell lines. This study presented an artificial intelligence pipeline of miRNA-based discovery of biomarkers, therapeutic targets, and repositioning drugs that can be applied to many cancer types.

NFATc2-dependent epigenetic downregulation of the TSC2/Beclin-1 pathway is involved in neuropathic pain induced by oxaliplatin.

Neuropathic pain is a common dose-limiting side effect of oxaliplatin, which hampers the effective treatment of tumors. Here, we found that upregulation of transcription factor NFATc2 decreased the expression of Beclin-1, a critical molecule in autophagy, in the spinal dorsal horn, and contributed to neuropathic pain following oxaliplatin treatment. Meanwhile, manipulating autophagy levels by intrathecal injection of rapamycin (RAPA) or 3-methyladenine (3-MA) differentially altered mechanical allodynia in oxaliplatin-treated or naïve rats. Utilizing chromatin immunoprecipitation-sequencing (ChIP-seq) assay combined with bioinformatics analysis, we found that NFATc2 negatively regulated the transcription of tuberous sclerosis complex protein 2 (TSC2), which contributed to the oxaliplatin-induced Beclin-1 downregulation. Further assays revealed that NFATc2 regulated histone H4 acetylation and methylation in the TSC2 promoter site 1 in rats' dorsal horns with oxaliplatin treatment. These results suggested that NFATc2 mediated the epigenetic downregulation of the TSC2/Beclin-1 autophagy pathway and contributed to oxaliplatin-induced mechanical allodynia, which provided a new therapeutic insight for chemotherapy-induced neuropathic pain.

Adaptation of prelimbic cortex mediated by IL-6/STAT3/Acp5 pathway contributes to the comorbidity of neuropathic pain and depression in rats.

BACKGROUND: The adaption of brain region is fundamental to the development and maintenance of nervous system disorders. The prelimbic cortex (PrL) participates in the affective components of the pain sensation. However, whether and how the adaptation of PrL contributes to the comorbidity of neuropathic pain and depression are unknown. METHODS: Using resting-state functional magnetic resonance imaging (rs-fMRI), genetic knockdown or overexpression, we systematically investigated the activity of PrL region in the pathogenesis of neuropathic pain/depression comorbid using the combined approaches of immunohistochemistry, electrophysiology, and behavior. RESULTS: The activity of PrL and the excitability of pyramidal neurons were decreased, and the osteoclastic tartrate-resistant acid phosphatase 5 (Acp5) expression in PrL neurons was upregulated following the acquisition of spared nerve injury (SNI)-induced comorbidity. Genetic knockdown of Acp5 in pyramidal neurons, but not parvalbumin (PV) neurons or somatostatin (SST) neurons, attenuated the decrease of spike number, depression-like behavior and mechanical allodynia in comorbidity rats. Overexpression of Acp5 in PrL pyramidal neurons decreased the spike number and induced the comorbid-like behavior in naïve rats. Moreover, the expression of interleukin-6 (IL-6), phosphorylated STAT3 (p-STAT3) and acetylated histone H3 (Ac-H3) were significantly increased following the acquisition of comorbidity in rats. Increased binding of STAT3 to the Acp5 gene promoter and the interaction between STAT3 and p300 enhanced acetylation of histone H3 and facilitated the transcription of Acp5 in PrL in the modeled rodents. Inhibition of IL-6/STAT3 pathway prevented the Acp5 upregulation and attenuated the comorbid-like behaviors in rats. CONCLUSIONS: These data suggest that the adaptation of PrL mediated by IL-6/STAT3/Acp5 pathway contributed to the comorbidity of neuropathic pain/depression induced by SNI.

NFATc2-dependent epigenetic upregulation of CXCL14 is involved in the development of neuropathic pain induced by paclitaxel.

BACKGROUND: The major dose-limiting toxicity of paclitaxel, one of the most commonly used drugs to treat solid tumor, is painful neuropathy. However, the molecular mechanisms underlying paclitaxel-induced painful neuropathy are largely unclarified. METHODS: Paw withdrawal threshold was measured in the rats following intraperitoneal injection of paclitaxel. The qPCR, western blotting, protein or chromatin immunoprecipitation, ChIP-seq identification of NFATc2 binding sites, and microarray analysis were performed to explore the molecular mechanism. RESULTS: We found that paclitaxel treatment increased the nuclear expression of NFATc2 in the spinal dorsal horn, and knockdown of NFATc2 with NFATc2 siRNA significantly attenuated the mechanical allodynia induced by paclitaxel. Further binding site analysis utilizing ChIP-seq assay combining with gene expression profile revealed a shift of NFATc2 binding site closer to TTS of target genes in dorsal horn after paclitaxel treatment. We further found that NFATc2 occupancy may directly upregulate the chemokine CXCL14 expression in dorsal horn, which was mediated by enhanced interaction between NFATc2 and p300 and consequently increased acetylation of histone H4 in CXCL14 promoter region. Also, knockdown of CXCL14 in dorsal horn significantly attenuated mechanical allodynia induced by paclitaxel. CONCLUSION: These results suggested that enhanced interaction between p300 and NFATc2 mediated the epigenetic upregulation of CXCL14 in the spinal dorsal horn, which contributed to the chemotherapeutic paclitaxel-induced chronic pain.

Oxaliplatin-induced neuropathic pain involves HOXA6 via a TET1-dependent demethylation of the SOX10 promoter.

Chemotherapy-induced neuropathic pain is a common dose-limiting side effect of cancer treatment but the underlying mechanisms are largely unknown. Here, we used a whole-genome expression microarray and gene ontology analysis to identify the upregulation of a sequence-specific DNA-binding protein, HOXA6, in the spinal dorsal horn on Day 10 after injection of rats with oxaliplatin. Genetic disruption of HOXA6 with siRNAs alleviated mechanical allodynia after oxaliplatin administration. Reduced representation bisulfite sequencing assays indicated that oxaliplatin decreased the methylation levels of the SOX10 promoter but not of HOXA6. TET1 was also upregulated by oxaliplatin. Genetic disruption of TET1 with siRNA blocked the promoter demethylation of SOX10 and the upregulation of HOXA6 and SOX10. Importantly, inhibition of SOX10 by intrathecal application of SOX10 siRNA ameliorated the mechanical allodynia induced by oxaliplatin and downregulated the expression of HOXA6. Consistently, overexpression of SOX10 through intraspinal injection of AAV-SOX10-EGFP produced mechanical allodynia and upregulated the expression of spinal dorsal horn HOXA6. Moreover, chromatin immunoprecipitation assays demonstrated that oxaliplatin increased the binding of SOX10 to the promoter region of HOXA6. Taken together, our data suggest that HOXA6 upregulation through the TET1-mediated promoter demethylation of SOX10 may contribute to oxaliplatin-induced neuropathic pain.

Upregulation of TRPC6 Mediated by PAX6 Hypomethylation Is Involved in the Mechanical Allodynia Induced by Chemotherapeutics in Dorsal Root Ganglion.

BACKGROUND: Although the action mechanism of antineoplastic agents is different, oxaliplatin, paclitaxel, or bortezomib as first-line antineoplastic drugs can induce painful neuropathy. In rodents, mechanical allodynia is a common phenotype of painful neuropathy for 3 chemotherapeutics. However, whether there is a common molecular involved in the different chemotherapeutics-induced painful peripheral neuropathy remains unclear. METHODS: Mechanical allodynia was tested by von Frey hairs following i.p. injection of vehicle, oxaliplatin, paclitaxel, or bortezomib in Sprague-Dawley rats. Reduced representation bisulfite sequencing and methylated DNA immunoprecipitation were used to detect the change of DNA methylation. Western blot, quantitative polymerase chain reaction, chromatin immunoprecipitation, and immunohistochemistry were employed to explore the molecular mechanisms. RESULTS: In 3 chemotherapeutic models, oxaliplatin, paclitaxel, or bortezomib accordantly upregulated the expression of transient receptor potential cation channel, subfamily C6 (TRPC6) mRNA and protein without affecting the DNA methylation level of TRPC6 gene in DRG. Inhibition of TRPC6 by using TRPC6 siRNA (i.t., 10 consecutive days) relieved mechanical allodynia significantly following application of chemotherapeutics. Furthermore, the downregulated recruitment of DNA methyltransferase 3 beta (DNMT3b) at paired box protein 6 (PAX6) gene led to the hypomethylation of PAX6 gene and increased PAX6 expression. Finally, the increased PAX6 via binding to the TPRC6 promoter contributes to the TRPC6 increase and mechanical allodynia following chemotherapeutics treatment. CONCLUSIONS: The TRPC6 upregulation through DNMT3b-mediated PAX6 gene hypomethylation participated in mechanical allodynia following application of different chemotherapeutic drugs.

Randomized Trial on Comparison of the Efficacy of Extracorporeal Shock Wave Therapy and Dry Needling in Myofascial Trigger Points.

OBJECTIVE: The aim of the study was to compare the efficacy of radial extracorporeal shock wave therapy and dry needling in the treatment of myofascial trigger points in the upper trapezius muscle. DESIGN: A total of 65 patients with myofascial trigger points were randomly divided into extracorporeal shock wave therapy group (n = 32) and dry needling group (n = 33). Patients received 3 wks of treatment at 1-wk intervals (in both groups). Visual analog scale, pressure pain threshold, Neck Disability Index, and shear modulus were evaluated before treatment, immediately after the first therapy, 1 mo, and 3 mos after the completion of the third therapy. RESULTS: Significant improvements of visual analog scale, pressure pain threshold, and Neck Disability Index scores were observed at all time points after treatment (P < 0.01) in both treatment groups. The shear modulus of myofascial trigger points was reduced in both dry needling group (P < 0.05) and extracorporeal shock wave therapy group (P < 0.01) immediately after the first treatment. Significant reductions in shear modulus were maintained up to 3-mo posttreatment in both groups (P < 0.01). There were no significant differences between the radial extracorporeal shock wave therapy group and dry needling group. CONCLUSIONS: The extracorporeal shock wave therapy is as effective as dry needling for relieving pain, improving function, and reducing shear modulus for patients with myofascial trigger points after a series of three treatments.

Comparison of two cohorts of medically at-risk adolescents engaging in substance use (cancer survivors and asthmatics): Clinical predictors for monitoring care.

BACKGROUND AND PURPOSE: Medically at-risk adolescents differ in their perception of severity and are vulnerable to substance use because of effects on their medical regimen. The intent in comparing two cohorts, adolescent survivors of cancer and teens with asthma, is to provide clinical predictors to help in monitoring those needing help with substance use decision making. METHODS: Baseline data were obtained from two randomized controlled trials for a decision-making program of research for medically at-risk adolescents. Multivariate analyses were used to identify clinical predictors for poor decision making as well as lifetime and current substance use (smoking, alcohol use, and marijuana use). CONCLUSIONS: Predictors for both cohorts for lifetime and current substance use were increasing age and risk motivation. A significant predictor for both cohorts for poor decision making related to substance use was risk motivation, measured as a more positive attitude for engaging in substance use. Negative modeling by peers and family members had an impact on teen survivors' decision making; but, this was not clear for teens with asthma. IMPLICATIONS FOR PRACTICE: Research is needed comparing other medically at-risk adolescents to determine which cohorts on the substance use spectrum are less resilient to peer and parent modeling, have unrealistic views of their decision-making skills, and need close monitoring and guidance.

Upregulation of tumor necrosis factor-alpha in the anterior cingulate cortex contributes to neuropathic pain and pain-associated aversion.

Injury associated pain involves subjective perception and emotional experience. The anterior cingulate cortex (ACC) is a key area involved in the affective component of pain processing. However, the neuroimmune mechanisms underlying enhanced ACC excitability following peripheral nerve injury are still not fully understood. Our previous work has shown that tumor necrosis factor-alpha (TNF-α) overexpression leads to peripheral afferent hyperexcitability and synaptic transmission potentiation in spinal cord. Here, we aimed to reveal the potential role of ACC TNF-α in ACC hyperexcitability and neuropathic pain. c-Fos, a widely used neuronal activity marker, was induced especially in contralateral ACC early [postoperative (PO) 1 h] and later (PO day 7 and 10) during the development of neuropathic pain. Spared nerve injury (SNI) elevated TNF-α level in contralateral ACC from PO day 5 to 14, delayed relative to decreased ipsilateral paw withdrawal threshold apparent from PO day 1 to 14. Microinjection of anti-TNF-α antibody into the ACC completely eliminated c-Fos overexpression and greatly attenuated pain aversion and mechanical allodynia induced by SNI, suggesting an important role of ACC TNF-α in the pain aversiveness and pain maintenance. Furthermore, modulating ACC pyramidal neurons via a Gi-coupled human M4 muscarinic receptor (hM4Di) or a Gq-coupled human M3 muscarinic receptor (hM3Dq), a type of designer receptors exclusively activated by designer drugs (DREADD), greatly changed the ACC TNF-α level and the mechanical paw withdrawal threshold. The positive interactions between TNF-α and ACC neurons might modulate the cytokine microenvironment thus contribute to the neuropathic pain.

TNF-α/STAT3 pathway epigenetically upregulates Nav1.6 expression in DRG and contributes to neuropathic pain induced by L5-VRT.

BACKGROUND: Studies showed that upregulation of Nav1.6 increased the neuronal excitability and participated in neuropathic pain in the dorsal root ganglion (DRG). However, the molecular mechanisms underlying Nav1.6 upregulation were not reported yet. METHODS: The paw withdrawal threshold was measured in the rodents following lumbar 5 ventral root transection (L5-VRT). Then qPCR, western blotting, immunoprecipitation, immunohistochemistry, and chromatin immunoprecipitation assays were performed to explore the molecular mechanisms in vivo and in vitro. RESULTS: We found that the levels of Nav1.6 and phosphorylated STAT3 were significantly increased in DRG neurons following L5-VRT, and TNF-α incubation also upregulated the Nav1.6 expression in cultured DRG neurons. Furthermore, immunoprecipitation and chromatin immunoprecipitation assays demonstrated that L5-VRT increased the binding of STAT3 to the Scn8a (encoding Nav1.6) promoter and the interaction between STAT3 and p300, which contributed to the enhanced transcription of Scn8a by increasing histone H4 acetylation in Scn8a promoter in DRG. Importantly, intraperitoneal injection of the TNF-α inhibitor thalidomide reduced the phosphorylation of STAT3 and decreased the recruitment of STAT3 and histone H4 hyperacetylation in the Scn8a promoter, thus subsequently attenuating Nav1.6 upregulation in DRG neurons and mechanical allodynia induced by L5-VRT. CONCLUSION: These results suggested a new mechanism for Nav1.6 upregulation involving TNF-α/STAT3 pathway activation and subsequent STAT3-mediated histone H4 hyperacetylation in the Scn8a promoter region in DRG, which contributed to L5-VRT-induced neuropathic pain.

Reduction of SIRT1 epigenetically upregulates NALP1 expression and contributes to neuropathic pain induced by chemotherapeutic drug bortezomib.

BACKGROUND: Bortezomib is a frequently used chemotherapeutic drug for the treatment of multiple myeloma and other nonsolid malignancies. Accumulating evidence has demonstrated that bortezomib-induced persistent pain serves as the most frequent reason for treatment discontinuation. METHODS: The von Frey test was performed to evaluate neuropathic pain behavior, and real-time quantitative reverse transcription polymerase chain reaction, chromatin immunoprecipitation, western blot, immunohistochemistry, and small interfering RNA were performed to explore the molecular mechanisms in adult male Sprague-Dawley rats. RESULTS: We found that application of bortezomib significantly increased the expression of NALP1 protein and mRNA levels in spinal dorsal horn neurons, and intrathecal application of NALP1 siRNA attenuated the bortezomib-induced mechanical allodynia. In addition, bortezomib also decreased the SIRT1 expression, and treatment with SIRT1 activator resveratrol ameliorated the NALP1 upregulation and mechanical allodynia induced by bortezomib. Meanwhile, knockdown of SIRT1 using the SIRT1 siRNA induced the NALP1 upregulation in dorsal horn and mechanical allodynia in normal animal. These results suggested that reduction of SIRT1 induced the NALP1 upregulation in dorsal horn neurons, and participated in bortezomib-induced mechanical allodynia. Importantly, we found that the binding of SIRT1 and NALP1 promoter region did not change before and after bortezomib treatment, but SIRT1 downregulation increased p-STAT3 expression. Furthermore, the activation of STAT3 enhanced the recruitment of p-STAT3 to the Nalp1 gene promoter, which increased the acetylation of histone H3 and H4 in NALP1 promoter regions and epigenetically upregulated NALP1 expression in the rodents with bortezomib treatment. CONCLUSION: These findings suggested a new epigenetic mechanism for NALP1 upregulation involving SIRT1 reduction and subsequent STAT3-mediated histone hyperacetylation in NALP1 promoter region in dorsal horn neurons, which contributed to the bortezomib-induced mechanical allodynia.

Upregulation of NLRP3 via STAT3-dependent histone acetylation contributes to painful neuropathy induced by bortezomib.

Painful neuropathy, as a severe side effect of chemotherapeutic bortezomib, is the most common reason for treatment discontinuation. However, the mechanism by which administration of bortezomib leads to painful neuropathy remains unclear. In the present study, we found that application of bortezomib significantly increased the expression of NOD-like receptor family pyrin domain containing 3 (NLRP3) and phosphorylated signal transducer and activator of transcription-3 (STAT3) in dorsal root ganglion (DRG). Intrathecal injection of NLRP3 siRNA significantly prevented the mechanical allodynia induced by bortezomib treatment, and intrathecal injection of recombinant adeno-associated virus vector encoding NLRP3 markedly decreased paw withdrawal threshold of naive rats. Furthermore, the expressions of p-STAT3 were colocalized with NLRP3-positive cells in DRG neurons, and inhibition of STAT3 by intrathecal injection of AAV-Cre-GFP into STAT3flox/flox mice or inhibitor S3I-201 suppressed the upregulation of NLRP3 and mechanical allodynia induced by bortezomib treatment. Chromatin immunoprecipitation further found that bortezomib increased the recruitment of STAT3, as well as the acetylation of histone H3 and H4, in the NLRP3 promoter region in DRG neurons. Importantly, inhibition of the STAT3 activity by using S3I-201 or DRG local deficiency of STAT3 also significantly prevented the upregulated H3 and H4 acetylation in the NLRP3 promoter region following bortezomib treatment. Altogether, our results suggest that the upregulation of NLRP3 in DRG via STAT3-dependent histone acetylation is critically involved in bortezomib-induced mechanical allodynia.

AKAP150 involved in paclitaxel-induced neuropathic pain via inhibiting CN/NFAT2 pathway and downregulating IL-4.

Antitubulin chemotherapeutics agents, such as paclitaxel, are effective chemotherapy drugs for cancer treatment. However, painful neuropathy is a major adverse effect limiting the wider application of chemotherapeutics. In this study, we found that A-kinase anchor protein 150 (AKAP150) was significantly upregulated after paclitaxel injection. Inhibition of AKAP150 via siRNA or AKAP150flox/flox in rodents alleviated the pain behavior induced by paclitaxel, and partly restored the decreased calcineurin (CN) phosphatase activity after paclitaxel treatment. Paclitaxel decreased the expression of anti-inflammatory cytokine interleukin-4 (IL-4), and intrathecal injections of IL-4 effectively alleviated paclitaxel-induced hypersensitivity and the frequency of dorsal root ganglion (DRG) neurons action potential. The decreased CN enzyme activity, resulted in reduced protein expression of nuclear factor of activated T cells 2 (NFAT2) in cell nuclei. Chromatin immunoprecipitation showed that, NFAT2 binds to the IL-4 gene promoter regulating the protein expression of IL-4. Overexpression of NFAT2 by intrathecal injection of the AAV5-NFAT2-GFP virus alleviated the pain behavior induced by paclitaxel via increasing the expression of IL-4. Knocked down AKAP150 by siRNA or AAV5-Cre-GFP partly restored the expression of IL-4 in DRG. Our results indicated that regulation of IL-4 via the CN/NFAT2 pathway mediated by AKAP150 could be a pivotal treatment target for paclitaxel-induced neuropathic pain and or other neuropsychiatric disorders.

Haptoglobin 2-2 Phenotype Is Associated With Increased Acute Kidney Injury After Elective Cardiac Surgery in Patients With Diabetes Mellitus.

BACKGROUND: Recent studies reported an association between the 2-2 phenotype of haptoglobin (Hp 2-2) and increased cardiorenal morbidity in nonsurgical diabetic patients. Our goal was to determine whether the Hp 2-2 phenotype was associated with acute kidney injury (AKI) after elective cardiac surgery in patients with diabetes mellitus. METHODS AND RESULTS: We prospectively enrolled 99 diabetic patients requiring elective cardiac surgery with cardiopulmonary bypass. Haptoglobin phenotypes were determined by gel electrophoresis. Cell-free hemoglobin, haptoglobin, and total serum bilirubin were quantified as hemolysis markers. The primary outcome was postoperative AKI, as defined by the Acute Kidney Injury Network classification. The incidence of AKI was significantly higher in Hp 2-2 patients compared with patients without this phenotype (non-Hp-2-2; 55.6% versus 27%, P<0.01). The need for renal replacement therapy was also significantly higher in the Hp 2-2 group (5 patients versus 1 patient, P=0.02). Thirty-day mortality (3 versus 0 patients, P=0.04) and 1-year mortality (5 versus 0 patients, P<0.01) were also significantly higher in patients with the Hp 2-2 phenotype. In multivariable analysis, Hp 2-2 was an independent predictor of postoperative AKI (P=0.01; odds ratio: 4.17; 95% confidence interval, 1.35-12.48). CONCLUSIONS: Hp 2-2 phenotype is an independent predictor of postoperative AKI and is associated with decreased short and long-term survival after cardiac surgery in patients with diabetes mellitus.

Activation of RAGE/STAT3 pathway by methylglyoxal contributes to spinal central sensitization and persistent pain induced by bortezomib.

Bortezomib is a first-line chemotherapeutic drug widely used for multiple myeloma and other nonsolid malignancies. Although bortezomib-induced persistent pain is easily diagnosed in clinic, the pathogenic mechanism remains unclear. Here, we studied this issue with use of a rat model of systemic intraperitoneal administration of bortezomib for consecutive 5days. Consisted with our previous study, we found that bortezomib treatment markedly induced mechanical allodynia in rats. Furthermore, we first found that bortezomib treatment significantly induced the upregulation of methylglyoxal in spinal dorsal horn of rats. Spinal local application of methylglyoxal also induced mechanical allodynia and central sensitization in normal rats. Moreover, administration of bortezomib upregulated the expression of receptors for advanced glycation end products (RAGE) and phosphorylated STAT3 (p-STAT3) in dorsal horn. Importantly, intrathecal injection of metformin, a known scavenger of methylglyoxal, significantly attenuated the upregulation of methylglyoxal and RAGE in dorsal horn, central sensitization and mechanical allodynia induced by bortezomib treatment, and blockage of RAGE also prevented the upregulation of p-STAT3, central sensitization and mechanical allodynia induced by bortezomib treatment. In addition, inhibition of STAT3 activity by S3I-201 attenuated bortezomib-induced mechanical allodynia and central sensitization. Local knockdown of STAT3 also ameliorated the mechanical allodynia induced by bortezomib administration. Our results suggest that accumulation of methylglyoxal may activate the RAGE/STAT3 signaling pathway in dorsal horn, and contributes to the spinal central sensitization and persistent pain induced by bortezomib treatment.

Epigenetic upregulation of CXCL12 expression mediates antitubulin chemotherapeutics-induced neuropathic pain.

Clinically, Microtubule-targeted agents-induced neuropathic pain hampers chemotherapeutics for patients with cancer. Here, we found that application of paclitaxel or vincristine increased the protein and mRNA expression of CXCL12 and frequency and amplitude of miniature excitatory post synaptic currents (mEPSCs) in spinal dorsal horn neurons. Spinal local application of CXCL12 induced the long-term potentiation of nociceptive synaptic transmission and increased the amplitude of mEPSCs. Inhibition of CXCL12 using the transgenic mice (CXCL12) or neutralizing antibody or siRNA ameliorated the mEPSC's enhancement and mechanical allodynia. In addition, paclitaxel and vincristine both could increase the phosphorylation of signal transducer and activator of transcription 3 (STAT3) and the acetylation of histone H4 in the CXCL12-expressing neurons. Immunoprecipitation and chromatin immunoprecipitation assays demonstrated that antitubulin chemotherapeutics increased the binding of STAT3 to the CXCL12 gene promoter and the interaction between STAT3 and p300, and contributed to the enhanced transcription of CXCL12 by increasing the acetylation of histone H4 in CXCL12 gene promoter. Inhibition of STAT3 by intrathecal injection of adeno-associated virus encoding Cre and green fluorescent protein into STAT3 mice or inhibitor S3I-201 into rats suppressed the CXCL12 upsurge by decreasing the acetylation of histone H4. Finally, blockade of CXCR4 but not CXCR7 ameliorated the paclitaxel- or vincristine-induced mechanical allodynia. Together, these results suggested that enhanced interaction between STAT3 and p300 mediated the epigenetic upregulation of CXCL12 in dorsal horn neurons, which contributed to the antitubulin chemotherapeutics-induced persistent pain.

TNF-α Differentially Regulates Synaptic Plasticity in the Hippocampus and Spinal Cord by Microglia-Dependent Mechanisms after Peripheral Nerve Injury.

Clinical studies show that chronic pain is accompanied by memory deficits and reduction in hippocampal volume. Experimental studies show that spared nerve injury (SNI) of the sciatic nerve induces long-term potentiation (LTP) at C-fiber synapses in spinal dorsal horn, but impairs LTP in the hippocampus. The opposite changes may contribute to neuropathic pain and memory deficits, respectively. However, the cellular and molecular mechanisms underlying the functional synaptic changes are unclear. Here, we show that the dendrite lengths and spine densities are reduced significantly in hippocampal CA1 pyramidal neurons, but increased in spinal neurokinin-1-positive neurons in mice after SNI, indicating that the excitatory synaptic connectivity is reduced in hippocampus but enhanced in spinal dorsal horn in this neuropathic pain model. Mechanistically, tumor necrosis factor-alpha (TNF-α) is upregulated in bilateral hippocampus and in ipsilateral spinal dorsal horn, whereas brain-derived neurotrophic factor (BDNF) is decreased in the hippocampus but increased in the ipsilateral spinal dorsal horn after SNI. Importantly, the SNI-induced opposite changes in synaptic connectivity and BDNF expression are prevented by genetic deletion of TNF receptor 1 in vivo and are mimicked by TNF-α in cultured slices. Furthermore, SNI activated microglia in both spinal dorsal horn and hippocampus; pharmacological inhibition or genetic ablation of microglia prevented the region-dependent synaptic changes, neuropathic pain, and memory deficits induced by SNI. The data suggest that neuropathic pain involves different structural synaptic alterations in spinal and hippocampal neurons that are mediated by overproduction of TNF-α and microglial activation and may underlie chronic pain and memory deficits. SIGNIFICANCE STATEMENT: Chronic pain is often accompanied by memory deficits. Previous studies have shown that peripheral nerve injury produces both neuropathic pain and memory deficits and induces long-term potentiation (LTP) at C-fiber synapses in spinal dorsal horn (SDH) but inhibits LTP in hippocampus. The opposite changes in synaptic plasticity may contribute to chronic pain and memory deficits, respectively. However, the structural and molecular bases of these alterations of synaptic plasticity are unclear. Here, we show that the complexity of excitatory synaptic connectivity and brain-derived neurotrophic factor (BDNF) expression are enhanced in SDH but reduced in the hippocampus in neuropathic pain and the opposite changes depend on tumor necrosis factor-alpha/tumor necrosis factor receptor 1 signaling and microglial activation. The region-dependent synaptic alterations may underlie chronic neuropathic pain and memory deficits induced by peripheral nerve injury.

The inhibition of spinal synaptic plasticity mediated by activation of AMP-activated protein kinase signaling alleviates the acute pain induced by oxaliplatin.

Our recent findings demonstrated that oxaliplatin entering CNS may directly induce spinal central sensitization, and contribute to the rapid development of CNS-related side effects including acute pain during chemotherapy. However, the mechanism is largely unclear. In the current study, we found that the amplitude of C-fiber-evoked field potentials was significantly increased and the expression of phosphorylated mammalian AMP-activated protein kinase α (AMPKα) was markedly decreased following high frequency stimulation (HFS) or single intraperitoneal injection of oxaliplatin (4mg/kg). Spinal local application of AMPK agonist metformin (25µg) prevented the long term potentiation (LTP) induction and the activation of mTOR/p70S6K signal pathway, and significantly attenuated the acute thermal hyperalgesia and mechanical allodynia following single oxaliplatin treatment. Importantly, we found that incubation of low concentration oxaliplatin at dose of 6.6nM (the detected concentration in CSF following a single intraperitoneal injection of oxaliplatin) also significantly inhibited the AMPKα activation and increased the amplitude of sEPSCs, the number of action potential, and the expression of p-mTOR and p-p70S6K in spinal cord slices. Metformin (25µg) or rapamycin (2µg) inhibited the increased excitability of dorsal horn neurons and the decrease of p-AMPKα expression induced by low concentration oxaliplatin incubation. Furthermore, spinal application of AMPK inhibitor compound C (5µg) induced the spinal LTP, thermal hyperalgesia and mechanical allodynia, and rapamycin attenuated the spinal LTP, the thermal hyperalgesia and mechanical allodynia following oxaliplatin treatment (i.p.). Local application of metformin significantly decreased the mTOR and p70S6K activation induced by tetanus stimulation or oxaliplatin (i.p.). These results suggested that the decreased AMPKα activity via negatively regulating mTOR/p70S6K signal pathway enhanced the synaptic plasticity and contributed to acute pain induced by low concentration of oxaliplatin entering CNS.

mir-500-Mediated GAD67 Downregulation Contributes to Neuropathic Pain.

UNLABELLED: Neuropathic pain is a common neurobiological disease involving multifaceted maladaptations ranging from gene modulation to synaptic dysfunction, but the interactions between synaptic dysfunction and the genes that are involved in persistent pain remain elusive. In the present study, we found that neuropathic pain induced by the chemotherapeutic drug paclitaxel or L5 ventral root transection significantly impaired the function of GABAergic synapses of spinal dorsal horn neurons via the reduction of the GAD67 expression. We also found that mir-500 expression was significantly increased and involved in the modulation of GAD67 expression via targeting the specific site of Gad1 gene in the dorsal horn. In addition, knock-out of mir-500 or using mir-500 antagomir rescued the GABAergic synapses in the spinal dorsal horn neurons and attenuated the sensitized pain behavior in the rats with neuropathic pain. To our knowledge, this is the first study to investigate the function significance and the underlying molecular mechanisms of mir-500 in the process of neuropathic pain, which sheds light on the development of novel therapeutic options for neuropathic pain. SIGNIFICANCE STATEMENT: Neuropathic pain is a common neurobiological disease involving multifaceted maladaptations ranging from gene modulation to synaptic dysfunction, but the underlying molecular mechanisms remain elusive. The present study illustrates for the first time a mir-500-mediated mechanism underlying spinal GABAergic dysfunction and sensitized pain behavior in neuropathic pain induced by the chemotherapeutic drug paclitaxel or L5 ventral root transection, which sheds light on the development of novel therapeutic options for neuropathic pain.