Paravertebrally-Injected Multifunctional Hydrogel for Sustained Anti-Inflammation and Pain Relief in Lumbar Disc Herniation.

Pain caused by lumbar disc herniation (LDH) severely compromises patients' quality of life. The combination of steroid and local anesthetics is routinely employed in clinics to alleviate LDH-induced pain. However, the approach only mediates transient efficacy and requires repeated and invasive lumbar epidural injections. Here a paravertebrally-injected multifunctional hydrogel that can efficiently co-load and controlled release glucocorticoid betamethasone and anesthetics ropivacaine for sustained anti-inflammation, reactive oxygen species (ROS)-removal and pain relief in LDH is presented. Betamethasone is conjugated to hyaluronic acid (HA) via ROS-responsive crosslinker to form amphiphilic polymer that self-assemble into particles with ropivacaine loaded into the core. Solution of drug-loaded particles and thermo-sensitive polymer rapidly forms therapeutic hydrogel in situ upon injection next to the herniated disc, thus avoiding invasive epidural injection. In a rat model of LDH, multifunctional hydrogel maintains the local drug concentration 72 times longer than free drugs and more effectively inhibits the expression of pro-inflammatory cytokines and pain-related molecules including cyclooxygenase-2 (COX-2) and prostaglandin E2 (PGE2). Therapeutic hydrogel suppresses the LDH-induced pain in rats for 12 days while the equivalent dose of free drugs is only effective for 3 days. This platform is also applicable to ameliorate pain caused by other spine-related diseases.

Mitochondrial control of proteasomal Psmb5 drives the differentiation of tissue-resident memory T cells in patients with rheumatoid arthritis.

OBJECTIVE: To explore T-cell-intrinsic mechanisms underpinning the mal-differentiation of tissue-resident memory T (Trm) cells in patients with rheumatoid arthritis (RA). METHODS: Circulating T cells from RA patients and healthy individuals were used for Trm cell differentiation. The role of Hobit in Trm differentiation was investigated through targeted silencing experiments. Psmb5 expression regulation was explored by identifying BRD2 as a key transcription factor, with the interaction validated through ChIP-qPCR. The impact of BRD2 succinylation on Trm differentiation was examined by manipulating succinyl-CoA levels in T cells. Humanized NSG chimeras representing synovitis provided insights into Trm infiltration in RA synovitis and were utilized for translational experiments. RESULTS: In RA patients, a notable predisposition of CD4+ T cells towards differentiation into Trm cells was observed, demonstrating a positive correlation with the Disease Activity Score 28. Remarkably, Hobit was a pivotal facilitator in the formation of RA CD4+ Trm cells. Mechanistic studies unveiled the dysregulation of proteasomal Psmb5 in T cells of RA patients as the key factor contributing to elevated Hobit protein levels. The deficiency of proteasomal Psmb5 was intricately linked to BRD2, with succinylation exerting a significant impact on Psmb5 transcription and Trm cell differentiation. This heightened BRD2 succinylation was attributed to elevated levels of mitochondrial succinyl-CoA in RA T cells. Consequently, targeting succinyl-CoA within CD4+ T cells controlled the inflammation of synovial tissues in humanized chimeras. CONCLUSION: Mitochondrial succinyl-CoA fosters the succinylation of BRD2, resulting in compromised transcription of proteasomal Psmb5 and the differentiation of Trm cells in RA.

Comparison of the total and hidden blood loss in patients undergoing single-level open and unilateral biportal endoscopic transforaminal lumbar interbody fusion: a retrospective case control study.

PURPOSE: This study aimed to compare total blood loss (TBL) and hidden blood loss (HBL) in patients undergoing single-level open transforaminal lumbar interbody fusion (O-TLIF) and unilateral biportal endoscopic transforaminal lumbar interbody fusion (ULIF). METHODS: A total of 53 patients who underwent ULIF and 53 patients who underwent O-TLIF from March 2020 to July 2022 were retrospectively reviewed. The Nadler's formula was employed to estimate the patient's blood volume (PBV), Gross's formula to estimate TBL, and Sehat's formula to estimate HBL. The obtained data were then analyzed with independent t test, chi-squared test, and analysis of covariance. RESULTS: TBL and measured blood loss (MBL) in ULIF group (326.86 ± 223.45 ml, 99.00 ± 72.81 ml) was significantly lower than O-TLIF group (427.97 ± 280.52 ml, 270.66 ± 102.34 ml). Nevertheless, the HBL in ULIF group was higher than that in O-TLIF group (227.86 ± 221.75 ml vs 157.31 ± 268.08 ml), however this was not statistically significant (p = 0.143). The HBL was 69.71 ± 23.72% of TBL in ULIF group and 36.76 ± 18.79% of TBL in O-TLIF group. Patients in ULIF group had lower TBL and MBL, shorter duration of drainage, lower postoperative anemia, and shorter postoperative hospital stay compared to those in O-TLIF group. CONCLUSIONS: Perioperative HBL should not be neglected in patients undergoing ULIF or O-TILF, as it accounts for a large percentage of TBL in both groups. ULIF is associated with lower TBL and MBL, postoperative anemia, shorter postoperative hospital stays compared with O-TLIF.

Melatonin attenuated chronic visceral pain by reducing Nav1.8 expression and nociceptive neuronal sensitization.

BACKGROUND: Irritable bowel syndrome (IBS) is a common functional gastrointestinal disorder, and its specific pathogenesis is still unclear. We have previously reported that TTX-resistant (TTX-R) sodium channels in colon-specific dorsal root ganglion (DRG) neurons were sensitized in a rat model of visceral hypersensitivity induced by neonatal colonic inflammation (NCI). However, the detailed molecular mechanism for activation of sodium channels remains unknown. This study was designed to examine roles for melatonin (MT) in sensitization of sodium channels in NCI rats. METHODS: Colorectal distention (CRD) in adult male rats as a measure of visceral hypersensitivity. Colon-specific dorsal root ganglion (DRG) neurons were labeled with DiI and acutely dissociated for measuring excitability and sodium channel current under whole-cell patch clamp configurations. Western blot and Immunofluorescence were employed to detect changes in expression of Nav1.8 and MT2. RESULTS: The results showed that rats exhibited visceral hypersensitivity after NCI treatment. Intrathecal application of melatonin significantly increased the threshold of CRD in NCI rats with a dose-dependent manner, but has no role in the control group. Whole-cell patch clamp recording showed that melatonin remarkably decreased the excitability and the density of TTX-R sodium channel in DRG neurons from NCI rats. The expression of MT2 receptor at the protein level was markedly lower in NCI rats. 8MP, an agonist of MT2 receptor, enhanced the distention threshold in NCI rats. Application of 8MP reversed the enhanced hypersensitivity of DRG neurons from NCI rats. 8MP also reduced TTX-R sodium current density and modulated dynamics of TTX-R sodium current activation. CONCLUSIONS: These data suggest that sensitization of sodium channels of colon DRG neurons in NCI rats is most likely mediated by MT2 receptor, thus identifying a potential target for treatment for chronic visceral pain in patients with IBS.

Upregulation of RAB7 is related to neuronal pyroptosis after spinal cord injury in rats.

Rab7 belongs to the Ras small GTPase superfamily, and abnormal expression of Rab7 can cause neuropathy and lipid metabolism diseases. Studies have shown that Rab7 plays a crucial role in the inner membrane translocase. However, the role of Rab7 in the regulatory mechanisms of cell survival in spinal cord injury remains unknown. We used a rat spinal cord injury (SCI) model to explore the cellular localization and expression of Rab7 after SCI in this study. Western blot analysis showed that Rab7 was expressed in the spinal cord tissue. On the first day, it significantly increased and peaked after SCI on the third day. Furthermore, western blotting also demonstrated that pyroptosis-related protein Gasdermin D (GSDMD), Caspase-1, apoptosis-associated speck-like protein (ASC) expression peaked after the third-day post-injury. Importantly, the immunohistochemistry analysis revealed that Rab7 was completely colocalized with ASC in neurons after SCI. These results suggested that Rab7 was colocalized with NeuN and ASC, involved in the pyroptosis of neurons, and closely related to the spinal cord after injury.

A new method for establishing operative channels in unilateral biportal endoscopic surgery: Technical notes and preliminary results.

BACKGROUND: The unilateral biportal endoscopic (UBE) technique has been widely used in spine surgery. At present, a traditional rigid working channel is available for the UBE system. A metal semicircular canal is located in the working channel. However, due to the metal material of the working channel, arthroscopy and instruments are constrained from moving in UBE surgery. Additionally, an assistant is needed during the procedure to hold the traditional working channel. OBJECTIVE: For simplicity of operation and convenient movement of the arthroscopy and instrument, we describe a new method for establishing operative channels in UBE surgery. METHODS: We retrospectively reviewed 50 patients who underwent unilateral biportal endoscopic discectomy (UBED) from February 2020 to August 2020 via our new method. The Oswestry Disability Index (ODI) and visual analogue scale (VAS) score were measured preoperatively and 1 month, 3 months, 6 months and 12 months postoperatively. Statistical comparisons were made using analysis of covariance and paired t tests. RESULTS: The VAS scores for back pain at the five time points were 5.20 ± 2.57, 1.96 ± 0.95, 1.50 ± 0.84, 1.64 ± 1.08 and 1.18 ± 0.39. The leg pain VAS scores were 7.02 ± 2.25, 2.02 ± 1.27, 1.48 ± 0.89, 1.32 ± 0.79 and 0.88 ± 0.52. The ODI values were 51.08 ± 19.97, 19.62 ± 15.51, 8.26 ± 7.40, and 7.54 ± 6.42 to 3.24 ± 1.10. The postoperative ODIs and VAS scores of low back pain and leg pain were significantly lower than those before surgery, and differences were statistically significant (all p< 0.05). The pressure of the closed outflow was significantly higher than that of the open outflow (37.35 ± 13.11 mm Hg vs. 24.55 ± 12.64 mm Hg p= 0.003). After we tightened the infusion strap to open the outflow, the pressure decreased significantly (26.4 ± 14.08 mm Hg vs. 37.35 ± 13.11 mm Hg p= 0.015). There were 2 cases of complications, including 1 case of postoperative recurrence and 1 case of dural tears. CONCLUSION: This study demonstrates the technical feasibility, safety, and efficacy of modified channel establishment in UBE surgery.

Retinoic Acid Receptor Gamma (RARγ) Promotes Cartilage Destruction through Positive Feedback Activation of NF-κB Pathway in Human Osteoarthritis.

Osteoarthritis (OA) is a severe inflammation-related disease which leads to cartilage destruction. The retinoic acid receptor gamma (RARγ) has been indicated to be involved in many inflammation processes. However, the role and mechanism of RARγ in cartilage destruction caused by inflammation in OA are still unknown. Here, we demonstrated that the RARγ was highly expressed in chondrocytes of OA patients compared with healthy people and was positively correlated with the damage degree of cartilage in OA. Cytokine TNF-α promoted the transcription and expression of RARγ through activating the NF-κB pathway in OA cartilage. In addition, the overexpression of RARγ resulted in the upregulation of matrix degradation and inflammation associated genes and downregulation of differentiation and collagen production genes in human normal chondrocyte C28/I2 cells. Mechanistically, overexpression of RARγ could increase the level of p-IκBα and p-P65 to regulate the expression of downstream genes. RARγ and IκBα also could interact with each other and had the same localization in C28/I2 cells. Moreover, the SD rats OA model induced by monosodium iodoacetate indicated that CD437 (RARγ agonist) and TNF-α accelerated the OA progression, including more severe cartilage layer destruction, larger knee joint diameter, and higher serum ALP levels, while LY2955303 (RARγ inhibitor) showed the opposite result. RARγ was also highly expressed in OA group and even higher in TNF-α group. In conclusion, RARγ/NF-κB positive feedback loop was activated by TNF-α in chondrocyte to promote cartilage destruction. Our data not only propose a novel and precise molecular mechanism for OA disease but also provide a prospective strategy for the treatment.

Ankylosing spondylitis complicated with andersson lesion in the lower cervical spine: A case report.

BACKGROUND: Andersson lesion (AL) is an uncommon complication in ankylosing spondylitis (AS), which is characterized by nonneoplastic bone destruction and often appears as bone destruction and sclerosis in the vertebral body and/or the area involving the intervertebral disc. According to the literature, Andersson lesion commonly occur in the thoracic and lumbar spine and rarely in the cervical spine. CASE SUMMARY: This case involved a 78-year-old man with a long history of AS who developed AL in the cervical spine (C5/6 and C6/7). One-stage anterior-posterior approach surgery was successfully performed. At the 6-month follow-up, the pain was significantly reduced, and the limb function was gradually improved. CONCLUSION: AL uncharacteristically appears in the cervical spine and tends to be misdiagnosed as vertebral metastases or spinal tuberculosis. Posterior combined with anterior surgery achieves solid biological stabilization in the treatment of AL bone destruction.

Circ-EGFR Functions as an Inhibitory Factor in the Malignant Progression of Glioma by Regulating the miR-183-5p/TUSC2 Axis.

Circular RNAs (circRNAs) have pivotal functions in regulating diverse biological processes of human tumors, including glioma. Herein, a novel circRNA epidermal growth factor receptor (circ-EGFR, hsa_circ_0080223) was researched in glioma. The molecular expression levels were analyzed via quantitative real-time polymerase chain reaction (qRT-PCR). Cell Counting Kit-8 (CCK-8) and colony formation assays were conducted to assess cell proliferation. Apoptosis was analyzed using flow cytometry. Cell migration and invasion were examined via transwell assay. Interaction relations between targets were verified using dual-luciferase reporter assay. Tumor Suppressor Candidate 2 (TUSC2) protein expression was examined by Western blot. In vivo experiment was performed by establishing xenograft model in mice. The qRT-PCR showed the downregulation of circ-EGFR and TUSC2 but the upregulation of microRNA-183-5p (miR-183-5p) in glioma samples. In vitro assays revealed that circ-EGFR overexpression induced the repression of cell proliferation, migration, and invasion but the promotion of apoptosis. Circ-EGFR was identified as a sponge of miR-183-5p and circ-EGFR-mediated glioma progression inhibition was abolished by miR-183-5p downregulation. Additionally, miR-183-5p targeted TUSC2 and miR-183-5p inhibitor impeded the development of glioma by upregulating the expression of TUSC2. Furthermore, circ-EGFR could regulate the TUSC2 level by sponging miR-183-5p. Glioma growth in vivo was also reduced by circ-EGFR via targeting the miR-183-5p/TUSC2 axis. Altogether, our results suggested that circ-EGFR inhibited the malignant progression of glioma by regulating the levels of miR-183-5p and TUSC2. Circ-EGFR may be a useful therapeutic target to antagonize the glioma progression.

Clinical comparison of three-dimensional exoscope vs. operative microscope in transforaminal lumbar interbody fusion: A retrospective case-control study.

Purpose: Here, we sought to determine the safety and feasibility of three-dimensional exoscope (3D EX). We compared data on surgery, complications, postoperative drainage, hematology, and clinical outcomes in the group that underwent transforaminal lumbar interbody fusion (TLIF) using an operative microscope (OM) relative to those treated using 3D EX. Methods: We retrospectively reviewed records on 74 patients who underwent one- or two-level TLIF from August 2019 to October 2020. Repeated measures analysis of variance was used to compare pre- and post-operative visual analogue scale (VAS) scores and oswestry disability index (ODI). We used analysis of covariance to compare pre- and post-operative erythrocyte count (RBC), hemoglobin (Hb), and hematocrit (Hct). Independent sample t-tests was used to compare postoperative drainage volume, total blood loss (TBL), visible blood loss (VBL) and hidden blood loss (HBL). Results: There were no significant differences in VAS and ODI scores between the two groups at all time points (all p > 0.05). RBC and HBL did not differ significantly between the two groups (all p > 0.05). There were significant differences in postoperative drainage volume,TBL, Hb, and Hct values when using 3D EX relative to OM in two-level TLIF (all p < 0.05), but not for one-level TLIF (all p > 0.05). The two groups differed significantly with regards to VBL when used for one- or two-level TLIF (all p < 0.05). Conclusion: Our data show that 3D EX is a suitable alternative to OM in TLIF. Relative to OM, 3D EX has important strengths in reducing perioperative bleeding in two-level TLIF.

The Role of Mitophagy in Regulating Cell Death.

Mitochondria are multifaceted organelles that serve to power critical cellular functions, including act as power generators of the cell, buffer cytosolic calcium overload, production of reactive oxygen species, and modulating cell survival. The structure and the cellular location of mitochondria are critical for their function and depend on highly regulated activities such as mitochondrial quality control (MQC) mechanisms. The MQC is regulated by several sets of processes: mitochondrial biogenesis, mitochondrial fusion and fission, mitophagy, and other mitochondrial proteostasis mechanisms such as mitochondrial unfolded protein response (mtUPR) or mitochondrial-derived vesicles (MDVs). These processes are important for the maintenance of mitochondrial homeostasis, and alterations in the mitochondrial function and signaling are known to contribute to the dysregulation of cell death pathways. Recent studies have uncovered regulatory mechanisms that control the activity of the key components for mitophagy. In this review, we discuss how mitophagy is controlled and how mitophagy impinges on health and disease through regulating cell death.

Methylation-mediated miR-214 regulates proliferation and drug sensitivity of renal cell carcinoma cells through targeting LIVIN.

LIVIN, a member of the inhibitor of apoptosis proteins (IAPs), is reported playing important roles in the development and progression of multiple human cancers. However, its underlined mechanisms in human renal cell carcinoma (RCC) are still needed to be clarified. In the present study, we reported that inhibition of miR-214 promoted the expression of LIVIN, then facilitated RCC cells growth and reduced the sensitivity of RCC cells to chemotherapeutic drugs. In constant, overexpression of miR-214 had contradictory effects. Further investigation showed that miR-214 was down-regulated in RCC because of abnormal methylation. In addition, DNA methyltransferase DNMT1, miR-214 and LIVIN are directly correlated in RCC patients. In conclusion, these results suggest that abnormal miR-214 methylation negatively regulates LIVIN, which may promote RCC cells growth and reduced the sensitivity of RCC cells to chemotherapeutic drugs.

Single-cell transcriptome-based multilayer network biomarker for predicting prognosis and therapeutic response of gliomas.

Occurrence and development of cancers are governed by complex networks of interacting intercellular and intracellular signals. The technology of single-cell RNA sequencing (scRNA-seq) provides an unprecedented opportunity for dissecting the interplay between the cancer cells and the associated microenvironment. Here we combined scRNA-seq data with clinical bulk gene expression data to develop a computational pipeline for identifying the prognostic and predictive signature that connects cancer cells and microenvironmental cells. The pipeline was applied to glioma scRNA-seq data and revealed a tumor-associated microglia/macrophage-mediated EGFR/ERBB2 feedback-crosstalk signaling module, which was defined as a multilayer network biomarker (MNB) to predict survival outcome and therapeutic response of glioma patients. We used publicly available clinical data sets from large cohorts of glioma patients to examine the prognostic significance and predictive accuracy of the MNB, which outperformed conventional gene biomarkers and other methods. Additionally, the MNB was found to be predictive of the sensitivity or resistance of glioma patients to molecularly targeted therapeutics. Moreover, the MNB was an independent and the strongest prognostic factor when adjusted for clinicopathologic risk factors and other existing gene signatures. The robustness of the MNB was further tested on additional data sets. Our study presents a promising scRNA-seq transcriptome-based multilayer network approach to elucidate the interactions between tumor cell and tumor-associated microenvironment and to identify prognostic and predictive signatures of cancer patients. The proposed MNB method may facilitate the design of more effective biomarkers for predicting prognosis and therapeutic resistance of cancer patients.

Differential regulatory network-based quantification and prioritization of key genes underlying cancer drug resistance based on time-course RNA-seq data.

Drug resistance is a major cause for the failure of cancer chemotherapy or targeted therapy. However, the molecular regulatory mechanisms controlling the dynamic evolvement of drug resistance remain poorly understood. Thus, it is important to develop methods for identifying key gene regulatory mechanisms of the resistance to specific drugs. In this study, we developed a data-driven computational framework, DryNetMC, using a differential regulatory network-based modeling and characterization strategy to quantify and prioritize key genes underlying cancer drug resistance. The DryNetMC does not only infer gene regulatory networks (GRNs) via an integrated approach, but also characterizes and quantifies dynamical network properties for measuring node importance. We used time-course RNA-seq data from glioma cells treated with dbcAMP (a cAMP activator) as a realistic case to reconstruct the GRNs for sensitive and resistant cells. Based on a novel node importance index that comprehensively quantifies network topology, network entropy and expression dynamics, the top ranked genes were verified to be predictive of the drug sensitivities of different glioma cell lines, in comparison with other existing methods. The proposed method provides a quantitative approach to gain insights into the dynamic adaptation and regulatory mechanisms of cancer drug resistance and sheds light on the design of novel biomarkers or targets for predicting or overcoming drug resistance.

Characterization of novel lnc RNAs in the spinal cord of rats with lumbar disc herniation.

BACKGROUND: Radicular pain, caused by a lesion or autologous nucleus pulposus (NP) implantation, is associated with alteration in gene expression of the pain-signaling pathways. lncRNAs have been shown to play critical roles in neuropathic pain. However, the mechanistic function of lncRNAs in lumbar disc herniation (LDH) remains largely unknown. Identifying different lncRNA expression under sham and NP-implantation conditions in the spinal cord is important for understanding the molecular mechanisms of radicular pain. METHODS: LDH was induced by implantation of autologous nucleus pulposus (NP), harvested from rat tail, in lumbar 5 and 6 spinal nerve roots. The mRNA and lncRNA microarray analyses demonstrated that the expression profiles of lncRNAs and mRNAs between the LDH and sham groups were markedly altered at 7 days post operation. The expression patterns of several mRNAs and lncRNAs were further proved by qPCR. RESULTS: LDH produced persistent mechanical and thermal hyperalgesia. A total of 19 lncRNAs was differentially expressed (>1.5-folds), of which 13 was upregulated and 6 was downregulated. In addition, a total of 103 mRNAs was markedly altered (>1.5-folds), of which 40 was upregulated and 63 downregulated. Biological analyses of these mRNAs further demonstrated that the most significantly upregulated genes in LDH included chemotaxis, immune response, and positive regulation of inflammatory responses, which might be important mechanisms underlying radicular neuropathic pain. These 19 differentially expressed lncRNAs have overlapping mRNAs in the genome, which are related to glutamatergic synapse, cytokine-cytokine receptor interaction, and the oxytocin-signalling pathway. CONCLUSION: Our findings revealed the alteration of expression patterns of mRNAs and lncRNAs in the spinal cord of rats in a radicular pain model of LDH. These mRNAs and lncRNAs might be potential therapeutic targets for the treatment of radicular pain.

Baicalein inhibits progression of osteosarcoma cells through inactivation of the Wnt/ß-catenin signaling pathway.

Osteosarcoma is a very common type of malignant bone tumor in children and young adults and aberrant activation of Wnt/ß-catenin signaling pathway has been discovered in osteosarcoma. The traditional Chinese medicine baicalein was proved to have anti-proliferative and anti-metastatic properties in osteosarcoma, but the mechanism remained poorly understood. In the present study, we assessed the effects of baicalein on osteosarcoma and detected the potential molecular mechanism. We found that baicalein significantly suppressed the proliferation of osteosarcoma cells in a concentration- and time-dependent manner. In additional, baicalein could induce apoptosis and cell cycle arrest and reduce cell motility. Moreover, the level of ß-catenin and its target genes, including c-myc, cyclinD1, and survivin significantly decreased in baicalein-treated osteosarcoma cells, whereas exogenous expression of ß-catenin could reverse the anti-proliferative and anti-metastatic effects of baicalein. Subsequently, we established a 143B xenograft tumor model and found that baicalein treatment significantly inhibited tumor growth accompanied with inhibiting Wnt/ß-catenin pathway. Thus, these findings suggest that baicalein may be a potentially effective Chinese herbal medicine for therapeutics of osteosarcoma and Wnt/ß-catenin signaling pathway may serve as an efficient molecular marker or predictive target for osteosarcoma.

Inhibition of cystathionine ß-synthetase suppresses sodium channel activities of dorsal root ganglion neurons of rats with lumbar disc herniation.

The pathogenesis of pain in lumbar disc herniation (LDH) remains poorly understood. We have recently demonstrated that voltage-gated sodium channels (VGSCs) in dorsal root ganglion (DRG) neurons were sensitized in a rat model of LDH. However, the detailed molecular mechanism for sensitization of VGSCs remains largely unknown. This study was designed to examine roles of the endogenous hydrogen sulfide synthesizing enzyme cystathionine ß-synthetase (CBS) in sensitization of VGSCs in a previously validated rat model of LDH. Here we showed that inhibition of CBS activity by O-(Carboxymethyl) hydroxylamine hemihydrochloride (AOAA) significantly attenuated pain hypersensitivity in LDH rats. Administration of AOAA also reduced neuronal hyperexcitability, suppressed the sodium current density, and right-shifted the V1/2 of the inactivation curve, of hindpaw innervating DRG neurons, which is retrogradely labeled by DiI. In vitro incubation of AOAA did not alter the excitability of acutely isolated DRG neurons. Furthermore, CBS was colocalized with NaV1.7 and NaV1.8 in hindpaw-innervating DRG neurons. Treatment of AOAA markedly suppressed expression of NaV1.7 and NaV1.8 in DRGs of LDH rats. These data suggest that targeting the CBS-H2S signaling at the DRG level might represent a novel therapeutic strategy for chronic pain relief in patients with LDH.

Hyperexcitability and sensitization of sodium channels of dorsal root ganglion neurons in a rat model of lumber disc herniation.

PURPOSE: Low back pain and sciatica are the most common symptoms of patients with lumbar disc herniation (LDH). The pathophysiology of lumbocrural pain and sciatica is not fully understood. The aim of the present study was to define the membrane properties and activities of voltage-gated sodium channels of dorsal root ganglion (DRG) neurons in a rat model of LDH. METHODS: LDH was established by transplantation of autologous nucleus pulposus (NP) to lumbar 5 and 6 spinal nerves (L5-L6 DRG) of adult male rats. Mechanical paw withdrawal threshold (PWT) and thermal paw withdrawal latency (PWL) were measured 1 day before and through 35 days after transplantation of NP. Changes in expression of VGSCs were determined by western blotting. L5-L6 DRGs neurons innervating the hindpaw were labeled with DiI and acutely dissociated for measuring excitability and sodium channel currents under whole-cell patch clamp configurations. RESULTS: NP transplantation significantly reduced the PWT and PWL in association with a significant reduction in rheobase and an increase in numbers of action potentials evoked by 2X and 3X rheobase current stimulation. Voltage-gated sodium current density was significantly enhanced in L5-L6 DRG neurons from LDH rats. The inactivation curve showed a leftward shift in LDH rats while activation curve did not significantly alter. However, NP transplantation remarkably enhanced expression of NaV1.7 and NaV1.8 in L5-L6 DRGs but not in T10-12 DRGs. CONCLUSION: These data suggest that NP application produces pain-related behavior and potentiates sodium current density of DRG neurons, which is most likely mediated by enhanced expression of NaV1.7 and NaV1.8.

Reversible ubiquitination shapes NLRC5 function and modulates NF-κB activation switch.

NLRC5 is an important regulator in innate immune responses. However, the ability of NLRC5 to inhibit NF-κB activation is controversial in different cell types. How dynamic modification of NLRC5 shapes NF-κB signaling remains unknown. We demonstrated that NLRC5 undergoes robust ubiquitination by TRAF2/6 after lipopolysaccharide treatment, which leads to dissociation of the NLRC5-IκB kinase complex. Experimental and mathematical analyses revealed that the K63-linked ubiquitination of NLRC5 at lysine 1,178 generates a coherent feedforward loop to further sensitize NF-κB activation. Meanwhile, we found USP14 specifically removes the polyubiquitin chains from NLRC5 to enhance NLRC5-mediated inhibition of NF-κB signaling. Furthermore, we found that different cell types may exhibit different sensitivities to NF-κB activation in response to NLRC5 ablation, possibly as a result of the various intrinsic levels of deubiquitinases and NLRC5. This might partially reconcile controversial studies and explain why NLRC5 exhibits diverse inhibitory efficiencies. Collectively, our results provide the regulatory mechanisms of reversible NLRC5 ubiquitination and its role in the dynamic control of innate immunity.

Sensitization of P2X3 receptors by cystathionine ß-synthetase mediates persistent pain hypersensitivity in a rat model of lumbar disc herniation.

Lumbar disc herniation (LDH) is a major cause of discogenic low back pain and sciatica, but the underlying mechanisms remain largely unknown. Hydrogen sulfide (H2S) is becoming recognized for its involvement in a wide variety of processes including inflammation and nociception. The present study was designed to investigate the roles of the H2S signaling pathway in the regulation of expression and function of purinergic receptors (P2XRs) in dorsal root ganglion (DRG) neurons from rats with LDH. LDH was induced by implantation of autologous nucleus pulposus (NP), harvested from rat tail, in lumbar 5 and 6 spinal nerve roots. Implantation of autologous NP induced persistent pain hypersensitivity, which was partially reversed by an intrathecal injection of A317491, a potent inhibitor of P2X3Rs and P2X2/3Rs. The NP induced persistent pain hypersensitivity was associated with the increased expression of P2X3Rs, but not P2X1Rs and P2X2Rs, receptors in L5-6 DRGs. NP implantation also produced a 2-fold increase in ATP-induced intracellular calcium signals in DRG neurons when compared to those of controls (P < 0.05). Interestingly, NP implantation significantly enhanced expression of the endogenous hydrogen sulfide producing enzyme, cystathionine-ß-synthetase (CBS). Systematic administration of O-(Carboxymethyl) hydroxylamine hemihydrochloride (AOAA), an inhibitor of CBS, suppressed the upregulation of P2X3R expression and the potentiation of ATP-induced intracellular calcium signals in DRG neurons (P < 0.05). Intrathecal injection of AOAA markedly attenuated NP induced- persistent pain hypersensitivity. Our results suggest that sensitization of P2X3Rs, which is likely mediated by CBS-H2S signaling in primary sensory neurons, contributes to discogenic pain. Targeting CBS/H2S-P2X3R signaling may represent a potential treatment for neuropathic pain caused by LDH.