Animal Studies

BRCA1 (Breast Cancer-Associated Protein 1) is a human tumor suppressor that functions as an ubiquitin (Ub) ligase enzyme (E3) and plays a key role in genomic stability and DNA repair. Heterodimerization of BRCA1 with BARD1 (BRCA1-associated RING domain protein 1) is known to increase its Ub ligase activity and is important for its stability, and cooperative activation of UbcH5c (Ub conjugating enzyme (E2)). Recent studies demonstrate the importance of ubiquitination of the nucleosomal H2A C-terminal tail by BRCA1/BARD1-UbcH5c in which its mutations inhibit ubiquitination, predispose cells to chromosomal instability and greatly increase the likelihood of breast and ovarian cancer development. Due to the lack of molecular-level insight on the flexible and dis-ordered H2A C-tail, its ubiquitination mechanism by BRCA1/BARD1-UbcH5c and its function and relationship to cancer susceptibility remain elusive. Here, we use molecular dynamics simulations to provide molecular-level insights into the dynamics of the less-studied H2A C-tail and BRCA1/BARD1-UbcH5c on the nucleosome surface and their effect on ubiquitination. Our results precisely identify the key interactions and residues that trigger conformational transitions of BRCA1/BARD1-UbcH5c, and characterize the important role of histone electrostatics in their dynamics. We provide a mechanistic basis for the H2A C-tail lysine approach to UbcH5c and show the role of H2A C-tail and UbcH5c dynamics in lysine ubiquitination. Furthermore, our data demonstrate the potential for ubiquitination based on the lysine position of the C-tail. Altogether, the findings of this study provide unrevealed insights into the mechanism of H2A C-tail ubiquitination and help us understand the communication between Ub ligase/Ub conjugating enzymes (E3/E2) and nucleosome to regulate ubiquitination machinery, paving the way for the development of effective treatments for cancer and chronic pain.

To investigate the role of DNA methylation in modulating chronic neuropathic pain (NPP), identify possible target genes of DNA methylation involved in this process, and preliminarily confirm the medicinal value of the DNA methyltransferases (DNMTs) inhibitor 5-azacytidine (5-AZA) in NPP by targeting gene methylation. Two rat NPP models, chronic constriction injury (CCI) and spinal nerve ligation (SNL), were used. The DNA methylation profiles in the lumbar spinal cord were assayed using an Arraystar Rat RefSeq Promoter Array. The underlying genes with differential methylation were then identified and submitted to Gene Ontology and pathway analysis. Methyl-DNA immunoprecipitation quantitative PCR (MeDIP-qPCR) and quantitative reverse transcription-PCR (RT-qPCR) were used to confirm gene methylation and expression. The protective function of 5-AZA in NPP and gene expression were evaluated via behavioral assays and RT-qPCR, respectively. Analysis of the DNA methylation patterns in the lumbar spinal cord indicated that 1205 differentially methylated fragments in CCI rats were located within DNA promoter regions, including 638 hypermethylated fragments and 567 hypomethylated fragments. The methylation levels of Grm4, Htr4, Adrb2, Kcnf1, Gad2, and Pparg, which are associated with long-term potentiation (LTP) and glutamatergic synapse pathways, were increased with a corresponding decrease in their mRNA expression, in the spinal cords of CCI rats. Moreover, we found that the intraperitoneal injection of 5-AZA (4 mg/kg) attenuated CCI- or SNL-induced mechanical allodynia and thermal hyperalgesia. Finally, the mRNA expression of hypermethylated genes such as Grm4, Htr4, Adrb2, Kcnf1, and Gad2 was reversed after 5-AZA treatment. CCI induced widespread methylation changes in the DNA promoter regions in the lumbar spinal cord. Intraperitoneal 5-AZA alleviated hyperalgesia in CCI and SNL rats, an effect accompanied by the reversed expression of hypermethylated genes. Thus, DNA methylation inhibition represents a promising epigenetic strategy for protection against chronic NPP following nerve injury. Our study lays a theoretical foundation for 5-AZA to become a clinical targeted drug.

Projections from the periaqueductal gray (PAG) to the rostral ventromedial medulla (RVM) are known to engage in descending pain modulation, but how the neural substrates of the PAG-RVM projections contribute to neuropathic pain remains largely unknown. In this study, we showed somatostatin-expressing glutamatergic neurons in the lateral/ventrolateral PAG that facilitate mechanical and thermal hypersensitivity in a mouse model of chemotherapy-induced neuropathic pain. We found that these neurons form direct excitatory connections with neurons in the RVM region. Inhibition of this PAG-RVM projection alleviates mechanical and thermal hypersensitivity associated with neuropathy, whereas its activation enhances hypersensitivity in the mice. Thus, our findings revealed that somatostatin neurons within the PAG-RVM axial are crucial for descending pain facilitation and can potentially be exploited as a useful therapeutic target for neuropathic pain. PERSPECTIVE: We report the profound contribution of somatostatin neurons within the PAG-RVM projections to descending pain facilitation underlying neuropathic pain. These results may help to develop central therapeutic strategies for neuropathic pain.

Intervertebral disc degeneration (IVDD) is a degenerative disease characterized by lower-back pain, causing disability globally. Antioxidant therapy is currently considered one of the most promising strategies for IVDD treatment, given the crucial role of reactive oxygen species (ROS) in IVDD pathogenesis. Herein, a ROS-responsive magnesium-containing microsphere (Mg@PLPE MS) was constructed for the antioxidative treatment of IVDD. The Mg@PLPE MS has a core-shell structure comprising poly(lactic-co-glycolic acid) (PLGA) and ROS-responsive polymer poly(PBT-co-EGDM) as the shell and a magnesium microparticle as the core. The poly(PBT-co-EGDM) can be destroyed by HO through the HO-triggered hydrophobic-to-hydrophilic transition, subsequently promoting an Mg-water reaction to produce H. Thus, Mg@PLPE MS provides a valuable platform for HO elimination and controlled H release. The generated H scavenge for ROS by reacting with noxious •OH. Notably, the Mg@PLPE MS exerted significant antioxidative and anti-inflammatory effects in a disc degeneration rat model and alleviated extracellular matrix degradation and disc cells apoptosis, thereby underlining its efficacy in IVDD treatment. The Mg@PLPE MS also exhibited robust biocompatibility and negligible toxicity, presenting the promise for the antioxidative treatment of IVDD in vivo. STATEMENT OF SIGNIFICANCE: Antioxidant therapy is currently considered one of the most promising strategies for intervertebral disc degeneration (IVDD) treatment, given the crucial role of reactive oxygen species (ROS) in IVDD pathogenesis. Here, ROS-responsive magnesium-containing microspheres (Mg@PLPE MSs) were constructed to alleviate IVDD through controlled release of hydrogen gas. The Mg@PLPE MSs can effectively scavenge overproduced ROS by simultaneously reacting with HO and •OH, thus creating a suitable microenvironment for inhibition of ECM degradation. As a result, Mg@PLPE MSs treated IVDD rats exhibit minimal nucleus pulposus decrease, less extracellular matrix degradation, minimal radial fissure of fibrous rings, and higher disc height index. Therefore, the as-prepared Mg@PLPE MS may shed a new light on clinical treatment of IVDD.

Neuropathic pain is a complex condition that seriously affects human quality of life. This study aimed to investigate the therapeutic mechanism of mesenchymal stem cell-derived extracellular vesicles (MSC-EVs) and try to discover new targets for alleviating neuropathic pain. Extracellular vesicles were isolated and identified via ultracentrifugation. BV-2 microglial cells were stimulated with lipopolysaccharide (LPS) in the presence or absence of MSC-EVs. Further, microglial activation and neuroinflammation were evaluated by flow cytometry, RT-qPCR, and ELISA. High-throughput sequencing analysis was performed to reveal the differentially expressed (DE) miRNAs in BV-2 microglia. Autophagy-related regulators were assessed by Western blotting and Immunofluorescence staining. Chronic constriction injury (CCI) model was used to induce neuropathic pain in rats, and the mechanical withdrawal threshold (MWT) was measured. High-throughput sequencing analysis identified 17 DE miRNAs, which were mainly enriched in PI3K-AKT and mTOR signaling pathways. MSC-EVs inhibited the activation of PI3K/AKT/mTOR signaling pathway in LPS-stimulated microglia. Moreover, MSC-EVs treatment enhanced the autophagy level in activated microglia, whereas autophagy inhibitor 3-MA reversed the suppressing effects of MSC-EVs on microglial activation and neuroinflammation. The MSC-EV-mediated transfer of miR-99b-3p was verified to promote microglial autophagy, and miR-99b-3p overexpression suppressed the expression of pro-inflammatory factors in activated microglia. During in vivo studies, intrathecal injection of MSC-EVs significantly up-regulated the expression of miR-99b-3p, and alleviated mechanical allodynia caused by activated microglia in the spinal cord dorsal horn of CCI rats. Moreover, MSC-EVs treatment repaired CCI-induced autophagic impairment by stimulating autophagy in the spinal cord. Collectively, our findings demonstrated that MSC-EVs had an analgesic effect on neuropathic pain via promoting autophagy, and these antinociceptive effects were at least in part caused by MSC-EV-mediated transfer of miR-99b-3p, thereby inhibiting microglial activation and pro-inflammatory cytokines expression.

Posterior hypothalamus (PH), an important part of the descending pain processing pathway, has been found to be activated in trigeminal autonomic cephalalgias. However, there are very few studies conducted and information regarding its implications in trigeminal neuropathic pain (TNP). Therefore, we aimed to ascertain whether optogenetic inhibition of PH could affect the outcomes of a chronic constriction injury in the infraorbital nerve (CCI-ION) rat model. Animals were divided into the TNP animal, sham, and naive-control groups. CCI-ION surgery was performed to mimic TNP symptoms, and the optogenetic or null virus was injected into the ipsilateral PH. In vivo single-unit extracellular recordings were obtained from both the ipsilateral ventrolateral periaqueductal gray (vlPAG) and contralateral ventral posteromedial (VPM) thalamus in stimulation "OFF" and "ON" conditions. Alterations in behavioral responses during the stimulation-OFF and stimulation-ON states were examined. We observed that optogenetic inhibition of the PH considerably improved behavioral responses in TNP animals. We found increased and decreased firing activity in the vlPAG and VPM thalamus, respectively, during optogenetic inhibition of the PH. Inhibiting PH attenuates trigeminal pain signal transmission by modulating the vlPAG and trigeminal nucleus caudalis, thereby providing evidence of the therapeutic potential of PH in TNP management.

Neuropathic pain has become a global public problem and health burden. Pharmacological interventions are the primary treatment, but the drug cure rate is low with side effects. There is an urgent need to develop novel treatment approaches. High frequency electrical stimulation (KHES) has been widely applied in clinical analgesia. However, its mechanism is poorly understood. In this study, datasets related to neuropathic pain were obtained from the GEO database. The differentially expressed genes (DEGs) and key genes were analyzed through functional enrichment analysis, showing that most of the pathways involve the inflammation. The MyD88 and NFκB pathways were further studied. KHES significantly alleviated mechanical and thermal allodynia in chronic constriction injury of the sciatic nerve mice. KHES also inhibited the increase in Myd88 and p-NFκB expression. The administration of NFκB pathway activator partly reversed the antinociceptive effects of KHES, and NFκB pathway inhibitor achieved analgesic effects similar to those of KHES. Therefore, KHES might be a novel intervention for the treatment of neuropathic pain.

Pregabalin is an anti-neuropathic pain drug inhibiting the α2δ subunit of the voltage-dependent calcium channel in the spinal cord. The aim of this study is to characterize the transport mechanism of pregabalin at the blood-spinal cord barrier (BSCB) by means of in vivo experiments in rats and in vitro studies using primary-cultured rat spinal cord endothelial cells. We isolated endothelial cells by culturing rat spinal cord tissue in the presence of puromycin, and confirmed the expression of BSCB markers such as Cd31, Mdr1a, and Claudin-5. The uptake of pregabalin by primary-cultured rat spinal cord endothelial cells was sodium-independent and was significantly inhibited by L-leucine, 2-aminobicyclo-(2,2,1)-heptane-2-carboxylic acid, and JPH203. These results suggest the involvement of L-type amino acid transporter (LAT) 1. LAT1 mRNA and protein was expressed in primary-cultured rat spinal cord endothelial cells, which is consistent with LAT1 expression at the BSCB. In the in vivo study, the transfer of pregabalin to rat spinal cord and brain was significantly decreased by the pre-administration of branched chain amino acids (BCAAs), which are endogenous substrates of LAT1. Our results indicate that pregabalin transport across the BSCB is mediated at least in part by LAT1 and is inhibited by plasma BCAAs.