Pregabalin inhibits purinergic P2Y2 receptor and TRPV4 to suppress astrocyte activation and to relieve neuropathic pain.

BACKGROUNDS: Transient receptor potential vanilloid 4 (TRPV4)-mediated astrocyte activation is critical to neuropathic pain. Pregabalin, a widely used drug to treat chronic pain, is reported to lower the intracellular calcium level. However, the molecular mechanism by which pregabalin decreases the intracellular calcium level remains unknown. Purinergic P2Y2 receptor-a member of the G protein-coupled receptor (GPCR) family-regulates calcium-related signal transduction in astrocyte activation. We investigated whether P2Y2 receptor is involved in the pharmacological effects of pregabalin on neuropathic pain. METHODS: Neuropathic pain was induced by chronic compression of the dorsal root ganglion (CCD) in rats. Paw withdrawal mechanical threshold (PWMT) was used for behavioral testing. Intracellular calcium concentration was measured using a fluorescent calcium indicator (Fluo-4 AM). RESULTS: We found that P2Y2 receptor protein was upregulated and astrocytes were activated in the experimental rats after CCD surgery. Lipopolysaccharide (LPS) increased the intracellular calcium concentration and induced astrocyte activation in cultured astrocytes but was prevented via P2Y2 receptor inhibitor AR-C118925 or pregabalin. Furthermore, plasmid-mediated P2Y2 receptor overexpression induced an elevation of the intracellular calcium levels and inflammation in astrocytes, which was abolished by the TRPV4 inhibitor HC-067047. AR-C118925, HC-067047, and pregabalin relieved neuropathic pain and inflammation in rats after CCD surgery. Finally, plasmid-mediated P2Y2 receptor overexpression induced neuropathic pain in rats, which was abolished by pregabalin administration. CONCLUSIONS: Pathophysiological variables that upregulated the P2Y2 receptor/TRPV4/calcium axis contribute to astrocyte activation in neuropathic pain. Pregabalin exerts an analgesic effect by inhibiting this pathway.

Immunogenicity and protective effects of recombinant bivalent COVID-19 vaccine in mice and rhesus macaques.

Coronavirus disease (COVID-19) is still spreading rapidly worldwide, and a safe, effective, and cheap vaccine is still required to combat the COVID-19 pandemic. Here, we report a recombinant bivalent COVID-19 vaccine containing the RBD proteins of the prototype strain and beta variant. Immunization studies in mice demonstrated that this bivalent vaccine had far greater immunogenicity than the ZF2001, a marketed monovalent recombinant protein COVID-19 vaccine, and exhibited good immunization effects against the original COVID-19 strain and various variants. Rhesus macaque challenge experiments showed that this bivalent vaccine drastically decreased the lung viral load and reduced lung lesions in SARS-CoV-2 (the causative virus of COVID-19)-infected rhesus macaques. In summary, this bivalent vaccine showed immunogenicity and protective efficacy that was far superior to the monovalent recombinant protein vaccine against the prototype strain and provided an important basis for developing broad-spectrum COVID-19 vaccines.

Obesity increases neuropathic pain via the AMPK-ERK-NOX4 pathway in rats.

This study focused on the relationship between extracellular-regulated kinase (ERK) and obesity-induced increases in neuropathic pain. We fed rats a high-fat diet to establish the obesity model, and rats were given surgery to establish the chronic compression of the dorsal root ganglia (CCD) model. U0126 was applied to inhibit ERK, and metformin or 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR) was applied to cause AMP-activated protein kinase (AMPK) activation. Paw withdrawal mechanical threshold (PWMT) were calculated to indicate the level of neuropathic pain. The data indicated that compared with normal CCD rats, the PWMT of obese CCD rats were decreased, accompanied with an increase of ERK phosphorylation, NAD(P)H oxidase 4 (NOX4) protein expression, oxidative stress and inflammatory level in the L4 to L5 spinal cord and dorsal root ganglia (DRG). Administration of U0126 could partially elevate the PWMT and reduce the protein expression of NOX4 and the above pathological changes in obese CCD rats. In vitro, ERK phosphorylation, NOX4 protein expression increased significantly in DRG neurons under the stimulation of palmitic acid (PA), accompanied with increased secretion of inflammatory factors, oxidative stress and apoptosis level, while U0126 partially attenuated the PA-induced upregulation of NOX4 and other pathological changes. In the rescue experiment, overexpression of NOX4 abolished the above protective effect of U0126 on DRG neurons in high-fat environment. Next, we explore upstream mechanisms. Metformin gavage significantly reduced neuropathic pain in obese CCD rats. For the mechanisms, activating AMPK with metformin (obese CCD rats) or AICAR (DRG neurons in a high-fat environment) not only inhibited the ERK-NOX4 pathway, but also improved oxidative stress and inflammation caused by high-fat. In conclusion, the AMPK-ERK-NOX4 pathway may has a pivotal role in mediating obesity-induced increases in neuropathic pain.

DOK3 is involved in microglial cell activation in neuropathic pain by interacting with GPR84.

Adaptor molecule downstream of kinase-3 (DOK3) is a vital regulator of innate immune responses in macrophages and B cells, and G-protein-coupled receptor 84 (GPR84) is significant in mediating the biosynthesis and maintenance of inflammatory mediators that are induced by neuropathic pain in microglia. In the present study, we determined the role of DOK3 in activating microglia-induced neuropathic pain and investigated the underlying mechanisms associated with GPR84. We found that knockdown of DOK3 in microglial cells dramatically reduced the levels of inflammatory factors, and we uncovered a physical association between DOK3 and GPR84 in the induction of inflammatory responses. We also observed that neuropathic pain and inflammatory responses induced by chronic constriction injury (CCI) of the sciatic nerve or intrathecal injection of a GPR84 agonist were compromised in DOK3-/- mice in vivo. Finally, enforced expression of DOK3 provoked inflammatory responses, and administration of pregabalin relieved neuropathic pain via inhibition of DOK3 expression. In conclusion, DOK3 induced neuropathic pain in mice by interacting with GPR84 in microglia. We hypothesize that targeting the adaptor protein DOK3 may open new avenues for pharmaceutical approaches to the alleviation of neuropathic pain in the spinal cord.

Effect of cGMP-activated aquaporin 1 on TRPV4 in rats with allodynia induced by chronic compression of the dorsal root ganglion.

BACKGROUND: The aim of this study was to investigate the effects of aquaporin 1 (AQP1) knockdown on allodynia in rats with chronic compression of the dorsal root ganglia (DRG) and the role of TRPV4 in these effects. METHODS: Adult male Wistar rats were subjected to chronic compression of the dorsal root ganglia (CCD) via surgery. Behavioral tests were performed to calculate the paw withdrawal mechanical threshold (PWMT). Gene silence was induced by injecting rats with lentivirus expressing AQP1 short hairpin RNA (shRNA, Lv-shAQP1). Western blot analyses were performed to examine AQP1 and TRPV4 protein expression. The concentration of cyclic guanosine monophosphate (cGMP) was determined via enzyme-linked immunosorbent assay. RESULTS: AQP1 protein levels in DRG neurons were significantly increased in CCD rats and were accompanied by a decrease in the PWMT. Lentivirus-mediated RNA interference of AQP1 decreased AQP1 protein expression in CCD rats and normalized their PWMT, but not in rats infected with lentivirus-expressing negative control short hairpin RNA. Furthermore, AQP1 was identified as a cGMP-gated channel. cGMP concentration was upregulated in CCD rats. This effect was attenuated by treatment with a cGMP inhibitor. Additionally, the cGMP inhibitor decreased the mechanical allodynia and AQP1 protein expression in CCD rats. Finally, levels of TRPV4 expression were upregulated in DRG neurons and the L4/L5 spinal cord following surgery, and these effects were reversed by treatment with Lv-shAQP1 or a cGMP inhibitor. CONCLUSION: AQP1 plays a vital role in CCD-induced allodynia as Lv-shAQP1 significantly reduced the allodynia in CCD rats by inhibiting TRPV4 expression.

[LAH-3, a transcriptional factor involved in osmotic regulation in Neurospora crassa].

[OBJECTIVE] In order to identify the function of lah-3 in osmotic regulation, we generated lah-3 deletion strain and analyzed its phenotype by osmostress treatment. [METHODS] We used homologous recombination to replace lah-3gene by hph gene and treated these cells with 4% NaCl and 1 M sorbitol to analyze the phenotype. Northern blot was used to detect the expressions of osmoresponsing genes. Western blot was used to examine the phosphorylation level of LAH-3 and OS-2 and the expression of OS-2. [RESULTS] In the deletion strain of transcription factor lah-3 gene, the expressions of osmoresponsing genes gcy-1, stl-1 and pck-1 were significantly reduced. Besides, the phosphorylation level of LAH-3 protein increased under the osmostress treatment. The phosphorylation of LAH-3 was not mediated by OS-2. The deletion of lah-3 did not affect the os-2 expression and the phosphorylation level of OS-2 upon osmostress. [ CONCLUSION] LAH-3 involved in osmoresponsing was independent of OS-2 MAPK pathway.