Roles of Thermosensitive Transient Receptor Channels TRPV1 and TRPM8 in Paclitaxel-Induced Peripheral Neuropathic Pain.

Paclitaxel, a microtubule-stabilizing chemotherapy drug, can cause severe paclitaxel-induced peripheral neuropathic pain (PIPNP). The roles of transient receptor potential (TRP) ion channel vanilloid 1 (TRPV1, a nociceptor and heat sensor) and melastatin 8 (TRPM8, a cold sensor) in PIPNP remain controversial. In this study, Western blotting, immunofluorescence staining, and calcium imaging revealed that the expression and functional activity of TRPV1 were upregulated in rat dorsal root ganglion (DRG) neurons in PIPNP. Behavioral assessments using the von Frey and brush tests demonstrated that mechanical hyperalgesia in PIPNP was significantly inhibited by intraperitoneal or intrathecal administration of the TRPV1 antagonist capsazepine, indicating that TRPV1 played a key role in PIPNP. Conversely, the expression of TRPM8 protein decreased and its channel activity was reduced in DRG neurons. Furthermore, activation of TRPM8 via topical application of menthol or intrathecal injection of WS-12 attenuated the mechanical pain. Mechanistically, the TRPV1 activity triggered by capsaicin (a TRPV1 agonist) was reduced after menthol application in cultured DRG neurons, especially in the paclitaxel-treated group. These findings showed that upregulation of TRPV1 and inhibition of TRPM8 are involved in the generation of PIPNP, and they suggested that inhibition of TRPV1 function in DRG neurons via activation of TRPM8 might underlie the analgesic effects of menthol.

Dihydromyricetin Alleviates Ischemic Brain Injury by Antagonizing Pyroptosis in Rats.

Ischemic stroke is a worldwide disease that seriously threatens human health, and there are few effective drugs to treat it. Dihydromyricetin (DHM) has anti-inflammatory, antioxidant, and antiapoptotic functions. We identified pyroptosis following ischemic stroke. Here, we investigated the effect of DHM on ischemic stroke and pyroptosis. In the first part of the experiment, Sprague-Dawley rats were randomly divided into the sham group and MCAO group. The MCAO model was established by occlusion of the middle cerebral artery for 90 min using a silica gel suture. The ischemic penumbra was used for mRNA sequencing 1 day after reperfusion. In the second part, rats were divided into the sham group, MCAO group, and DHM group. DHM was injected intraperitoneally at the same time as reperfusion starting 90 min after embolization for 7 consecutive days. The changes in pyroptosis were observed by morphological and molecular methods. The transcriptomics results suggested the presence of NLRP3-mediated pyroptotic death pathway activation after modeling. The Longa score was increased after MCAO and decreased after DHM treatment. 2,3,5-Triphenyltetrazolium chloride (TTC) staining showed that DHM could reduce the infarct volume induced by MCAO. Nissl staining showed disordered neuronal arrangement and few Nissl bodies in the MCAO group, but this effect was reversed by DHM treatment. Analysis of pyroptosis-related molecules showed that the MCAO group had serious pyroptosis, and DHM effectively reduced pyroptosis. Our results demonstrate that DHM has a neuroprotective effect on ischemic stroke that is at least partly achieved by reducing pyroptosis.

Integrated multiomics analysis reveals changes in liver physiological function in Aqp9 gene knockout mice.

Aquaporin 9 (AQP9) is the main channel by which blood glycerol enters the liver, where it plays key roles in osmotic pressure regulation and energy metabolism. Previous studies have shown that AQP9 is involved in the pathogenesis of many liver diseases. In this study, we aimed to clarify the role of AQP9 in maintaining the physiological environment of the liver using Aqp9-/- mice. We constructed Aqp9 knockout mice and used comprehensive multiomics analysis to elucidate the potential molecular effects of AQP9 expression on liver tissue. Knockout of Aqp9 reduced mouse body weight by affecting glycerol metabolism and led to hepatocyte death and inflammatory cell infiltration, which was confirmed by transcriptomics, proteomics and metabolomics. Moreover, knockout of Aqp9 triggered immune and inflammatory responses, leading to scattered and mild liver cell pyroptosis and compensatory liver cell proliferation.

High-Altitude Hypoxia Exposure Induces Iron Overload and Ferroptosis in Adipose Tissue.

High altitude (HA) has become one of the most challenging environments featuring hypobaric hypoxia, which seriously threatens public health, hence its gradual attraction of public attention over the past decade. The purpose of this study is to investigate the effect of HA hypoxia on iron levels, redox state, inflammation, and ferroptosis in adipose tissue. Here, 40 mice were randomly divided into two groups: the sea-level group and HA hypoxia group (altitude of 5000 m, treatment for 4 weeks). Total iron contents, ferrous iron contents, ROS generation, lipid peroxidation, the oxidative enzyme system, proinflammatory factor secretion, and ferroptosis-related biomarkers were examined, respectively. According to the results, HA exposure increases total iron and ferrous iron levels in both WAT and BAT. Meanwhile, ROS release, MDA, 4-HNE elevation, GSH depletion, as well as the decrease in SOD, CAT, and GSH-Px activities further evidenced a phenotype of redox imbalance in adipose tissue during HA exposure. Additionally, the secretion of inflammatory factors was also significantly enhanced in HA mice. Moreover, the remarkably changed expression of ferroptosis-related markers suggested that HA exposure increased ferroptosis sensitivity in adipose tissue. Overall, this study reveals that HA exposure is capable of inducing adipose tissue redox imbalance, inflammatory response, and ferroptosis, driven in part by changes in iron overload, which is expected to provide novel preventive targets for HA-related illness.

CircSV2b participates in oxidative stress regulation through miR-5107-5p-Foxk1-Akt1 axis in Parkinson's disease.

As a novel type of non-coding RNAs, covalently closed circular RNAs (circRNAs) are ubiquitously expressed in eukaryotes. Emerging studies have indicated that dysregulation of circRNAs was related to neurological diseases. However, the biogenesis, regulation, function, and mechanism of circRNAs in Parkinson's disease (PD) remain largely unclear. In this study, thirty-three differentially expressed circRNAs (DECs) were detected by RNA-sequencing between the MPTP-induced PD mice model and the wild-type mice. Quantitative real-time PCR was used to determine the RNA level of DECs in the striatum (STR), substantia nigra pars compacta (SNpc), and serum exosomes, and it was found that circSV2b was downregulated in PD mice. Then, functional experiments in vivo were employed to explore the effect of circSV2b in PD. For the mechanism study, dual-luciferase reporter, fluorescence in situ hybridization (FISH), RNA immunoprecipitation (RIP), RNA pull-down, gene editing, and CUT & Tag were performed in vitro to confirm that circSV2b directly sponged miR-5107-5p and alleviated the suppression of the expression of the target gene Foxk1, and then positively regulated Akt1 transcription. In vivo, the mechanistic analysis demonstrated that circSV2b overexpression resisted oxidative stress damage through the ceRNA-Akt1 axis in PD models. Taken together, these findings suggested that the miR-5107-5p-Foxk1-Akt1 axis might serve as a key target of circSV2b overexpression in PD treatment, and highlighted the significant change of circSV2b in serum exosomes. Therefore, circSV2b might be a novel biomarker for the diagnosis and treatment of PD.

A Preliminary Study on the Effect of Hydrogen Gas on Alleviating Early CCl4-Induced Chronic Liver Injury in Rats.

As a small-molecule reductant substance, hydrogen gas has an obvious antioxidant function. It can selectively neutralize hydroxyl radicals (•OH) and peroxynitrite (ONOO•) in cells, reducing oxidative stress damage. The purpose of this study was to investigate the effect of hydrogen gas (3%) on early chronic liver injury (CLI) induced by CCl4 and to preliminarily explore the protective mechanism of hydrogen gas on hepatocytes by observing the expression of uncoupling protein 2 (UCP2) in liver tissue. Here, 32 rats were divided into four groups: the control group, CCl4 group, H2 (hydrogen gas) group, and CCl4 + H2 group. The effect of hydrogen gas on early CLI was observed by serological tests, ELISA, hematoxylin and eosin staining, and oil red O staining. Immunohistochemical staining and Western blotting were used to observe the expression of UCP2 in liver tissues. We found that CCl4 can induce significant steatosis in hepatocytes. When the hydrogen gas was inhaled, hepatocyte steatosis was reduced, and the UCP2 expression level in liver tissue was increased. These results suggest that hydrogen gas might upregulate UCP2 expression levels, reduce the generation of intracellular oxygen free radicals, affect lipid metabolism in liver cells, and play a protective role in liver cells.

Aquaporin 9 Represents a Novel Target of Chronic Liver Injury That May Antagonize Its Progression by Reducing Lipotoxicity.

In recent years, chronic liver injury has become a common disease that harms human health. Its clinical manifestations are hepatic steatosis and secondary chronic steatohepatitis, which can quickly transform into liver fibrosis and cirrhosis if not treated in time. Therefore, this study is aimed at searching for new therapeutic targets of chronic liver injury and clarifying the molecular mechanisms of the new targets involved in chronic liver injury. After aquaporin 9 was identified as a target by proteomics, Aqp9-/- mice were constructed using the CRISPR/Cas9 system. Biochemical and morphological tests were used to verify the effect of Aqp9 knockout on early chronic liver injury. Proteomics, molecular biology, and morphology experiments were used to screen and verify the effects of Aqp9 knockout on its downstream pathway. Through the above experiments, we demonstrated that aquaporin 9 could be used as an intervention target for antagonizing the development of early chronic liver injury and its gene knockout affected downstream inflammation, oxidative stress, apoptosis, and pyroptosis by alleviating hepatic lipotoxicity.

Nogo-A Induced Polymerization of Microtubule Is Involved in the Inflammatory Heat Hyperalgesia in Rat Dorsal Root Ganglion Neurons.

The microtubule, a major constituent of cytoskeletons, was shown to bind and interact with transient receptor potential vanilloid subfamily member 1 (TRPV1), and serves a pivotal role to produce thermal hyperalgesia in inflammatory pain. Nogo-A is a modulator of microtubule assembly and plays a key role in maintaining the function of TRPV1 in inflammatory heat pain. However, whether the microtubule dynamics modulated by Nogo-A in dorsal root ganglion (DRG) neurons participate in the inflammatory pain is not elucidated. Here we reported that the polymerization of microtubules in the DRG neurons, as indicated by the acetylated α-tubulin, tubulin polymerization-promoting protein 3 (TPPP3), and microtubule numbers, was significantly elevated in the complete Freund's adjuvant (CFA) induced inflammatory pain. Consistent with our previous results, knock-out (KO) of Nogo-A protein significantly attenuated the heat hyperalgesia 72 h after CFA injection and decreased the microtubule polymerization via up-regulation of phosphorylation of collapsin response mediator protein 2 (CRMP2) in DRG. The colocalization of acetylated α-tubulin and TRPV1 in DRG neurons was also reduced dramatically in Nogo-A KO rats under inflammatory pain. Moreover, the down-regulation of TRPV1 in DRG of Nogo-A KO rats after injection of CFA was reversed by intrathecal injection of paclitaxel, a microtubule stabilizer. Furthermore, intrathecal injection of nocodazole (a microtubule disruptor) attenuated significantly the CFA-induced inflammatory heat hyperalgesia and the mechanical pain in a rat model of spared nerve injury (SNI). In these SNI cases, the Nogo-A and acetylated α-tubulin in DRG were also significantly up-regulated. We conclude that the polymerization of microtubules promoted by Nogo-A in DRG contributes to the development of inflammatory heat hyperalgesia mediated by TRPV1.

Dihydromyricetin ameliorates chronic liver injury by reducing pyroptosis.

BACKGROUND: Chronic liver injury (CLI) is now a worldwide disease. However, there is no effective treatment. Pyroptosis plays an essential role in CLI. Dihydromyricetin (DHM) resists oxidation and protects the liver. We hypothesize that the beneficial effect of DHM on CLI is related to its effect on the expression of pyroptosis-related molecules. Therefore, we studied the influence of DHM on CLI and pyroptosis. AIM: To study the role of pyroptosis in the pathogenesis of CLI and the therapeutic mechanism of DHM. METHODS: Thirty-two mice were randomly divided into four groups: The control group was injected with olive oil, the carbon tetrachloride (CCl4) group was injected with CCl4, the vehicle group was injected with hydroxypropyl-ß-cyclodextrin while injecting CCl4 and the DHM group was injected with DHM while injecting CCl4. After four weeks of treatment, liver tissues from the mice were stained with hematoxylin and eosin, and oil red O. Blood was collected from the angular vein for serological analysis. The severity of CLI was estimated. Some liver tissue was sampled for immunohistochemistry, Western blotting and quantitative reverse transcription PCR to observe the changes in pyroptosis-related molecules. RESULTS: Serum total cholesterol, low density lipoprotein, aspartate aminotransferase (AST) and alanine aminotransferase (ALT) in the CCl4 group were higher than those in the control group, and serum total cholesterol, low density lipoprotein, AST and ALT in the DHM group were lower than those in the vehicle group. Hematoxylin and eosin and oil red O staining showed that there were more lipid droplets in the CCl4 group than in the control group, and there were fewer lipid droplets in the DHM group than in the vehicle group. Western blotting showed that the expression of the pyroptosis-related molecules caspase-1, NOD-, LRR- and pyrin domain-containing 3 (NLRP3) and gasdermin D (GSDMD)-N in the CCl4 group was higher than that in the control group, while expression of these proteins in the DHM group was lower than that in the vehicle group. Quantitative reverse transcription PCR results showed that the expression of the pyroptosis-related genes caspase-1, NLRP3, GSDMD and interleukin-1ß (IL-1ß) in the CCl4 group was higher than that in the control group, while there was no significant change in NLRP3 and caspase-1 expression in the DHM group compared with that in the vehicle group, and the expression of GSDMD and IL-1ß was decreased. CONCLUSION: DHM improves CCl4-induced CLI and regulates the pyroptosis pathway in hepatocytes. DHM may be a potential therapeutic agent for CLI.

Identification of a new functional domain of Nogo-A that promotes inflammatory pain and inhibits neurite growth through binding to NgR1.

Nogo-A is a key inhibitory molecule to axon regeneration, and plays diverse roles in other pathological conditions, such as stroke, schizophrenia, and neurodegenerative diseases. Nogo-66 and Nogo-Δ20 fragments are two known functional domains of Nogo-A, which act through the Nogo-66 receptor (NgR1) and sphingosine-1-phosphate receptor 2 (S1PR2), respectively. Here, we reported a new functional domain of Nogo-A, Nogo-A aa 846-861, was identified in the Nogo-A-specific segment that promotes complete Freund's adjuvant (CFA)-induced inflammatory pain. Intrathecal injection of its antagonist peptide 846-861PE or the specific antibody attenuated the CFA-induced inflammatory heat hyperalgesia. The 846-861 PE reduced the content of transient receptor potential vanilloid subfamily member 1 (TRPV1) in dorsal root ganglia (DRG) and decreased the response of DRG neurons to capsaicin. These effects were accompanied by a reduction in LIMK/cofilin phosphorylation and actin polymerization. GST pull-down and fluorescence resonance energy transfer (FRET) assays both showed that Nogo-A aa 846-861 bound to NgR1. Moreover, we demonstrated that Nogo-A aa 846-861 inhibited neurite outgrowth from cortical neurons and DRG explants. We concluded that Nogo-A aa 846-861 is a novel ligand of NgR1, which activates the downstream signaling pathways that inhibit axon growth and promote inflammatory pain.

Nogo-A promotes inflammatory heat hyperalgesia by maintaining TRPV-1 function in the rat dorsal root ganglion neuron.

Nogo-A is a key inhibitory molecule of axon regeneration in oligodendrocytes. However, little is known about its role in adult neurons. In this study, we showed an important function of Nogo-A on regulation of inflammatory pain in dorsal root ganglion (DRG) neurons. In adult rats with complete Freund's adjuvant (CFA) hind paw inflammation, DRG neurons showed a significant increase in Nogo-A expression. Disruption of Nogo-A signaling with Nogo-66 receptor antagonist peptide, Nogo-A blocking antibody, Nogo-A short hairpin RNA, or Nogo-A gene knockout attenuated CFA-induced inflammatory heat hyperalgesia. Moreover, disruption of Nogo-A signaling suppressed the function and expression in DRG neurons of the transient receptor potential vanilloid subfamily member (TRPV)-1 channel, which is known to be the endogenous transducer of noxious heat during inflammation. These effects were accompanied with a reduction in LIM domain kinase (LIMK)/cofilin phosphorylation and actin polymerization. Similar disruption of actin filament architecture by direct action of Latrunculin A reduced the TRPV-1 activity and up-regulation of TRPV-1 protein caused by CFA. We conclude that Nogo-A plays an essential role in the development of inflammatory heat hyperalgesia, partly through maintaining TRPV-1 function via activation of the LIMK/cofilin pathway, which regulates actin filament dynamics. These findings support a therapeutic potential of modulating Nogo-A signaling in pain management.-Hu, F., Liu, H.-C., Su, D.-Q., Chen, H.-J., Chan, S.-O., Wang, Y., Wang, J. Nogo-A promotes inflammatory heat hyperalgesia by maintaining TRPV-1 function in the rat dorsal root ganglion neuron.

Neuronal Nogo-A in New-born Retinal Ganglion Cells: Implication for the Formation of the Age-related Fiber Order in the Optic Tract.

Nogo-A is highly expressed in oligodendrocytes in the adult central nervous system (CNS). Recently it was found that Nogo-A is also expressed in some neuronal types during development. Here, we examined the expression pattern of Nogo-A in both the retina and optic tract (OT) of mouse embryos from E12 to E15. After perturbation of its function in the OT for 5 hr in the brain slice culture system using a Nogo-A specific antibody or antagonist of its receptor (NEP1-40), the optic nerve fibers and growth cones were traced with DiI. We showed that most Tuj-1 positive new-born neurons at E12 were Nogo-A positive. At E15, retinal neurons reduced the Nogo-A expression. It was worth noting that some projecting axons expressed Nogo-A along the retinofugal pathway. On the basis of their specific locations within the superficial half of the OT and the colocalization with GAP-43 (a marker for the newly born growth cones and axons), we concluded that those Nogo-A positive axons were the newly arrived retinal fibers. Blocking the function of Nogo-A with Nogo-A antibody or NEP1-40 resulted in the shift of DiI labeled axons and growth cones from the superficial half to the whole depth of the OT. These results indicate that Nogo-A in the newly born retinal ganglion cells (RGCs) and their axons are involved in sorting out the newly arrived axons to the subpial region of the OT. Anat Rec, 299:1027-1036, 2016. © 2016 Wiley Periodicals, Inc.