Keratinocyte TLR2 and TLR7 contribute to chronic itch through pruritic cytokines and chemokines in mice.

Although neuronal Toll-like receptors (TLRs) (e.g., TLR2, TLR3, and TLR7) have been implicated in itch sensation, the roles of keratinocyte TLRs in chronic itch are elusive. Herein, we evaluated the roles of keratinocyte TLR2 and TLR7 in chronic itch under dry skin and psoriasis conditions, which was induced by either acetone-ether-water treatment or 5% imiquimod cream in mice, respectively. We found that TLR2 and TLR7 signaling were significantly upregulated in dry skin and psoriatic skin in mice. Chronic itch and epidermal hyperplasia induced by dry skin or psoriasis were comparably reduced in TLR2 and TLR7 knockout mice. In the dry skin model, the enhanced messenger RNA (mRNA) expression levels of pruritic CXCL1/2, IL-31, IL-33, ST2, IL-6, IL-17A, TNF-α, and IFN-γ were inhibited in TLR2-/- mice, while CXCL2, IL-31, and IL-6 were inhibited in TLR7-/- mice. In psoriasis model, the enhanced mRNA expression levels of pruritic CXCL1/2, IL-31, IL-33, ST2, IL-6, and TNF-α were inhibited in TLR2-/- mice, while CXCL1/2, IL-31, IL-33, ST2, IL-6, IL-17A, and TNF-α were inhibited in TLR7-/- mice. Incubation with Staphylococcus aureus (S. aureus) peptidoglycan (PGN-SA) (a TLR2 agonist), imiquimod (a TLR7 agonist), and miR142-3p (a putative TLR7 agonist) were sufficient to upregulate the expression of pruritic cytokines or chemokines in cultured keratinocyte HaCaT cells. Finally, pharmacological blockade of C-X-C Motif Chemokine Receptor 1/2 and high mobility group box protein 1 dose-dependently attenuated acute and chronic itch in mice. Together, these results indicate that keratinocyte TLR2 and TLR7 signaling pathways are distinctly involved in the pathogenesis of chronic itch.

LINC00365 inhibited lung adenocarcinoma progression and glycolysis via sponging miR-429/KCTD12 axis.

This study researched the function of long non-coding RNA LINC00365 in lung adenocarcinoma (LAD) progression. LINC00365, miR-429, and KCTD12 expression in the LAD clinical tissues and cells were detcetd by qRT-PCR and Western blot. LINC00365, miR-429, and KCTD12 effects on H1975 cells malignant phenotype were detected by cell counting kit-8 assay, clone formation experiment, Transwell experiment, and glycolysis. Dual luciferase reporter gene assay and RNA pull-down assay were implemented. LINC00365 effect on H1975 cells in vivo growth was detected. LINC00365 was low expressed in the LAD patients and cells, associating with poor outcome. LINC00365 up-regulation attenuated H1975 cells proliferation, migration, invasion, glycolysis and in vivo growth. LINC00365 inhibited KCTD12 expression by sponging miR-429. miR-429 up-regulation and KCTD12 down-regulation partial reversed LINC00365 inhibition on H1975 cells malignant phenotype. Thus, LINC00365 inhibited LAD progression and glycolysis via targeting miR-429/KCTD12 axis. LINC00365 might be a potential candidate for LAD target treatment clinically.

TNF-α/TNFR1 Signaling is Required for the Full Expression of Acute and Chronic Itch in Mice via Peripheral and Central Mechanisms.

Increasing evidence suggests that cytokines and chemokines play crucial roles in chronic itch. In the present study, we evaluated the roles of tumor necrosis factor-alpha (TNF-α) and its receptors TNF receptor subtype-1 (TNFR1) and TNFR2 in acute and chronic itch in mice. Compared to wild-type (WT) mice, TNFR1-knockout (TNFR1-KO) and TNFR1/R2 double-KO (DKO), but not TNFR2-KO mice, exhibited reduced acute itch induced by compound 48/80 and chloroquine (CQ). Application of the TNF-synthesis inhibitor thalidomide and the TNF-α antagonist etanercept dose-dependently suppressed acute itch. Intradermal injection of TNF-α was not sufficient to evoke scratching, but potentiated itch induced by compound 48/80, but not CQ. In addition, compound 48/80 induced TNF-α mRNA expression in the skin, while CQ induced its expression in the dorsal root ganglia (DRG) and spinal cord. Furthermore, chronic itch induced by dry skin was reduced by administration of thalidomide and etanercept and in TNFR1/R2 DKO mice. Dry skin induced TNF-α expression in the skin, DRG, and spinal cord and TNFR1 expression only in the spinal cord. Thus, our findings suggest that TNF-α/TNFR1 signaling is required for the full expression of acute and chronic itch via peripheral and central mechanisms, and targeting TNFR1 may be beneficial for chronic itch treatment.

Antioxidants Attenuate Acute and Chronic Itch: Peripheral and Central Mechanisms of Oxidative Stress in Pruritus.

Itch (pruritus) is one of the most disabling syndromes in patients suffering from skin, liver, or kidney diseases. Our previous study highlighted a key role of oxidative stress in acute itch. Here, we evaluated the effects of antioxidants in mouse models of acute and chronic itch and explored the potential mechanisms. The effects of systemic administration of the antioxidants N-acetyl-L-cysteine (NAC) and N-tert-butyl-α-phenylnitrone (PBN) were determined by behavioral tests in mouse models of acute itch induced by compound 48/80 or chloroquine, and chronic itch by treatment with a mixture of acetone-diethyl-ether-water. We found that systemic administration of NAC or PBN significantly alleviated compound 48/80- and chloroquine-induced acute itch in a dose-dependent manner, attenuated dry skin-induced chronic itch, and suppressed oxidative stress in the affected skin. Antioxidants significantly decreased the accumulation of intracellular reactive oxygen species directly induced by compound 48/80 and chloroquine in the cultured dorsal root ganglia-derived cell line ND7-23. Finally, the antioxidants remarkably inhibited the compound 48/80-induced phosphorylation of extracellular signal-regulated kinase in the spinal cord. These results indicated that oxidative stress plays a critical role in acute and chronic itch in the periphery and spinal cord and antioxidant treatment may be a promising strategy for anti-itch therapy.

Hydrogen sulfide-induced itch requires activation of Cav3.2 T-type calcium channel in mice.

The contributions of gasotransmitters to itch sensation are largely unknown. In this study, we aimed to investigate the roles of hydrogen sulfide (H2S), a ubiquitous gasotransmitter, in itch signaling. We found that intradermal injection of H2S donors NaHS or Na2S, but not GYY4137 (a slow-releasing H2S donor), dose-dependently induced scratching behavior in a µ-opioid receptor-dependent and histamine-independent manner in mice. Interestingly, NaHS induced itch via unique mechanisms that involved capsaicin-insensitive A-fibers, but not TRPV1-expressing C-fibers that are traditionally considered for mediating itch, revealed by depletion of TRPV1-expressing C-fibers by systemic resiniferatoxin treatment. Moreover, local application of capsaizapine (TRPV1 blocker) or HC-030031 (TRPA1 blocker) had no effects on NaHS-evoked scratching. Strikingly, pharmacological blockade and silencing of Cav3.2 T-type calcium channel by mibefradil, ascorbic acid, zinc chloride or Cav3.2 siRNA dramatically decreased NaHS-evoked scratching. NaHS induced robust alloknesis (touch-evoked itch), which was inhibited by T-type calcium channels blocker mibefradil. Compound 48/80-induced itch was enhanced by an endogenous precursor of H2S (L-cysteine) but attenuated by inhibitors of H2S-producing enzymes cystathionine γ-lyase and cystathionine ß-synthase. These results indicated that H2S, as a novel nonhistaminergic itch mediator, may activates Cav3.2 T-type calcium channel, probably located at A-fibers, to induce scratching and alloknesis in mice.

Adrenergic ß2-receptor mediates itch hypersensitivity following heterotypic chronic stress in rats.

Chronic stress is widely considered to trigger or enhance itch, especially for pruritic dermatitis. However, the molecular mechanisms linking chronic stress and itch are still unknown. The present study aimed to elucidate the role of adrenergic signaling in itch hypersensitivity following heterotypic chronic intermittent stress (HIS) in rats. HIS significantly increased hindlimb scratching, but not forepaw swiping, induced by intradermal injection of 5-hydroxytryptamine (5-HT) in the rat cheek. Coadministration of stress mediators such as norepinephrine or epinephrine dose-dependently increased both 5-HT-induced hindlimb scratching and 5-HT-induced forepaw swiping. HIS-induced itch hypersensitivity was attenuated by blockade of sympathetic signaling through guanethidine treatment, and systemic administration of the ß-adrenoceptor antagonist propranolol and the ß2-adrenoceptor antagonist butoxamine, but not on treatment with an α-adrenoceptor antagonist phentolamine and a ß1-adrenoceptor antagonist atenolol. Moreover, HIS selectively increased the expression of ß2-adrenoceptors and proinflammatory factors [tumor necrosis factor-α (TNF-α), interleukin-1ß (IL-1ß), and nerve growth factor (NGF)] in rat skin. The ß-blockers propranolol and butoxamine abolished the upregulation of proinflammatory factors. The ß2-adrenoceptor agonist terbutaline was sufficient to enhance the skin expression of TNF-α and IL-1ß and to increase 5-HT-induced scratching in naive rats. Pretreatment with TNF-α could increase 5-HT-induced scratching. Together, these results demonstrate that ß2-adrenoceptors mediate itch hypersensitivity following chronic stress by inducing proinflammatory factors, such as TNF-α, in the skin.

Activated microglia contribute to neuronal apoptosis in Toxoplasmic encephalitis.

BACKGROUND: A plethora of evidence shows that activated microglia play a critical role in the pathogenesis of the central nervous system (CNS). Toxoplasmic encephalitis (TE) frequently occurs in HIV/AIDS patients. However, knowledge remains limited on the contributions of activated microglia to the pathogenesis of TE. METHODS: A murine model of reactivated encephalitis was generated in a latent infection with Toxoplasma gondii induced by cyclophosphamide. The neuronal apoptosis in the CNS and the profile of pro-inflammatory cytokines were assayed in both in vitro and in vivo experiments. RESULTS: Microglial cells were found to be activated in the cortex and hippocampus in the brain tissues of mice. The in vivo expression of interleukin-6 (IL-6), interleukin-1ß (IL-1ß), tumor necrosis factor-α (TNF-α), and inducible nitric oxide synthase (iNOS) were up-regulated in TE mice, and accordingly, the neuronal apoptosis was significantly increased. The results were positively correlated with those of the in vitro experiments. Additionally,apoptosis of the mouse neuroblastoma type Neuro2a (N2a) remarkably increased when the N2a was co-cultured in transwell with microglial cells and Toxoplasma tachyzoites. Both in vivo and in vitro experiments showed that minocycline (a microglia inhibitor) treatment notably reduced microglial activation and neuronal apoptosis. CONCLUSIONS: Activated microglia contribute to neuronal apoptosis in TE and inhibition of microglia activation might represent a novel therapeutic strategy of TE.

[Microglial activation and inflammatory cytokine expression in the brain of chronic Toxoplasma gondii-infected mice].

OBJECTIVE: To investigate microglial activation and inflammatory cytokine expression in chronic Toxoplasma gondii infection. METHODS: Thirty mice were randomly divided into chronic T. gondii infection group and normal control group. Each mouse in infection group was infected orally with 30 cysts of the TgCtwh6 strain. Normal group received 0.3 ml normal saline. On the 60th day after infection, immunohistochemical staining was performed to assess the number of microglia and morphological change. The expression of inflammatory cytokines (IL-1beta, IL-6, and TNF-alpha) was measured by RT-PCR. The expression of iNOS was determined by Western blotting and immunofluorescence. RESULTS: Immunohistochemistry analysis showed that the number of Iba-1 positive cells in the cortex and hippocampus of infection group (16.5 +/- 0.8 and 17.9 +/- 1.1) was higher than that of the control (8.4 +/- 0.2 and 10.3 +/- 0.8)(P < 0.05). Iba-1 positive cells (i.e. microglia) had larger cell bodies and ramified morphology. RT-PCR result indicated that mRNA level of IL-1beta, IL-6, and TNF-alpha in infection group (0.862 +/- 0.169, 0.407 +/- 0.158, and 0.305 +/- 0.073) was significantly higher than that of the control (0.149 +/- 0.030, 0.037 +/- 0.008, and 0.001 +/- 0.001) (P < 0.05). The iNOS protein expression in infection group (0.252 +/- 0.164) was higher than that of the control (0.0433 +/- 0.004) (P < 0.05). Immunofluorescence demonstrated that iNOS protein released by activated microglia. CONCLUSION: Chronic T. gondii infection caused microglial activation, which up-regulate the level of IL-1beta, IL-6, TNF-alpha, and iNOS.

[Cloning, expression and prediction of protein structure of Ts3 gene of Cysticercus cellulosae].

OBJECTIVE: To discover a new gene of candidate molecule for the diagnosis of cysticercosis. METHODS: Cysticercus cellulosae cDNA library was immunoscreened with pooled serum of cysticercosis patients. The encoded protein of a new gene, Ts3, was analyzed through biology software (EXPASY) on line. The new gene was cloned, the prokaryotic expression vector pET28a (+)-Ts3 was constructed, and the expressed result was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting. RESULTS: The new gene Ts3 (GenBank accession No. EU338456) was obtained with 531 bp and an ORF of C. cellulosae. With relatively stable structure and physicochemical properties, the new gene was anon-transmembrane protein and contained more protein kinase C phosphorylation site. To induce its expression, the Ts3 gene was cloned into the vector pET28a (+), and a protein with a relative molecular mass Mr21000 was obtained which was recognized by the sera of cysticercosis cases. CONCLUSION: The Ts3 gene of C. cellulosae was expressed, and the protein shows adequate antigenicity.

Transfection of mouse macrophage metalloelastase gene into murine CT-26 colon cancer cells suppresses orthotopic tumor growth, angiogenesis and vascular endothelial growth factor expression.

A cDNA fragment coding for domains I and II of mouse macrophage metalloelastase (MME) was transfected into murine CT-26 colon cancer cells that are MME deficient. An orthotopic implantation model was established by using MME-transfected cells. In MME-transfected primary tumors, it demonstrated that tumor growth and microvessel formation were significantly inhibited compared with the controls. The expression of vascular endothelial growth factor (VEGF) mRNA and protein was significantly lower in MME-transfected group compared with those in the controls. Our data show that both MME and VEGF gene expression is highly associated with the vascularity of tumors, which may depend on a balance between MME and VEGF expression.