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.

Synthesis and cytotoxic activity of ethyl ferulate derivatives as potent anti-inflammatory agents.

While a range of pharmacological agents are currently used to alleviate inflammation, the clinical administration of many of these anti-inflammatory drugs is associated with high rates of adverse side effects that make them poorly suited to long-term use. Therefore, there is a critical need for the development of novel anti-inflammatory agents. Natural compounds and derivatives like ethyl ferulate have risen to prominence as a foundation for many drug discovery efforts owing to their structural diversity and wide-ranging biological activities. In the present study, 24 ethyl ferulate derivatives were synthesized. Their anti-inflammatory activity was evaluated in vitro using RAW264.7 cells and CCK-8, ELISA, and Western blotting assays. These analyses revealed that most of the synthesized compounds exhibited moderate to high anti-inflammatory activities. In particular, c10 and c23 exerted more pronounced activity than ethyl ferulate or dexamethasone with respect to the suppression of tumour necrosis factor-α production by RAW264.7 cells through the targeting of the NF-κB and MAPK signalling pathways, suggesting that these compounds warrant further investigation.

Cardioprotective effect of ethanol extracts of Sugemule-3 decoction on isoproterenol-induced heart failure in Wistar rats through regulation of mitochondrial dynamics.

ETHNOPHARMACOLOGICAL RELEVANCE: Sugemule-3 decoction (SD-3) is a commonly used prescription in Mongolian medicine which composed of the herbs Baidoukou (the fruit of Amomum compactum Sol. ex Maton), Baijusheng (the fruit of Lactuca sativa L.) and Biba (Piper longum L.). SD-3 has remarkable effect on the cardiovascular diseases, but its pharmacological mechanism has not been elucidated. AIM OF THIS STUDY: To evaluate the cardioprotective effects and the potential mechanisms of the ethanol extracts of SD-3 against isoproterenol (ISO)-induced heart failure (HF) in rats. MATERIAL AND METHODS: The ethanol extracts of SD-3 were prepared and analyzed by LC-ESI-MS/MS. One hundred male Wistar rats were randomly divided into five groups: control, ISO (HF) and different doses of SD-3 (0.4, 0.2, 0.1 g/kg/d) groups. HF model rats were established by intraperitoneal injecting of ISO. The left ventricular function was evaluated by echocardiography. Myocardial injury and fibrosis were examined by hematoxylin-eosin (HE) and Masson staining. Western-blot analysis was performed to determine the protein expression of apoptosis and mitochondrial dynamics in all the groups. Moreover, the structural changes in the mitochondria of cardiomyocytes were also observed by transmission electron microscopy. RESULTS: Fifteen compounds were detected in the ethanol extracts of SD-3, include piperine, piperanine, etc. Rats administered with ISO showed a significant decline in the left ventricular function. The cardiac histopathological changes such as local necrosis, interstitial edema, and cardiac fibrosis were also observed in the ISO group. The treatment with SD-3 significantly inhibited these effects of ISO. ISO was found to increase the protein expression of Bax, cleaved-PARP and cleaved-caspase-3, -7 -9, destroy the balance between mitochondrial fusion and fission, and alter the mitochondrial morphology. The ethanol extracts of SD-3 could rebalance mitochondrial fusion and fission, and ameliorates the morphological abnormalities induced by ISO in mitochondria. CONCLUSION: The current study demonstrated that ethanol extracts of SD-3 improved isoprenaline-induced cardiac hypertrophy and fibrosis through inhibiting cardiomyocyte apoptosis and regulating the mitochondrial dynamics.

Pteryxin attenuates LPS-induced inflammatory responses and inhibits NLRP3 inflammasome activation in RAW264.7 cells.

ETHNOPHARMACOLOGICAL RELEVANCE: Pteryxin is a natural coumarin compound that is found in "Qianhu", a traditional Chinese medicine, which possesses heat-clearing and detoxifying functions according to the theory of Traditional Chinese Medicine. Despite its medicinal effects, its anti-inflammatory and mechanisms of actions have not been established. AIM OF THIS STUDY: This study aims to evaluate the anti-inflammatory property and reveal the possible anti-inflammatory mechanisms of pteryxin. MATERIAL AND METHODS: LPS-induced RAW 264.7 macrophages and LPS-induced zebrafish model were used for the anti-inflammatory activity determination of pteryxin. The level of NO, PEG2, TNF-α and IL-6 were measured by ELISA. The accumulation of NO and ROS was stained and observed by a fluorescence microscopy. The nuclear translocation of NF-κB p65 and formation of NLRP3 inflammasome complex in LPS-induced RAW 264.7 macrophage cells were analyzed by immunofluorescence assay. The expression level of iNOS, IL-6, COX-2, TNF-α, p-p38, p38, ERK, JNK, p-ERK, p-JNK, IKK, IκB-α, p-IKK, p-IκB-α, p65, NLRP3, p-p65, Caspase 1 (p 20), ASC, and GAPDH were determined by Western blotting. RESULTS: Lipopolysaccharide (LPS)-induced prostaglandin E2 (PGE2) and nitric oxide (NO) secretions were found to be downregulated by pteryxin. Moreover, pteryxin significantly suppressed inflammatory factor secretion in LPS-treated RAW 264.7 cells. Mechanistically, pteryxin significantly downregulated NF-κB/MAPK activation. Moreover, pteryxin inhibited caspase-1 and NLRP3 activation and formation of ASC specks in RAW 264.7 cells, implying that pteryxin inhibits inflammasome assembly, which is a signal for NLRP3 inflammasome activation. In conclusion, pteryxin blocks NF-κB/MAPK signaling, and suppresses the initiation and activation of NLRP3 thereby preventing inflammation. CONCLUSION: Pteryxin is a potential treatment option for inflammatory-related diseases.

Formalin Itch Test: Low-Dose Formalin Induces Histamine-Independent, TRPA1-Mediated Itch in Mice.

Chronic itch is a common distressing symptom of many diseases, which reduced patient's quality of life. The mechanistic study on itch and screening for new anti-itch drugs require the development of new pre-clinical itch animal models. Herein, we established an acute itch model by intradermal (i.d.) injection of low-dose formalin into the neck or cheek in mice. In mice, i.d. injection of formalin (0.1-5%) in the nape of the neck evoked robust scratching behavior in a dose-dependent manner and the dose-response curves showed an inverted "U" shape. I.d. injection of formalin (0.3-0.6%) into the cheek evoked scratching in mice but wiping in rats, while formalin (1.25-5%) induced mixed wiping and scratching behavior in both mice and rats. Further, we found that 0.3% formalin-induced scratching was histamine-independent and significantly attenuated by transient receptor potential ion channel A1 (TRPA1) inhibitor (HC030031) or in TRPA1 knockout (KO) mice, but not affected by transient receptor potential ion channel V1 (TRPV1) inhibitor (capsazepine) or in TRPV1 KO mice. Additionally, 0.3% formalin-induced up-regulation of phosphorylation of extracellular regulated protein kinases (p-ERK) in the dorsal root ganglion (DRG) and scratching were suppressed by intrathecal injection of MEK inhibitor U0126 in mice. Incubation of 0.03% formalin induced the accumulation of intracellular reactive oxygen species (ROS) in the cultured DRG-derived cell line ND7-23, and formalin-induced itch was suppressed by antioxidants in mice. Finally, perfusion of 0.03% formalin induced elevation of intracellular calcium in a subset of primary cultured DRG neurons of mice. Thus, these results indicate that low-dose formalin induced non-histaminergic itch by activation of TRPA1 in mice, which may be employed as a useful acute itch model for screening potential anti-itch drugs.

Silenced long non-coding RNA activated by DNA damage elevates microRNA-495-3p to suppress atherosclerotic plaque formation via reducing Krüppel-like factor 5.

OBJECTIVE: Atherosclerosis (AS) is an inflammatory disease and the formation of atherosclerotic plaque plays a critical role in AS progression. We aimed to investigate the effect of long non-coding RNA (lncRNA) activated by DNA damage (NORAD)/microRNA-495-3p (miR-495-3p)/Krüppel-like factor 5 (KLF5) axis on atherosclerotic plaque formation. METHODS: The ApoE-/- mice were fed a high-fat diet to construct AS mouse models and the modeled mice were treated with altered NORAD, miR-495-3p or KLF5. NORAD, miR-495-3p and KLF5 expression in mouse aorta tissues were evaluated, and the levels of inflammatory factors, oxidative stress factors, endothelial function indices and blood lipid in mice were all determined. The atherosclerotic plaque area, lipid deposition area, collagen fibers and CD68 expression in mouse aorta tissues were assessed. The regulatory relation between NORAD and miR-495-3p, and the target relation between miR-495-3p and KLF5 were confirmed. RESULTS: NORAD and KLF5 were increased whereas miR-495-3p was decreased in atherosclerotic mouse aortas. Inhibited NORAD or elevated miR-495-3p suppressed inflammation, oxidative stress, endothelial dysfunction, blood lipid level, atherosclerotic plaque area, collagen fibers and CD68 expression in atherosclerotic mouse aortas. Effects of elevated miR-495-3p on atherosclerotic mice could be reversed by up-regulation of KLF5. NORAD served as a sponge of miR-495-3p and miR-495-3p directly targeted KLF5. CONCLUSION: Silenced NORAD elevated miR-495-3p to suppress atherosclerotic plaque formation via reducing KLF5. Findings in our research may be helpful for exploring molecular mechanisms of AS.

The Role of Transient Receptor Potential A1 and G Protein-Coupled Receptor 39 in Zinc-Mediated Acute and Chronic Itch in Mice.

Itching is a common symptom of many skin or systemic diseases and has a negative impact on the quality of life. Zinc, one of the most important trace elements in an organism, plays an important role in the regulation of pain. Whether and how zinc regulates itching is largely unclear. Herein, we explored the role of Zn2+ in the regulation of acute and chronic itch in mice. It is found that intradermal injection (i.d.) of Zn2+ dose-dependently induced acute itch and transient receptor potential A1 (TRPA1) participated in Zn2+-induced acute itch in mice. Moreover, the pharmacological analysis showed the involvement of histamine, mast cells, opioid receptors, and capsaicin-sensitive C-fibers in Zn2+-induced acute itch in mice. Systemic administration of Zn2+ chelators, such as N,N,N',N'-Tetrakis(2-pyridylmethyl)ethylenediamine (TPEN), pyrithione, and clioquinol were able to attenuate both acute itch and dry skin-induced chronic itch in mice. Quantitative polymerase chain reaction (Q-PCR) analysis showed that the messenger RNA (mRNA) expression levels of zinc transporters (ZIPs and ZnTs) significantly changed in the dorsal root ganglia (DRG) under dry skin-induced chronic itch condition in mice. Activation of extracellular signal-regulated kinase (ERK) pathway was induced in the DRG and skin by the administration of zinc or under dry skin condition, which was inhibited by systemic administration of Zn2+ chelators. Finally, we found that the expression of GPR39 (a zinc-sensing GPCR) was significantly upregulated in the dry skin mice model and involved in the pathogenesis of chronic itch. Together, these results indicated that the TRPA1/GPR39/ERK axis mediated the zinc-induced itch and, thus, targeting zinc signaling may be a promising strategy for anti-itch therapy.

LINC01087 is Highly Expressed in Breast Cancer and Regulates the Malignant Behavior of Cancer Cells Through miR-335-5p/Rock1.

PURPOSE: Long non-coding RNA is involved in the genesis and development of various tumors, and it has been found through database screening that LINC01087 is highly expressed in breast cancer (BC), but mechanisms of LINC01087 in BC are still under investigation. Therefore, this study aimed to study relevant mechanisms of LINC01087 in BC to provide potential therapeutic targets for the disease in clinic practice. PATIENTS AND METHODS: The qRT-PCR assay was applied to determine the LINC01087 expression in BC, and the cell counting kit-8 (CCK8) assay, transwell assay, and flow cytometry were used to analyze the proliferation, apoptosis, and invasion of breast cancer cells (BCCs), respectively. The Western blot assay was used to determine the ROCK1 expression, and the luciferase reporter gene assay, RNA-binding protein immunoprecipitation (RIP), and RNA pull-down assays were applied to study the interaction between LINC01087 and miR-335-5p. Moreover, tumor xenotransplantation was conducted in nude mice to explore the effects of LINC01087 on BCCs. RESULTS: The qRT-PCR assay revealed that the LINC01087 expression in BC tissues was higher than that in corresponding tumor-adjacent tissues, and survival analysis revealed an unfavorable prognosis of patients with high expression of LINC01087. Down-regulation of LINC01087 could slow down the proliferation, invasion, and migration of BCCs and accelerate apoptosis of them in vitro. Luciferase reporter gene assay results revealed that LINC01087 enhanced the expression of ROCK1 by regulating miR-335-5p, and LINC01087 could be adopted as a miR-335-5p sponge to inhibit ROCK1 expression. CONCLUSION: LINC01087 is overexpressed in cases with BC, and patients with high expression of it suffer a poor survival. Furthermore, LINC01087 can act as a miR-335-5p sponge to affect the expression of ROCK1 and affect the invasion and migration of BCCs.