Interleukin-6 mRNA Research Articles

Elevated serotonin alters whole-blood expression of serotonin receptor and metabolism genes in the lactating dairy cow

Serotonin's role as a hormone has been demonstrated through modulating calcium metabolism, energy homeostasis, and immune function in the preweaned and lactating bovine. Recently, manipulation of the serotonergic axis in calves via administration of 5-hydroxy-l-tryptophan (5-HTP), the serotonin precursor, elicited immunomodulatory effects and regulated serotonin transport and metabolism genes in leukocytes. However, serotonin's ability to modulate whole-blood gene expression in lactating dairy cows is unknown. Our objective was to explore the effect of infusing 5-HTP on whole-blood expression of genes regulating serotonin transport (reuptake transporter), signaling (receptors), metabolism (synthesis and degradation), and cytokines in dairy cows. Twelve lactating multiparous Holstein dairy cows were blocked by parity, milk production, and days in milk in a randomized complete block design and randomly assigned to intravenous infusion of either 1 L of 1.5 mg/kg 5-HTP dissolved in saline (n = 6) or 1 L of saline solution (n = 6) for 3 consecutive days. Whole blood was collected at 48, 56, and 72 h relative to termination of first infusion to analyze whole-blood gene expression. Infusion of 5-HTP increased whole-blood mRNA expression of monoamine oxidase-A and serotonin receptor 7 across the experimental period. Forty-eight hours from termination of first infusion, the mRNA of monoamine oxidase-A, serotonin reuptake transporter, and serotonin receptor 7 were increased in blood relative to the control. Interleukin-8 concentrations and mRNA were unchanged in response to 5-HTP infusion. Collectively, these data suggest that infusion of 5-HTP may alter mRNA of serotonin metabolism, transport, and signaling genes in whole blood of lactating dairy cows.

Human Islet Expression Levels of Prostaglandin E2 Synthetic Enzymes, But Not Prostaglandin EP3 Receptor, Are Positively Correlated with Markers of β-Cell Function and Mass in Nondiabetic Obesity.

Elevated islet production of prostaglandin E2 (PGE2), an arachidonic acid metabolite, and expression of prostaglandin E2 receptor subtype EP3 (EP3) are well-known contributors to the β-cell dysfunction of type 2 diabetes (T2D). Yet, many of the same pathophysiological conditions exist in obesity, and little is known about how the PGE2 production and signaling pathway influences nondiabetic β-cell function. In this work, plasma arachidonic acid and PGE2 metabolite levels were quantified in a cohort of nondiabetic and T2D human subjects to identify their relationship with glycemic control, obesity, and systemic inflammation. In order to link these findings to processes happening at the islet level, cadaveric human islets were subject to gene expression and functional assays. Interleukin-6 (IL-6) and cyclooxygenase-2 (COX-2) mRNA levels, but not those of EP3, positively correlated with donor body mass index (BMI). IL-6 expression also strongly correlated with the expression of COX-2 and other PGE2 synthetic pathway genes. Insulin secretion assays using an EP3-specific antagonist confirmed functionally relevant upregulation of PGE2 production. Yet, islets from obese donors were not dysfunctional, secreting just as much insulin in basal and stimulatory conditions as those from nonobese donors as a percent of content. Islet insulin content, on the other hand, was increased with both donor BMI and islet COX-2 expression, while EP3 expression was unaffected. We conclude that upregulated islet PGE2 production may be part of the β-cell adaption response to obesity and insulin resistance that only becomes dysfunctional when both ligand and receptor are highly expressed in T2D.

Potential targets of Euodiae Fructus in treatment of insomnia based on network pharmacology

The acupoint application of Euodiae Fructus at Yongquan(KI1) can significantly improve the sleep quality of patients with insomnia with berberine as the main effective component for the efficacy. Nineteen active compounds and 203 drug targets were screened out from Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform(TCMSP). After comparison with GeneCards and Online Mendelian Inheritance in Man(OMIM), 24 common genes of diseases and drugs were obtained. STRING 11.0 was used to construct a protein-protein interaction(PPI) network of the overlapping genes, and Matthews correlation coefficient(MCC) was employed to screen the core genes, which were then subjected to enrichment analysis with gene ontology(GO) and Kyoto encyclopedia of genes and genomes(KEGG). The results revealed that the main compounds of Euodiae Fructus, such as berberine and rutaecarpine, participated in the biological processes(such as neurotransmitter receptor activity) by regulating C-reactive protein(CRP), estrogen receptor 1(ESR1), 5-hydroxytryptamine(5-HT) receptor, and interleukin-6(IL-6) to exert sedative, anxiolytic, and antidepressant effects. Sixty 4-week-old SPF mice were randomly divided into a control group, a model group, a positive drug(diazepam tablets) group, and low-, medium-, and high-dose berberine groups. Medication with corresponding drugs was performed for one week. The results demonstrated that berberine was potent in reducing the activities and standing times of mice, down-regulating the levels of CRP and IL-6 mRNA in the hypothalamus, and up-regulating the expression of 5-HT(P<0.01); however, no significant effect on ESR1 was observed. The network of Euodiae Fructus in treating insomnia was constructed by network pharmacology and verified by tests. The findings indicated that the therapeutic efficacy of Euodiae Fructus in treating insomnia was achieved by participating in multiple biological processes, such as neurotransmitter receptor activity, which provided a scientific basis for its clinical application.

Inhibitory Effect of Korean Red Ginseng Extract on Interleukin-8 Expression in Helicobacter pylori-Infected Gastric Epithelial Cells

ObjectivesInfection of Helicobacter pylori (H. pylori), a gram-negative bacterium, leads to various gastric diseases, such as gastritis, peptic ulcer and gastric cancer. H. pylori increases cytokine release and activates inflammatory mediators in gastric mucosa. Particularly, H. pylori upregulates the inflammatory chemokine interleukin-8 (IL-8), which are activated by oxidative stress. IL-8 can cause severe inflammation of the stomach and gastric cancer. Korean red ginseng is the steamed root of 6-year-old Korean ginseng (Panax ginseng Meyer). Ginsenosides, triterpene glycosides, are the active components of Korean red ginseng. Ginsenosides have antioxidant, anti-inflammatory, and antitumor activities. The present study is aimed at determining whether Korean red ginseng extract inhibits H. pylori-induced IL-8 expression in gastric epithelial cells. MethodsThe human gastric epithelial cell line AGS was used. Gastric epithelial AGS cells were treated with Korean red ginseng extract, and infected with H. pylori (NCTC 11,637). Reactive oxygen species (ROS) levels were determined using dichlorofluorescein fluorescence. NADPH oxidase activity was measured using lucigenin chemiluminescence. IL-8 mRNA expression was measured by using real-time PCR. NADPH oxidase subunits were determined in cytosolic extract and membrane extract by using Western blotting. ResultsH. pylori increased NADPH oxidase activity, ROS levels, and upregulated IL-8 expression in gastric epithelial cells. Korean red ginseng extract inhibited IL-8 expression by suppressing NADPH oxidase activity and reducing ROS levels in gastric epithelial cells. H. pylori induced translocation of NADPH oxidase cytosolic subunits to membrane, which is a marker of NADPH oxidase activation, in AGS cells. Korean red ginseng extract inhibited translocation of cytosolic subunits of NADPH oxidase to membrane in AGS cells. ConclusionsKorean red ginseng may be beneficial for preventing H. pylori-associated gastric inflammation by inhibiting oxidative stress and IL-8 expression. Funding SourcesThis study was supported by a Brain Korea 21 FOUR Project, Yonsei University, Seoul, Republic of Korea.

Palmitic Acid, but Not Lauric Acid, Induces Metabolic Inflammation, Mitochondrial Fragmentation, and a Drop in Mitochondrial Membrane Potential in Human Primary Myotubes

The chain length of saturated fatty acids may dictate their impact on inflammation and mitochondrial dysfunction, two pivotal players in the pathogenesis of insulin resistance. However, these paradigms have only been investigated in animal models and cell lines so far. Thus, the aim of this study was to compare the effect of palmitic (PA) (16:0) and lauric (LA) (12:0) acid on human primary myotubes mitochondrial health and metabolic inflammation. Human primary myotubes were challenged with either PA or LA (500 μM). After 24 h, the expression of interleukin 6 (IL-6) was assessed by quantitative polymerase chain reaction (PCR), whereas Western blot was used to quantify the abundance of the inhibitor of nuclear factor κB (IκBα), electron transport chain complex proteins and mitofusin-2 (MFN-2). Mitochondrial membrane potential and dynamics were evaluated using tetraethylbenzimidazolylcarbocyanine iodide (JC-1) and immunocytochemistry, respectively. PA, contrarily to LA, triggered an inflammatory response marked by the upregulation of IL-6 mRNA (11-fold; P < 0.01) and a decrease in IκBα (32%; P < 0.05). Furthermore, whereas PA and LA did not differently modulate the levels of mitochondrial electron transport chain complex proteins, PA induced mitochondrial fragmentation (37%; P < 0.001), decreased MFN-2 (38%; P < 0.05), and caused a drop in mitochondrial membrane potential (11%; P < 0.01) compared to control, with this effect being absent in LA-treated cells. Thus, LA, as opposed to PA, did not trigger pathogenetic mechanisms proposed to be linked with insulin resistance and therefore represents a healthier saturated fatty acid choice to potentially preserve skeletal muscle metabolic health.

LncRNA NEAT1 Knockdown Alleviates Lipopolysaccharide-Induced Acute Lung Injury by Modulation of miR-182-5p/WISP1 Axis.

Accumulating evidence has demonstrated the vital roles of long non-coding RNAs (lncRNAs) in acute lung injury (ALI). In this study, we aimed to explore the effect of Nuclear Paraspeckle Assembly Transcript 1 (NEAT1) on ALI development. The ALI mice and cell models were constructed using lipopolysaccharide (LPS)-induced method. The concentrations of tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6) and interleukin-1β (IL-1β) were measured by enzyme-linked immunosorbent assay (ELISA). The levels of TNF-α mRNA, IL-6 mRNA, IL-1β mRNA, NEAT1, miR-182-5p, and WNT-inducible secreted protein 1 (WISP1) mRNA were determined by quantitative real-time polymerase chain reaction (qRT-PCR) assay. Cell viability was evaluated by Cell Counting Kit-8 (CCK-8) assay. The level of lactate dehydrogenase (LDH) and the activity of caspase-3 were measured by specific kits. The interaction between miR-182-5p and NEAT1 or WISP1 was investigated by dual-luciferase reporter and RNA immunoprecipitation (RIP) assays. Protein levels were measured by Western blot assay. NEAT1 level was elevated in LPS-induced ALI mice and LPS-stimulated MH-S cells. LPS treatment repressed MH-S cell viability and promoted apoptosis and inflammation, while NEAT1 silencing restored the impacts. For mechanism analysis, NEAT1 was identified as the sponge for miR-182-5p to positively regulate WISP1 expression. Moreover, NEAT1 knockdown could accelerate cell viability and inhibit cell apoptosis and inflammation in LPS-induced MH-S cells by elevating miR-182-5p and decreasing WISP1 in LPS-exposed MH-S cells. In addition, NEAT1 deficiency blocked the activation of NF-κB pathway caused by LPS in MH-S cells. NEAT1 overexpression restrained cell viability and facilitated cell apoptosis and inflammation in LPS-exposed MH-S cells through miR-182-5p/WISP1 axis.

Hepatic stellate cell-derived exosomes modulate macrophage inflammatory response

BackgroundHepatic stellate cell (HSC) differentiation/activation is central to liver fibrosis and is innately linked to the immune response to liver injury. Exosomes (EXOs) are important means of communication between cell populations. This study sought to characterize EXO release from HSCs and the effect of HSC-EXOs on macrophage cytokine release/function. MethodsLiver from a rat fibrosis model was analyzed for EXO expression and localization. Quiescent and culture-activated rat and mouse HSCs and activated human HSCs were analyzed for microRNA expression. Mouse, rat, and human HSCs were culture-activated and EXOs purified from culture medium prior to addition to macrophages, and interleukin-6 (IL-6) and tumor necrosis factor-α (TNFα) mRNA and protein measured. The effect of activated HSC-EXOs on macrophage migration was assayed. ResultsActivation of rat HSCs led to increased EXO production in vivo, an effect mirrored by in vitro rat HSC culture-activation. Culture activation of mouse and rat HSCs led to altered EXO microRNA profiles, with a similar microRNA profile detected in activated human HSCs. Addition of activated HSC-EXOs to macrophages stimulated IL-6 and TNFα mRNA expression and protein secretion in mouse and human macrophages, but not for rat HSC-EXO-macrophages. Addition of human EXOs to macrophages stimulated migration, effects mirrored by the direct addition of rhIL-6 and rhTNFα. ConclusionsHSC-EXOs associate with macrophages and stimulate cytokine synthesis-release and macrophage migration. Constructing a comprehensive understanding of EXO interactions between liver cell populations in the setting of inflammation/fibrosis increases the potential for developing new diagnostic/therapeutic approaches.

Induction of the Proinflammatory Chemokine Interleukin-8 Is Regulated by Integrated Stress Response and AP-1 Family Proteins Activated during Coronavirus Infection.

Infection induces the production of proinflammatory cytokines and chemokines such as interleukin-8 (IL-8) and IL-6. Although they facilitate local antiviral immunity, their excessive release leads to life-threatening cytokine release syndrome, exemplified by the severe cases of coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. In this study, we investigated the roles of the integrated stress response (ISR) and activator protein-1 (AP-1) family proteins in regulating coronavirus-induced IL-8 and IL-6 upregulation. The mRNA expression of IL-8 and IL-6 was significantly induced in cells infected with infectious bronchitis virus (IBV), a gammacoronavirus, and porcine epidemic diarrhea virus, an alphacoronavirus. Overexpression of a constitutively active phosphomimetic mutant of eukaryotic translation initiation factor 2α (eIF2α), chemical inhibition of its dephosphorylation, or overexpression of its upstream double-stranded RNA-dependent protein kinase (PKR) significantly enhanced IL-8 mRNA expression in IBV-infected cells. Overexpression of the AP-1 protein cJUN or its upstream kinase also increased the IBV-induced IL-8 mRNA expression, which was synergistically enhanced by overexpression of cFOS. Taken together, this study demonstrated the important regulatory roles of ISR and AP-1 proteins in IL-8 production during coronavirus infection, highlighting the complex interactions between cellular stress pathways and the innate immune response.

Knockout of the Cannabinoid Receptor 2 Gene Promotes Inflammation and Hepatic Stellate Cell Activation by Promoting A20/Nuclear Factor-κB (NF-κB) Expression in Mice with Carbon Tetrachloride-Induced Liver Fibrosis.

BackgroundThis study aimed to investigate the effect of deleting the cannabinoid receptor 2 (CB2) gene on the development of hepatic fibrosis induced by carbon tetrachloride (CCl4) in mice via regulating inflammation.Material/MethodsThe DNA was extracted from the tails of mice to identify whether the cannabinoid receptor 2 gene was successfully knocked out. A liver fibrosis model was established by an intraperitoneal injection of CCl4 into mice. Hepatic damage and hepatic fibrosis were evaluated by detecting serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), and staining paraffin sections of liver tissue with hematoxylin-eosin (HE). The secretion and distribution of collagen in liver tissue were observed by Masson staining. Western blot analysis was performed to detect the expression of α-smooth muscle actin (α-SMA), transforming growth factor-β1 (TGF-β1), tumor necrosis factor alpha-induced protein 3 (A20), phosphorylated nuclear factor-κB p65 (p-NF-κB p65), tumor necrosis factor alpha (TNF-α), and interleukin-6 (IL-6) in liver tissue. Reverse transcription-polymerase chain reaction (RT-PCR) was used to detect the expression of IL-6 and TNF-α mRNA in liver tissue.ResultsCompared with the control mice, the mice with CB2 knockout that were exposed to CCl4 exhibited increased liver damage, liver fibrosis, and upregulated α-SMA, TGF-β1, A20, and p-NF-κB p65 protein levels. IL-6 and TNF-α protein levels and mRNA levels were upregulated.ConclusionsThe deletion of the CB2 gene promoted the activation of hepatic stellate cells in mice with liver fibrosis and aggravated liver fibrosis by up-regulating the protein expression of A20 and p-NF-κB p65 and inducing inflammatory response, potentially providing new insight into the treatment of liver fibrosis.

FTY720 Inhibits the Development of Collagen-Induced Arthritis in Mice by Suppressing the Recruitment of CD4+ T Lymphocytes.

BackgroundFingolimod (FTY720), a novel immunomodulator, was found to suppress the severity of collagen-induced arthritis (CIA) in mice. However, the potential molecular mechanisms are still unknown, and the effect of FTY720 on the recruitment of immune cells in the affected joints in the CIA model is not clear.Materials and MethodsFollowing the oral administration of FTY720 (2 mg/kg) was treated into CIA mice per day for 35 days, intravital microscopy and immunofluorescence assays were performed to examine immune cell recruitment in the affected joints. Human MH7A synoviocytes were stimulated with tumour necrosis factor (TNF)-α and incubated with FTY720. Interleukin-1β (IL-1β), interleukin-6 (IL-6), and interleukin-8 (IL-8) mRNA and protein expression were evaluated using RT-PCR and enzyme-linked immunosorbent assay, respectively. Signal transduction pathway protein expression was measured by Western blotting. Nuclear translocation of nuclear factor (NF)-κB was also analyzed by fluorescence microscopy.ResultsIn vivo experiments showed that FTY720 inhibited the recruitment of CD4+ lymphocytes in the affected joints of CIA mice. FTY720 reduced the secretion of IL-1β, IL-6, and IL-8 from TNF-α-stimulated MH7A cells in a dose-dependent manner. FTY720 also inhibited TNF-α-induced phosphorylation of NF-κBp65 and IκBα, as well as NF-κBp65 nuclear translocation, in a dose- and time-dependent manner. Interestingly, FTY720 blocked PI3K/Akt, the upstream targets of the NF-κB pathway.ConclusionOur findings demonstrated that oral administration of FTY720 exerted beneficial effects in CIA mice by inhibiting CD4+ T lymphocyte recruitment to the affected joints. Our data also indicated that FTY720 inhibited TNF-α-induced inflammation by suppressing the AKT/PI3K/NF-κB pathway in MH7A cells.

Suppressor of cytokine signalling 3 (SOCS3) expressed in podocytes attenuates glomerulonephritis and suppresses autoantibody production in an imiquimod-induced lupus model

ObjectiveRecently, podocytes have been recognised not only as a physical barrier to prevent urinary protein loss but also as producers of proinflammatory cytokines. However, the roles of podocytes in the...

SOCS3 Limits Pro‐Inflammatory Signature in Septic Endothelium

During sepsis, the innate immune system releases multiple inflammatory cytokines in a process known as the cytokine storm, which results in a severe and persistent inflammatory response. The cytokine storm affects practically all aspects of endothelial cell function and is thought to be the key factor in the progression from sepsis to organ failure. Interleukin 6 (IL-6), a major mediator of the cytokine storm during shock, activates JAK kinases to phosphorylate the transcription factor STAT3. In turn, STAT3 promotes expression of SOCS3, which leads to a potent negative regulation of this pathway. The aim of this study is to determine the role of SOCS3 in the regulation of the intensity and duration of IL-6 signaling in the endothelium by assessing the response to endotoxin of mice lacking SOCS3 specifically in the endothelial cells (SOCS3iEKO). Two weeks after tamoxifen induced SOCS3 deletion, we induced severe acute inflammation by a single IP injection of LPS. While this treatment was not lethal in control mice, SOCS3iEKO mice died in less than 24 hours after injection, with death starting at ~16 hours post-treatment. Furthermore, a severity score applied at 15 hours showed an increased severity in the endotoxemic mice, which was further aggravated by the SOCS3 deficiency. We also detected a 10-fold increase in whole tissue IL-6 mRNA levels in kidney, lung and liver in these mice, suggesting persistent IL-6 activation in the SOCS3iEKO mice. Collectively, our data suggest that the loss of endothelial SOCS3 exacerbates the LPS-induced pro-thrombotic and type I interferon-like transcriptional response. We detected increased levels tissue factor, as well as MX1, IRF8 and OASL1expression in endotoxin-treated mice that was further aggravated by the loss of endothelial SOCS3. Bioinformatic analysis of gene expression (RNA-Seq) from HUVEC treated with IL-6 showed a similar activation of an IFN-like response. We observed increased NETosis in SOCS3iEKO kidneys upon LPS. Consistently, SOCS3iEKO mice showed severe kidney failure in response to LPS, as determined by direct glomerular filtration rate assays, and confirmed by increased plasma BUN levels. Fluorescent dextran assays showed kidney cortex areas devoid of fluorescence, suggesting tissue hypoperfusion. In summary, levels of endothelial SOCS3 are critical to regulating the extent of the inflammatory response, and overactivation of this pathway in the endothelium of SOCS3iEKO mice leads to endotheliopathy that will promote kidney failure and lethality.

Fibrinogen activates IκBα signaling in neurons via binding to its receptors, ICAM‐1 and PrP C

Mild traumatic brain injury (TBI) is the most common type of brain injury that is a major health issue. The goal of the current study was to extend our previous observations that during TBI, extravasated fibrinogen (Fg) deposited in vasculo/astrocyte interfaces and was associated with neurodegeneration and short-term memory (STM) reduction. We have previously shown that an elevated level of Fg, called hyperfibrinogenemia, causes astrocyte activation via interactions with intercellular adhesion molecule-1 (ICAM-1) and cellular prion protein (PrPC). However, the direct effect of Fg on neurons is not known. Primary mouse brain cortical neurons from C57BL/6 mice (Lonza) were grown in Primary Neural Basal Medium and PNGMTM Single QuotsTM. Twenty-four well plates, with #1 glass-bottomed coverslips coated with Poly-D-Lysine (30 µg/ml) and laminin (200 µg/ml) were used for seeding neurons at the density of 200,000 cells/well. After the required 7-day growth in complete medium, neurons were treated with various doses of Fg for 24 hours unless otherwise indicated. Each experimental group contained hirudin (1 U/ml) to inhibit the conversion of Fg to fibrin via thrombin. In parallel, experiments to test neuronal viability using a live/dead assay were performed without the presence of hirudin. Interactions of Fg with neuronal ICAM-1 and PrPC were tested using a proximity ligation assay (PLA), which allows highly specific and sensitive immunofluorescent detection to be visualized and consequently protein-protein interactions in situ to be objectively quantified. Real time PCR was performed to investigate the expression of mRNAs for interleukin 6 (IL-6) and inhibitor of kappa Bα (IκBα). The levels of IκBα mRNA reflect stimulation of nuclear factor-kappa B (NF-κB) transcription. PLA confirmed Fg-ICAM-1 and Fg-PrPC interactions on the surface of neurons. An increased expression of neuronal IκBα and IL-6 was induced after treatment with Fg for 2hr or 24 hr, respectively. Data showed that Fg, in the presence or absence of hirudin, caused increased neuronal death. Thus, Fg binding to its neuronal receptors, ICAM-1 and PrPC may activate NF-κB, triggered by IκBα signaling, that leads to neuroinflammation and increased neuronal death. This study is a first attempt to define the molecular mechanisms by which Fg causes neuroinflammation and neurodegeneration that resulted in the cognitive decline previously seen during mild-to-moderate TBI in our studies.

Respiratory virus induced IL‐6 expression is regulated by an extragenic suppressor

Interleukin‐6 (IL‐6) is a pleiotropic inflammatory cytokine that plays a pivotal role in the activation and regulation of proper immune response during respiratory viral infections. Aberrant IL‐6 expression, however, may promote virus survival and/or exacerbation of clinical symptoms, such as the inflammatory cytokine storms observed in severe COVID‐19 patients infected with SARS‐CoV‐2. IL‐6 is now regarded as a prominent target for clinical intervention but less is known about how IL‐6 itself is regulated in various disorders.Using precision nuclear run‐on next generation sequencing (PRO‐Seq) to measure the transcriptionally engaged RNA polymerases (RNA Pol II) in human primary small airway epithelial cells (hSAECs), we have discovered an upstream enhancer‐like region in the IL‐6 gene locus. This is a region that resides within an open chromatin domain. It has DNase I hypersensitivity and enriches in activating H3K27Ac mark, but lack of active transcription mark H3K36me3. Interestingly, it displays increased ratio of H3K4me1 over H3K4me3, a phenomenon usually associated with active enhancers. We were therefore surprised to observe that rhinovirus (RV) infection disrupts active RNA Pol II binding in this region, despite the fact that RNA Pol II is increased in the body of the IL‐6 gene. These findings suggest that the upstream sequence may be functioning as a suppressor of IL‐6 expression. To test this possibility, we targeted the KRAB transcriptional silencer to the upstream sequence using CRISPRi (KRAB/dCas9 directed by sgRNA). hSAECs expressing control sgRNA or the sequence‐specific sgRNA were infected with RV and IL‐6 mRNA measured by Q‐RT‐PCR. Intriguingly, the sequence‐specific sgRNA enhanced RV‐ induced IL‐6 expression. These results suggest that the upstream element is not an enhancer but a functionally active suppressor of IL‐6 expression and that the mechanism of IL‐6 expression involves a previously unknown de‐repression step. It will advance us to understand how dynamic interactions between cis‐regulatory elements regulate the threshold, magnitude and duration of IL‐6 gene transcription in response to distinct factors.

Activated IL-6 signaling contributes to the pathogenesis of, and is a novel therapeutic target for, CALR-mutated MPNs

Calreticulin (CALR), an endoplasmic reticulum–associated chaperone, is frequently mutated in myeloproliferative neoplasms (MPNs). Mutated CALR promotes downstream JAK2/STAT5 signaling through interaction with, and activation of, the thrombopoietin receptor (MPL). Here, we provide evidence of a novel mechanism contributing to CALR-mutated MPNs, represented by abnormal activation of the interleukin 6 (IL-6)-signaling pathway. We found that UT7 and UT7/mpl cells, engineered by clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) to express the CALR type 1–like (DEL) mutation, acquired cytokine independence and were primed to the megakaryocyte (Mk) lineage. Levels of IL-6 messenger RNA (mRNA), extracellular-released IL-6, membrane-associated glycoprotein 130 (gp130), and IL-6 receptor (IL-6R), phosphorylated JAK1 and STAT3 (p-JAK1 and p-STAT3), and IL-6 promoter region occupancy by STAT3 all resulted in increased CALR DEL cells in the absence of MPL stimulation. Wild-type, but not mutated, CALR physically interacted with gp130 and IL-6R, downregulating their expression on the cell membrane. Agents targeting gp130 (SC-144), IL-6R (tocilizumab [TCZ]), and cell-released IL-6 reduced proliferation of CALR DEL as well as CALR knockout cells, supporting a mutated CALR loss-of-function model. CD34+ cells from CALR-mutated patients showed increased levels of IL-6 mRNA and p-STAT3, and colony-forming unit–Mk growth was inhibited by either SC144 or TCZ, as well as an IL-6 antibody, supporting cell-autonomous activation of the IL-6 pathway. Targeting IL-6 signaling also reduced colony formation by CD34+ cells of JAK2V617F-mutated patients. The combination of TCZ and ruxolitinib was synergistic at very low nanomolar concentrations. Overall, our results suggest that target inhibition of IL-6 signaling may have therapeutic potential in CALR, and possibly JAK2V617F, mutated MPNs.

Ellagitannins from Rosa roxburghii suppress poly(I:C)-induced IL-8 production in human keratinocytes.

The anti-inflammatory effects of a 50% aqueous extract of Rosa roxburghii fruit (RRFE) and two ellagitannins (strictinin and casuarictin) isolated from the RRFE were evaluated in a cell model of skin inflammation induced by self-RNA released from epidermal cells damaged by UV ray (UVR) irradiation. The RRFE inhibited interleukin-8 (IL-8) mRNA expression in normal human epidermal keratinocytes (NHEKs) stimulated with polyinosinic:polycytidylic acid (poly(I:C)), a ligand of toll-like receptor-3 (TLR-3). The plant-derived anti-inflammatory agents, dipotassium glycyrrhizinate (GK2) and allantoin, had no influence on the IL-8 expression. The purified compounds, strictinin and casuarictin, inhibited the IL-8 mRNA expression and IL-8 release induced in NHEKs by poly(I:C). These ellagitannins were thus found to be responsible for the biological activity exhibited by the RRFE. This study demonstrates that RRFE and isolated RRFE compounds show promise as ingredients for products formulated to improve skin disorders induced by UVR irradiation.

IL-8 as a Potential Therapeutic Target for Periodontitis and Its Inhibition by Caffeic Acid Phenethyl Ester In Vitro.

Salivary levels of interleukin-8 (IL-8) are elevated in patients with periodontitis. Caffeic acid phenethyl ester (CAPE) improves the periodontal status in subjects. However, whether CAPE can reduce IL-8 expression is unclear. We collected saliva to determine proinflammatory cytokine levels and used subgingival calculus and surrounding tissues from patients with periodontitis for oral microbiota analysis via 16s ribosomal RNA gene sequencing. THP-1 cells were stimulated with sterile-filtered saliva from patients, and target gene/protein expression was assessed. IL-8 mRNA expression was analyzed in saliva-stimulated THP-1 cells treated with CAPE and the heme oxygenase-1 (HO-1) inhibitor tin-protoporphyrin (SnPP). In 72 symptomatic individuals, IL-8 was correlated with periodontal inflammation (bleeding on probing, r = 0.45; p < 0.001) and disease severity (bleeding on probing, r = 0.45; p < 0.001) but not with the four oral microbiota species tested. Reduced salivary IL-8 secretion was correlated with effective periodontitis treatment (r = 0.37, p = 0.0013). In THP-1 cells, saliva treatment induced high IL-8 expression and IKK2 and nuclear factor-κB (NF-κB) phosphorylation. However, the IKK inhibitor BMS-345541, NF-κB inhibitor BAY 11-7082, and CAPE attenuated saliva-induced IL-8 expression. CAPE induced HO-1 expression and inhibited IKK2, IκBα, and NF-κB phosphorylation. Blocking HO-1 decreased the anti-inflammatory activity of CAPE. The targeted suppression of IL-8 production using CAPE reduces inflammation and periodontitis.

Electroacupuncture improves lung function by suppressing mucin-5AC mediated EGFR-p38MAPK signaling and inflammation reaction in chronic obstructive pulmonary disease rats

To observe the effect of electroacupuncture (EA) at "Zusanli"(ST36) on the expression of epidermal growth factor receptor (EGFR), tumor necrosis factor α(TNF-α) transfer growth factor α(TGF-α), interleukin-8(IL-8), p38 mitogen-activated protein kinases (p38MAPK), mucin-5AC (MUC5AC) and other related factors in chronic obstructive pulmonary disease (COPD) rats, so as to reveal its underlying mechanisms in improving COPD. A total of thirty male SD rats were randomly divided into normal control, model and EA groups, with 10 rats in each group. The COPD model was replicated using a combined method of tracheal infusion of lipopolysaccharide (LPS) and forced smoke-inhaling. EA (1-3 mA, 4 Hz/20 Hz) was applied to bilateral ST36 for 30 min, once daily for two consecutive weeks. The lung ventilation activities including the forced vital capacity (FVC) and forced expiratory volume (FEV) at 0.1 and 0.3 s (FEV0.1, FEV0.3) were detected. Histopathological changes of the middle lobe and bronchus of the right lung were observed after H.E. staining. The contents of TGF-α, TNF-α and IL-8 in the serum, bronchoalveolar lavage fluid (BALF) and superior lobe of the right lung were assayed by using ELISA, and the expression levels of EGFR, p38MAPK and MUC5AC proteins (inferior lobe of the left lung) and mRNAs (inferior lobe of the right lung) detected using Western blot, immunohistochemistry (strept avidin-biotin complex, SABC method) and real-time quantitative PCR, respectively. Compared with the normal group, the FVC, FEV0.1, FEV0.3, FEV0.1/FVC and FEV0.3/FVC levels were significantly decreased (P<0.01), while the contents of TNF-α, TGF-α and IL-8 in the serum, BALF and lung tissues, expression levels of EGFR, p38MAPK and MUC5AC mRNAs and proteins, and the immunoactivity of EGFR, p38MAPK and MUC5AC in the lung tissues were significantly increased in the model group (P<0.01). After EA intervention, the decreased levels of the FVC, FEV0.1, FEV0.3, FEV0.1/FVC and FEV0.3/FVC, and the increased levels of the abovementioned genes and proteins were all reversed in the EA group (P<0.01, P<0.05). After modeling, the bronchial walls were thickened, with enlarged alveolar cavities, fractured alveolar walls, obvious inflammatory cell infiltration, and rich mucus secretion in the lumen, which was relatively milder in the EA group. EA of ST36 can improve the ventilation function in COPD rats, which may be associated with its function in down-regulating the levels of TNF-α, TGF-α, IL-8, EGFR, p38MAPK and MUC5AC mRNAs and proteins in the lung tissues, inhibiting EGFR-p38MAPK signaling mediated expression of MUC5AC.

Effects and mechanisms of Xuezhikang on preventing contrast-induced acute kidney injury in diabetic rats

Objective: To explore the effects and mechanisms of Xuezhikang on preventing contrast-induced nephropathy (CIN) in diabetic rats. Methods: Streptozotocin (65 mg/kg) was injected intraperitoneally to establish a diabetes model in 7-week-old male Sprague-Dawley (SD) rats. After 4 weeks of modeling, 24 diabetic rats were randomly divided into 4 groups: sham group, CIN group, CIN+vehicle (Veh) group and Xuezhikang group. All animals were sacrificed at 24 hours after administration of contrast. Blood and kidney tissues were collected to detect biochemical, inflammation-related, oxidative stress-related and pathological indicators. Results: After administration of contrast agent, the renal function-related indicators were decreased in Xuezhikang group compared with CIN+Veh group [serum creatinine (SCr): (59.3±3.3) μmol/L vs (73.2±4.1) μmol/L; blood urea nitrogen (BUN): (13.8±0.5) mmol/L vs (16.3±0.6) mmol/L; serum neutrophil gelatinase-associated lipocalin (sNGAL): (41.4±2.0) ng/ml vs (54.9±4.4) ng/ml; urinary kidney injury moleculer-1 (uKIM-1): (11.1±0.5) ng/ml vs (16.6±0.5) ng/ml] (all P<0.05). Histological analysis showed that the severity of renal tubule dilatation, brush border loss and renal tubular cell necrosis in Xuezhikang group was better than that of CIN+Veh group. Additionally, the oxidative stress-related indicators of Xuezhikang group improved compared with those of CIN+Veh group [malondialdehyde (MDA): (12.1±0.7) nmol/mg vs (15.5±0.8) nmol/mg, superoxide dismutase (SOD): (35.0±2.2) U/mg vs (23.7±3.4) U/mg, renal nitrite: (1.7±0.1) nmol/mg vs (1.2±0.1) nmol/mg, all P<0.05]. Meanwhile, Xuezhikang pretreatment downregulated the mRNA and protein expression of tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) (both P<0.05). Conclusion: The current study suggests that Xuezhikang protects against CIN in diabetic rats by inhibiting oxidative stress and inflammation.

Punicalagin inhibits pro-inflammatory cytokines induced by influenza A virus

IntroductionInfluenza A virus causes severe pneumonia in humans, leading to high morbidity and mortality. Studies have shown that an excessive immune response in overproduction of pro-inflammatory cytokines can exacerbate complications of influenza virus infection. Thus immune-targeted therapies are an appropriate strategy to control the disease. This study was conducted to investigate the effect of punicalagin on expression and concentration levels of tumor necrosis factor alpha (TNFα), interleukin 6 (IL-6) and interleukin1-β in Madin-Darby Canine Kidney (MDCK) cells. MethodsIn this experimental study, confluent MDCK cells were infected with influenza A virus (H1N1; PR8) and treated with punicalagin at different concentrations. After incubation, mRNA expression and concentration levels of TNFa, IL-6 and IL1-β were investigated using real-time polymerase chain reaction (PCR) and enzyme-linked immunesorbent assay (ELISA), respectively. ResultsThe results of quantitative Real-time PCR showed a significant increase in TNFa, IL-6 and IL1-β mRNA expression in control virus compared to cell control, and a significant, dose-dependent decrease in these expression levels in punicalagin-treated virus infected cells compared to control virus (P < 0.05). The ELISA results also confirmed the dose-dependent decrease in TNFa, IL-6 and IL1-β concentrations in protein levels in punicalagin treated, virus infected cells compared to control virus (P < 0.05). ConclusionsOur results demonstrated the anti-inflammatory effects of punicalagin, and therefore suggests that the compound can be used as an appropriate treatment of choice to control cytokines-induced inflammation in influenza A virus infection.