IL-6 mRNA Levels Research Articles

Cannabis Use Associates With Reduced Proviral Burden and Inflammatory Cytokine in Tissues From Men With Clade C HIV-1 on Suppressive Antiretroviral Therapy.

Human immunodeficiency virus 1 (HIV-1) tissue reservoirs remain the main obstacle against an HIV cure. Limited information exists regarding cannabis's effects on HIV-1 infections in vivo, and the impact of cannabis use on HIV-1 parenchymal tissue reservoirs is unexplored. To investigate whether cannabis use alters HIV-1 tissue reservoirs, we systematically collected 21 postmortem brain and peripheral tissues from 20 men with subtype C HIV-1 and with suppressed viral load enrolled in Zambia, 10 of whom tested positive for cannabis use. The tissue distribution and copies of subtype C HIV-1 LTR, gag, env DNA and RNA, and the relative mRNA levels of cytokines IL-1β, IL-6, IL-10, and TGF-β1 were quantified using PCR-based approaches. Utilizing generalized linear mixed models we compared persons with HIV-1 and suppressed viral load, with and without cannabis use. The odds of tissues harboring HIV-1 DNA and the viral DNA copies in those tissues were significantly lower in persons using cannabis. Moreover, the transcription levels of proinflammatory cytokines IL-1β and IL-6 in lymphoid tissues of persons using cannabis were also significantly lower. Our findings suggested that cannabis use is associated with reduced sizes and inflammatory cytokine expression of subtype C HIV-1 reservoirs in men with suppressed viral load.

Epstein-Barr Virus Promotes Inflammatory Cytokine Production in Human Gingival Fibroblasts

BackgroundPeriodontitis is one of the most common chronic oral inflammatory diseases. Over the past decade, herpes viruses, particularly Epstein-Barr virus (EBV), have been considered promising pathogenic candidates for periodontitis. However, the specific mechanism by which EBV contributes to the development of periodontitis is still unknown. This study aimed to explore the mechanism of EBV underlying the inflammatory response in human gingival fibroblasts (HGFs). Materials and methodsHGFs were stimulated with different concentrations of EBV (104, 105, 106, 107, and 108 DNA copies/mL) for 0, 8, 24, or 48 hours. The mRNA levels of interleukin (IL)-1β, tumour necrosis factor-α (TNF-α), IL-8, monocyte chemoattractant protein-1 (MCP-1), and Toll-like receptor 9 (TLR9) were measured using quantitative real-time polymerase chain reaction (PCR). Enzyme-linked immunosorbent assays (ELISAs) were performed for determining the mRNA and protein levels of IL-1β, TNF-α, IL-8, and MCP-1. Real-time PCR and ELISA were performed to determine the protein levels of IL-1β, TNF-α, IL-8, and MCP-1. Activation of the TLR9/myeloid differentiation factor 88 (MyD88)/nuclear factor kappa B (NF-κB) pathway was evaluated using western blotting. ResultsThe expressions of IL-1β, TNF-α, IL-8, and MCP-1 were significantly upregulated in HGFs under EBV stimulation in a concentration- and time-dependent manner. EBV promoted TLR9 and MyD88 expression and induced NF-κB transcription. On the contrary, the upregulation of these factors and the activation of NF-κB pathway were drastically inhibited by TLR9 antagonists. ConclusionsOur findings demonstrate that EBV promotes the production of inflammatory cytokines IL-1β and TNF-α and chemokines IL-8 and MCP-1 in HGFs through the TLR9/MyD88/NF-κB pathway.

Microbial colony sequencing combined with metabolomics revealed the effects of chronic hexavalent chromium and nickel combined exposure on intestinal inflammation in mice

The pollution and toxic effects of hexavalent chromium [Cr(VI)] and divalent nickel [Ni(II)] have become worldwide public health issues. However, the potential detailed effects of chronic combined Cr(VI) and Ni exposure on colonic inflammation in mice have not been reported. In this study, 16S rDNA sequencing, metabolomics data analysis, qPCR and other related experimental techniques were used to comprehensively explore the mechanism of toxic damage and the inflammatory response of the colon in mice under the co-toxicity of chronic hexavalent chromium and nickel. The results showed that long-term exposure to Cr(VI) and/or Ni resulted in an imbalance of trace elements in the colon of mice with significant inflammatory infiltration of tissues. Moreover, Cr(VI) and/or Ni poisoning upregulated the expression levels of IL-6, IL-18, IL-1β, TNF-α, IFN-γ, JAK2 and STAT3 mRNA, and downregulated IL-10 mRNA, which was highly consistent with the trend in protein expression. Combined with multiomics analysis, Cr(VI) and/or Ni could change the α diversity and β diversity of the gut microbiota and induce significant differential changes in metabolites such as Pyroglu-Glu-Lys, Val-Asp-Arg, stearidonic acid, and 20-hydroxyarachidonic acid. They are also associated with disorders of important metabolic pathways such as lipid metabolism and amino acid metabolism. Correlation analysis revealed that there was a significant correlation between gut microbes and metabolites (P < 0.05). In summary, based on the advantages of comprehensive analysis of high-throughput sequencing sets, these results suggest that chronic exposure to Cr(VI) and Ni in combination can cause microbial flora imbalances, induce metabolic disorders, and subsequently cause colonic damage in mice. These data provide new insights into the toxicology and molecular mechanisms of Cr(VI) and Ni.

Impact of ibrutinib on inflammation in a mouse model of Graves' orbitopathy.

Bruton's tyrosine kinase (BTK) and interleukin (IL)-2 Inducible T-cell Kinase (ITK) inhibitors have anti-inflammatory properties. We investigated the therapeutic effect of ibrutinib, an orally bioavailable BTK/ITK inhibitor, in a mouse model of Graves' orbitopathy (GO). Genetic immunization was performed through intramuscular administration of the recombinant plasmid, pCMV6-hTSHR cDNA, to 8-week-old female BALB/c mice. Serum levels of T3, T4, and thyroid-stimulating hormone receptor (TSHR) antibodies (TRAbs) were quantified using enzyme-linked immunosorbent assay. Histopathological changes in orbital tissues were examined using immunohistochemistry (IHC) staining for TSHR and various inflammatory markers. Following successful genetic immunization, ibrutinib was orally administered daily for 2 weeks in the GO model mice. After treatment, the mRNA and protein expression levels of BTK, ITK, IL-1β, and IL-6 in orbital tissues were evaluated using real-time PCR and Western blotting. In total, 20 mice were sacrificed to confirm successful genetic immunization. The GO mouse group exhibited significantly increased serum T3, T4, and TRAb levels. IHC revealed increased expression of TSHR, IL-1β, IL-6, transforming growth factor-β1, interferon-γ, CD40, CD4, BTK, and ITK in the GO mouse model. The orbital inflammation was significantly attenuated in ibrutinib-treated mice. The mRNA and protein expression levels of BTK, ITK, IL-1β, and IL-6 in orbital tissue were lower in ibrutinib-treated GO mouse group compared to the phosphate-buffered saline-treated GO mouse group. The GO mouse model demonstrated enhanced BTK and ITK expression. Ibrutinib, a BTK/ITK inhibitor, suppressed the inflammatory cytokine production. These findings highlight the potential involvement of BTK/ITK in the inflammatory pathogenesis of GO, suggesting its role as a novel therapeutic target.

Typical structural characteristics and hepatoprotective effects of novel high Fischer ratio oligopeptides from Antarctic krill on acute alcoholic liver injury.

High Fischer ratio oligopeptides derived from Antarctic krill (HFOPs-AK) were screened, and their hepatoprotective effects and potential mechanisms were investigated. Herein, HFOPs-AK, with a Fischer ratio of 29 g/g (40.22 mol/mol) (MW < 1000 Da), were prepared via two-step enzymatic hydrolysis using chymotrypsin and flavourzyme and aromatic amino acid removal. Seventy-eight characteristic peptides were identified from HFOPs-AK through UHPLC-Q/TOF, with peptides containing Leu, Val, or Ile accounting for 79%. High hepatoprotective peptides were purified using GFC and RP-HPLC and identified as SDELGW and LLGWDDM. Furthermore, a murine model of acute liver injury induced by alcohol was successfully established. It was demonstrated that the oral administration of HFOPs-AK (800 mg per kg bw per d) remarkably increased the contents of ADH and ALDH compared with the model group, reaching 3.40 and 5.10 U mg-1 prot, respectively. Further, it was revealed that HFOPs-AK could effectively mitigate hepatic oxidative stress by increasing the levels of GSH-Px (p< 0.01) and decreasing the level of MDA (p < 0.05). Additionally, HFOPs-AK (800 mg per kg bw per d) attenuated liver inflammation by down-regulating the mRNA levels of TNF-α, IL-1β, and IL-6 by 40.45%, 38.48%, and 35.83%, respectively. Therefore, HFOPs-AK may have the potential as a new nutritional supplement for the treatment of alcoholic liver injury.

Resveratrol Shows Potent Senescence Reversal in Experimental Cellular Models of Particular Matter 2.5-induced Cellular Senescence in Human Dermal Papilla Cells.

Particular matter 2.5 (PM2.5) pollution is associated with senescence induction. Since the impact of PM2.5 on stem cell senescence and potential compounds capable of reversing this process are largely unknown, this study aimed to examine the senescence effects of PM2.5 on dermal papilla (DP) stem cells. Additionally, we explored the reversal of these effects using natural product-derived substances, such as resveratrol (Res) or Emblica fruits, soybean, and Thunbergia Laurifolia (EST) extract. Cell senescence was determined using the β-Galactosidase (SA-β-gal) assay. The senescence-associated secretory phenotype (SASP) was detected using real-time RT-PCR. For senescence markers, the mRNA and protein levels of p21 and p16 were measured using real-time RT-PCR and immunofluorescence analysis. Subtoxic concentration of PM2.5 (50 μg/ml) induced senescence in DP cells. Resveratrol (50, 100 μM) and plant extracts (400, 800 μg/ml) reversed PM2.5-induced cell senescence. Treatment with Res or EST significantly decreased SA-β-gal staining in PM2.5-treated cells. Furthermore, Res and EST decreased the mRNA levels of SASP, including IL1α, IL7, IL8, and CXCL1. DP cells exposed to PM2.5 exhibited an increase in p21 and p16 mRNA and protein levels, which could be reversed by the addition of Res or EST. Res and EST could reduce p21 and p16 in senescent cells approximately 3- and 2-fold, respectively, compared to untreated senescent cells. PM2.5 induced senescence in human DP stem cells. Res and EST extract potentially reverse the senescence phenotypes of such cells.

Anti-inflammatory effects of Mentha pulegium L. extract on human peripheral blood mononuclear cells are mediated by TLR-4 and NF-κB suppression

There is great interest in evaluating the anti-inflammatory properties of new herbal products. Thus, the effects of Mentha pulegium L. extract on gene and protein expressions of pro-inflammatory mediators and transcription factors were determined.The hydro-ethanolic extract of Mentha pulegium L. was obtained and optimal non-cytotoxic concentrations of the extract were determined by MTT assay. Then, three different concentrations of Mentha pulegium L. (10, 30, and 90 μg/mL) were used to pre-treat the lipopolysaccharide (LPS)-stimulated and non-stimulated peripheral blood mononuclear cells (PBMCs) of 10 healthy individuals. Finally, the tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), IL-6, Toll-like receptor-4 (TLR-4), nuclear factor-kappa B (NF-κB) p65, activator protein-1 (AP-1), inducible nitric oxide synthase (iNOS), and cyclooxygenase-2 (COX-2) gene expressions and TNF-α, IL-1β, IL-6, TLR-4, prostaglandin E2 (PGE2), and COX-2 protein levels were measured.MTT results showed that there is no significant difference in cell viability among 10, 20, 40, and 80 μg/mL concentrations of Mentha pulegium L. extract at 24, 48, and 72 h (P > 0.05). The IC50 values were 236.1, 147.0, and 118.0 μg/mL after 24, 48, and 72 h respectively. TNF-α, IL-1β, IL-6, TLR-4, iNOS, and NF-κB p65 mRNA levels in the pre-treated LPS-stimulated PBMCs were concentration-dependently reduced (P < 0.01 for TNF-α, TLR-4, and NF-κB p65; P < 0.05 for IL-1β, IL-6, and iNOS). Also, the protein levels of pro-inflammatory mediators decreased and these differences were significant for TNF-α, IL-1β, and TLR-4 (P < 0.001, P < 0.01, and P < 0.001, respectively).Mentha pulegium L. extract decreased the expression and biosynthesis of pro-inflammatory mediators. These effects are mainly mediated by TLR-4 and NF-κB suppression. Thus, Mentha pulegium L. could be useful in treating or ameliorating chronic inflammatory diseases.

Structurally diverse stilbenes from Gnetum parvifolium and their anti-neuroinflammatory activities

Phytochemical investigation on the aerial parts of Gnetum parvifolium led to the isolation of 15 new and eight known structurally diverse stilbenes. The isolated compounds comprised (E)- or (Z)-stilbene (1–6, 15–20), dihydrostilbene (21), phenylbenzofuran (7, 8, 22), benzylated stilbene (9–11), benzylated stilbene dimer (12), and nitrogen-containing stilbene (13a, 13b, 14) types. The structures of the new compounds (1–12, 13a, 13b, 14) were established through spectroscopic analyses and experimental and calculated ECD data. Compound 12 is the first stilbene dimer connected through a benzyl group. In the anti-neuroinflammatory activity assay, compounds 4, 5, 9–11, 13b, and 16–21 displayed significant inhibitory effects against LPS-induced NO release in BV-2 microglial cells, with IC50 values of 0.35–16.1 μM. Compound 10 had the most potent activity (IC50 = 0.35 μM), and the further research indicated that it could decrease the mRNA levels of iNOS, IL-1β, IL-6, and TNF-α in a dose-dependent manner.

Increased Expression of Neprilysin Is Associated with Inflammation in Preeclampsia

Preeclampsia (PE) is associated with a finely tuned equilibrium between trophoblast cell invasion and fetal-maternal immunological tolerance. An imbalance between proinflammatory (IL-6) and anti-inflammatory (IL-10) cytokines is a hallmark of PE. Neprilysin (NEP), a membrane-bound metalloprotease, is vulnerable to the inflammatory environment and plays a significant role in modulating vascular tone. The aim of this study was to determine the correlation between NEP (mRNA and protein) levels and the inflammatory status in PE patients compared to healthy pregnant women and to identify the role of NEP in evaluating the severity of preeclampsia. The study group comprised 52 pregnant women with PE while the control group comprised 47 normotensive pregnant women. After a caesarean section, placental tissue samples from patients and controls were collected to measure the expression levels of IL-6, TGF-β, IL-10, and NEP mRNA. In addition, an enzyme-linked immunosorbent assay was used to assess the quantity of NEP protein in blood samples. Our results revealed a significant positive correlation between NEP (mRNA and protein) and proinflammatory markers IL-6 and TGF-β levels in patients compared to controls and a significant inverse correlation between NEP and anti-inflammatory cytokine IL-10. Moreover, this is the first study to find a strong positive correlation between NEP level and PE severity. In conclusion, in PE patients, there is a substantial relationship between NEP, the degree of inflammation, and PE severity. NEP could act as a potential biomarker for diagnosis and prognosis of PE.

Reduction of IL-6, IL-8 and β2-ADRENOCEPTOR mRNA levels in circulating polymorphonuclear leukocytes after adenotonsillectomy in children with Obstructive Sleep Apnea Syndrome

BackgroundObstructive Sleep Apnea Syndrome (OSAS) affects approximately 1–5% of children and is linked to cardiovascular, metabolic, and neurobehavioral complications. Dysregulation of inflammatory process and sympathetic nervous system overstimulation leading to increased catecholamine production may contribute to OSAS pathogenesis. Polymorphonuclear Neutrophils (PMN), key cells in the inflammatory process, express adrenergic receptors, including β2-adrenergic receptor (ADRB2), which modulate their functions through an autocrine/paracrine loop. In this pilot study, we aimed to investigate the relationship between OSAS severity, ADRB2 expression in PMN and patient's inflammatory profile before and after adenotonsillectomy. Patients/methodsIn this pilot study we enrolled OSAS pediatric patients in which ADRB2, IL-6 and IL-8 mRNA expression levels were evaluated in circulating PMN by RT-PCR. Results9 OSAS pediatric patients, ranged from 3 to 8 years of age, were enrolled in the study. We found that adenotonsillectomy significantly reduced ADRB2 as well as IL-6, IL-8 mRNA expression levels in PMN. ConclusionsThese findings offer valuable insights into the underlying immune and inflammatory mechanisms of OSAS and open the way for the development of novel therapeutic approaches.

Acacetin protects against sepsis-induced acute lung injury by facilitating M2 macrophage polarization via TRAF6/NF-κB/COX2 axis.

Acute lung injury (ALI) is the leading cause of death in patients with sepsis syndrome and without effective protective or therapeutic treatments. Acacetin, a natural dietary flavonoid, reportedly exerts several biological effects, such as anti-tumor, anti-inflammatory, and anti-oxidative effects. However, acacetin's effect and underlying mechanism on sepsis-induced ALI remain unclear. Here, the mouse model was established to explore the impact of acacetin on sepsis-induced ALI. Acacetin significantly increased ALI murine survival and attenuated lung injury in histological examinations. Additionally, acacetin down-regulated myeloperoxidase activity, protein concentration, and number of neutrophils and macrophages in bronchoalveolar lavage fluid. Subsequently, inflammatory cytokines, including TNF-α, IL-1β, and IL-6, were examined. Results showed that acacetin dramatically suppressed the production of TNF-α, IL-1β, and IL-6. These above results indicated that acacetin attenuated sepsis-induced ALI by inhibiting the inflammatory response. Moreover, acacetin inhibited the expression of markers for M1-type (iNOS, CD86) macrophages and promoted the expression of markers for M2-type (CD206, Arg1) macrophages by western blot. In addition, acacetin down-regulated the expression TRAF6, NF-κB, and Cyclooxygenase-2 (COX2) by western blot. The high concentration of acacetin had a better effect than the low concentration. Besides, over-expression of TRAF6 up-regulated the expression of COX2, CD86, and iNOS, and the ratio of p-NF-κB to NF-κB increased the mRNA levels of TNF-α, IL-1β, and IL-6, down-regulated the expression of CD206 and Arg1. The effects of TRAF6 were the opposite of acacetin. And TRAF6 could offset the impact of acacetin. This study demonstrated that acacetin could prevent sepsis-induced ALI by facilitating M2 macrophage polarization via TRAF6/NF-κB/COX2 axis.

4-amino-2-trifluoromethyl-phenyl retinate alleviates lipopolysaccharide-induced acute myocardial injury through activation of the KLF4/p62 axis

Ferroptosis plays a pivotal role in the pathological process of sepsis-induced cardiomyopathy (SIC). All-trans retinoic acid (ATRA) enhances the host immune response to lipopolysaccharides (LPS). This study investigated the role of 4-amino-2-trifluoromethyl-phenyl retinate (ATPR), a derivative of ATRA, in myocardial injury caused by sepsis. Male C57BL/6 mice were intraperitoneally injected with LPS to establish a sepsis model. H9c2 cells were stimulated by LPS to establish an injury model. We observed that ATPR improved myocardial injury in mice, which was presented in terms of an increased glutathione (GSH) level and reduced production of malondialdehyde (MDA), as well as an increased number of mitochondrial cristae and maintenance of the mitochondrial membrane integrity. ATPR improved cardiac function in the LPS-injured mice. It inhibited the inflammatory response as evidenced by the decreasing mRNA levels of TNF-α and IL-6. The elevated protein expression levels of Nrf2, SLC7A11, GPX4, and FTH1 in mice and H9c2 cells showed that ATPR inhibited ferroptosis. Immunoprecipitation of LPS-stimulated H9c2 cells demonstrated that ATPR increased the interaction between p62 and Keap1. ATPR upregulated the KLF4 and p62 protein expression. However, the inhibition of Nrf2 by ML385 reduced the protective effect of ATPR in LPS-treated H9c2 cells. Furthermore, we used siRNA to knock down KLF4 in H9c2 cells and found that the KLF4 knockdown eliminated the inhibition of ferroptosis by ATPR in H9c2 cells. Therefore, ATPR alleviates LPS-induced myocardial injury by inhibiting ferroptosis via the KLF4/p62 axis.

Anxiolytic and antidepressant like effects of Zamzam water in STZ-induced diabetic rats, targeting oxidative stress, neuroinflammation, BDNF/ERK/CREP pathway with modulation of hypothalamo-pituitary-adrenal axis.

Recent studies have reported a strong relationship between diabetes and anxiety- and depression-like behaviors; however, there is a lack of information on the underlying pathophysiology. Alkaline Zamzam water (ZW), which is rich in several trace elements, has neuroprotective properties. This study aimed to investigate the anxiolytic and antidepressant effects of ZW against diabetes-induced behavioral changes and shed light on the possible underlying mechanisms. Forty-eight rats were divided into four experimental groups (n = 12): group I (control group), group II (Zamzam water group), group III (diabetic group), and group IV (diabetic + Zamzam water group). Diabetes was induced by an intraperitoneal injection of 60 mg/kg streptozotocin (STZ). At the end of the experiment, the forced swimming test (FST) was used to assess depression-like effects. The elevated plus maze test (EPMT) and open field test (OFT) were performed to evaluate anxiety-like behavior. Blood levels of the hypothalamic-pituitary-adrenal (HPA) axis were measured, and prefrontal cortex and hippocampal tissue samples were removed for histological, immunohistochemical, ELISA, and Q-PCR analyses. ZW significantly decreased the immobility time in the FST, indicating an antidepressant effect (p < 0.001). Additionally, ZW significantly improved the OFT and open field entry (OFE) percentages in the EPMT, increasing center crossing and decreasing grooming and fecal boli in the OFT. This indicated an anxiolytic-like effect in diabetic rats with histological improvement. Interestingly, ZW significantly increased prefrontal cortical and hippocampal levels of antioxidant enzymes and the Nrf2/HO-1 pathway. It also modulated the HPA axis by increasing cortisol and corticotropin-releasing hormone (CRH) levels, with a decrease in ACTH and an increase in monoamine neurotransmitters. Furthermore, diabetic rats that received ZW showed a decrease in the inflammatory markers TNF-α and GFAP by immunohistochemistry and in the mRNA levels of NFκB, IL-1β, and IL6. In addition, ZW downregulated the expression of the BDNF/ERK2/CREP pathway. Our results suggested a neuroprotective effect of ZW against diabetes-induced anxiety- and depression-like behaviors and explored the underlying mechanisms. These findings suggest a promising therapeutic strategy for patients with diabetes who experience anxiety and depression.

Ginsenoside Rg1 Attenuates Eccentric Exercise-Induced Muscle Damage via the Modulation of Lipid Peroxidation and Inflammation

Eccentric exercise (EE) may lead to skeletal muscle injury, including oxidative stress and inflammation induction. Ginsenoside Rg1, a glycosylated triterpene present in the traditional Chinese medicine ginseng, was previously shown to prevent the development of multiple diseases through the attenuation of oxidative stress and inflammation. Therefore, this article hopes to investigate whether Rg1 exhibits anti-oxidant and anti-inflammatory effects in eccentric exercise-induced muscle damage (EEIMD). Additionally, Adult male Wistar rats were intraperitoneally injected with Rg1 (20 or 40 mg/kg) every day before EE for 5 consecutive days. The impact of Rg1 administration on levels of serum creatine kinase was evaluated, followed by observation of histological muscle damage through H&amp;E staining. To assess protein nitrotyrosylation, lipid peroxidation and leukocyte infiltration in rat skeletal muscles, the levels of nitrotyrosine, MDA and MPO protein were analysed through western blotting analysis. The inflammatory response was evaluated by detecting iNOS, COX-2, IL-1β, IL-6, MCP-1 and TNF-α mRNA and protein levels in rat skeletal muscles. The regulation of Rg1 on the NF-κB pathway was examined through the analysis of phosphorylated NF-κB p65 and IκBα protein levels. Result display, EE resulted in elevated serum creatine kinase levels, widespread leukocyte infiltration, and notable muscle cell vacuolization and fragmentation in muscles. Furthermore, EE increased nitrotyrosine, MDA, MPO, iNOS, COX-2, IL-1β, IL-6, MCP-1, and TNF-α levels in rats. However, these changes were reversed by Rg1 treatment. Furthermore, EE-induced upregulation in phosphorylated NF-κB p65 and IκBα levels was counteracted by Rg1. Overall, ginsenoside Rg1 plays an anti-oxidant and anti-inflammatory role in EEIMD through suppressing this NF-κB signaling pathway.

Integrating GEO, network pharmacology, and in vitro assays to explore the pharmacological mechanism of Bruceae Fructus against laryngeal cancer

The goal of this study is to look into the pharmacological mechanism of Bruceae Fructus in conjunction with GEO, network pharmacology, and in vitro assays for the treatment of laryngeal cancer to provide theoretical support for its therapeutic use. The active components and matching targets of Bruceae Fructus were retrieved from the TCMSP database, while genes linked with laryngeal cancer were obtained from the GEO, GeneCards, DisGeNET, and DrugBank databases. Besides, the components and targets were supplemented by literatures in PubMed database. Cytoscape software was used to create the active ingredients–target network diagram. The String database was used to build the PPI network. Following that, the core targets were subjected to GO enrichment and KEGG pathway analysis using the DAVID database. Finally, AutoDock was used to perform molecular docking between the core components and the core targets. To investigate the biological effects of beta-sitosterol, the viability of laryngeal cancer cells was assessed after beta-sitosterol therapy using the MTS technique. Following that, how beta-sitosterol affected colony formation after 14 days of culture of treated cells was researched. Flow cytometry was utilized to detect apoptosis to examine the influence of beta-sitosterol on laryngeal cancer cell apoptosis, and then detected mRNA and protein expression levels of 10 key genes by RT-qPCR and Western Blot assay. There were 1258 laryngeal cancer–related genes and 15 Bruceae Fructus components, with beta-sitosterol and luteolin serving as key components. Bruceae Fructus’ primary targets against laryngeal cancer were IL6, JUN, TNF, IL2, IL4, IFNG, RELA, TP53, CDKN1A, and AKT1. GO enrichment yielded 41 CC, 78 MF, and 383 BP. Platinum drug resistance, the PI3K-Akt signaling pathway, the p53 signaling pathway, apoptosis, the HIF-1 signaling pathway, and 147 additional pathways have been added to KEGG. The results of molecular docking revealed that the core components had a high affinity for the core target. The results of the cell experiment indicate that beta-sitosterol suppressed Hep-2 cell activity in a concentration-dependent manner. Besides, beta-sitosterol has powerful antiproliferative properties in Hep-2 cells. Flow cytometry results showed that beta-sitosterol promoted laryngeal cancer cell apoptosis in a concentration-dependent manner. The results of RT-qPCR and Western Blot assay showed that the mRNA and protein expression levels of TP53, JUN, TNF-α, CDKN1A, and IL-2 were significantly up-regulated after beta-sitosterol treatment, while the mRNA and protein expression levels of RELA, AKT1, IL-6, IFNG, and IL-4 were significantly down-regulated. This study integrating GEO, network pharmacology, and in vitro assays investigated the probable mechanism of Bruceae Fructus’ anti-laryngeal cancer activity, which can give a theoretical foundation for additional future animal experiments.

Amplification of poly(I:C)-induced interleukin-6 production in human bronchial epithelial cells by priming with interferon-γ

Proinflammatory cytokine interleukin (IL)-6 was associated with disease severity in patients with COVID-19. The mechanism underlying the excessive IL-6 production by SARS-Cov-2 infection remains unclear. Respiratory viruses initially infect nasal or bronchial epithelial cells that produce various inflammatory mediators. Here, we show that pretreatment of human bronchial epithelial cells (NCl-H292) with interferon (IFN)-γ (10 ng/mL) markedly increased IL-6 production induced by the toll-like receptor (TLR) 3 agonist poly(I:C) (1 µg/mL) from 0.4 ± 0.1 to 4.1 ± 0.4 ng/mL (n = 3, P < 0.01). A similar effect was observed in human alveolar A549 and primary bronchial epithelial cells. TLR3 knockdown using siRNA in NCl-H292 cells diminished the priming effects of IFN-γ on poly(I:C)-induced IL-6 production. Furthermore, the Janus kinase (JAK) inhibitor tofacitinib (1 µM) inhibited IFN-γ-induced upregulation of TLR3, and suppressed poly(I:C)-induced IL-6 production. Quantitative chromatin immunoprecipitation revealed that IFN-γ stimulated histone modifications at the IL-6 gene locus. Finally, IFN-γ priming significantly increased lung IL-6 mRNA and protein levels in poly(I:C)-administrated mice. Thus, priming bronchial epithelial cells with IFN-γ increases poly(I:C)-induced IL-6 production via JAK-dependent TLR3 upregulation and chromatin remodeling at the IL-6 gene locus. These mechanisms may be involved in severe respiratory inflammation following infection with RNA viruses.

8-Oxoguanine DNA Glycosylase 1 Inhibition Suppresses Inflammatory Responses in Sickle Cell Disease

Background: The continuous production of reactive oxygen species (ROS) and oxidative stress contribute to ongoing inflammation and the pathology of sickle cell disease (SCD). ROS are known to mediate DNA damage through oxidation of nucleotide bases with guanine being the primary target. Although guanine base lesions vary according to the nature of the oxidants, 7,8-dihydro-8-oxoguanine (8-oxoG) is the most frequent oxidation product of DNA, which is repaired by 8-Oxoguanine-DNA glycosylase-1 (OGG1) via the DNA base excision repair pathway (OGG1-BER). The release of the 8-oxoG base from the genome by OGG1-BER is a prerequisite for increasing expression of pro-inflammatory cytokines and immune response. OGG1 inhibition has been shown to attenuate proinflammatory responses via the mitochondrial DNA-cGAS-STING axis in mice (Qin et al, 2020), a pathway also involved in the activation of neutrophils in SCD (Tumburu et al, 2021). Here, we evaluate the effect of OGG1 inhibition on expression of pro-inflammatory cytokines and immune response in SCD. Methods: Blood samples were obtained from ethnic-matched healthy controls (HbAA) and patients with SCD (HbSS) enrolled under protocols NCT03049475 and NCT00047996. CD14+ monocytes were isolated from PBMC of HbAA and HbSS subjects using EasySep™ Human Monocyte Isolation Kit (STEMCELL Technologies). 8-oxoG was measured by staining CD14+ monocytes with anti-8-oxoG antibody; OGG1 and pro-inflammatory cytokine levels were evaluated by qRT-PCR and western blotting. After differentiation, macrophages derived from CD14+ monocytes were treated with or without 10 µM SU0268 (OGG1 inhibitor) for 1 hour and then incubated with plasma from healthy controls or HbSS patients in steady or crisis status, followed by qRT-PCR quantitation of IL-1ꞵ, IL-6, TNF-α, and OGG1 mRNA levels. These measurements were repeated after OGG1 knockdown using OGG1 siRNA in macrophages derived from CD14+ monocytes. We repeated the study on peritoneal macrophages (PMs) from Townes HbAA and HbSS mice that were collected 4 days after a 3% Brewer's thioglycolate medium intraperitoneal injection. The level of 8-oxoG was measured by staining PMs with anti-8-oxoG antibody. The mRNA and protein level of OGG1 and pro-inflammatory cytokines were evaluated by qRT-PCR and western blotting. PMs from HbAA mice were treated with 10 µM SU0268 (OGG1 inhibitor) or vehicle for 1 hour, before incubation with plasma from HbAA or HbSS mice, the mRNA levels of IL-1ꞵ, IL-6, TNF-α, and OGG1 were measured by qRT-PCR. Results: CD14+ monocytes isolated from human HbSS PBMC showed increased levels of 8-oxoG nuclear staining when compared with human HbAA CD14+ monocytes, consistent with an increased oxidative burden in SCD patients (Fig 1A). The mRNA and protein levels of OGG1 were also increased in HbSS monocytes, suggestive of a potential therapeutic target of OGG1. Consistent with previous studies, mRNA levels of pro-inflammatory cytokines IL-1ꞵ, IL-6, and TNF-α were increased in SS compared with AA monocytes. To investigate the role of OGG1 in regulating cytokine expression, we treated the macrophages derived from CD14+ monocytes with SU0268, a selective OGG1 inhibitor (Tahara et al, 2018). Macrophages treated with plasma from HbSS patients (steady and crisis status) showed increased expression of OGG1, IL-1ꞵ, IL-6, and TNF-α compared with cells treated with normal plasma, and the increase was largely blocked by SU0268 pretreatment (Fig1B). Further, siRNA knockdown of OGG1 decreased the expression of IL-1ꞵ and TNF-α, confirming the role of OGG1 in regulating pro-inflammatory cytokine expression. We then evaluated the effect of OGG1 in regulating cytokines expression in cells form SCD mice. The levels of 8-oxoG and OGG1, as well as cytokines (IL-1ꞵ, IL-6, and TNF-α), were increased in PMs obtained from HbSS mice compared with macrophages obtained from HbAA mice. Similar to the results obtained from human macrophages, murine cells treated with plasma from SS mice showed increased expression of IL-1ꞵ, IL-6, TNF-α and OGG1 compared with cells treated with plasma from HbAA mice, and pretreatment with SU0268 largely blocked their increased expression. Conclusion: OGG1 has a regulatory role in the expression of pro-inflammatory cytokines in SCD. OGG1 inhibition could be a potential therapeutic approach to control sickle cell inflammation.

Metformin attenuates inflammation and boosts autophagy in the liver and intestine of chronologically aged rats

BackgroundOur previous studies found that autophagy levels in liver and intestinal segments of naturally aging rats were downregulated, and the expression of pro-inflammatory factors was increased. The increased expression of pro-inflammatory factors might be related to the downregulation of autophagy. AMPK is the most critical upstream targeting and regulating molecule of autophagy, and Metformin, as an agonist of AMPK, has the effects of anti-inflammation and anti-aging. We pretreated 29-month-old naturally aging rats with Metformin for a short period and observed the changes in autophagy levels and pro-inflammatory factors in the liver, ileum, and colon after 31 days of intervention and preliminarily investigated the mechanism of its action. Methods29-month-old SPF male Wistar rats were divided into three groups: The control group, the Metformin 100 mg/kg intervention group, and the Metformin 250 mg/kg intervention group, with eight rats in each group. At 29 months, different concentrations of Metformin (100 mg/kg, 250 mg/kg) were given by gavage once a day until 30 months, and the control group was kept generally until 30 months. Western Blot was used to assess the expression levels of AMPK, P-AMPK, LC3, and P62 proteins in the liver and intestinal tissues. Intestinal and liver tissues were immunofluorescence labeled for LC3 and P62 proteins. Moreover, RT-qPCR was conducted to detect the expression levels of pro-inflammatory factors IL-1β, TNF-α, IL-6, and MMP-9 mRNA in liver and intestinal tissues. ResultsShort-term Metformin pretreatment (31 days) in naturally aging rats (29 months old) increased autophagy levels and down-regulated the expression of various pro-inflammatory cytokines (IL-1β, TNF-α, MMP-9, and IL-6) in various intestinal segments and the liver—the expression of LC3II protein enriched with the increase of Metformin concentration. The level of P62 protein decreased with the accumulation of Metformin concentration. And a higher concentration of Metformin was associated with increased expression of P-AMPK protein. ConclusionsMetformin intervention can boost the autophagy level in the liver and intestine and reduce the expression of aging-related inflammatory factors in aged rats, and these effects may be related to the increase of the AMPK phosphorylation level.

PCSK9 ablation attenuates Aβ pathology, neuroinflammation and cognitive dysfunctions in 5XFAD mice

BackgroundIncreasing evidence highlights the importance of novel players in Alzheimer’s disease (AD) pathophysiology, including alterations of lipid metabolism and neuroinflammation. Indeed, a potential involvement of Proprotein convertase subtilisin/kexin type 9 (PCSK9) in AD has been recently postulated. Here, we first investigated the effects of PCSK9 on neuroinflammation in vitro. Then, we examined the impact of a genetic ablation of PCSK9 on cognitive performance in a severe mouse model of AD. Finally, in the same animals we evaluated the effect of PCSK9 loss on Aβ pathology, neuroinflammation, and brain lipids. MethodsFor in vitro studies, U373 human astrocytoma cells were treated with Aβ fibrils and human recombinant PCSK9. mRNA expression of the proinflammatory cytokines and inflammasome-related genes were evaluated by q-PCR, while MCP-1 secretion was measured by ELISA. For in vivo studies, the cognitive performance of a newly generated mouse line - obtained by crossing 5XFADHet with PCSK9KO mice – was tested by the Morris water maze test. After sacrifice, immunohistochemical analyses were performed to evaluate Aβ plaque deposition, distribution and composition, BACE1 immunoreactivity, as well as microglia and astrocyte reactivity. Cholesterol and hydroxysterols levels in mouse brains were quantified by fluorometric and LC-MS/MS analyses, respectively. Statistical comparisons were performed according to one- or two-way ANOVA, two-way repeated measure ANOVA or Chi-square test. ResultsIn vitro, PCSK9 significantly increased IL6, IL1B and TNFΑ mRNA levels in Aβ fibrils-treated U373 cells, without influencing inflammasome gene expression, except for an increase in NLRC4 mRNA levels. In vivo, PCSK9 ablation in 5XFAD mice significantly improved the performance at the Morris water maze test; these changes were accompanied by a reduced corticohippocampal Aβ burden without affecting plaque spatial/regional distribution and composition or global BACE1 expression. Furthermore, PCSK9 loss in 5XFAD mice induced decreased microgliosis and astrocyte reactivity in several brain regions. Conversely, knocking out PCSK9 had minimal impact on brain cholesterol and hydroxysterol levels. ConclusionsIn vitro studies showed a pro-inflammatory effect of PCSK9. Consistently, in vivo data indicated a protective role of PCSK9 ablation against cognitive impairments, associated with improved Aβ pathology and attenuated neuroinflammation in a severe mouse model of AD. PCSK9 may thus be considered a novel pharmacological target for the treatment of AD.

Tristetraprolin in cardiomyocytes has a protective role in pressure-overloaded hearts

Abstract Background Elevated circulating levels of TNFα, IL-6 and the soluble TNF receptors predict adverse outcomes in patients with heart failure. However, the targeted anti TNF approaches resulted in worsening heart failure or death. The entire scenario of how the expression levels of inflammatory cytokines are maintained in failing hearts must be elucidated to develop novel and effective treatments for heart failure. Although transcriptional control is a determinant of the kinetics of proinflammatory cytokine gene expression, the stability of the mRNA also has a key function in coordinating immune responses. Tristetraprolin (TTP) has been shown to control mRNA decay by associating with the 3’- untranslated region and critical for the decay of the mRNAs for cytokines. The role of TTP in nonimmune cells such as cardiomyocytes remains to be elucidated. Purpose The purpose of this study is to examine the in vivo role of proinflammatory cytokine degradation by TTP in cardiomyocytes during cardiac remodelling under pressure overload. Methods Cardiomyocyte-specific TTP–deficient mice were generated. The mice were subjected to pressure overload by means of transverse aortic constriction (TAC) to induce heart failure. Cardiac remodelling was assessed by echocardiography as well as histological and molecular analyses 4 weeks after operation. Cytokine mRNA levels were examined by quantitative PCR. Results Cardiomyocyte-specific TTP–deficient mice showed no cardiac phenotypes under baseline conditions. However, the mice exhibited severe systolic dysfunction, indicated by left ventricular fractional shortening, after 4 weeks of pressure overload by means of TAC compared with control littermates (fractional shortening, control littermates 35.8% versus cardiomyocyte-specific TTP-deficient mice 19.9%, p = 0.000). In addition, those hearts showed increases in collagen deposition and the mRNA levels of Col1a2 and Nppb (1.4 times (p = 0.023), 1.5 times (p = 0.033), and 2.0 times (p = 0.020), respectively) compared to corresponding control hearts. Four weeks after TAC, the Il6 mRNA level was upregulated 2.7 times (p = 0.000), but not other cytokine mRNAs, including Tnfα, in cardiomyocyte-specific TTP–deficient hearts compared to controls. Conclusions The degradation of cytokine mRNA by TTP in cardiomyocytes plays a protective role in failing hearts and the TTP–mediated pathway in cardiomyocytes might be a therapeutic target to treat patients with heart failure.