TRIP6 promotes inflammatory damage via the activation of TRAF6 signaling in a murine model of DSS-induced colitis

Background TRIP6 is a zyxin family member that serves as an adaptor protein to regulate diverse biological processes. In prior reports, TRIP6 was shown to play a role in regulating inflammation. However, its in vivo roles and mechanistic importance in colitis remain largely elusive. Herein, we therefore employed TRIP6-deficient (TRIP6−/−) mice in order to explore the mechanistic importance of TRIP6 in a dextran sodium sulfate (DSS)-induced model of murine colitis. Findings Wild-type (TRIP6+/+) mice developed more severe colitis following DSS-mediated disease induction relative to TRIP6−/− mice, as evidenced by more severe colonic inflammation and associated crypt damage. TRIP6 expression in wild-type mice was significantly elevated following DSS treatment. Mechanistically, TRIP6 binds to TRAF6 and enhances oligomerization and autoubiquitination of TRAF6. This leads to the activation of NF-κB signaling and the expression of pro-inflammatory cytokines such as TNFα and IL-6, in the in vivo mouse model of colitis. Conclusions These in vivo data demonstrate that TRIP6 serves as a positive regulator of DSS-induced colitis through interactions with TRAF6 resulting in the activation of inflammatory TRAF6 signaling, highlighting its therapeutic promise as a protein that theoretically can be targeted to prevent or treat colitis.

Ginsenoside Rg3 alleviates septic liver injury by regulating the lncRNA TUG1/miR-200c-3p/SIRT1 axis

Background Studies have shown that ginsenoside R3 (Rg3) plays a protective role in sepsis-induced organ injuries and mitochondrial dysfunction. Long noncoding RNA (lncRNA) taurine-upregulated gene 1 (TUG1) is regarded as a regulator in sepsis. However, the association between TUG1 and Rg3 remains elusive. Methods A sepsis mouse model was established by caecal ligation and puncture (CLP), and liver injury was induced by haematoxylin-eosin (H&E) staining. Lipopolysaccharide (LPS) was used to induce hepatocyte damage. The expression levels of TUG1, microRNA (miR)-200a-3p, and silencing information regulator 1 (SIRT1) were examined by quantitative real-time polymerase chain reaction (qRT–PCR) assays. Cell viability was monitored using the Cell Counting Kit-8 (CCK-8) assay. MitoSOX Red staining and CBIC2 (JC-1) dye were employed to detect mitochondrial reactive oxygen species (ROS) and mitochondrial transmembrane potential (MTP) levels, respectively. The interaction between miR-200a-3p and TUG1 or SIRT1 was confirmed via dual-luciferase reporter or RNA immunoprecipitation (RIP) assay. Results Rg3 upregulated TUG1 expression in liver tissues of CLP mice and LPS-induced hepatocytes. Rg3 could activate autophagy to improve mitochondrial dysfunction in LPS-treated hepatocytes, which was partially reversed by TUG1 depletion or miR-200a-3p overexpression. Importantly, TUG1 targeted miR-200a-3p to activate the SIRT1/AMP-activated protein kinase (AMPK) pathway in LPS-treated hepatocytes. Moreover, gain of TUG1 ameliorated mitochondrial dysfunction in LPS-treated hepatocytes by sequestering miR-200a-3p. Conclusion Our study revealed that Rg3 increased TUG1 expression and reduced miR-200a-3p expression to stimulate the SIRT1/AMPK pathway, thereby enhancing autophagy to improve sepsis-induced liver injury and mitochondrial dysfunction.

TGF-β gene polimorphisms as risk factors for asthma control among clinic patients

Background TGF-β and its receptors play a crucial role in asthma pathogenesis, bronchial hyperreactivity, and bronchial remodeling. Expression of isoforms 1–3 of TGFβ cytokine is influenced by tagging polymorphisms in the TGFβ1, TGFβ2 and TGFβ3 gene, and these SNPs may be associated with the risk of asthma development and severity as well as with other diseases. Polymorphic forms of TGF-β1, TGF-β2 and TGF-β3 genes regulate the degree of bronchial inflammation, deterioration of lung functional parameters in spirometry and elevated level of total IgE. All this results in intensification of disease symptoms. According to current GINA 2020 guidelines, the Asthma Control Test (ACT™) should be applied to assess asthma symptoms. Methods An analysis of polymorphisms localized in TGF-β1, TGF-β2 and TGF-β3 genes was conducted on 652 DNA samples with an application of the MassARRAY® system using the mass spectrometry technique MALDI TOF MS. The degree of asthma control was evaluated with ACT™. Results The occurrence of the T / C genotype in rs8109627 (p = 0.0171) in the TGF-β1 gene is significantly associated with a higher ACT result (controlled asthma) in a multivariate linear regression analysis model after using backward stepwise selection of variables. In addition, in the linear model for prediction of ACT score we showed SNP rs8109627 (p = 0.0497) in the TGF-β1 gene (improvement of the disease control - controlled asthma) and rs2796822 (p = 0.0454) in the TGF-β2 gene (deterioration of the diseases control - uncontrolled asthma) significantly modify the degree of asthma control. Discussion We described clinical significance of two SNPs in two genes TGF-β1 and TGF-β2, as yet unknown. We proved that the use of both genotypes and MAC allows to create a moderately correct prognostic model which is about 70% efficient on the entire set of analyzed SNPs in TGF-β1, TGF-β2, and TGF-β3 genes.

Downregulation of lncRNA-PVT1 participates in the development of progressive chronic kidney disease among patients with congestive heart failure

Background Congestive heart failure (CHF) is a major cause of the development of progressive chronic kidney disease (CKD), while the mechanism is still unknown. LncRNA PVT1 contributes to kidney injury. This study aimed to explore the role of PVT1 in the development of CKD in CHF patients. Methods Expression of PVT1 in plasma samples of CHF patients with and without CKD was determined by RT-qPCR. The diagnostic value of plasma PVT1 for CKD was evaluated by ROC curve analysis. The predictive value of PVT1 for the development of CKD in CHF patients was analyzed by a 2-year follow-up study. Changes in PVT1 expression in CKD patients during treatment were analyzed by RT-qPCR and reflected by heatmaps. Results Plasma PVT1 was downregulated in CHF and further downregulated in CHF patients complicated with progressive CKD. ROC curve analysis showed that plasma PVT1 levels could be used to distinguish CHF patients complicated with CKD from CHF patients without CKD and healthy controls. During a 2-year follow-up, patients with high CHF levels had a low incidence of progressive CKD among CHF patients. Moreover, with the treatment of progressive CKD, plasma PVT1 was upregulated. Conclusions LncRNA-PVT1 downregulation may participate in the development of progressive CKD among patients with CHF.

Therapeutic potential of a novel semi-synthetic-sulfated-polysaccharide to suppress inflammatory mediators in P. gingivalis LPS stimulated human monocytes/macrophages

Background Chronic periodontitis is associated with an increased risk for systemic conditions such as cardiovascular disease, diabetes, and osteoporosis. During chronic periodontitis, endotoxin (lipopolysaccharide, LPS) produced by P. gingivalis provokes monocyte accumulation and differentiation into macrophages and increased secretion of pro-inflammatory cytokines and matrix metalloproteases (MMPs). While normal levels of MMPs are important in cellular function, increased levels of cytokines and MMPs can cause connective tissue destruction. Results In the current study, we investigated the therapeutic capability of a novel semi-synthetic sulfated polysaccharide (SAGE) on the production of cytokines and MMPs by cultured human mononuclear cells and macrophages stimulated with endotoxin LPS produced by P. gingivalis, a periodontally-relevant cell culture model. Our research demonstrated SAGE inhibited the LPS induced synthesis of inflammatory mediators including TNF-α, IL-1β, PGE2, and MMP-9 in this periodontal-relevant cell culture model. In addition, TLR-2 and TLR-4 levels were also reduced with the SAGE treatment. Conclusions The therapeutic potential of this novel semi-synthetic sulfated polysaccharide compound may help to prevent tissue damage and bone loss in patients with periodontal disease or other inflammatory diseases.

USP10 alleviates sepsis-induced acute kidney injury by regulating Sirt6-mediated Nrf2/ARE signaling pathway

Background Severe sepsis, a major health problem worldwide, has become one of the leading causes of death in ICU patients. Further study on the pathogenesis and treatment of acute kidney injury (AKI) is of great significance to reduce high mortality rate of sepsis. In this study, the mechanism by which ubiquitin specific peptidase 10 (USP10) reduces sepsis-induced AKI was investigated. Ligation and perforation of cecum (CLP) was employed to establish C57BL/6 mouse models of sepsis. Hematoxylin-eosin (H&E) staining was performed to detect renal injury. The concentrations of serum creatinine (Cr), urea nitrogen (BUN) and cystatin C (Cys C) were determined using a QuantiChrom™ Urea Assay kit. RT-qPCR and western blot were conducted to assess the USP10 expression level. DHE staining was used to detect reactive oxygen species (ROS) levels. H2O2, MDA and SOD levels were assessed using corresponding colorimetric kits. Western blot was used to examine the expression levels of Bcl-2, Bax, cleaved caspase-3, Sirt6, Nrf2 and HO-1. MTT assay was used to determine cell viability, whereas TUNEL staining and flow cytometry were used to assess cell apoptosis. Results In this study, we found that USP10 was decreased in CLP-induced mouse renal tissues. We identified that USP10 alleviated renal dysfunction induced by CLP. Moreover, USP10 was found to reduce oxidative stress, and abated LPS-induced renal tubular epithelial cell injury and apoptosis. Finally, we discovered that USP10 promoted activation of the NRF2/HO-1 pathway through SIRT6 and attenuated LPS-induced renal tubular epithelial cell injury. Conclusions This study found that USP10 activates the NRF2/ARE signaling through SIRT6. USP10 alleviates sepsis-induced renal dysfunction and reduces renal tubular epithelial cell apoptosis and oxidative stress.

Increased levels of S100A8/A9, IL-1ß and IL-18 as a novel biomarker for recurrent tonsillitis

Background Acute tonsillitis represents one of the most frequent reasons patients seek primary medical care and otorhinolaryngology consultation. Therefore, recurrent episodes of acute tonsillitis (RAT), also called chronic tonsillitis, exhaust a substantial amount of medical and financial resources. Diagnosis of tonsillitis depends on a physical examination, which therefore does not allow for a reliable differentiation between viral and bacterial infection. However, the frequency of bacterial infections during the previous three years is currently being used as the major deciding factor in patient selection for tonsillectomy. The aim of the present study was to determine an objective biomarker to help in the identification of patients suffering from recurrent tonsillitis. Results By analyzing a panel of cytokines and chemokines in serum and saliva of patients with RAT compared to healthy controls, increased levels of IL-1ß (153.7 ± 48.5 pg/ml vs 23.3 ± 6.6 pg/ml, p = 0.021), IL-18 (120.2 ± 16.5 vs 50.6 ± 9.3 pg/ml, p = 0.007) and/or S100A8/A9 (996 ± 102 ng/ml vs 546 ± 86 ng/ml, p = 0.042) could be observed in patients suffering from RAT. Cut-off values of these parameters were determined and combined to a new RAT-score allowing for reliable identification of patients suffering from recurrent tonsillitis with a sensitivity of 95% and a specificity of 88%. Conclusion The RAT-score represents the first objective criterion as a tool for the diagnosis of recurrent tonsillitis and it also improves patient selection for tonsillectomy.

Bullatacin triggers immunogenic cell death of colon cancer cells by activating endoplasmic reticulum chaperones

Background It is well accepted that the immune system efficiently contributes to positive outcomes of chemotherapeutic cancer treatment by activating immunogenic cell death (ICD). However, only a limited number of ICD-inducing compounds are well characterized at present; therefore, identification of novel ICD inducers is urgently needed for cancer drug discovery, and the need is becoming increasingly urgent. Methods Herein, we assessed the antitumour activity of bullatacin by MTS assay and apoptosis assay. ICD biomarkers, such as calreticulin (CRT), high-mobility group protein B1 (HMGB-1), heat shock protein (HSP)70, HSP90 and ATP, were assessed by Western blotting, ELISA and flow cytometry. Western blot and qPCR assays were performed to explore the underlying mechanisms of bullatacin-induced ICD. Flow cytometry was used to detect macrophage phagocytosis. Results First, bullatacin induced apoptosis in both SW480 cells and HT-29 cells in a time-dependent manner at 10 nM, as assessed by flow cytometry. Moreover, Western blot and flow cytometry assays showed that CRT and HSP90 (biomarkers of early ICD) significantly accumulated on the cell membrane surface after approximately 6 h of treatment with bullatacin. In addition, ELISAs and Western blot assays showed that the second set of hallmarks required for ICD (HMGB1, HSP70 and HSP90) were released in the conditioned media of both SW480 and HT-29 cells after 36 h of treatment. Furthermore, qPCR and Western blot assays indicated that bullatacin triggered ICD via activation of the endoplasmic reticulum stress (ERS) signalling pathway. Finally, bullatacin promoted macrophage phagocytosis. Conclusion This study documents that bullatacin, a novel ICD inducer, triggers immunogenic tumour cell death by activating ERS even at a relatively low concentration in vitro.

The functional characterization of phosphorylation of tristetraprolin at C-terminal NOT1-binding domain

Background Tristetraprolin (TTP) family proteins contain conserved tandem CCCH zinc-finger binding to AU-rich elements and C-terminal NOT1-binding domain. TTP is phosphorylated extensively in cells, and its mRNA destabilization activity is regulated by protein phosphorylation. Methods We generated an antibody against phospho-Serine316 located at the C-terminal NOT1-binding site and examined TTP phosphorylation in LPS-stimulated RAW264.7 cells. Knockout of TTP was created in RAW264.7 cells using CRISPR/Cas9 gene editing to explore TTP functions. Results We demonstrated that Ser316 was phosphorylated by p90 ribosomal S6 kinase 1 (RSK1) and p38-activated protein kinase (MK2) and dephosphorylated by Protein Phosphatase 2A (PP2A). A phosphorylation-mimic mutant of S316D resulted in dissociation with the CCR4-NOT deadenylase complex through weakening interaction with CNOT1. Furthermore, Ser316 and serines 52 and 178 were independently contributed to the CCR4-NOT complex recruitment in the immunoprecipitation assay using phosphor-mimic mutants. In RAW264.7 macrophages, TTP was induced, and Ser316 was phosphorylated through RSK1 and MK2 by LPS stimulation. Knockout of TTP resulted in TNFα mRNA increased due to mRNA stabilization. Overexpression of non-phosphorylated S316A TTP mutant can restore TTP activity and lead to TNFα mRNA decreased. GST pull-down and RNA pull-down analyses demonstrated that endogenous TTP with Ser316 phosphorylation decreased the interaction with CNOT1. Conclusions Our results suggest that the TTP-mediated mRNA stability is modulated by Ser316 phosphorylation via regulating the TTP interaction with the CCR4-NOT deadenylase complex.