TNF Protein Levels Research Articles

Sitagliptin, an anti‐diabetic drug, inhibits the pro‐inflammatory microglia reactivity via dipeptidyl peptidase‐4 (DPP4)

Aims/Purpose: Diabetic retinopathy is characterized by a chronic low‐grade retinal neuroinflammation triggered by diabetes and mediated, at least in part, by microglial cells. We previously showed that sitagliptin, an oral drug for type 2 diabetes, developed to inhibit dipeptidyl peptidase 4 (DPP4) and increase insulin release, can prevent retinal neuroinflammation in diabetic rat models and the LPS‐induced morphological changes of microglial cells in retinal organotypic cultures. However, the mechanisms underlying these effects are not clarified. We hypothesize that sitagliptin is able to directly inhibit microglia reactivity via Dpp4.Methods: BV‐2 microglia cell line was exposed to a non‐toxic dose of lipopolysaccharide (LPS, 0.1 μg/mL) in the presence or absence of 200 μM sitagliptin. The expression of Dpp4, iNOS, IL‐1 beta and TNF were assessed by Western Blot and/or RT‐qPCR. The BV‐2 phagocytic efficiency was assessed using fluorescent microbeads. The metabolic activity and proliferation of BV‐2 cells were assessed by resazurin assay and Edu staining, respectively. Dpp4 deletion in BV‐2 cells was generated using Crispr‐cas9 method.Results: Sitagliptin inhibited the LPS‐induced increase in protein levels of iNOS, IL‐1 beta and TNF to 39.9 ± 6.3%, 47.2 ± 8.1% and 34.0 ± 7.2%, respectively, comparing to LPS. Sitagliptin reduced the LPS‐induced increase in phagocytic efficiency by approximately 40%. Sitagliptin did not affect the metabolism and proliferation of BV‐2. BV‐2 cells express Dpp4. Dpp4 deletion does not affect the LPS‐induced phagocytosis but abrogated the effect of sitagliptin on the reduction of BV‐2 phagocytic efficiency (approx. 320% as in LPS).Conclusions: Sitagliptin exerts direct anti‐inflammatory effects on microglia, mediated by DPP4. These results suggest that sitagliptin can potentially be used in retinal pathologies characterized by microglia‐mediated neuroinflammation.Support.FCT‐Portugal: PEst UID/NEU/04539/2013, UIDB/04539/2020 and UIDP/04539/2020 and PTDC/NEU‐OSD/1113/2012. COMPETE‐FEDER.

Construction of the miRNA/Pyroptosis-Related Molecular Regulatory Axis in Abdominal Aortic Aneurysm: Evidence From Transcriptome Data Combined With Multiple Machine Learning Approaches Followed by Experiment Validation.

Background: Abdominal aortic aneurysm (AAA) represents a permanent and localized widening of the abdominal aorta, posing a potentially lethal risk of aortic rupture. Several recent studies have highlighted the role of pyroptosis, a pro-inflammatory programed cell death, as critical molecular regulators in AAA occurrence, progression, and rupture. However, the potential effects of pyroptosis in AAA and its upstream microRNA (miRNA) have not been comprehensively clarified. Methods: Through a search of the gene expression omnibus (GEO) database, the expression profiles of mRNAs (GSE7084, GSE57691, and GSE98278) and miRNAs (GSE62179) and corresponding clinical features were downloaded, respectively. Expression profiles of 15 AAA and 10 normal vascular samples were consecutively collected for in vitro experimentation and subsequent analysis. Various machine learning techniques were employed to identify hub pyroptosis-related genes (PRGs), leading to the development of a predictive model termed the PRG classifier. Quantitative real-time-polymerase chain reaction (qRT-PCR), western blot (WB), and enzyme-linked immunosorbent assay (ELISA) were used to confirm the expression of the hub PRGs. The diagnostic and predictive capabilities of the model were comprehensively evaluated in GEO and hospital cohorts. Then, the crucial immune cell infiltration and molecular pathways implicated in the initiation and rupture of AAA and their association with pyroptosis were explored. Lastly, a miRNA/hub pyroptosis-related molecular regulatory axis was constructed using the TargetScan dataset, which was further explored through loss-of-function assays. Results: Differential analysis, enrichment score analysis, and principal component analysis (PCA) revealed that pyroptosis-related molecules were significantly involved in the occurrence of AAA. Utilizing multiple machine learning algorithms, eight key PRGs (cysteinyl aspartate specific proteinase [CASP]1, infiltrating lymphocyte [IL]1B, IL18, IL6, NOD-, LRR- and pyrin domain-containing protein [NLRP]1, NLRP2, NLRP3, and tumor necrosis factor [TNF]) were integrated to establish a PRG classifier. Demonstrating robust diagnostic capabilities (area under curve [AUC] > 0.90), the PRG classifier provided clinical insights across two GEO datasets and effectively differentiated small AAA from large AAA, elective stable AAA (eAAA), and ruptured AAA (rAAA), respectively. qRT-PCR, WB, and ELISA verified the mRNA and protein expression of the hub PRGs. Notably, in hospital cohorts, a substantial positive link was unveiled between the PRG classifier and AAA risk factors (hypertension history, diastolic pressure, triglyceride levels, and aneurysm diameter). Furthermore, immune cell infiltration and functional enrichment analysis revealed significant associations of the PRG classifier/PRGs with M2 macrophage infiltration, activated dendritic cells, and enrichment scores of the cytosolic deoxyribonucleic acid (DNA) sensing pathway and tryptophan metabolism, potentially mediating AAA onset and rupture. Finally, based on 90 differentially expressed miRNAs (DEmiRNAs) and eight hub PRGs through TargetScan dataset, a hsa-miR-331-3p/TNF regulatory axis was constructed, wherein upregulation of hsa-miR-331-3p expression significantly reduced TNF and CASP1 protein levels. Conclusion: A predictive model (PRG classifier) incorporating eight PRGs through multiple machine learning algorithms was developed and validated. This model may stand as a potent tool for diagnosing AAA and assessing disease severity. The identification of the cytosolic DNA sensing pathway and the hsa-miR-331-3p/TNF interaction axis may represent crucial targets for AAA treatment, offering deeper insights into its potential pathogenesis.

Relation of STAT3 rs1053005 Variation and miR-452-3p with Osteoarthritis Susceptibility and Severity and the Clinical Response to High-Molecular-Weight Hyaluronic Acid Injection in Osteoarthritis Patients

Polymorphisms in the 3′ untranslated region of STAT3 mRNA can derange STAT3 gene expression via modifying the microRNA-binding site. This study aimed to examine the impact of STAT3 rs1053005 variation and miR-452-3p expression on osteoarthritis (OA) susceptibility and severity and the efficacy of intra-articular high-molecular-weight hyaluronic acid (HMW-HA) injection as a therapy option for knee OA. Two hundred and fifty-eight OA patients and 200 healthy controls were enrolled in the study. STAT3 genotyping and STAT3 and miR-452-3p expression were carried out using allelic-discrimination PCR and quantitative real-time PCR. Functional assessment and pain evaluation were performed for all patients. Eighty-three patients received HMW-HA injections, and multiple follow-up visits were performed. STAT3 mRNA was upregulated, and expression was positively associated with plasmin, TNF-α, MMP-3, and STAT3 serum levels, whereas miR-452-3p was downregulated and negatively associated with the previously mentioned parameters in OA patients. Osteoarthritis patients had a lower prevalence of the minor allele of the rs1053005 variant (p < 0.001). Plasmin, TNF, MMP-3, and STAT3 mRNA and protein levels were significantly decreased, and miR-452-3p expression was significantly increased in the GG genotype compared to AG and AA genotypes. HMW-HA injection improved OA patients’ clinical scores with concomitant decreased STAT3 levels and enhanced expression of miR-452-3p. More efficient improvement was observed in rs1053005 AG + GG genotype carriers vs. AA genotype carriers. The G allele of STAT3 rs1053005 (A/G) polymorphism was associated with decreased OA susceptibility and severity and enhanced clinical response to HMW-HA injection, possibly via enhancing miR-452-3p binding and a subsequent decrease in STAT3 expression.

MAGED4B is a Poor Prognostic Marker of Stomach Adenocarcinoma and a Potential Therapeutic Target for Stomach Adenocarcinoma Tumorigenesis.

Gastric cancer is the second most common cause of cancer death worldwide with poor overall prognosis. It is important to study the molecular mechanism of stomach adenocarcinoma (STAD). MAGED4B, a member of the melanoma antigen gene (MAGE) family, is highly expressed in many tumor cells and is associated with tumor progression. Its prognostic value in and the function of the encoded protein are still unclear. The data of 415 STAD tissues was retrieved from TCGA database, and the expression level of MAGED4B mRNA was evaluated. The correlation between the expression of MAGED4B mRNA and the progression free survival (PFS) time of STAD patients was evaluated by Kaplan Meier analysis. The STAD cell lines with overexpressed and silent MAGED4B were constructed, and the effects of MAGED4B on the viability, migration and proliferation were evaluated by the CCK-8, scratch test and EDU test. The flow cytometry was used to detect apoptosis with overexpressed and silent MAGED4B under the cisplatin treatment, and WB was used to detect the expressions of related proteins, such as TNF-α. The expression level of MAGED4B mRNA in the STAD tissues was higher than that in the normal tissues, and its high expression was related to poor PFS. The overexpression of MAGED4B in the STAD cell lines can promote the vitality, motility and proliferation of the STAD cells, while the silencing of MAGED4B can inhibit the above three cell functions of the STAD cells. The overexpression of MAGED4B can reduce the cisplatin induced apoptosis and increase the cisplatin IC50; the silencing of MAGED4B can promote the cisplatin induced apoptosis and reduce the cisplatin IC50. The overexpression of MAGED4B reduced the protein levels of TRIM27 and TNF- α. MAGED4B could be a valuable prognostic biomarker and a therapeutic target for gastric adenocarcinoma of great interest.

Wnt 5a mediated inflammatory injury of renal tubular epithelial cells dependent on calcium signaling pathway in Trichloroethylene sensitized mice

Patients with trichloroethene-induced Trichloroethylene hypersensitivity syndrome (THS) often present kidney injury. However, the role of Wnt 5a/Ca2+ pathway in renal tubular injury in Trichloroethylene (TCE) sensitized mice remains unclear. This study aimed to investigate how Wnt 5a/Ca2+ pathway induced renal tubular epithelial cell injury in TCE sensitized mice. A total of 84 female BALB/c Specific Pathogen Free mice aged 6–8 weeks were used to establish TCE sensitized mouse models. Renal histology and serum levels of α1-MG and β2-MG were used to assess the renal injury. The renal protein levels of Wnt 5a, ROR2, FZD5, PLC, p-CaMKII, IκB α, p-IκB α, NF-κB(p65), TNF α, IL 6 and IL 1β were measured. The levels of serum α1-MG and β2-MG and TNF α, IL 6 and IL 1β levels in the kidney tissue were significantly increased in TCE sensitized positive group. However, Box5 pretreatment inhibited the expression of PLC, p-CaMKII, p65 and attenuated the injury of renal tubular epithelial cells and suppressed the upregulated expression of the above cytokines. In addition, KN93 also reduced nuclear translocation of p65 and renal injury as well as the elevated cytokines by inhibiting CaMKII. These data identify Wnt 5a binding to ROR2 and FZD5, p65 nuclear translocation, and inflammatory cytokine release as a novel mechanism for renal tubular epithelial cells injury by sensitization with TCE. Box5 or KN93 pretreatment can block the expression of inflammatory cytokines and reduce the injury of renal tubular epithelial cells.

Inflammation-related transcripts define “high” and “low” subgroups of individuals with schizophrenia and bipolar disorder in the midbrain

Dopamine dysregulation in schizophrenia may be associated with midbrain inflammation. Previously, we found elevated levels of pro-inflammatory cytokine mRNAs in the post-mortem midbrain of people with schizophrenia (46%) but not from unaffected controls (0%) using a brain cohort from Sydney, Australia. Here, we measured cytokine mRNAs and proteins in the midbrain in the Stanley Medical Research Institute (SMRI) array cohort (N = 105). We tested if the proportions of individuals with schizophrenia and with high inflammation can be replicated, and if individuals with bipolar disorder with elevated midbrain cytokines can be identified. mRNA levels of 7 immune transcripts from post-mortem midbrain tissue were measured via RT-PCR and two-step recursive clustering analysis was performed using 4 immune transcripts to define “high and low” inflammatory subgroups. The clustering predictors used were identical to our earlier midbrain study, and included: IL1B, IL6, TNF, and SERPINA3 mRNA levels. 46% of schizophrenia cases (16/35 SCZ), 6% of controls (2/33 CTRL), and 29% of bipolar disorder cases (10/35 BPD) were identified as belonging to the high inflammation (HI) subgroups [χ2 (2) = 13.54, p < 0.001]. When comparing inflammatory subgroups, all four mRNAs were significantly increased in SCZ-HI and BPD-HI compared to low inflammation controls (CTRL-LI) (p < 0.05). Additionally, protein levels of IL-1β, IL-6, and IL-18 were elevated in SCZ-HI and BPD-HI compared to all other low inflammatory subgroups (all p < 0.05). Surprisingly, TNF-α protein levels were unchanged according to subgroups. In conclusion, we determined that almost half of the individuals with schizophrenia were defined as having high inflammation in the midbrain, replicating our previous findings. Further, we detected close to one-third of those with bipolar disorder to be classified as having high inflammation. Elevations in some pro-inflammatory cytokine mRNAs (IL-1β and IL-6) were also found at the protein level, whereas TNF mRNA and protein levels were not concordant.

Acute Intoxication With Alcohol Reduces Trauma-Induced Proinflammatory Response and Barrier Breakdown in the Lung via the Wnt/β-Catenin Signaling Pathway.

BackgroundTrauma is the third leading cause of mortality worldwide. Upon admission, up to 50% of traumatized patients are acutely intoxicated with alcohol, which might lead to aberrant immune responses. An excessive and uncontrolled inflammatory response to injury is associated with damage to trauma-distant organs. We hypothesize that, along with inflammation-induced apoptosis, the activation of the Wnt/β-catenin signaling pathway would cause breakdown of the lung barrier and the development of lung injury after trauma. It remains unclear whether ethanol intoxication (EI) prior to trauma and hemorrhagic shock will attenuate inflammation and organ injury.MethodsIn this study, 14 male C57BL/6J mice were randomly assigned to two groups and exposed either to EtOH or to NaCl as a control by an oral gavage before receiving a femur fracture (Fx) and hemorrhagic shock, followed by resuscitation (THFx). Fourteen sham animals received either EtOH or NaCl and underwent surgical procedures without THFx induction. After 24 h, oil red O staining of fatty vacuoles in the liver was performed. Histological lung injury score (LIS) was assessed to analyze the trauma-induced RLI. Gene expression of Cxcl1, Il-1β, Muc5ac, Tnf, and Tnfrsf10b as well as CXCL1, IL-1β, and TNF protein levels in the lung tissue and bronchoalveolar lavage fluid were determined by RT-qPCR, ELISA, and immunohistological analyses. Infiltrating polymorphonuclear leukocytes (PMNLs) were examined via immunostaining. Apoptosis was detected by activated caspase-3 expression in the lung tissue. To confirm active Wnt signaling after trauma, gene expression of Wnt3a and its inhibitor sclerostin (Sost) was determined. Protein expression of A20 and RIPK4 as possible modulators of the Wnt signaling pathway was analyzed via immunofluorescence.ResultsSignificant fatty changes in the liver confirmed the acute EI. Histopathology and decreased Muc5ac expression revealed an increased lung barrier breakdown and concomitant lung injury after THFx versus sham. EI prior trauma decreased lung injury. THFx increased not only the gene expression of pro-inflammatory markers but also the pulmonary infiltration with PMNL and apoptosis versus sham, while EI prior to THFx reduced those changes significantly. EI increased the THFx-reduced gene expression of Sost and reduced the THFx-induced expression of Wnt3a. While A20, RIPK4, and membranous β-catenin were significantly reduced after trauma, they were enhanced upon EI.ConclusionThese findings suggest that acute EI alleviates the uncontrolled inflammatory response and lung barrier breakdown after trauma by suppressing the Wnt/β-catenin signaling pathway.

Extract of Corallodiscus flabellata attenuates renal fibrosis in SAMP8 mice via the Wnt/\u03b2-catenin/RAS signaling pathway

BackgroundFibrosis is one of the most common pathological features of the aging process of the kidney, and fibrosis in aging kidneys also aggravates the process of chronic kidney disease (CKD). Corallodiscus flabellata B. L. Burtt (C. flabellata, CF) is a commonly used botanical drug in Chinese folklore. However, few studies have reported its pharmacological effects. This study aimed to explore the effect of CF ethanol extract on renal fibrosis in SAMP8 mice and identify potentially active compounds.MethodsSenescence-accelerated mouse-prone 8 (SAMP8) were used as animal models, and different doses of CF were given by gavage for one month. To observe the degree of renal aging in mice using β-galactosidase staining. Masson staining and the expression levels of Col-I, α-SMA, and FN were used to evaluate the renal fibrosis in mice. The protein expression levels of Nrf2 pathway and Wnt/β-catenin/RAS pathway in the kidney were measured. And β-galactosidase (β-gal) induced NRK-52E cells as an in vitro model to screen the active components of CF.ResultsThe CF ethanol extract significantly inhibited the activity of renal β-galactosidase and the expression levels of Col-I, α-SMA, and FN in SAMP8 mice, and improved Masson staining in SAMP8 mice. CF remarkably reduced urinary protein, creatinine, urea nitrogen and serum levels of TNF-α and IL-1β in SAMP8 mice, and significantly increased the levels of SOD and GSH-Px. Moreover, CF activated the Nrf2 pathway and blocked the Wnt/β-catenin/RAS pathway in the kidneys of mice. Besides, 3,4-dihydroxyphenylethanol (SDC-0-14, 16) and (3,4-dihydroxyphenylethanol-8-O-[4-O-trans-caffeoyl-β-D-apiofuranosyl-(1→3)-β-D-glucopyranosyl (1→6)]-β-D-glucopyranoside (SDC-1-8) were isolated from CF, which reduced the senescence of NRK-52E cells, and maybe the active ingredients of CF playing the anti-aging role.ConclusionsOur experiments illuminated that CF ethanol extract may ameliorate renal fibrosis in SAMP8 mice via the Wnt/β-catenin/RAS pathway. And SDC-0-14,16 and SDC-1-8 may be the material basis for CF to exert anti-renal senescence-related effects.

Age of Rats Affects the Degree of Retinal Neuroinflammatory Response Induced by High Acute Intraocular Pressure.

Purpose To investigate whether retinal neuroinflammatory response was affected by aging in a rat model of acute glaucoma. Methods Young adult and aged rats were randomly assigned into normal control, 45 mmHg, 60 mmHg, and 90 mmHg groups. Intraocular pressure (IOP) of rats was acutely elevated to 45 mmHg, 60 mmHg, and 90 mmHg, respectively. Three days after high IOP treatment, loss of retinal ganglion cells (RGCs), formation of proinflammatory microglia/macrophages and neurotoxic astrocytes, and deposition of complement C3 in the retina were detected by immunofluorescence. ELISA was used to assess the protein levels of proinflammatory cytokines TNF and IL-1β in the retina. Results Compared with young adult retinae, (1) loss of RGCs was more severe in aged retinae under the same IOP treatment, (2) microglia/macrophages were more prone to adopt proinflammatory phenotype in aged retinae in response to elevated IOP, (3) high IOP treatment induced astrogliosis, formation of neurotoxic astrocytes, and deposition of complement C3 more easily in aged retinae, and (4) aged retinae induced higher levels of proinflammatory cytokines TNF and IL-1β under the same IOP treatment. Conclusion Our data indicated that aging affects the degree of retinal neuroinflammatory response initiated by ocular hypertension, which may contribute to the age-related susceptibility of RGCs to elevated IOP.

Changes in Cerebrospinal Fluid Balance of TNF and TNF Receptors in Naïve Multiple Sclerosis Patients: Early Involvement in Compartmentalised Intrathecal Inflammation

An imbalance of TNF signalling in the inflammatory milieu generated by meningeal immune cell infiltrates in the subarachnoid space in multiple sclerosis (MS), and its animal model may lead to increased cortical pathology. In order to explore whether this feature may be present from the early stages of MS and may be associated with the clinical outcome, the protein levels of TNF, sTNF-R1 and sTNF-R2 were assayed in CSF collected from 122 treatment-naïve MS patients and 36 subjects with other neurological conditions at diagnosis. Potential correlations with other CSF cytokines/chemokines and with clinical and imaging parameters at diagnosis (T0) and after 2 years of follow-up (T24) were evaluated. Significantly increased levels of TNF (fold change: 7.739; p < 0.001), sTNF-R1 (fold change: 1.693; p < 0.001) and sTNF-R2 (fold change: 2.189; p < 0.001) were detected in CSF of MS patients compared to the control group at T0. Increased TNF levels in CSF were significantly (p < 0.01) associated with increased EDSS change (r = 0.43), relapses (r = 0.48) and the appearance of white matter lesions (r = 0.49). CSF levels of TNFR1 were associated with cortical lesion volume (r = 0.41) at T0, as well as with new cortical lesions (r = 0.56), whilst no correlation could be found between TNFR2 levels in CSF and clinical or MRI features. Combined correlation and pathway analysis (ingenuity) of the CSF protein pattern associated with TNF expression (encompassing elevated levels of BAFF, IFN-γ, IL-1β, IL-10, IL-8, IL-16, CCL21, haptoglobin and fibrinogen) showed a particular relationship to the interaction between innate and adaptive immune response. The CSF sTNF-R1-associated pattern (encompassing high levels of CXCL13, TWEAK, LIGHT, IL-35, osteopontin, pentraxin-3, sCD163 and chitinase-3-L1) was mainly related to altered T cell and B cell signalling. Finally, the CSF TNFR2-associated pattern (encompassing high CSF levels of IFN-β, IFN-λ2, sIL-6Rα) was linked to Th cell differentiation and regulatory cytokine signalling. In conclusion, dysregulation of TNF and TNF-R1/2 pathways associates with specific clinical/MRI profiles and can be identified at a very early stage in MS patients, at the time of diagnosis, contributing to the prediction of the disease outcome.

Effect of docosahexaenoic acid plus insulin on atherosclerotic human endothelial cells

BackgroundAtherosclerosis is touted as one of the most critical consequences of diabetes mellitus indicated by local inflammation of endothelial cells. The Effect of Omega 3 fatty acids, mainly docosahexaenoic acid (DHA), has been investigated in cells after exposure to high doses of lipids. The current experiment aimed to address the modulatory effects of docosahexaenoic acid and insulin in palmitic-treated human endothelial cells.MethodsHuman umbilical vein endothelial cells were treated with 1 mM palmitic acid, 50 μM insulin, 50 μM docosahexaenoic acid, and their combination for 48 h. Cell survival rate and apoptosis were measured using MTT and flow cytometry assays. The Griess assay detected NO levels. Protein levels of TNF-α, IL-6, and NF-κB were studied using ELISA and immunofluorescence imaging. The expression of genes participating in atherosclerosis was monitored using PCR array analysis.ResultsOil Red O staining showed the inhibitory effect of DHA and insulin to reduce the intracellular accumulation of palmitic acid. Both DHA and Insulin blunted palmitic acid detrimental effects on HUVECs indicated by an increased survival rate (p < 0.05). The percent of apoptotic cells was decreased in palmitic-treated cells received insulin and DHA compared to palmitic-treated group (p < 0.05). Based on our data, DHA and Insulin diminished the production of all inflammatory cytokines, TNF-α, IL-6, and NF-κB, in palmitic-treated cells (p < 0.05). Similar to these data, NO production was also decreased in all groups treated with insulin and DHA compared to the palmitic-treated cells (p < 0.05). PCR array analysis revealed the modulatory effect of DHA and insulin on the expression of atherosclerosis-related genes pre-treated with palmitic acid compared to the control group (p < 0.05).ConclusionDHA and Insulin could alter the dynamic growth and dysfunctional activity of human endothelial cells after treatment with palmitic acid. Taken together, Omega 3 fatty acids, along with insulin, could dictate specific cell behavior in endothelial cells in vitro.

Novel Combinatorial Regimen of Garcinol and Curcuminoids for Non-alcoholic Steatohepatitis (NASH) in Mice

Non-alcoholic steatohepatitis (NASH) is a progressive form of Non-alcoholic fatty liver disease (NAFLD), a chronic liver disease with a significant unmet clinical need. In this study, we examined the protective effects of Garcinia indica extract standardized to contain 20% w/w of Garcinol (GIE) and 95% Curcuminoids w/w from Curcuma longa (Curcuminoids) in a Stelic animal model (STAM) of NASH. The STAM mice developed steatosis, hepatocyte ballooning, and inflammation, which were significantly reduced by the combination of GIE and Curcuminoids, resulting in a lower NAFLD activity score. The treatment reduced fibrosis as observed by Sirius red staining, liver hydroxyproline content and mRNA levels of TGF- β and collagen in the liver. Immunostaining with alpha-smooth muscle actin (α SMA) revealed a significant reduction in hepatic stellate cells. Intriguingly, the combination regimen markedly decreased the mRNA levels of MCP1 and CRP and both mRNA and protein levels of TNF-α. NF-kB, reduced the hepatic and circulating FGF21 levels and altered the nonenzymatic (glutathione) and enzymatic antioxidant markers (Glutathione peroxidase, and superoxide dismutase). Our results suggest that the combination of GIE and Curcuminoids can reduce the severity of NASH by reducing steatosis, fibrosis, oxidative stress, and inflammation. The results suggest that the combinatorial regimen could be an effective supplement to prevent the progression of liver steatosis to inflammation and fibrosis in NASH.

Clinical significance of miR-19b-3p in patients with sepsis and its regulatory role in the LPS-induced inflammatory response

BackgroundMicroRNAs (miRNAs) play important roles in the development and progression of sepsis. This study investigated the clinical value of miR-19b-3p in sepsis patients, and explored its role in regulating inflammatory responses in HUVECs cells.Methods103 patients with sepsis and 98 healthy individuals were recruited. qRT-PCR was used for the measurement of miR-19b-3p level. Cell viability was evaluated using CCK-8. The protein levels of TNF-α and IL-6 were measured using ELISA. Receiver operating characteristic (ROC) curve and logistic regression analysis were constructed to evaluate the diagnostic and prognostic values of miR-19b-3p in sepsis patients.ResultsMiR-19b-3p level was significantly reduced in the serum from patients with sepsis compared with healthy controls (P < 0.001). Sepsis patients in the survival group had significantly high miR-19b-3p levels compared with the non-survival group (P < 0.001). MiR-19b-3p was of a good value in predicting sepsis risk, and was an independent prognostic factor for 28-day survival in sepsis patients (OR = 3.226, 95% CI 1.076–9.670, P = 0.037). MiR-19b-3p level was negatively associated with serum levels of IL-6 (r = − 0.852, P < 0.001) and TNF-α (r = − 0.761, P < 0.001). Overexpression of miR-19b-3p alleviated LPS-induced inflammatory response of HUVECs, which was reflected by the decrease of the levels of IL-6 and TNF-α induced by LPS treatment (P < 0.001).ConclusionMiR-19b-3p might be a potential biomarker for the early diagnosis and prognosis of sepsis patients. Overexpression of miR-19b-3p alleviated sepsis-induced inflammatory responses.

Improvement of human pancreatic islet quality after co-culture with human adipose-derived stem cells

The aim of this study was to investigate whether co-culture of human islets with adipose-derived stem cells (ASCs) can improve islet quality and to evaluate which factors play a role in the protective effect of ASCs against islet dysfunction. Islets and ASCs were cultured in three experimental groups for 24 h, 48 h, and 72 h: 1) indirect co-culture of islets with ASC monolayer (Islets/ASCs); 2) islets alone; and 3) ASCs alone. Co-culture with ASCs improved islet viability and function in all culture time-points analyzed. VEGFA, HGF, IL6, IL8, IL10, CCL2, IL1B, and TNF protein levels were increased in supernatants of islet/ASC group compared to islets alone, mainly after 24 h. Moreover, VEGFA, IL6, CCL2, HIF1A, XIAP, CHOP, and NFKBIA genes were differentially expressed in islets from the co-culture condition compared to islets alone. In conclusion, co-culture of islets with ASCs promotes improvements in islet quality.

Deciphering the chemical profile and pharmacological mechanisms of Baihu-Guizhi decoction using ultra-fast liquid chromatography-quadrupole-time-of-flight tandem mass spectrometry coupled with network pharmacology-based investigation

BackgroundBaihu-Guizhi decoction (BHGZD) has been extensively used for the treatment of rheumatoid arthritis (RA) with a satisfying therapeutic effect. However, the material basis and the underlying mechanisms of BHGZD against RA have not been fully elucidated. PurposeTo investigate the chemical profile and the pharmacological mechanisms of BHGZD against RA. MethodsThe chemical constituents containing in BHGZD were identified using UFLC-Q-TOF-MS/MS system, and the corresponding putative targets were predicted. Then, the differentially expressed genes (DEGs) between adjuvant-induced arthritis (AIA) and normal control groups were identified using microarray analysis. After constructing the interaction network of “RA-related gene-BHGZD putative target”, BHGZD candidate targets against RA were screened by topological analysis and further experimentally validated based on AIA rat model. ResultsA total of 41 chemical constituents were identified in the water extract of BHGZD, which were predicted to hit 1312 putative targets. Additionally, 26 DEGs between the AIA and normal control groups were defined as “RA-related genes”, which were functionally involved into the imbalance of “inflammation-immune” system during RA progression. On the basis of the topological importance in the network of “RA-related gene-BHGZD putative target”, 177 BHGZD candidate targets against RA were identified. Among them, TLR4, c-Fos/AP-1, IL2 and TNF had direct interactions with each other and also function as crucial components of toll-like receptor and T cell receptor signaling pathways, which may play important roles in maintaining the balance of "inflammation-immune" system. Experimentally, we verified that BHGZD dose-dependently attenuated the severity, pathological changes, as well as mechanical, cold, and heat hypersensitivities during RA progression based on the AIA rat model. Further western blot analysis demonstrated that BHGZD significantly reduced the protein levels of TLR4, c-Fos/AP-1, IL2 and TNF, which were induced by RA modeling, in the inflamed joints of AIA rats (all p<0.05). ConclusionThis study combining the chemical and transcriptomic profilings, target prediction, network calculation and experimental validations identifies the chemical constituents containing in BHGZD and offers the convincing evidence that BHGZD may ameliorate RA partially by restoring the balance of "inflammation-immune" system and subsequently reversing the pathological events during RA progression through regulating the TLR4-c-Fos-IL2-TNF axis.

Initial In Vitro Development of a Potassium-Based Intra-Articular Injection for Osteoarthritis.

The long-term goal of this work is to develop a potassium (K+)-based intra-articular (IA) injection for osteoarthritis treatment. Within this context, the objectives of this study were to (1) demonstrate that hyperosmolar K+ solutions can suppress proinflammatory macrophage activation and (2) evaluate the therapeutic potential of a hyperosmolar K+ solution relative to a clinically utilized drug-based (methylprednisolone acetate [MPA]-a corticosteroid) or cell-based (human mesenchymal stem cell [hMSC]) IA injectable. A 3D in vitro model with poly(ethylene glycol) diacrylate hydrogels encapsulated with proinflammatory interferon-gamma (IFN)-stimulated macrophages (M(IFN)s) was utilized. Long-term changes in cell phenotype in response to short-term stimulation (i.e., mimicking an IA injection) were assessed following treatment with 80 mM K+ gluconate, hMSCs, or MPA. Addition of 80 mM K+ gluconate to culture media significantly reduced iNOS and TNF protein levels in M(IFN)s. Furthermore, short-term stimulation with K+ gluconate elicited a significant increase in the anti/proinflammatory cytokine profile in M(IFN)s, a response that is not noticed with either clinically utilized MPA or an hMSC injectable. Hyperosmolar K+ solutions are capable of attenuating proinflammatory macrophage activation. Moreover, when evaluated in an in vitro setting mimicking an IA injection, K+ performed significantly better than hMSCs or the corticosteroid MPA. Cumulatively, these results support further development and application of a K+-based IA injection toward osteoarthritis research.

Increased pro-inflammatory cytokine gene expression in peripheral blood mononuclear cells of patients with polyneuropathies.

Distinct cytokine expression patterns have been reported in biomaterial of patients with polyneuropathies (PNP). We investigated gene expression profiles of pro- and anti-inflammatory cytokines in peripheral blood mononuclear cells (PBMC) of patients with neuropathies of different etiologies. We prospectively studied 97 patients with neuropathies and compared data between diagnostic subgroups and healthy controls. Gene expression of a panel of pro- and anti-inflammatory cytokines was analyzed (interleukin-1 [IL-1], IL-2, IL-6, IL-8, tumor necrosis factor alpha [TNF], IL-4, and IL-10) in PBMC samples. Furthermore, protein levels of IL-6, IL-8, and TNF were measured in supernatant of PBMC stimulated with lipopolysaccharide (LPS). PNP were associated with higher PBMC gene expression of IL-1 (p<0.05), IL-2 (p<0.05), IL-8 (p<0.001), and TNF (p<0.01) compared to healthy controls. Inflammatory neuropathies were associated with higher gene expression of IL-8 (p<0.001) and TNF (p<0.05) and lower gene expression of IL-10 (p<0.05) compared to healthy controls. More pro-inflammatory cytokines were elevated in painful neuropathy (IL-1, IL-2 [p<0.05], IL-8 [p<0.001] and TNF [p<0.05]) than in painless neuropathy (IL-8 [p<0.01] and TNF [p<0.01]) compared to healthy controls, while IL-10 expression was higher in treatment naïve patients with painless neuropathy compared to patients with painful neuropathy (p<0.05). Disease duration positively correlated with IL-6 gene expression (p<0.01). Supernatant protein levels of IL-6, IL-8, and TNF did not differ between groups. Systemic gene expression of pro-inflammatory cytokines is increased in patients with neuropathies and may be influenced by the presence of neuropathic pain.

Ultrasound-Mediated Delivery of RNA to Colonic Mucosa of Live Mice

It is a challenge to deliver nucleic acids to gastrointestinal (GI) tissues due to their size and need for intracellular delivery. They are also extremely susceptible to degradation by nucleases, which are ubiquitous in the GI tract. We investigated whether ultrasound, which can permeabilize tissue through a phenomenon known as transient cavitation, can be used to deliver RNA to the colonic mucosa of living mice. We investigated delivery of fluorescently labeled permeants to colon tissues of Yorkshire pigs exvivo and mice invivo. Colon tissues were collected and fluorescence was measured by confocal microscopy. We then evaluated whether ultrasound is effective in delivering small interfering (si)RNA to C57BL/6 mice with dextran sodium sulfate-induced colitis. Some micewere given siRNA against tumor necrosis factor (Tnf) mRNA for 6 days; colon tissues were collected and analyzed histologically and TNF protein levels measured by enzyme-linked immunosorbent assay. Feces were collected and assessed for consistency and occult bleeding. We delivered mRNA encoding firefly luciferase to colons of healthy C57BL/6 mice. Exposure of exvivo pig colon tissues to 20 kHz ultrasound for 1 minute increased the level of delivery of 3 kDa dextran 7-fold compared with passive diffusion (P= .037); 40 kHz ultrasound application for 0.5 seconds increased the delivery 3.3-fold in living mice (P= .041). Confocal microscopy analyses of colon tissues from pigs revealed regions of punctuated fluorescent dextran signal, indicating intracellular delivery of macromolecules. In mice with colitis, ultrasound delivery of unencapsulated siRNA against Tnf mRNA reduced protein levels of TNF in colon tissues, compared with mice with colitis given siRNA against Tnf mRNA without ultrasound (P ≤ .014), and reduced features of inflammation (P≤4.1× 10-5). Separately, colons of mice administered an mRNA encoding firefly luciferase with ultrasound and the D-luciferin substrate had levels of bioluminescence 11-fold greater than colons of mice given the mRNA alone (P= .0025). Ultrasound exposures of 40 kHz ultrasound for 0.5 seconds were well tolerated, even in mice with acute colitis. Ultrasound can be used to deliver mRNAs and siRNAs to the colonic mucosa of mice and knock down expression of target mRNAs.

Immunomodulation of Inflammatory Markers in Activated Macrophages by Leaf Extracts of Gingko Biloba

Much recent attention has been given to traditional medicines and natural products with potential and promising anti-inflammatory properties. Leaf extract of Ginkgo biloba GbE-761, possesses several clinical beneficial effects and is now used in a broad spectrum of pharmacological actions. However, mechanisms associated with its anti-inflammatory effect are not very much clear. Previously we established that GbE-761 exerts a neuroprotective effect against ischemic brain injury through an anti-apoptotic mechanism. In this study we investigated the anti-inflammatory effect of GbE-761 in LPS-activated murine macrophage. Our results demonstrate that GbE-761 potently inhibited LPS-induced NO and PGE2 production and these observations were consistent with the inhibition in the protein and mRNA expression levels of inducible iNOS and COX-2 enzymes in a dose dependent manner. Not only this, GbE-761 attenuated the production of pro-inflammatory cytokines like IL-1 , IL-6 and TNFin LPS-activated murine macrophages in concentration dependent manner. In addition we reported inhibition in the protein and mRNA expression levels of IL-1 , IL-6 and TNF. Furthermore, GbE-761 inhibited the NFB activation induced by LPS. Together these results suggest that anti-inflammatory properties of GbE-761 extract might be the results of inhibition of iNOS and COX-2 by upregulating anti-oxidative enzymes and inhibition of pro-inflammatory cytokines (IL-1 , IL-6 and TNF) expression through the down-regulation of NF-kB activation in murine macrophages.

Altered monocyte expression and expansion of non-classical monocyte subset in IgA nephropathy patients

The main defect of immunoglobulin A nephropathy (IgAN) lies within the immune system and in peripheral blood mononuclear cells rather than in the kidney. Previously, we found an altered gene expression in monocytes compared with B and T cells isolated from IgAN patients; thus, our aim here has been to study this subset at a genome-wide and functional level. A total of 39 IgAN patients and 37 healthy blood donors (HBDs) were included in this study, and microarray technology was used to evaluate global gene expression differences in monocytes isolated from IgAN patients and HBDs. Aberrantly expressed genes and pathways were then validated on an independent set of IgAN patients with RT-PCR western blot and flow cytometric analysis. Gene expression differences in monocytes from IgAN patients and HBDs primarily involved apoptosis signalling, mitochondrial dysfunction, tnfr2/1 and death receptor signalling. Both the extrinsic and intrinsic apoptotic pathways seem to be implicated; in particular, the protein levels of NDUFS3 and TNFRSF1A were upregulated thus confirming the altered mitochondrial and death receptor homeostasis. Furthermore, the basal intracellular protein levels of TNF in monocytes were lower in IgAN patients compared with HBDs. We validated at protein level an enhanced apoptotic phenotype and a different subset distribution in monocytes from IgAN patients. We found that the non-classical monocyte subset (CD14(+)CD16(+)) was significantly expanded in all IgAN patients tested even though the total monocyte count remained unchanged. Our findings demonstrate, for the first time, an aberrant modulation of the mitochondrial respiratory system in monocytes isolated from IgAN patients. Furthermore, the aberrant expansion of the (CD14(+)CD16(+)) subset could explain the enhanced apoptotic phenotype seen in these cells thus revealing their potential role in the pathogenesis of IgAN.