Cytokine Protein Expression Research Articles

Immunomodulatory Properties of Multi-Strain Postbiotics on Human CD14+ Monocytes

The ability of probiotics, comprising live microbiota, to modulate the composition of intestinal microbiomes has been connected to modulation of the central nervous system (Gut–Brain axis), neuroendocrine system (Gut–Skin axis), and immune response (Gut–Immune axis). Less information is known regarding the ability of postbiotics (cell wall components and secreted metabolites derived from live organisms) to regulate host immunity. In the present study, we tested postbiotics comprising single strains of bacteria and yeast (Lactobacillus acidophilus 16axg, Lacticaseibacillus rhamnosus 18fx, Saccharomyces cerevisiae var. boulardii 16mxg) as well as combinations of multiple strains for their ability to stimulate cytokine production by human CD14+ monocytes. We quantified cytokine gene and protein expression levels in monocytes following stimulation with postbiotics. Both heat-killed L. acidophilus and L. rhamnosus stimulated naïve monocytes without significant differences between them. Heat-killed S. boulardii stimulated less cytokine production compared to postbiotic bacteria at the same concentration. Interestingly, the addition of heat-killed yeast to heat-killed L. acidophilus and L. rhamnosus resulted in an enhancement of immune stimulation. Thus, heat-killed postbiotics have immune-modulating potential, particularly when bacteria and yeast are combined. This approach may hold promise for developing targeted interventions that can be fine-tuned to modulate host immune response with beneficial health impact.

P38α-eIF6-Nsun2 axis promotes ILC3's rapid response to protect host from intestinal inflammation.

Group 3 innate lymphoid cells (ILC3s) are important for maintaining gut homeostasis. Upon stimulation, ILC3s can rapidly produce cytokines to protect against infections and colitis. However, the regulation of ILC3 quick response is still unclear. Here, we find that eIF6 aggregates with Nsun2 and cytokine mRNA in ILC3s at steady state, which inhibits the methyltransferase activity of Nsun2 and the nuclear export of cytokine mRNA, resulting in the nuclear reservation of cytokine mRNA. Upon stimulation, phosphorylated p38α phosphorylates eIF6, which in turn releases Nsun2 activity, and promotes the nuclear export of cytokine mRNA and rapid cytokine production. Genetic disruption of p38α, Nsun2, or eIF6 in ILC3s influences the mRNA nuclear export and protein expression of the protective cytokines, thus leading to increased susceptibility to colitis. Together, our data identify a crucial role of the p38α-eIF6-Nsun2 axis in regulating rapid ILC3 immune response at the posttranscriptional level, which is critical for gut homeostasis maintenance and protection against gut inflammation.

In Vitro Assessment of the Neuroprotective Effects of Pomegranate (Punica granatum L.) Polyphenols Against Tau Phosphorylation, Neuroinflammation, and Oxidative Stress.

Oxidative stress and chronic inflammation, at both the systemic and the central level, are critical early events in atherosclerosis and Alzheimer's disease (AD). To investigate the oxidative stress-, inflammation-, and Tau-phosphorylation-lowering effects of pomegranate polyphenols (PPs) (punicalagin, ellagic acid, peel, and aril extracts). We used flow cytometry to quantify the protein expression of proinflammatory cytokines (IL-1β) and anti-inflammatory mediators (IL-10) in THP-1 macrophages, as well as M1/M2 cell-specific marker (CD86 and CD163) expression in human microglia HMC3 cells. The IL-10 protein expression was also quantified in U373-MG human astrocytes. The effect of PPs on human amyloid beta 1-42 (Aβ1-42)-induced oxidative stress was assessed in the microglia by measuring ROS generation and lipid peroxidation, using 2',7'-dichlorofluorescein diacetate (DCFH-DA) and thiobarbituric acid reactive substance (TBARS) tests, respectively. Neuronal viability and cell apoptotic response to Aβ1-42 toxicity were assayed using the MTT (3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide) assay and the annexin-V-FITC apoptosis detection kit, respectively. Finally, flow cytometry analysis was also performed to evaluate the ability of PPs to modulate Aβ1-42-induced Tau-181 phosphorylation (pTau-181). Our data indicate that PPs are significantly (p < 0.05) effective in countering Aβ1-42-induced inflammation through increasing the anti-inflammatory cytokines (IL-10) in U373-MG astrocytes and THP1 macrophages and decreasing proinflammatory marker (IL-1β) expression in THP1 macrophages. The PPs were also significantly (p < 0.05) effective in inducing the phenotypic transition of THP-1 macrophages and microglial cells from M1 to M2 by decreasing CD86 and increasing CD163 surface receptor expression. Moreover, our treatments have a significant (p < 0.05) beneficial impact on oxidative stress, illustrated in the reduction in TBARS and ROS generation. Our treatments have significant (p < 0.05) cell viability improvement capacities and anti-apoptotic effects on human H4 neurons. Furthermore, our results suggest that Aβ1-42 significantly (p < 0.05) increases pTau-181. This effect is significantly (p < 0.05) attenuated by arils, peels, and punicalagin and drastically reduced by the ellagic acid treatment. Overall, our results attribute to PPs anti-inflammatory, antioxidant, anti-apoptotic, and anti-Tau-pathology potential. Future studies should aim to extend our knowledge of the potential role of PPs in Aβ1-42-induced neurodegeneration, particularly concerning its association with the tauopathy involved in AD.

CSWT improves ventricular function by promoting angiogenesis through Rho/ROCK/ERK1/2 signal pathway in infarcted myocardium in rats

Abstract Objective This study aimed to investigate the effects of extracorporeal cardiac shock wave therapy (CSWT) on myocardial ischemia and cardiac function in rats with myocardial infarction, and to explore its impact on the expression of myocardial vascular endothelial growth factor and the extent of myocardial fibrosis, thereby elucidating the mechanism of angiogenesis. Methods A rat model of myocardial infarction was induced by ligating the left anterior descending branch of the coronary artery, confirmed by echocardiography. Animals were randomly assigned to five groups: sham operation group, myocardial infarction group, myocardial infarction + CSWT group, myocardial infarction + Y-27632 group, and myocardial infarction + CSWT + Y-27632 group. Cardiac function was evaluated by echocardiography on the 16th and 72nd day post-surgery. Myocardial morphology was assessed by HE staining, and the extent of myocardial fibrosis was determined by Masson's trichrome staining. Immunohistochemical staining for factor VIII was conducted to observe angiogenesis in each infarcted area. Western blotting was employed to detect the protein expression of extracellular regulated kinase (ERK1/2) and pro-angiogenic cytokines in the infarct border zone. Results CSWT treatment significantly reduced myocardial fibrosis and improved cardiac function compared to the myocardial infarction group. Additionally, CSWT upregulated ERK expression and angiogenesis-related factors, promoting angiogenesis. The beneficial effects of CSWT were abolished by inhibiting the Rho/ROCK/ERK1/2 pathway. Furthermore, the area of myocardial fibrosis was significantly reduced in the MI+Y-27632 group compared to the myocardial infarction group (p &amp;lt; 0.01), and left ventricular systolic function index was significantly higher in the myocardial infarction group. Conclusions CSWT may promote angiogenesis post-myocardial infarction by activating the Rho/ROCK/ERK1/2 pathway. Additionally, Y-27632 may improve cardiac function in rats with myocardial infarction, potentially through mitigating myocardial fibrosis.echo images of ratsprotein expression by WB

In situ assessment of neuroinflammatory cytokines in different stages of ovine natural prion disease.

According to the neuroinflammatory hypothesis, a cytokine-mediated host innate immune response may be involved in the mechanisms that contribute to the process of neurodegeneration. Specifically, regarding prion diseases, some experimental murine models have evidenced an altered profile of inflammatory intermediaries. However, the local inflammatory response has rarely been assessed, and never in tissues from different natural models throughout the progression of neurodegeneration. The aim of this study was to use immunohistochemistry (IHC) to in situ assess the temporal protein expression of several cytokines in the cerebellum of sheep suffering from various clinical stages of scrapie. Clear changes in the expression of most of the assessed markers were observed in the affected sheep compared to the healthy control sheep, and from different stages. In summary, this preliminary IHC study focusing in the Purkinje cell layer changes demonstrate that all cytokines or respective receptors studied (IL-1, IL-1R, IL-2R, IL-6, IL-10R, and TNFαR) except for IFNγR are disease-associated signaling proteins showing an increase or decrease in relation to the progression of clinical disease. In the future, this study will be extended to other inflammatory mediators and brain regions, focusing in particular on the release of these inflammatory mediators by astroglial and microglial populations.

MTORC2 inhibition by JR-AB2-011 improves IL-1β-induced inflammation, catabolic response, and apoptosis in human chondrocytes through IκB-α/NF-κB p65.

Osteoarthritis (OA) is a very common chronic joint condition marked by inflammation and cartilage loss. mTOR is a well-known mediator of inflammation, cell survival, and aging; however, its role in OA has not been determined. To explore the role of mTORC2 in OA-and associated pathological changes, we examined the contribution of mTORC2-mediated Akt, rictor and IκB-α/NF-κB p65 pathway in interleukin (IL)-1β-treated human chondrocytes. We focused on the protein expression of proinflammatory cytokines and catabolic and apoptotic factors, including TNF-α, IL-6, iNOS, MMP13, Bax, and caspase3, which may occur through this signalling pathway in IL-1β-treated chondrocytes. Chondrocytes were cultured and treated with either 2 ng/mL IL‑1β alone or in combination with increasing concentrations of JR-AB2-011 (50, 100, or 250 µM), a selective mTORC2 inhibitor. The protein levels of phosphorylated (p)‑Akt, Akt, rictor, p-NF-κB p65, NF-κB p65, IκB-α, p-IκB-α, iNOS, MMP13, Bax, and caspase3 were evaluated by Western blotting. In IL-1β-stimulated chondrocytes, mTORC2 activity was increased with increased phosphorylation of Akt and expression of rictor. IL-1β increased the expression of p-IκBα, p-NF-κB p65, NF-κB p65, IL-6, TNF-α, iNOS, Bax, and caspase3 proteins and decreased the expression of IκB-α. All of these IL-1β-induced alterations were prevented by JR-AB2-011. The main novel finding in the present study is that selective mTORC2 inhibition by JR-AB2-011 prevents the inflammatory, catabolic, and apoptotic responses induced by IL-1β via modulation of IκB-α/NF-κB activity. Therefore, we demonstrated a previously unknown function of mTORC2 inhibition that seems to be a potential therapeutic target for OA.

CEACAM1 increased the lymphangiogenesis through miR-423-5p and NF- kB in Non-Small Cell Lung Cancer

BackgroundLung cancer causes significant mortality, with invasion and metastasis being the main features that cause most cancer deaths. Lymph node metastasis is the primary metastatic route in non-small cell carcinoma (NSCLC) and influences the staging and prognosis of NSCLC. Cumulative studies have reported that Carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1) is involved in the progression of various cancers. However, few studies have discussed the function of CEACAM1 in lymphangiogenesis in NSCLC. Here, we examined how CEACAM1 influences lymphangiogenesis in NSCLC. MethodsA total of 30 primary squamous cell carcinoma (LUSC) patients diagnosed with LN metastasis were prospectively selected. LUSC tumor tissues, para-cancerous tissues, and positive lymph node tissues were harvested. The expression and subcellular location of CEACAM1, CD31, and LVYE1 in clinical samples were detected by immunohistochemistry. Next, the CEACAM1 and hsa-miR-423-5p expressions were detected by qPCR. The protein expression of lymphangiogenesis-associated proteins and critical cytokines of the NF–κB pathway in HDLECs was detected by Western blot. A tube formation assay was performed to detect the lymphangiogenesis in different groups. The interaction between CEACAM1 and hsa-miR-423-5p was verified using a dual luciferase assay. ResultsCEACAM1 was found to be a potential gene associated with lung cancer prognosis. It was positively correlated with angiogenesis and lymphangiogenesis. Then, we detected the function of CEACAM1 in lymphangiogenesis and found that CEACAM1 promoted lymphangiogenesis. hsa-miR-423-5p overexpression inhibited lymphangiogenesis via targeting CEACAM1. Finally, we observed that CEACAM1 can activate the NF–κB pathway and, therefore, promote lymphangiogenesis. ConclusionWe found that CEACAM1 enhanced lymphangiogenesis in NSCLC via NF-kB activation and was repressed by miR-423-5p. This suggests the value of CEACAM1 as a new therapeutic marker in NSCLC.

1,25‑Dihydroxyvitamin D3 mitigates the adipogenesis induced by bisphenol A in 3T3-L1 and hAMSC through miR-27-3p regulation

PurposeEndocrine-disrupting compounds, including bisphenol A (BPA), may promote obesity influencing basal metabolic rate and shifting metabolism towards energy storage. The role of 1,25‑Dihydroxyvitamin D3 (VitD) in counteracting adipogenesis is still a matter of debate. Thus, the current study aims to investigate whether and how VitD exposure during adipogenesis could prevent the pro-adipogenic effect of BPA in two adipocyte models, mouse 3T3-L1 cell line and human adipose-derived mesenchymal stem cells (hAMSC).Methods3T3-L1, mouse pre-adipocytes and human adipose-derived mesenchymal stem cells (hAMSC) were treated with VitD (10−7 M) and BPA (10−8 M and 10−9 M), alone or in combination, throughout the differentiation in mature adipocytes. Cellular lipid droplet accumulation was assessed by Oil Red O staining, mRNA and protein expression of key adipogenic markers, transcription factors, and cytokines were investigated by RT-qPCR and WB, respectively. miRNAs involved in the regulation of adipogenic transcription factors were evaluated by RT-qPCR, and highly potent steric-blocking oligonucleotides (miRNA inhibitors) were used to modulate miRNAs expression.ResultsPre-adipocytes express VitD receptor (VDR) in basal condition, but during the differentiation process VDR expression reduces if not stimulated by the ligand. VitD significantly decreases lipid accumulation, with a consequent reduction in adipogenic marker expression, and counteracts the pro-adipogenic effect of BPA in 3T3-L1 and hAMSC during differentiation. This effect is associated to the increased expression of miR-27a-3p and miR-27b-3p. The blocking of miR-27a-3p and miR-27b-3p through miRNA inhibitors prevents the anti-adipogenic effect of VitD in both cell models.ConclusionsThese results suggest that in cultured 3T3-L1 and hAMSC VitD induces an anti-adipogenic effect and prevents BPA pro-adipogenic effect by triggering at least in part epigenetic mechanisms involving miR-27-3p.

Effect of 1800 MHz radiofrequency field exposure on cytokine and signal transduction protein expression in differentiated THP-1 cells

Purpose To evaluate the effects of 1800 MHz continuous wave (CW) and global system for mobile communications (GSM) modulated radiofrequency electromagnetic field (RFEMF) exposures on signal transduction (ST) protein and cytokine expression in differentiated human-derived monocytic THP-1 cells. Materials and methods THP-1 cells were differentiated into adherent macrophage-like cells using phorbol 12-myristate 13-acetate (PMA). Following differentiation, cells were exposed to 1800 MHz CW or GSM modulated RFEMF for 0.5, 4, or 24 h at a specific absorption rate (SAR) of 0 (sham) or 2.0 W/kg. Concurrent positive controls (lipopolysaccharide for cytokines; anisomycin for ST proteins) and negative controls were included in each experiment. The expression levels of cytokines (GM-CSF, IFN-γ, IL-1β, IL-6, IL-10, TNF-α) from culture media and phosphorylated and total ST proteins (CREB, JNK, NF-κB, p38, ERK1/2, Akt, p70S6k, STAT3, STAT5) from cell lysates were assessed using Milliplex magnetic bead array panels. Results No consistent effect of RFEMF exposure was observed in differentiated THP-1 cells. A statistically significant effect of overall exposure condition was observed for IL-6 with GSM modulation (P = 0.042), but no difference between RFEMF and sham for any exposure condition remained following adjustment for multiple comparisons (P ≥ 0.128). No statistically significant effect of exposure condition was detected for any other cytokine evaluated with either of the RFEMF modulations (P ≥ 0.078). There were no statistically significant changes in expression levels for any of the ST proteins under any studied exposure condition (P ≥ 0.320). Conclusions In this study, no evidence of changes were observed in differentiated human derived THP-1 cells following exposure of up to 24 h to 1800 MHz RFEMF at SARs of 0 and 2.0 W/kg on the expression of ST proteins or cytokines.

The Therapeutic Potential of Ganoderma lucidum Karst and Ziziphus jujuba Mill for Postsurgical Adhesion Band Formation

Background: Intra-abdominal adhesions are a commonly occurring postoperative complication following abdominopelvic surgery. Peritoneal adhesion formation can lead to infertility, chronic pelvic pain, and intestinal obstruction. The anti-inflammatory and anti-oxidant activities of Ganoderma lucidum Karst (G. lucidum) and Ziziphus jujuba Mill (Z. jujuba) have been reported. Here, we have explored the therapeutic potential of Ganoderma lucidum Karst (G. lucidum) and Z. jujuba against postsurgical adhesion band formation. The NC3Rs ARRIVE guidelines were followed during experimental studies. Methods: G. lucidum powder (800 mg/kg) and Z. jujuba powder (400 mg/kg) were administered using oral gavage to different groups of male albino Wistar rats. After ten days of treatment, macroscopic evidence for peritoneal adhesions and adhesion band score were determined. Furthermore, the anti-inflammatory and antifibrosis effects of G. lucidum and Z. jujuba were assessed using histopathology, ELISA, and real-time polymerase chain reaction methods. Also, alterations in some oxidative stress parameters were evaluated. Results: G. lucidum and Z. jujuba significantly decreased adhesion bands and were associated with a reduction in inflammatory cell infiltration into damaged tissues and the mRNA and protein expression of inflammatory cytokines via modulation of TNF-α, IL-6, IL-1β, and TGF-β. Moreover, both agents inhibited fibrotic adhesion band formation by decreasing collagen deposition and reducing profibrotic marker expression, Col1A1, at the peritoneum adhesion tissues. G. lucidum and Z. jujuba improved some antioxidant factors in rats’ adhesion tissues. The result of LC-MS showed that Ganoderma lucidum Karst and Ziziphus jujuba Mill consist of components with antioxidant activity, including ganoderic acid, lucidenic acid, quercetin, linoleic acid, malic acid, and benzoic acid. Conclusion: The results have demonstrated the therapeutic potential of G. lucidum and Z. jujuba in reducing peritoneal adhesion through anti-inflammatory and anti-fibrotic properties, indicating their promising value as a new therapeutic approach in preventing postsurgical adhesion.

Expression patterns of immune checkpoint proteins and Plasmodium falciparum-induced cytokines in chronic hepatitis B virus-infected and uninfected individuals: A cross-sectional study.

Chronic hepatitis B virus (CHB) infection remains a major public health problem. The American Association for the Study of Liver Diseases (AASLD) 2018 Hepatitis B Guidelines provide that CHB individuals not requiring antiviral therapy yet are monitored to determine the need for antiviral therapy in the future; however, these tests do not include measurement of cytokines and immune cell characterization. This case-control study compared the cytokine and immune checkpoint protein expression profiles between CHB individuals not yet on antiviral treatment and hepatitis B virus (HBV)-negative individuals. CD4 and CD8 T cells from CHB and HBV-negative individuals were characterized for immune checkpoint proteins programmed cell death-1 (PD1), T cell Immunoglobulin domain and mucin domain-containing protein 3 (TIM-3), and cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) (CD152), and a memory marker CXCR3 (CD183) using flow cytometry. Malaria-induced cytokine expression levels were determined by stimulating their blood cells with Plasmodium falciparum 3D7 strain antigens (CSP, AMA-1, and TRAP) in whole blood assays, and cytokine levels were measured using a 13-plex Luminex kit. HBV-negative and CHB individuals had comparable levels of CD4+ and CD8+ T cells. However, a proportion of the CD4+ and CD8+ populations from both groups, which were CXCR3+, expressed PD-1 and CD152. The ability to produce cytokines in response to malaria antigen stimulation was not significantly different between the groups. These findings support excluding CHB individuals from antiviral therapy at this stage of infection. However, CHB individuals require regular monitoring to determine the need for later antiviral treatment.

In vitro Study on the Immunomodulatory and Anti-inflammatory Effect of Soregen® Technology Water in Broiler and Layer Hens Peripheral Blood Mononuclear Cells

Background: Peripheral blood mononuclear cells (PBMCs) represent an attractive tissue source in pharmacogenomics and molecular and immunologic studies. In order to investigate the changes in biological effects, water samples were exposed to a specific quantum entanglement signal from the SoRegen chip for 48 hours. This study aimed to evaluate the immunomodulatory and anti-inflammatory effects of SoRegen Technology in broiler and layer hens PBMCs. Methods: Six-week-old Arbor Acress broiler chickens and 66-week-old layer hens Lohmann were used for the establishment of an in vitro cell culture model with chicken PBMCs. Result: The result indicates that post-treated water media enhanced the proliferative properties of broiler and layer hens PBMCs. The mRNA and protein expression of immune-modulating and pro-inflammatory cytokines: IL-2, IL-6, iNOS, IFNγ, TNFα and NFκB exhibited a marked increase following stimulation of the cells by LPS and Con A when compared with the non-stimulated cells. However, stimulated cells grown in structured water media showed a strikingly decreased expression of pro-inflammatory cytokines, except for the upregulation of IFNg expression in broiler PBMCs but not in layer hens PBMCs. It can be concluded that SoRegen® Technology water had anti-inflammatory activities with potential clinical immunomodulatory effects in younger chicks.

Antiretroviral drug dolutegravir induces inflammation at the mouse brain barriers.

Integrase strand transfer inhibitors (INSTIs) based antiretroviral therapy (ART) is currently used as first-line regimen to treat HIV infection. Despite its high efficacy and barrier to resistance, ART-associated neuropsychiatric adverse effects remain a major concern. Recent studies have identified a potential interaction between the INSTI, dolutegravir (DTG), and folate transport pathways at the placental barrier. We hypothesized that such interactions could also occur at the two major blood-brain interfaces: blood-cerebrospinal fluid barrier (BCSFB) and blood-brain barrier (BBB). To address this question, we evaluated the effect of two INSTIs, DTG and bictegravir (BTG), on folate transporters and receptor expression at the mouse BCSFB and the BBB invitro, exvivo and invivo. We demonstrated that DTG but not BTG significantly downregulated the mRNA and/or protein expression of folate transporters (RFC/SLC19A1, PCFT/SLC46A1) in human and mouse BBB models invitro, and mouse brain capillaries exvivo. Our invivo study further revealed a significant downregulation in Slc19a1 and Slc46a1 mRNA expression at the BCSFB and the BBB following a 14-day DTG oral treatment in C57BL/6 mice. However, despite the observed downregulatory effect of DTG in folate transporters/receptor at both brain barriers, a 14-day oral treatment of DTG-based ART did not significantly alter the brain folate level in animals. Interestingly, DTG treatment robustly elevated the mRNA and/or protein expression of pro-inflammatory cytokines and chemokines (Cxcl1, Cxcl2, Cxcl3, Il6, Il23, Il12) in primary cultures of mouse brain microvascular endothelial cells (BBB). DTG oral treatment also significantly upregulated proinflammatory cytokines and chemokine (Il6, Il1β, Tnfα, Ccl2) at the BCSFB in mice. We additionally observed a downregulated mRNA expression of drug efflux transporters (Abcc1, Abcc4, and Abcb1a) and tight junction protein (Cldn3) at the CP isolated from mice treated with DTG. Despite the structural similarities, BTG only elicited minor effects on the markers of interest at both the BBB and BCSFB. In summary, our current data demonstrates that DTG but not BTG strongly induced inflammatory responses in a rodent BBB and BCSFB model. Together, these data provide valuable insights into the mechanism of DTG-induced brain toxicity, which may contribute to the pathogenesis of DTG-associated neuropsychiatric adverse effect.

G-Protein Coupled Receptor 1 Is Involved in Tetrachlorobisphenol A-Induced Inflammatory Response in Jurkat Cells.

Estrogens can affect the immune inflammatory response through estrogen receptor alpha (ERα), but the specific role of estrogen member receptor G-protein coupled receptor 1 (GPER1) in this process remains unclear. In this study, we evaluated the effects of tetrachlorobisphenol A (TCBPA), which has estrogen activity, on immune inflammatory-related indicators of Jurkat cells, as well as investigated the role of GPER1 in these effects. The results showed that TCBPA at lower concentrations significantly promoted the viability of Jurkat cells, whereas higher concentrations decreased cell viability. TCBPA at concentrations ranging from 1 to 25 μM increased the intracellular reactive oxygen species (ROS) levels. Additionally, treatment with 10 μM TCBPA increased the protein expression of ERα and GPER1, elevated the phosphorylation of protein kinase B (p-Akt), and upregulated the mRNA levels of GPER1, Akt, and phosphoinositide 3-kinase (PI3K) genes. Treatment with 10 μM TCBPA also upregulated the protein or gene expression of pro-inflammatory cytokines, such as interleukins (IL1β, IL2, IL6, IL8, IL12α) and tumor necrosis factor alpha (TNFα) in Jurkat cells. Furthermore, pretreatment with a GPER1 inhibitor G15 significantly reduced the mRNA levels of Akt induced by 10 μM TCBPA. Moreover, the upregulation of mRNA expression of RelA (p65), TNFα, IL6, IL8, and IL12α induced by 10 μM TCBPA was also significantly attenuated after G15 pretreatment. These findings suggest that TCBPA upregulates the expression of genes related to inflammatory responses by activating the GPER1-mediated PI3K/Akt signaling pathway. This study provides new insights into the mechanism of TCBPA-induced inflammatory response.

Impact of Zinc Oxide on the Development of Aspergillus-Induced Maxillary Sinusitis Rabbit Model.

Aspergillus fumigatus is commonly found in the airway and is associated with airway inflammatory diseases. Zinc oxide (ZO) is known to be an essential microelement that facilitates fungal survival, growth, and proliferation. This study aimed to investigate the impact of ZO on A. fumigatus-induced fungal sinusitis in rabbits. Twenty-eight New Zealand white rabbits were divided into four groups for this study. Group 1 (6 sides) was treated with intramaxillary phosphate buffer saline (PBS) served as the negative control, Group 2 (6 sides) received intramaxillary PBS and ZO, Group 3 (8 sides) was treated with intramaxillary A. fumigatus alone, and Group 4 (8 sides) treated with intramaxillary A. fumigatus with ZO. After 4 and 12 weeks, sinus mucosal cytokine and transcription factor expressions were determined. A histological analysis was performed to determine inflammatory cell infiltration, number of secretory cells, and mucosal thickness. Fungal biofilm formation was determined using confocal laser microscopy. The intramaxillary instillation of A. fumigatus conidia led to an increase in protein and mRNA expression of interleukin (IL)-1β and IL-8 in the maxillary sinus mucosa. They were associated with mitogen-activated protein kinase and activator protein-1. Furthermore, intramaxillary instillation of fungal conidia resulted in significant enhancement of inflammatory cell infiltration, epithelial thickening, and fungal biofilm formation. However, intramaxillary ZO did not have a significant impact on A. fumigatus-induced cytokine protein and mRNA expression, and inflammatory cell infiltration and epithelial thickness in sinonasal mucosa. While intramaxillary instillation of A. fumigatus increased mucosal inflammation, cytokine production, and biofilm formation, the intramaxillary application of ZO did not have a significant influence on inflammation in the maxillary sinus mucosa.

Rac1 promotes the lipopolysaccharide-induced inflammatory response and contraction-associated proteins (CAPs) expression in mouse uterine smooth muscle cells

Activation of the maternal immune system leads to a downstream cascade of proinflammatory events that culminate in the activation of spontaneous uterine contractions, which is associated with preterm birth. Ras-related C3 botulinum toxin substrate 1 (Rac1) is a crucial protein related to cell contraction and inflammation. The main purpose of this study was to explore the role and function of Rac1′s regulation of inflammation through in- vivo and in-vitro experiments. Rac1 inhibitor was used in animal model of preterm birth and cells isolated from the uterine tissues of pregnant mice on gestational day 16 were transfected with adenovirus to knockdown or overexpress Rac1 and treated with the Calcium–calmodulin-dependent protein kinase II (CaMKII) inhibitor KN93. The expression of Rac1, uterine contraction-associated proteins (CAPs) (COX-2 and Connexin43), and inflammatory cytokines, were assessed by Western blotting and RTPCR.LPS upregulated Rac1, COX-2 and Connexin43 expression in uterine smooth muscle cells (USMCs). The expression of inflammatory cytokines, COX-2, and Connexin43 was significantly decreased in shRac1-transfected cells compared with cells stimulated with LPS only. Rac1 overexpression led to an increase in the expression of inflammatory cytokines, COX-2, and Connexin43. Furthermore, after Rac1 overexpression, KN93 reduced the expression of uterine contraction-associated proteins and inflammatory cytokines. It is thought that the effect of Rac1 on inflammatory cytokine and contraction-associated protein expression in USMCs is mediated by CaMKII. Rac1 can modulate the expression of contraction-associated proteins and inflammatory cytokines through the CaMKII pathway. Rac1 could be an effective therapeutic target for improving the outcome of preterm birth.

Adrenomedullin improved endothelial dysfunction via receptor-Akt pathway in rats with obesity-related hypertension.

Obesity-related hypertension (OH) is accompanied by obvious endothelial dysfunction, which contributes to increased peripheral vascular resistance and hypertension. Adrenomedullin (ADM), a multifunctional active peptide, is elevated in obese humans. The OH rats induced by high fat diet (HFD) for 28 weeks and the human umbilical vein endothelial cells (HUVECs)-treated by palmitic acid (PA) were used to investigate the effects of ADM on endothelial dysfunction and the underlying mechanisms. Vascular reactivity was assessed using mesenteric arteriole rings, and the protein expression levels were examined by Western blot analysis. Compared with the control rats, OH rats exhibited hypertension and endothelial dysfunction, along with reduced eNOS protein expression and Akt activation, and increased protein expression of proinflammatory cytokines and ROS levels. Four-week ADM administration improved hypertension and endothelial function, increased eNOS protein expression and Akt activation, and attenuated endothelial inflammation and oxidative stress in OH rats. In vitro experiment, the antagonism of ADM receptors with ADM22-52 and the suppression of Akt signaling with A6730 significantly blocked ADM-caused increase of NO content and activation of eNOS and Akt, and inhibited the anti-inflammatory and anti-oxidant effect of ADM in PA-stimulated HUVECs. These data indicate that endothelial dysfunction in OH rats is partially attributable to the decreased NO level, and the increased inflammation and oxidative stress. ADM improves endothelial function and exerts hypotensive effect depending on the increase of NO, and its anti-inflammatory and anti-oxidant effect via receptor-Akt pathway.

Stryphnodendron adstringens have a modulatory effect on inflammatory cytokines markers of in vitro activated macrophages

Aim: The purpose of this study is to conduct a comprehensive investigation into the modulatory effects of Stryphnodendron adstringens (Mart.; S. adstringens), a Brazilian wound-healing plant, on the expression of inflammatory cytokines. This will be achieved using an in vitro protocol with the commercial macrophage cell line RAW 264.7. Methods: The macrophage inflammatory response was induced by the natural antigen phytohemagglutinin (PHA), with and without supplementation of different concentrations of S. adstringens extract. The effects on cell proliferation rate and the concentration and production of transcripts of pro-inflammatory cytokines interleukin 1β (IL-1β), IL-6, tumor necrosis factor-alpha (TNF-α), and interferon-gamma (IFN-γ), as well as the anti-inflammatory cytokine IL-10, were assessed using spectrophotometric, immunoassay, and quantitative reverse transcription-polymerase chain reaction (qRT-PCR) techniques. Results: S. adstringens extract at all concentrations tested here reduced the cellular proliferation rate of activated macrophages. Extracts at concentrations of 0.49 mg/mL and 0.99 mg/mL decreased the protein and gene expression of pro-inflammatory cytokines, exhibiting the opposite effect concerning IL-10. Conclusions: The findings suggest that the wound-healing action of S. adstringens may encompass differential modulation of inflammation associated with tissue injury.

Early administration of umbilical cord blood cells following brief high tidal volume ventilation in preterm sheep: a cautionary tale

BackgroundUmbilical cord blood (UCB) cells are a promising treatment for preterm brain injury. Access to allogeneic sources of UCB cells offer the potential for early administration to optimise their therapeutic capacities. As preterm infants often require ventilatory support, which can contribute to preterm brain injury, we investigated the efficacy of early UCB cell administration following ventilation to reduce white matter inflammation and injury.MethodsPreterm fetal sheep (0.85 gestation) were randomly allocated to no ventilation (SHAM; n = 5) or 15 min ex utero high tidal volume ventilation. One hour following ventilation, fetuses were randomly allocated to i.v. administration of saline (VENT; n = 7) or allogeneic term-derived UCB cells (24.5 ± 5.0 million cells/kg; VENT + UCB; n = 7). Twenty-four hours after ventilation, lambs were delivered for magnetic resonance imaging and post-mortem brain tissue collected. Arterial plasma was collected throughout the experiment for cytokine analyses. To further investigate the results from the in vivo study, mononuclear cells (MNCs) isolated from human UCB were subjected to in vitro cytokine-spiked culture medium (TNFα and/or IFNγ; 10 ng/mL; n = 3/group) for 16 h then supernatant and cells collected for protein and mRNA assessments respectively.ResultsIn VENT + UCB lambs, systemic IFNγ levels increased and by 24 h, there was white matter neuroglial activation, vascular damage, reduced oligodendrocytes, and increased average, radial and mean diffusivity compared to VENT and SHAM. No evidence of white matter inflammation or injury was present in VENT lambs, except for mRNA downregulation of OCLN and CLDN1 compared to SHAM. In vitro, MNCs subjected to TNFα and/or IFNγ displayed both pro- and anti-inflammatory characteristics indicated by changes in cytokine (IL-18 & IL-10) and growth factor (BDNF & VEGF) gene and protein expression compared to controls.ConclusionsUCB cells administered early after brief high tidal volume ventilation in preterm fetal sheep causes white matter injury, and the mechanisms underlying these changes are likely dysregulated responses of the UCB cells to the degree of injury/inflammation already present. If immunomodulatory therapies such as UCB cells are to become a therapeutic strategy for preterm brain injury, especially after ventilation, our study suggests that the inflammatory state of the preterm infant should be considered when timing UCB cells administration.

Functional and Molecular Analysis of Human Osteoarthritic Chondrocytes Treated with Bone Marrow-Derived MSC-EVs.

Osteoarthritis (OA) is a degenerative joint disease, causing impaired mobility. There are currently no effective therapies other than palliative treatment. Mesenchymal stromal cells (MSCs) and their secreted extracellular vesicles (MSC-EVs) have shown promise in attenuating OA progression, promoting chondral regeneration, and modulating joint inflammation. However, the precise molecular mechanism of action driving their beneficial effects has not been fully elucidated. In this study, we analyzed MSC-EV-treated human OA chondrocytes (OACs) to assess viability, proliferation, migration, cytokine and catabolic protein expression, and microRNA and mRNA profiles. We observed that MSC-EV-treated OACs displayed increased metabolic activity, proliferation, and migration compared to the controls. They produced decreased proinflammatory (Il-8 and IFN-γ) and increased anti-inflammatory (IL-13) cytokines, and lower levels of MMP13 protein coupled with reduced expression of MMP13 mRNA, as well as negative microRNA regulators of chondrogenesis (miR-145-5p and miR-21-5p). In 3D models, MSC-EV-treated OACs exhibited enhanced chondrogenesis-promoting features (elevated sGAG, ACAN, and aggrecan). MSC-EV treatment also reversed the pathological impact of IL-1β on chondrogenic gene expression and extracellular matrix component (ECM) production. Finally, MSC-EV-treated OACs demonstrated the enhanced expression of genes associated with cartilage function, collagen biosynthesis, and ECM organization and exhibited a signature of 24 differentially expressed microRNAs, associated with chondrogenesis-associated pathways and ECM interactions. In conclusion, our data provide new insights on the potential mechanism of action of MSC-EVs as a treatment option for early-stage OA, including transcriptomic analysis of MSC-EV-treated OA, which may pave the way for more targeted novel therapeutics.