TNF-α Secretion Research Articles

Clinical-grade human skin-derived ABCB5+ mesenchymal stromal cells exert anti-apoptotic and anti-inflammatory effects in vitro and modulate mRNA expression in a cisplatin-induced kidney injury murine model.

Acute kidney injury (AKI) is characterized by a rapid reduction in renal function and glomerular filtration rate (GFR). The broadly used anti-cancer chemotherapeutic agent cisplatin often induces AKI as an adverse drug side effect. Therapies targeted at the reversal of AKI and its potential progression to chronic kidney disease or end-stage renal disease are currently insufficiently effective. Mesenchymal stromal cells (MSCs) possess diverse immunomodulatory properties that confer upon them significant therapeutic potential for the treatment of diverse inflammatory disorders. Human dermal MSCs expressing ATP-Binding Cassette member B5 (ABCB5) have shown therapeutic efficacy in clinical trials in chronic skin wounds or recessive dystrophic epidermolysis bullosa. In preclinical studies, ABCB5+ MSCs have also shown to reverse metabolic reprogramming in polycystic kidney cells, suggesting a capacity for this cell subset to improve also organ function in kidney diseases. Here, we aimed to explore the therapeutic capacity of ABCB5+ MSCs to improve renal function in a preclinical rat model of cisplatin-induced AKI. First, the anti-apoptotic and immunomodulatory capacity was compared against research-grade adipose stromal cells (ASCs). Then, cross-species immunomodulatory capacity was checked, testing first inhibition of mitogen-driven peripheral blood mononuclear cells and then modulation of macrophage function. Finally, therapeutic efficacy was evaluated in a cisplatin AKI model. First, ABCB5+ MSCs suppressed cisplatin-induced apoptosis of human conditionally-immortalized proximal tubular epithelial cells in vitro, most likely by reducing oxidative stress. Second, ABCB5+ MSCs inhibited the proliferation of either human or rat peripheral blood mononuclear cells, in the human system via the Indoleamine/kynurenine axis and in the murine context via nitric oxide/nitrite. Third, ABCB5+ MSCs decreased TNF-α secretion after lipopolysaccharide stimulation and modulated phagocytosis and in both human and rat macrophages, involving prostaglandin E2 and TGF-β1, respectively. Fourth, clinical-grade ABCB5+ MSCs grafted intravenously and intraperitoneally to a cisplatin-induced AKI murine model exerted modulatory effects on mRNA expression patterns toward an anti-inflammatory and pro-regenerative state despite an apparent lack of amelioration of renal damage at physiologic, metabolic, and histologic levels. Our results demonstrate anti-inflammatory and pro-regenerative effects of clinical grade ABCB5+ MSCs in vitro and in vivo and suggest potential therapeutic utility of this cell population for treatment or prevention of cisplatin chemotherapy-induced tissue toxicity.

Effect of the ketone beta-hydroxybutyrate on markers of inflammation and immune function in adults with type 2 diabetes.

Pre-clinical and cell culture evidence supports the role of the ketone beta-hydroxybutyrate (BHB) as an immunomodulatory molecule that may inhibit inflammatory signalling involved in several chronic diseases such as type 2 diabetes (T2D), but studies in humans are lacking. Therefore, we investigated the anti-inflammatory effect of BHB in humans across three clinical trials. To investigate if BHB suppressed pro-inflammatory cytokine secretion, we treated LPS-stimulated leukocytes from overnight-fasted adults at risk for T2D with BHB (Study 1). Next (Study 2), we investigated if exogenously raising BHB acutely in vivo by ketone monoester supplementation (KME) in adults with T2D would suppress pro-inflammatory plasma cytokines. In Study 3, we investigated the effect of BHB on inflammation via ex vivo treatment of LPS-stimulated leukocytes with BHB and in vivo thrice-daily pre-meal KME for 14 days in adults with T2D. Ex vivo treatment with BHB suppressed LPS-stimulated IL-1β, TNF-α, and IL-6 secretion and increased IL-1RA and IL-10 (Study 1). Plasma IL-10 increased by 90 min following ingestion of a single dose of KME in T2D, which corresponded to peak blood BHB (Study 2). Finally, 14 days of thrice-daily KME ingestion did not significantly alter plasma cytokines or leukocyte subsets including monocyte and T-cell polarization (Study 3). However, direct treatment of leukocytes with BHB modulated TNF-α, IL-1β, IFN-γ, and MCP-1 secretion in a time- and glucose-dependent manner (Study 3). Therefore, BHB appears to be anti-inflammatory in T2D, but this effect is transient and is modulated by the presence of disease, glycaemia, and exposure time.

Argininyl-Fructosyl-Galactose: A Novel Amino Acid Derivative improves Cisplatin-induced Intestinal Toxicity via Reactive Oxygen Species- mediated Apoptosis Pathway.

Based on the Maillard reaction principle of red ginseng, this study innovatively synthesized a new amino acid derivative by combining arginine with lactose through simulated synthesis and was separated and purified through repeated silica gel and polyacrylamide gel (Bio-gel P-II) column chromatography. The work was aimed at elucidating the synthesis of a novel amino acid derivative and investigating the intestinal protective activity of the novel amino acid derivative and possible molecular mechanism by establishing the intestinal injury model induced by cisplatin in mice. The purity and molecular weight of the amino acid derivatives were determined to be by electrospray ionization mass spectrometry (ESI-MS). Subsequently, by establishing cisplatin (20 mg/kg)-induced intestinal injury in vivo for 10 days and IEC-6 cell model. The biochemical indexes and histopathological analysis were used to evaluate the oxidative stress and inflammatory and pathological changes of intestinal tissue in mice. The protein expression levels of p-Nuclear transcription factor-κB (p-NF-κB), cleaved caspase 3/caspase 3, cleaved caspase 9/caspase-9, Bcl-2, Bax, cytochrome C, phosphatidylinositol 3-kinase (PI3K), Protein Kinase B (Akt), p-PI3K, p-Akt were quantified through immunofluorescence staining and western blot analysis. The new amino acid derivatives of chemical structure were identified to be 1- (arginine-Nαgroup)-1-deoxy-4-O-(β-D-galactopyranosyl)-D-fructose, named Argininylfructosyl- galactose (AFGA, C18H34N4O12). The results showed that pretreatment with a single AFGA dose remarkably alleviated cisplatin-evoked intestinal oxidative stress injury, and the levels of reactive oxygen species (ROS) were lessening in IEC-6 cells (p<0.05, p<0.01) and could effectively reduce the secretion of TNF-α and IL-1β in serum and the expression level of NF-κB protein in intestinal tissues (p<0.01). Meantime, AFGA also significantly suppressed the caspase 3, caspase 9, cytochrome C and Bax protein expression in intestinal tissue in mice (p<0.01), and regulated the PI3K/Akt pathway (p<0.05, p<0.01). Importantly, the molecular docking results of AFGA also suggested a better binding ability with the above-mentioned related target proteins. The results clearly revealed AFGA as a potential multifunctional therapeutic agent with a clear protective effect against cisplatin-induced intestinal injury may be related to the PI3K/Akt signaling pathway.

Development of a tetrahydroindazolone-based HDAC6 inhibitor with in-vivo anti-arthritic activity

Histone deacetylase 6 (HDAC6) induces the expression of pro-inflammatory cytokines in macrophages; therefore, HDAC inhibitors may be beneficial for the treatment of macrophage-associated immune disorders and chronic inflammatory diseases, including atherosclerosis and rheumatoid arthritis. Structure-activity relationship studies were conducted on various phenyl hydroxamate HDAC6 inhibitors with indolone/indazolone-based bi- or tricyclic ring moieties as the cap group aiming to develop novel anti-arthritic drug candidates. Several compounds exhibited nanomolar activity and HDAC6 selectivity greater than 500-fold over HDAC1. Compound 21, a derivative with the tetrahydroindazolone cap group, is a potent HDAC6 inhibitor with an IC50 of 18 nM and 217-fold selectivity over HDAC1 and showed favorable oral bioavailability in animals. Compound 21 increases the acetylation level of tubulin without affecting histone acetylation in cutaneous T-cell lymphoma cells and inhibits TNF-α secretion in LPS-stimulated macrophage cells. The anti-arthritic effects of compound 21 were evaluated using a rat adjuvant-induced arthritis (AIA) model. Treatment with compound 21 significantly reduced the arthritis score, and combination treatment with methotrexate showed a synergistic effect in AIA models. We identified a novel HDAC6 inhibitor, compound 21, with excellent in vivo anti-arthritic efficacy, which can lead to the development of oral anti-arthritic drugs.

Syk/BLNK/NF-κB signaling promotes pancreatic injury induced by tacrolimus and potential protective effect from rapamycin

BackgroundThe treatment of tacrolimus-induced post-transplantation diabetes mellitus (PTDM) has become a hot topic to improve the long-term survival of organ transplant patients, however whose pathogenesis has not been fully elucidated. In pancreas, the up-regulation of NF-κB has been reported to stimulate cytokine IL-1β/TNF-α secretion, inducing pancreatic injury, meanwhile other studies have reported the inhibitory effect of rapamycin on NF-κB. PurposeThe aim of this study was to clarify the mechanism of tacrolimus-induced pancreatic injury and to explore the potential effect from small dose of sirolimus. MethodsWistar rats were randomly divided normal control (NC) group, PTDM group, sirolimus intervention (SIR) group. Transcriptomic analysis was used to screen potential mechanism of PTDM. Biochemical index detections were used to test the indicators of pancreatic injury. Pathological staining, immumohistochemical staining, immunofluorescent staining, western blot were used to verify the underlying mechanism. ResultsCompared with NC group, the level of insulin was significant reduction (P < 0.01), inversely the level of glucagon was significantly increase (P < 0.01) in PTDM group. Transcriptomic analysis indicated Syk/BLNK/NF-κB signaling was significantly up-regulated in PTDM group. Pathological staining, immumohistochemical staining, immunofluorescent staining, western blot verified Syk/BLNK/NF-κB and TNF-α/IL-1β were all significantly increased (P < 0.05 or P < 0.01), demonstrating the mechanism of tacrolimus-induced pancreatic injury via Syk/BLNK/NF-κB signaling. In addition, compared with PTDM group, the levels of weight, FPG, AMY, and GSP in SIR group were significant ameliorative (P < 0.05 or P < 0.01), and the expressions of p-NF-κB, TNF-α/IL-1β in SIR group were significantly reduction (P < 0.05 or P < 0.01), showing Syk/BLNK/NF-κB signaling promoted pancreatic injury induced by tacrolimus and potential protective effect from rapamycin reducing NF-κB. ConclusionSyk/BLNK/NF-κB signaling promotes pancreatic injury induced by tacrolimus and rapamycin has a potentially protective effect by down-regulating NF-κB. Further validation and clinical studies are needed in the future.

MCT1 gene silencing enhances the immune effect of dendritic cells on cervical cancer cells.

Dendritic cells (DCs) are a key class of immune cells that migrate to the draining lymph nodes and present processed antigenic peptides to lymphocytes after being activated by external stimuli, thereby establishing adaptive immunity. Moreover, DCs play an important role in tumor immunity. The aim of the study was to investigate whether MCT1 gene silencing in DCs affects their ability to mount an immune response against cervical cancer cells. We silenced the expression of MCT1 in DCs from mouse bone marrow (BM) by infection with adenovirus. The surface antigen profile of DCs was analyzed by flow cytometry and cytokine secretion was evaluated using enzyme-linked immunosorbent assay (ELISA) following sodium lactate (sLA) exposure and lipopolysaccharide (LPS) stimulation. Then, various groups of DC-induced cytotoxic T lymphocytes (CTLs) were prepared and their cytotoxicity against U14 was tested. Without sLA exposure, silencing MCT1 did not affect the expression of CD1a, CD80, CD83, CD86, and major histocompatibility complex class II (MHCII) in DCs after LPS challenge. Similar results were found for interleukin (IL)-6, IL-12 p70 and tumor necrosis factor alpha (TNF-α). After sLA exposure, silencing MCT1 significantly decreased the expression of CD1a, CD80, CD83, CD86, and MHCII in DCs after the LPS challenge, as well as the secretion of IL-6, IL-12 p70 and TNF-α. In addition, sLA exposure significantly reduced the toxicity and inhibited the proliferation of DC-induced CTLs compared to U14 cells in vitro and in vivo. However, silencing MCT1 significantly attenuated the changes caused by sLA exposure. At the same time, in the absence of sLA, silencing MCT1 did not affect the toxicity nor inhibit the proliferation of DC-induced CTLs on U14 cells. Lactate exposure reduces the immune effect of DCs on cervical cancer cells, but MCT1 gene silencing attenuates these alterations.

High-throughput screening identification of novel immunomodulatory combinations for the generation of tolerogenic dendritic cells.

Tolerogenic Dendritic Cells (tolDCs) have an exceptional promise as a potential therapy for autoimmune disease and transplantation rejection. TolDCs are a unique phenotype of antigen presenting cells (APCs) that can influence naïve T cells into antigen specific T regulatory cells (Tregs), which can re-establish tolerance against auto/allo-antigens in the long term. Despite their promise, tolDCs have not found clinical success. Most strategies seek to generate tolDCs ex vivo by differentiating naïve dendritic cells (DCs) with immunosuppressive agents. Recently, we developed a tolDC generation strategy, which we call Push/Pull Immunomodulation (PPI). In PPI, DCs are treated with combinations of toll-like-receptor (TLR) agonists and immunomodulatory agents, which generate more robust, Treg-inducing tolDCs than previous strategies. Here, we seek to identify more potent and clinically viable PPI formulations using data from a high-throughput screening project. Over 40,000 combinations of pathogen-associated molecular patterns (PAMPs) and immunomodulatory small molecules were screened using a modified murine macrophage line, RAW dual cells, to observe the effect of these combinations on two major immune regulatory transcription factors, NF-κB and IRF. Combinations were further screened for inflammatory cytokine activity using a human monocyte cell line, THP-1, then on murine DCs. Leading candidates were co-cultured with T cells to assess antigen specific T cell responses. From this data, we identified 355 combinations that showed low or moderate IRF activity, low NF-κB activity, low inflammatory cytokine generation and good viability: all hallmarks of tolerogenic potential. We further screened these 355 combinations using bone marrow derived DCs (BMDCs) and identified 10 combinations that demonstrated high IL-10 (tolerogenic) and low TNF-α (inflammatory) secretion. After further optimizing these combinations, we identified two combinations that generate robust tolDCs from BMDCs ex vivo. We further show that these PPI-tolDCs can also generate antigen specific Tregs but do not increase overall Treg populations. These second-generation PPI formulations have significant potential to generate robust tolDCs and strong antigen specific Tregs.

Structural Optimization and Biological Evaluation of Isoxazolo[5,4-d]pyrimidines as Selective Toll-Like Receptor 7 Agonists.

Toll-like receptors (TLRs) are components of innate immunity that play a crucial role in several diseases, including chronic inflammatory and infectious diseases, autoimmune diseases, and cancer. In particular, TLR7 has been identified as a key player in the innate immune response against viral infections and small-molecule TLR7 agonists have shown potential for vaccine therapy, for treatment of asthma and allergies, and as anticancer drugs. Inspired by our previous discovery of selective TLR7 agonists, our goal was to develop and introduce a new chemotype of TLR7 agonists by replacing the quinazoline ring with a new heterocycle isoxazolo[5,4-d]pyrimidine. Here, we report design, optimized synthesis, and structure-activity relationship studies of a novel class of TLR7 agonists based on the 6-(trifluoromethyl)isoxazolo[5,4-d]pyrimidine-4-amine scaffold that demonstrate high selectivity and low micromolar potencies. The best-in-class agonist 21a, with an EC50 value of 7.8 μM, also proved to be noncytotoxic and induced secretion of cytokines, including IL-1β, IL-12p70, IL-8, and TNF-α, indicating its potential to modulate the immune response.

Halogenation of common phenolic household and personal care product ingredients enhances their AhR-modulating capacity

The Aryl Hydrocarbon Receptor (AhR), a ligand-activated transcription factor, orchestrates responses to numerous structurally diverse endogenous and exogenous ligands. In addition to binding various xenobiotics, AhR also recognizes endocrine disruptors, particularly those featuring chlorinated or brominated aromatic structures. There is limited data available on the impact of common household and personal care product ingredients let alone their halogenated transformation products. Herein we bridge this knowledge gap by preparing a library of chlorinated and brominated parabens, bisphenols, UV filters, and nonylphenols. An evaluation of total of 125 compounds for agonistic and antagonistic activity on AhR unveiled a low micromolar agonist, Cl2BPAF with an EC50 of 13 μM. Moreover, our study identified several AhR antagonists, with BrBzP emerging as the most potent with an IC50 of 8.9 μM. To further investigate the functional implications of these compounds, we subjected the most potent agonist and antagonist to a functional assay involving cytokine secretion from peripheral blood mononuclear cells and compared their activity with the commercially available AhR agonist and antagonist. Cl2BPAF exhibited an overall immunosuppressive effect by reducing the secretion of proinflammatory cytokines, including IL-6, IFN-γ, and TNF-α, while BrBzP displayed opposite effects, leading to an increase of those cytokines. Notably, the immunomodulatory effects of Cl2BPAF surpassed those of ITE, a bona fide AhR agonist, while the impact of BrBzP exceeded that of CH223191, a bona fide AhR antagonist. In summary, our study underscores the potential influence of halogenated transformation products on the AhR pathway and, consequently, their role in shaping the immune responses.

Vikil 20 Suppresses the Proliferation of Prostate Cancer (PC-3) Cells and Quenches Free Radicals In Vitro.

Vikil 20 is an herbal formula produced in Ghana and is widely marketed as a product to boost immunity as well as for general well-being. However, the pharmacological effect of this herbal preparation has not been proven scientifically. Therefore, this study was aimed at investigating the antioxidative as well as the anti-prostate cancer effects of the product. To assess the antioxidative effect of Vikil 20, the DPPH and ABTS activities were investigated. The total phenolic content was investigated using the Folin-Ciocalteu method. The cytotoxic effect of Vikil 20 against prostate cancer (PC-3) cells as well as normal (RAW 264.7) cells was investigated using the MTT assay whereas its anti-metastatic effect was analyzed using the cell migration assay. The effect of Vikil 20 on cell adhesion was analyzed via the cell adhesion assay whereas its effect on TNF-α secretion was investigated using a TNF-α detection kit. Vikil 20 demonstrated significant antioxidant effects by suppressing 57.61% and 92.88% respectively of DPPH and ABTS radicals at 1000 µg/mL with total phenolic contents of 140.45 mg GAE/g. Vikil 20 suppressed the proliferation of PC-3 cells by reducing the number of viable cells to 49.5% while sparing the RAW, 264.7 cells. Further, Vikil 20 significantly suppressed both cellular migration and adhesion of prostate cancer cells. Finally, suppression of cellular migration and adhesion is associated with a reduction in TNF-α secretion by PC-3 cells. Taken together, Vikil 20 was found to possess significant antioxidant and anti-prostate cancer effects in vitro.

DSP-0509, a TLR7 agonist, exerted synergistic anti-tumor immunity combined with various immune therapies through modulating diverse immune cells in cancer microenvironment.

Toll-like receptor 7 (TLR7) acts as a crucial component of the innate immune system. Upon TLR7 binding to its ligand, myeloid cells, including dendritic cells (DCs) and macrophages, are activated and play vital roles in initiating adaptive immunity. Consequently, TLR7 agonists have been employed in cancer immunotherapy. We have synthesized DSP-0509, a systemic injectable TLR7 agonist, and in this investigation, we examined the effects of DSP-0509 on tumor-infiltrating lymphocytes (TILs) utilizing single-cell RNA sequencing (scRNA-seq) in a mouse model bearing tumors. Our results demonstrated that DSP-0509 induced an expansion of immune cell populations, such as Natural Killer (NK) cells, CD4+ T cells, and CD4+ regulatory T cells (Tregs). Subsequently, we combined an Indoleamine 2,3-dioxygenase 1 (IDO1) inhibitor with DSP-0509 to enhance the antitumor efficacy by reducing Tregs, as DSP-0509 led to an increase in Treg presence within tumors. Our findings demonstrated that this combination therapy effectively reduced Treg infiltration within the tumor, leading to enhanced antitumor activity. To further prevent CD8+ T cell exhaustion, we combined DSP-0509 with an anti-PD-1 antibody and assessed the alterations in TILs using scRNA-seq. Our results indicated that the combination treatment significantly increased the cluster of CD8+ T cells expressing Gzmb, Prf1, Ctla4, and Icos, when compared to the administration of DSP-0509 alone. Additionally, we observed a marked rise in the M1-like macrophage cluster in the combination treatment group compared to the group receiving only DSP-0509. To validate the potential of modulating myeloid cells within the tumor to enhance antitumor efficacy, we combined DSP-0509 with an inhibitor targeting the receptor tyrosine kinase AXL. In bone marrow derived macrophages (BMDMs), the AXL inhibitor further amplified DSP-0509-stimulated TNFα secretion while reducing IL-10 secretion. As a final step, we evaluated the antitumor activity by combining DSP-0509 and the AXL inhibitor in an in vivo tumor model, which demonstrated increased efficacy. In summary, our study elucidated the effects of DSP-0509 on immune activity within the tumor microenvironment. These findings provided valuable insights that pave the way for the development of novel combination immunotherapy strategies.

Aloe vera peel-derived nanovesicles display anti-inflammatory properties and prevent myofibroblast differentiation.

Aloe vera (AV) is a medicinal plant, most known for its beneficial effects on a variety of skin conditions. Its known active compounds include carbohydrates and flavonoids such as quercetin and kaempferol, among others. In the past decade, plant nanovesicles (NVs) have gained considerable interest as interkingdom communicators, presenting an opportunity for clinical standardization of natural products. In this study, we aimed to assess the potential of AVpNVs for the treatment of burn wounds. AVpNVs were isolated and characterized regarding vesicle yield (nanoparticle tracking analysis) and structure (transmission electron microscopy and atomic force microscopy), as well as their protein content with proteomics. We assessed key characteristics for treating burn wounds in vitro, such as the anti-inflammatory potential in LPS-stimulated macrophages and keratinocytes, and the effect of AVpNVs on myofibroblast differentiation and contraction. AVpNVs presented a homogenous NV population, vesicular shape, and NV-associated protein markers. AVpNVs significantly decreased the secretion of pro-inflammatory cytokines TNFα, IL-1β, and IL-6. Furthermore, AVpNVs inhibited myofibroblast differentiation and significantly decreased their contractile potential in collagen matrices. Observed effects were linked to proteins identified in the isolates through proteomics analysis. AVpNVs displayed characteristics as an inflammatory modulator, while simultaneously diminishing myofibroblast differentiation and contraction. Novel strategies for burn wound treatment seek to decrease scarring on a cellular and molecular level in the early stages of wound healing, which makes AVpNVs a promising candidate for future plant-vesicle-based treatments.

The Characteristics and Biological Activities of Niosome-Entrapped Salicylic Acid-Contained Oleoresin from Dipterocarpus alatus for Skin Product Applications.

Salicylic acid (SA) is widely renowned for its efficacy as a beneficial ingredient for skincare, especially for acne and uneven skin texture. The salicylic acid (SA) niosome formulation combined with the essential component of oleoresin from Dipterocarpus alatus Roxb. ex G. Don or Yang-Na (ODA) was developed and investigated for its physical characteristics, biological effects, and stability. The findings demonstrated that SA combined with ODA in the niosome formulation F4 enhanced the entrapment efficiency of SA, as well as the physical properties and stability of the formulation. Furthermore, the release pattern of this combined formulation indicated sustained release of SA. The permeation of SA was higher in the presence of ODA compared to SA-niosome formulations without ODA. Moreover, this F4 could downregulate the secretion of iNOS, COX-2, and TNF-α including anti-Propionibacterium acnes activities. Consequently, the incorporation of ODA into the niosome formulation has the potential to improve the entrapment efficiency of SA, facilitating controlled release and enhancing permeation, nitric oxide inhibition capabilities, and anti-P. acnes activity. Therefore, F4 has the potential to be developed as a topical product for the combined treatment of inflammation and P. acnes-associated conditions in the future.

Anti-Inflammatory Effect of Extracts of Inonotus obliquus and Microalgae

Chaga mushroom (Inonotus obliquus) and marine microalgae are two emerging natural products with many potential physiological health benefits. The aim of this study was to investigate the anti-inflammatory effects of two extracts prepared from Chaga mushroom and microalgae using lipopolysaccharide-stimulated RAW 264.7 murine macrophage cell model. The Chaga mushroom extract dose-dependently reduced the production of proinflammatory biomarkers of interleukin (IL)-6 and tumor necrosis factor-alpha (TNF-α). At a high concentration of 500 µg/L, Chaga mushroom extract significantly suppressed cyclooxygenase-2 levels. Similarly, the extract of microalgae suppressed the secretion of IL-6 and TNF-α by lipopolysaccharide-induced macrophages. Both extracts had no significant impact on the secretion of anti-inflammatory IL-4 production. These results suggest that extracts of Chaga mushroom and microalgae can be used in developing anti-inflammatory natural health products.

Unusual immunosuppressive pyridine-containing bisnor- (c23), tetranor- (c21) and pentanor- (c20) sesterterpenoids from Tibetan Leucosceptrum canum.

An unusual pyridine-containing sesterterpenoid, leucosceptrodine (1), and five new nor-leucosceptrane sesterterpenoids, including bisnor- (C23, 2), tetranor- (C21, 3) and pentanor- (C20, 4-6) skeletons, were isolated from the leaves of Tibetan Leucosceptrum canum. Their structures including their absolute configurations were determined by extensive spectroscopic analyses and quantum chemical calculations. A single crystal of one epimer (5) was crystallized from a pair of inseparable epimers, and its structure including its absolute configuration was determined by X-ray crystallographic analysis. The immunosuppressive activities of compounds 1-4 with different potencies were evaluated by inhibiting the secretion of cytokines TNF-α and IL-6 in LPS-induced RAW264.7 macrophages.

Sirtuin 3 ameliorates inflammatory bowel disease via inhibiting intestinal inflammation and oxidative stress.

Sirtuin 3 involved in development of various diseases, but its role in inflammatory bowel disease is still unknown. We used inflammatory bowel disease biopsies, colitis animal model, and vitro cells RAW264.7 to study the role of Sirtuin 3 in the pathophysiology of inflammatory bowel disease. Sirtuin 3 negatively correlated with intestinal TNF-α. Sirt3 was less pronounced in pediatric and adult inflammatory bowel disease patients compared with corresponding control group. Sirtuin 3 activator Honokiol suppressed dextran sulfate sodium induced colonic manifestations, while Sirt3 inhibitor caused opposite results. Honokiol inhibited colonic oxidative stress by and reduced intestinal permeability. Honokiol repressed inflammatory response by reducing macrophage infiltration, pro-inflammatory cytokines TNF-α, IL-1β, and IL-6 levels, and inhibiting activation of NF-κB p65 in the colitis mice. However, Sirt3 inhibitor amplified colonic oxidative stress and inflammatory response. In vitro study, Sirt3 inhibitor or siRNA Sirtuin 3 activated NF-κB p65 and enhanced TNF-α, IL-1β, and IL-6 secretion from LPS stimulated RAW264.7, while Honokiol remarkably attenuated these pro-inflammatory cytokines secretion. Finally, knockdown of Sirt3 in Caco-2 cells enhanced TNF-α induced intestinal barrier integrity injury. Sirtuin 3 negatively regulates inflammatory bowel disease progression via reducing colonic inflammation and oxidative stress. Sirtuin 3 is a promising therapeutic target in clinical application for inflammatory bowel disease therapy.

The mechanism of metallothionein MT1M-mediated PI3K/AKT signaling pathway in the regulation of diabetic retinopathy

Metallothionein (MT1M) is associated with tumors and autoimmune diseases. However, its role in DR has not yet been elucidated. DR and normal rat retinal endothelial cells (RECs) were isolated and cultured. DR Rat RECs achieved gene regulation by transfecting MT1M plasmid. PCR and MTT were used to detect MT1M expression, cell proliferation. Flow cytometry was used to detect cell apoptosis. Lactate dehydrogenase (LDH), superoxide dismutase (SOD) and reactive oxygen species (ROS) were detected by MTT. The expression of VEGF and PI3K/AKT signaling pathway was detected by Western blot. The levels of inflammatory factors TNF-α and IL-1β were detected by ELISA. The results showed that the expression of MT1M was reduced in the RECs of DRC rats compared to the normal control group, cell proliferation was enhanced, SOD activity was reduced, LDH and ROS levels were increased, TNF-α and IL-1β secretion increased, and vascular endothelial growth factor (VEGF), PI3K/AKT expression increased (P &lt; 0.05). However, transfection with MT1M plasmid could significantly inhibit cell proliferation, increase SOD activity, reduce LDH and ROS levels, reduce TNF-α and IL-1β secretion, and reduce VEGF and PI3K/AKT expression (P &lt;0.05). The expression of MT1M is reduced in RECs of DR rats. Up-regulation of MT1M can regulate DR3K/AKT signaling pathway and oxidative/antioxidant balance, alter VEGF expression, inhibit inflammation, regulate the growth and proliferation of RECs, and delay DR lesions.

Serum amyloid A contributes to radiation-induced lung injury by activating macrophages through FPR2/Rac1/NF-κB pathway.

Patients who receive thoracic radiotherapy may suffer from radiation-induced lung injury, but the treatment options are limited as the underlying mechanisms are unclear. Using a mouse model of right thorax irradiation with fractionated doses of X-rays for three consecutive days (8 Gy/per day), this study found that the thoracic irradiation (Th-IR) induced tissue injury with aberrant infiltration of macrophages, and it significantly increased the secretion of TNF-α, IL-1β, IL-6, TGF-β1 and serum amyloid A (SAA) in mice. Interestingly, SAA could activate macrophages and then induce epithelial-mesenchymal transition (EMT) of lung epithelial cells and fibrosis progression in lung tissue. Mechanistically, SAA enhanced the transient binding of FPR2 to Rac1 protein and further activated NF-κB signaling pathway in macrophages. Inhibition of FPR2 significantly reduced pulmonary fibrosis induced by SAA administration in mice. In addition, cimetidine could reduce the level of SAA release after irradiation and attenuate the lung injury induced by SAA or Th-IR. In conclusion, our results demonstrated that SAA activated macrophages via FPR2/Rac1/NF-κB pathway and might contribute to the Th-IR induced lung injury, which may provide a new strategy to attenuate radiation-induced adverse effects during radiotherapy.

Differential effects of Fucus vesiculosus fucoidan on fibroblast and macrophage cell lines inflammatory activation

It is estimated that more than one-third of people aged over 16 reported living with inflammation-related chronic illness. Fucoidan, sulfated polysaccharide, has been proven to modulate immune response and inhibit inflammation in both in vitro and in vivo research. In the present study the effect of commercially available fucoidan from Fucus vesiculosus (with purity 97%) on immune response was investigated. Two cell lines, human fibroblasts (HDFa) and mouse macrophages (J774A.1) were used in the study. Cells were activated with pro-inflammatory stimulus (LPS/IFN-γ or PMA) and treated with fucoidan. Results of NO inhibition assay revealed that fucoidan has anti-inflammatory activity and significantly reduces the level of NO produced by the cells. A significant decrease in secretion of IL-6 from both cell lines was observed. Moreover, fucoidan inhibits IL-12 production by mouse macrophages. The obtained results indicate that fucoidan not only acts as anti-inflammatory compound, but also has immunomodulatory activity. Enhanced secretion of TNF-α and IL-8 from macrophages and fibroblasts, respectively, was also noted. The antioxidant activity of fucoidan has also been studied. Fucoidan demonstrates dose-dependent scavenging properties by DPPH assay. Fucoidan's simultaneous ability to reduce inflammation, activate macrophages, and remove free radicals may be used as a tool to fight chronic inflammation disorders. These all contribute to the effective immunoprevention of cancer or other diseases related to the impaired function of the immune cells.

Pro-inflammatory activation of monocytes in patients with immunoinflammatory rheumatic diseases

The pathogenesis of immunoinflammatory rheumatic diseases (IRDs) is based on chronic inflammation, one of the key mechanisms of which may be abnormal activation of macrophages, leading to further disruption of the immune system.The aim – to evaluate the pro-inflammatory activation of circulating monocytes in patients with IRDs.Material and methods. The study included 149 participants: 53 patients with rheumatoid arthritis (RA), 45 – with systemic lupus erythematosus (SLE), 34 – with systemic scleroderma (SSc) and 17 participants without IRD, aged 30 to 65 years. Basal and lipolysaccharide (LPS)-stimulated secretion of monocytes was studied in a primary culture of monocytes obtained by immunomagnetic separation from blood. Quantitative assessment of the cytokines tumor necrosis factor-α (TNF-α), interleukin 1β (IL-1β) and the monocyte chemoattractant protein-1 (MCP-1) was carried out in the culture fluid by ELISA. Pro-inflammatory activation of monocytes was calculated as the ratio of LPS-stimulated and basal secretions.Results. It was shown that the basal secretion of all studied cytokines was significantly increased in all groups of patients with IRDs, except for the secretion of IL-1β in the SLE group, compared with the control. LPS-stimulated secretion of TNF-α was increased and MCP-1 was decreased in patients with IRDs compared to the control group; LPS-stimulated IL-1β secretion only in the SSc group was significantly different from the control group. In the RA group, monocyte activation was reduced for all cytokines compared to the control, in the SLE group – for TNF-α and MCP-1, in the SSc group – for MCP-1.Conclusion. The decrease in pro-inflammatory activation of monocytes in patients with IRDs is due to a high level of basal secretion of cytokines, which can lead to disruption of the adequate immune response in these diseases and is an important link in the pathogenesis of chronic inflammation.