Inhibition Of TNF-α Expression Research Articles

Abstract P3018: SCARNA20 Promotes M2 Macrophage Polarization Under Endotoxemia And Reduces Inflammation

Background: Macrophage polarization plays a key role in the inflammatory process. M1 pro-inflammatory and M2 anti-inflammatory phenotypes can down-regulate each other and may contribute to the pathogenesis of various diseases. Small Cajal Body Associated RNAs (SCARNAs) play a key role in alternative splicing and maturation of mRNAs. Our recent studies found that a combination of Procainamide (P, DNMT1 inhibitor) and Tubastatin A (T, HDAC6 inhibitor) reduces inflammation upon lipopolysaccharide (LPS) stimuli. Hypothesis: We hypothesize that the P+T treatment increases the expression of SCARNA20 hence reducing the proinflammatory response and promoting M2 macrophage polarization after LPS challenge. Methods and Results: To elucidate SCARNAs of interest during the inflammatory response, we treated macrophages with LPS (1μg/mL) and P (400nM/mL) + T (2.5μM/mL) for 1hr and performed RNA sequencing. Based on the sequencing results and qRT-PCR validation, SCARNA20 was the most significantly differentially expressed SCARNA between conditions (Fig. A) . The expression of TNFα was significantly reduced with the P+T treatment (Fig. B) . SCARNA20 overexpression (OE) showed an increase in the expression of the M2 marker CD163 and a reduction in the expression of the M1 marker CD80 (Fig. C) . Additionally, TNFα expression was significantly reduced in the SCARNA20 OE samples after the LPS challenge without treatment with P+T (Fig. D) . Finally, SCARNA20 knockdown macrophages (Fig. E) treated with P+T did not suppress TNFα expression (Fig. F) . Conclusions: We report for the first time that SCARNA20 is an important regulator of M2 macrophage polarization and pro-inflammatory response.

Alleviation of Cognitive Impairment-like Behaviors, Neuroinflammation, Colitis, and Gut Dysbiosis in 5xFAD Transgenic and Aged Mice by Lactobacillus mucosae and Bifidobacterium longum.

Neuropsychiatric disorders including Alzheimer's disease (AD) may cause gut inflammation and dysbiosis. Gut inflammation-suppressing probiotics alleviate neuropsychiatric disorders. Herein, to understand whether anti-inflammatory probiotics Lactobacillus mucosae NK41 and Bifidobacterium longum NK46, which suppressed tumor necrosis factor (TNF)-α expression in lipopolysaccharide (LPS)-stimulated macrophages, could alleviate cognitive impairment, we first examined their effects on cognitive function, gut inflammation, and gut microbiota composition in 5xFAD-transgenic mice. Oral administration of NK41 or NK46 decreased cognitive impairment-like behaviors, hippocampal amyloid-β (Aβ), TNF-α and interleukin (IL)-1β expression, hippocampal NF-κB+Iba1+ cell population, and Aβ accumulation, while hippocampal brain-derived neurotropic factor (BDNF) and IL-10 expression and BDNF+NeuN+ cell population increased. They also decreased TNF-α and IL-1β expression and NF-κB+CD11c+ cell population in the colon. They also reduced fecal and blood LPS levels and gut Proteobacteria and Verrucomicrobia populations (including Akkkermansiaceae), which are positively associated with hippocampal TNF-α and fecal LPS levels and negatively correlated with hippocampal BDNF level. However, they increased Odoribactericeae, which positively correlated with BDNF expression level and TNF-α to IL-10 expression ratio. The combination of NK41 and NK46 (4:1, NKc), which potently inhibited TNF-α expression in LPS-stimulated macrophages, additively alleviated cognitive impairment-like behaviors in 5xFAD-transgenic or aged mice. NKc increased hippocampal BDNF+NeuN+ cell population and BDNF expression in 5xFAD-transgenic or aged mice, while hippocampal TNF-α and IL-1β expression decreased. NKc also decreased TNF-α and IL-1β expression in the colon and LPS levels in the blood and feces. These findings suggest that gut bacteria and its product LPS may be closely connected with occurrence of cognitive impairment and neuroinflammation and the combination of NK41 and NK46 can additively alleviate cognitive impairment and neuroinflammation by inducing NF-κB-suppressed BDNF expression and suppressing LPS-producing gut bacteria.

The potency of propolis extract as anti-inflammatory agent for stimulation of reparative dentin formation

Background: As an effort to treat a deep caries lesion with a diagnosis of reversible pulpitis, one of the treatment options is pulp capping. For decades, calcium hydroxide has been considered the most acceptable pulp capping material and has been routinely used in dental practice, but it also has some weakness. To overcome that, alternative materials are needed that can provide a better effect. Recently, a lot of research has been done using natural ingredients, including Propolis. Propolis has been known to have strong antimicrobial and anti inflammatory properties. Therefore, it is necessary to conduct a deeper study regarding the potency of propolis extract as an anti inflammatory agent for the stimulation of reparative dentin formation so that it can be considered to be used as a pulp capping material.
 Objective: The aim of this literature review is to highlight the potency of propolis extract as anti inflammatory agent in the natural stimulation of reparative dentin formation.
 Methods: The search strategy consist of an electronic research using databases such as PubMed, Google Scholar, Science Direct, dan Springer Link for studies published from 2003 to 2021 regarding the potency of propolis extract as anti inflammatory agent for stimulation of reparative dentin formation, including review articles and research articles.
 Results: Propolis and its constituents have been proven to be used as a potent natural anti inflammatory, it also considered to have the ability to promote reparative dentin formation. However, there are some literatures stated that reparative dentin does not form with the application of propolis, and the causes may vary.
 Conclusion: Propolis extract has a potency as an anti inflammatory agent in dental pulp inflammation by inhibiting TNFα expression, and increasing TGF-β1 expression, as well as its potency in stimulating the formation of reparative dentin. Based on this review, further studies are needed.

Regulation of inflammatory macrophages by oral mineralized metal-organic framework nanoparticles for the synergistic treatment of ulcerative colitis and liver injury

The exacerbation of liver injury has been identified as a severe complication of ulcerative colitis (UC), due to its ability to trigger a systemic inflammatory response. The aggravation of both ulcerative colitis and liver injury is primarily attributed to the presence of inflammatory macrophages, which exhibit elevated expression of tumor necrosis factor alpha (TNF-α). Considerable efforts are being devoted to the development of drugs capable of effectively inhibiting TNF-α expression in inflammatory macrophages, with the aim of mitigating the severity of this complex disease. Glutathione (GSH) suppressed inflammation by neutralizing reactive oxygen species (ROS) and mitigating cellular damage. In this study, we designed biomimetic mineralized metal–organic framework nanoparticles encapsulating siTNF-α and GSH (ZIF-8/siTNF-α/GSH@CaCO3, ZTGC) for the purpose of reprogramming inflammatory macrophages. In this study, we verified the co-occurrence of ulcerative colitis and liver injury. The calcium carbonate present in ZTGC provided protection against degradation in the gastrointestinal environment, facilitating effective and sustained release of siTNF-α in inflammatory macrophages. Oral administration of ZTGC mitigated ulcerative colitis, ameliorated liver injury, and diminished systemic inflammation in vivo. These findings suggest that mineralized metal–organic framework nanoparticles have the potential to serve as an effective oral administration for the synergistic treatment of inflammatory diseases. This innovative approach provides new insights into the “two in one” treatment of these complications.

Immune-enhancing activity of polysaccharides and flavonoids derived from Phellinus igniarius YASH1.

Introduction: Phellinus igniarius (P. igniarius) (Sanghuang) is a widely used traditional Chinese medicine fungus, and its natural products have great potential for clinical application in immune enhancement. This study aimed to explore the immune-enhancing activity and underlying mechanisms of the polysaccharides and flavonoids derived from Phellinus igniarius (P. igniarius) and to provide a theoretical and experimental basis for the development of novel drugs. Methods: Wild P. igniarius YASH1 from the Loess Plateau in Yan'an region was collected, and polysaccharides and total flavonoids were extracted, isolated and identified from mycelium and sporophore. In vitro antioxidant activity was detected through the scavenging activity of hydroxyl radicals and total antioxidant capacity. Cell Counting Kit-8 and trypan blue detection kit were used to detect the effect of extract polysaccharides and flavonoids on the proliferation and phagocytosis ability of immune cells. To assess the effect of the drugs on cytokine secretion by immune cells and immune recovery in immunocompromised mice, the expression of interleukin (IL)-2, IL-6, interferon (IFN)-γ, and tumor necrosis factor (TNF)-α were examined at the cellular and animal levels. The species composition, abundance of gut microbiota and the altered content of short-chain fatty acids in the feces were analyzed to elucidate the possible mechanisms of drugs by 16S ribosomal RNA (rRNA) amplifiers sequencing and liquid chromatography-tandem mass spectrometry (LC-MS/MS). Results: Both polysaccharides and flavonoids derived from mycelium or sporophore had antioxidant activity and may stimulate the expression and secretion of IL-2, IL-6, and IFN-γ in immune cells while inhibiting TNF-α expression and secretion and increasing IL-2, IL-6, and IFN- γ expression levels in mice. Furthermore, polysaccharides and flavonoids from mycelium and sporophore showed different effects on the metabolic response of intestinal short-chain fatty acids (SCFAs) in mice, and the use of these drugs remarkably changed the species composition and abundance of intestinal flora in mice. Discussion: Polysaccharides and flavonoids from P. igniarius YASH1 mycelium and sporophore have in vitro antioxidant activity, and they affect the promotion of cell proliferation, stimulation of IL-2, IL-6, and IFN-γ secretion, and inhibition of TNF-α expression in immune cells. Polysaccharides and flavonoids from P. igniarius YASH1 may enhance immunity in immunocompromised mice and remarkably affect the intestinal flora and content of SCFAs.

Potential of siRNA-Bearing Subtilosomes in the Treatment of Diethylnitrosamine-Induced Hepatocellular Carcinoma

Therapeutics, based on small interfering RNA (siRNA), have demonstrated tremendous potential for treating cancer. However, issues such as non-specific targeting, premature degradation, and the intrinsic toxicity of the siRNA, have to be solved before they are ready for use in translational medicines. To address these challenges, nanotechnology-based tools might help to shield siRNA and ensure its specific delivery to the target site. Besides playing a crucial role in prostaglandin synthesis, the cyclo-oxygenase-2 (COX-2) enzyme has been reported to mediate carcinogenesis in various types of cancer, including hepatocellular carcinoma (HCC). We encapsulated COX-2-specific siRNA in Bacillus subtilis membrane lipid-based liposomes (subtilosomes) and evaluated their potential in the treatment of diethylnitrosamine (DEN)-induced hepatocellular carcinoma. Our findings suggested that the subtilosome-based formulation was stable, releasing COX-2 siRNA in a sustained manner, and has the potential to abruptly release encapsulated material at acidic pH. The fusogenic property of subtilosomes was revealed by FRET, fluorescence dequenching, content-mixing assay, etc. The subtilosome-based siRNA formulation was successful in inhibiting TNF-α expression in the experimental animals. The apoptosis study indicated that the subtilosomized siRNA inhibits DEN-induced carcinogenesis more effectively than free siRNA. The as-developed formulation also suppressed COX-2 expression, which in turn up-regulated the expression of wild-type p53 and Bax on one hand and down-regulated Bcl-2 expression on the other. The survival data established the increased efficacy of subtilosome-encapsulated COX-2 siRNA against hepatocellular carcinoma.

Whole-Transcriptome Analysis Highlights Adenylyl Cyclase Toxins-Derived Modulation of NF-κB and ERK1/2 Pathways in Macrophages.

Edema toxin (ET), one of the main toxic factors of Bacillus anthracis (B. anthracis), is a kind of potent adenylate cyclase (AC). B. anthracis has adapted to resist macrophage microbicidal mechanisms in part by secreting ET. To date, there is limited information on the pathogenic mechanisms used by ET to manipulate macrophage function, especially at the transcriptome level. We used RNA sequencing to study transcriptional changes in RAW264.7 cells treated with ET. We aimed to identify molecular events associated with the establishment of infection and followed changes in cellular proteins. Our results indicate that ET inhibited TNF-α expression in the RAW264.7 mouse macrophage cell line by activating the cAMP/PKA pathway. ET challenge of macrophages induced a differential expression of genes that participate in multiple macrophage effector functions such as cytokine production, cell adhesion, and the inflammatory response. Furthermore, ET influenced the expression of components of the ERK1/2, as well as the NF-αB signaling pathways. We also showed that ET treatments inhibit the phosphorylation of the ERK1/2 protein. ET also attenuated NF-αB subunit p65 phosphorylation and transcriptional activity of NF-αB via the cAMP/PKA pathway in macrophages. Since the observed modulatory effects were characteristic only of the bacterial exotoxin ET, we propose this may be a mechanism used by B. anthracis to manipulate macrophages and establish systemic infection.

Lactate attenuates astrocytic inflammation by inhibiting ubiquitination and degradation of NDRG2 under oxygen–glucose deprivation conditions

BackgroundBrain lactate concentrations are enhanced in response to cerebral ischemia and promote the formation of reactive astrocytes, which are major components of the neuroinflammatory response and functional recovery, following cerebral ischemia. NDRG2 is upregulated during reactive astrocyte formation. However, its regulation and function are unclear. We studied the relationship between lactate and NDRG2 in astrocytes under conditions of ischemia or oxygen–glucose deprivation (OGD).MethodsWe examined astrocytic NDRG2 expression after middle cerebral artery occlusion (MCAO) using western blot and immunofluorescence staining. Under hypoxia conditions, we added exogenous L-lactate sodium (lactate) to cultured primary astrocytes to explore the effects of lactate on the ubiquitination modification of NDRG2. We profiled the transcriptomic features of NDRG2 silencing in astrocytes after 8 h of OGD conditions as well as exogenous lactate treatment by performing RNA-seq. Finally, we evaluated the molecular mechanisms of NDRG2 in regulating TNFα under OGD conditions using western blot and immunohistochemistry.ResultsReactive astrocytes strongly expressed NDRG2 in a rat model of MCAO. We also showed that lactate stabilizes astrocytic NDRG2 by inhibiting its ubiquitination. NDRG2 inhibition in astrocytes increased inflammation and upregulated immune-associated genes and signaling pathways. NDRG2 knockdown induced TNFα expression and secretion via c-Jun phosphorylation.ConclusionsWe revealed that under OGD conditions, lactate plays an important anti-inflammatory role and inhibits TNFα expression by stabilizing NDRG2, which is beneficial for neurological functional recovery. NDRG2 may be a new therapeutic target for cerebral ischemia.

Quality marker prediction in Trillium tschonoskii based on UHPLC-MS chemical characterisation and network pharmacology.

As a folk herbal medicine, Trillium tschonoskii has been used for thousands of years. However, due to the complexity of the chemical constituents of this herb, few investigations have acquired a comprehensive understanding of its quality markers. This study was conducted to characterise the chemical composition of T. tschonoskii and identify its potential quality markers. A systematic analytical method based on ultra-high-performance liquid chromatography coupled with mass spectrometry (UHPLC-MS) was used to characterise the constituents of T. tschonoskii. Multivariate statistical analysis was performed to investigate the chemical differences between different tissues, as well as the relationship between chemical compositions and habitats. The potential quality markers were predicted via network pharmacology and molecular docking, then confirmed by cellular assays. A total of 77 compounds were co-isolated and identified, and among them, 26 were discovered from the genus Trillium for the first time. Ten batches of roots/rhizomes were explicitly clustered into five groups according to the climate types of the habitats, and the clusters of the fruits and roots/rhizomes from the same plants were independent due to the significant difference in chemical composition. Diosgenin had a good docking affinity with the relevant targets within the IL-17 pathway and cytokine pathway and could significantly inhibit TNF-α expression in hypoxic brain microvascular endothelial cells (BMECs). This is the first study to establish the chemical composition profile of T. tschonoskii by UHPLC-MS systematically, and diosgenin was confirmed to be a potential quality marker of T. tschonoskii for the treatment of headaches.

Green synthesis of silver nanoparticles reduced with Trigonella foenum-graecum and their effect on tumor necrosis factor-α in MCF7 cells.

Silver nanoparticles (AgNPs) are known to exhibit anti-inflammatory and anticancer activities. They have been reported to reduce the levels of tumor necrosis factor (TNF) - a proinflammatory cytokine involved in cell proliferation, differentiation, and apoptosis - in cell lines. As patients with breast cancer have been reported to have higher serum TNF levels, we aimed at developing a novel treatment for breast cancer by evaluating the effect of Trigonella foenum-graecum extract (TFG)-reduced AgNPs on the MCF-7 cell line, which serves as a model of human breast cancer. TFG-capped AgNPs were synthesized using a green reduction method, in which TFG reduced silver nitrate to generate AgNPs-TFG. The particle size, surface charge, ultraviolet (UV)-visible (VIS) spectra, surface morphology, % yield, and in vitro Ag+ release of the formulated AgNPs-TFG were evaluated. Additionally, the prepared NPs were examined for cytotoxicity using real-time polymerase chain reaction (real-time PCR), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, and enzyme-linked immunosorbent assay (ELISA). The prepared AgNPs-TFG were uniform, small, discrete, and non-aggregated with a particle size of 22.5±0.75 nm and ζ-potential of -47.45±0.666 mV. The yield of AgNPs-TFG was 224.545±3.9 µM. Furthermore, the AgNP-TFG thin film exhibited a prolonged release of Ag+ in phosphate buffer for up to 11 h. AgNPs-TFG suppressed TNF-α expression at mRNA and protein levels in MCF-7 cells. Additionally, the formulated AgNPs-TFG did not exhibit any toxicity toward MCF-7 cells. This study showed that AgNP-TFG could effectively inhibit TNF-α. These results provide significant insights for developing new therapeutic strategies for cancer and other inflammatory illnesses.

Sustainable Release of Propranolol Hydrochloride Laden with Biconjugated-Ufasomes Chitosan Hydrogel Attenuates Cisplatin-Induced Sciatic Nerve Damage in In Vitro/In Vivo Evaluation.

Peripheral nerve injuries significantly impact patients’ quality of life and poor functional recovery. Chitosan–ufasomes (CTS–UFAs) exhibit biomimetic features, making them a viable choice for developing novel transdermal delivery for neural repair. This study aimed to investigate the role of CTS–UFAs loaded with the propranolol HCl (PRO) as a model drug in enhancing sciatica in cisplatin-induced sciatic nerve damage in rats. Hence, PRO–UFAs were primed, embedding either span 20 or 60 together with oleic acid and cholesterol using a thin-film hydration process based on full factorial design (24). The influence of formulation factors on UFAs’ physicochemical characteristics and the optimum formulation selection were investigated using Design-Expert® software. Based on the optimal UFA formulation, PRO–CTS–UFAs were constructed and characterized using transmission electron microscopy, stability studies, and ex vivo permeation. In vivo trials on rats with a sciatic nerve injury tested the efficacy of PRO–CTS–UFA and PRO–UFA transdermal hydrogels, PRO solution, compared to normal rats. Additionally, oxidative stress and specific apoptotic biomarkers were assessed, supported by a sciatic nerve histopathological study. PRO–UFAs and PRO–CTS–UFAs disclosed entrapment efficiency of 82.72 ± 2.33% and 85.32 ± 2.65%, a particle size of 317.22 ± 6.43 and 336.12 ± 4.9 nm, ζ potential of −62.06 ± 0.07 and 65.24 ± 0.10 mV, and accumulatively released 70.95 ± 8.14% and 64.03 ± 1.9% PRO within 6 h, respectively. Moreover, PRO–CTS–UFAs significantly restored sciatic nerve structure, inhibited the cisplatin-dependent increase in peripheral myelin 22 gene expression and MDA levels, and further re-established sciatic nerve GSH and CAT content. Furthermore, they elicited MBP re-expression, BCL-2 mild expression, and inhibited TNF-α expression. Briefly, our findings proposed that CTS–UFAs are promising to enhance PRO transdermal delivery to manage sciatic nerve damage.

Metformin abrogates pathological TNF-α-producing B cells through mTOR-dependent metabolic reprogramming in polycystic ovary syndrome.

B cells contribute to the pathogenesis of polycystic ovary syndrome (PCOS). Clinically, metformin is used to treat PCOS, but it is unclear whether metformin exerts its therapeutic effect by regulating B cells. Here, we showed that the expression level of tumor necrosis factor-alpha (TNF-α) in peripheral blood B cells from PCOS patients was increased. Metformin used in vitro and in vivo was able to reduce the production of TNF-α in B cells from PCOS patients. Administration of metformin improved mouse PCOS phenotypes induced by dehydroepiandrosterone (DHEA) and also inhibited TNF-α expression in splenic B cells. Furthermore, metformin induced metabolic reprogramming of B cells in PCOS patients, including the alteration in mitochondrial morphology, the decrease in mitochondrial membrane potential, Reactive Oxygen Species (ROS) production and glucose uptake. In DHEA-induced mouse PCOS model, metformin altered metabolic intermediates in splenic B cells. Moreover, the inhibition of TNF-α expression and metabolic reprogramming in B cells of PCOS patients and mouse model by metformin were associated with decreased mTOR phosphorylation. Together, TNF-α-producing B cells are involved in the pathogenesis of PCOS, and metformin inhibits mTOR phosphorylation and affects metabolic reprogramming, thereby inhibiting TNF-α expression in B cells, which may be a new mechanism of metformin in the treatment of PCOS.

A Molecular Insight into the Role of Antioxidants in Nonalcoholic Fatty Liver Diseases.

Nonalcoholic fatty liver disease (NAFLD) defines fat accumulation in the liver, and it is commonly associated with metabolic syndromes like diabetes and obesity. Progressive NAFLD leads to nonalcoholic steatohepatitis (NASH) and ultimately causes cirrhosis and hepatocellular carcinoma, and NASH is currently a frequent cause of liver transplantation. Oxidative stress is often contributed to the progression of NAFLD, and hence, antioxidants such as silymarin, silybin, or silibinin, pentoxifylline, resveratrol, and vitamins A, C, and E are used in clinical trials against NAFLD. Silymarin induces the peroxisome proliferator-activated receptor α (PPARα), a fatty acid sensor, which promotes the transcription of genes that are required for the enzymes involved in lipid oxidation in hepatocytes. Silybin inhibits sterol regulatory element-binding protein 1 and carbohydrate response element-binding protein to downregulate the expression of genes responsible for de novo lipogenesis by activating AMP-activated protein kinase phosphorylation. Pentoxifylline inhibits TNF-α expression and endoplasmic reticulum stress-mediated inflammatory nuclear factor kappa B (NF-κB) activation. Thus, it prevents NAFLD to NASH progression. Resveratrol inhibits methylation at Nrf-2 promoters and NF-κB activity via SIRT1 activation in NAFLD conditions. However, clinically, resveratrol has not shown promising beneficial effects. Vitamin C is beneficial in NAFLD patients. Vitamin E is not effectively regressing hepatic fibrosis. Hence, its combination with antifibrotic agents is used as an adjuvant to produce a synergistic antifibrotic effect. However, to date, none of these antioxidants have been used as a definite therapeutic agent in NAFLD patients. Further, these antioxidants should be studied in NAFLD patients with larger populations and multiple endpoints in the future.

Differential regulation of lipopolysaccharide-induced IL-1β and TNF-α production in macrophages by palmitate via modulating TLR4 downstream signaling

Diabetic patients are susceptible to infectious diseases. Bacterial invasion activates immune cells such as macrophages through interaction between LPS and TLR4, and induces the expression of inflammatory mediators, including IL-1β and TNF-α, which play key roles in the elimination of infections. Unregulated overproduction or underproduction of these cytokines has been reported as a major factor in the development of septic shock, immune deficiency, and autoimmunity. Recent studies found that metabolic abnormalities of diabetes, such as hyperglycemia and dyslipidemia, played a major role in modulating the immune response. In this study, we studied the effects of palmitic acid (PA) pretreatment on LPS-induced IL-1β and TNF-α production and LPS-TLR4 signaling in macrophages. Compared with control, PA pretreatment significantly increased LPS-induced TNF-α production and secretion in macrophages. In contrast, LPS-induced IL-1β production and secretion was significantly suppressed by PA pretreatment. PA pretreatment did not affect the expression levels of TLR4 or Myd88, or the endocytosis of TLR4 in macrophages. However, PA pretreatment significantly suppressed the phosphorylation level and nuclear translocation of NF-κB, and the phosphorylation level of ERK1/2, whereas increased the phosphorylation levels of p38 and JNK. The activation of IKK which was upstream of NF-κB and ERK1/2 was attenuated, while the activation of TAK1 which was upstream of JNK and p38 was augmented by PA pretreatment. Inhibitors of NF-κB, MEK1/2, and p38 significantly decreased IL-1β expression, while JNK and p38 pathway inhibitors significantly inhibited TNF-α expression. The differential regulation of LPS-induced TNF-α and IL-1β production by PA was associated with cellular metabolism of PA, because inhibiting metabolism of PA with etomoxir or pretreatment with Br-PA which cannot be metabolized reversed these effects. We also showed that PA treatment increased acetylated IKK level which might contribute to the suppressed activation of IKK. The present study showed that LPS-induced production of TNF-α and IL-1β was regulated by different TLR4 downstream pathways in macrophages. PA differentially affected LPS-induced production of TNF-α and IL-1β in macrophages through differentially modulating these pathways. Further experiments will be needed to determine how these phenomena lead to the impaired immune response in patients with diabetes.

Glyceraldehyde-3-phosphate dehydrogenase present in extracellular vesicles from Leishmania major suppresses host TNF-alpha expression

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) fulfills various physiological roles that are unrelated to its glycolytic function. However, to date, the nonglycolytic function of GAPDH in trypanosomal parasites is absent from the literature. Exosomes secreted from Leishmania, like entire parasites, were found to have a significant impact on macrophage cell signaling and function, indicating cross talk with the host immune system. In this study, we demonstrate that the Leishmania GAPDH (LmGAPDH) protein is highly enriched within the extracellular vesicles (EVs) secreted during infection. To understand the function of LmGAPDH in EVs, we generated control, overexpressed, half-knockout (HKO), and complement cell lines. HKO cells displayed lower virulence compared with control cells when macrophages and BALB/c mice were infected with them, implying a crucial role for LmGAPDH in Leishmania infection and disease progression. Furthermore, upon infection of macrophages with HKO mutant Leishmania and its EVs, despite no differences in TNFA mRNA expression, there was a considerable increase in TNF-α protein expression compared with control, overexpressed, and complement parasites as determined by ELISA, RT-PCR, and immunoblot data. In vitro protein translation studies suggest that LmGAPDH-mediated TNF-α suppression occurs in a concentration-dependent manner. Moreover, mRNA binding assays also verified that LmGAPDH binds to the AU-rich 3′-UTR region of TNFA mRNA, limiting its production. Together, these findings confirmed that the LmGAPDH contained in EVs inhibits TNF-α expression in macrophages during infection via posttranscriptional repression.

Paediatric assessment of immune responses in 3 to 5 years olds having intestinal nematode infection and vaccinated with valent10-PCV

Valent 10 - Pneumococcal conjugate vaccine (PCV10) introduction into routine immunization schedule has resulted in reduction of the incidence of Invasive Pneumococcal disease (IPD) in children in Kenya. This study assessed immunological effect of nematode infection in children aged 3 to 5 years vaccinated with PCV10. The study subjects were from Mukuru Kwa Njenga settlement in Nairobi County, Kenya. The children were medically examined for the presence of other infectious diseases by a qualified clinical officer. Questionnaires on the PCV10 vaccination history and deworming history of children were administered to the parents or guardians of children with the help of community health workers. The intensity of nematode infections was examined in the faecal samples using Kato-Katz technique and expressed as eggs per gram (EPG) of faeces. Serum samples were obtained from blood collected during sampling and used in Flow cytometry to determine levels of IL-2, IL-4, IL-6, IL-10, TNF-α and IFN-γ in cellular immune responses, whereas, ELISA was used to determine the levels of IgG and IgE antibodies in humoral responses. Results showed 73.4% of children completed the PCV10 vaccination dose schedule, 21.6% of children sampled had suffered invasive pneumococcal infections post PCV10 vaccination. Ascaris lumbricoides was the only species identified in 34.9% of children having light to moderate infection intensity with mean intensity of 17808EPG. Significant IL-6 levels expressed in children having nematode infection impacted increased secretion of IgG and IgE, thus, an indication that the humoral responses were significantly stimulated. Results indicate suppression of TNF-α expression in the category of children who had nematode infection and were also vaccinated with PCV10, however, a higher expression of IFN-γ in the same category implied a negative impact on PCV10 responses. The study also indicated that presence of nematodes and vaccination had a profound influence on the expression of IL-2, however, PCV10 vaccination and intestinal nematode infection did not significantly impact the expression of IL-4 and IL-10 cytokines. Immune responses due to infection with Ascaris lumbricoides stimulated multiple effector mechanisms influencing induction of cellular responses in PCV10 vaccination. Further investigation of other key cytokines as well as other parasites and pneumonia vaccines in other vulnerable individuals is therefore necessary in the management of concomitant infections in order to reduce the suppression of immune responses often resulting in difficulties in disease management.

A Network Pharmacology-Based Investigation to the Pharmacodynamic Material Basis and Mechanisms of the Anti-Inflammatory and Anti-Viral Effect of Isatis indigotica.

PurposeIsatis indigotica (Ii) is a cruciferous herb that is widely distributed in China, and its roots and leaves have been used in two renowned antipyretic detoxicate crude drugs in Chinese Pharmacopoeia, Radix (R) and Folium (F) Isatidis. However, the pharmacodynamic material basis and underlying mechanisms of the herbal efficacy remained to be elucidated.MethodsUltra-performance liquid chromatography-quadrupole-time-of-flight mass spectrometry (UPLC-Q-TOF-MS) was adopted for the chemical profiling of R and F Isatidis. The active ingredients were screened out through the prediction of gastrointestinal absorption and druglikeness analysis using SwissADME. A herb-ingredient-target network was constructed through target prediction of the herbal active ingredients and anti-inflammation or anti-viral properties, followed by protein–protein interaction analysis. Then, the potential relevant signaling pathways were predicted by pathway enrichment. Finally, for verification, RAW 264.7 cell line was adopted to examine the anti-inflammatory and anti-viral activities of 6 representative ingredients in Ii.ResultsSeventy-three compounds have been identified from Ii through UPLC-Q-TOF-MS. A total of 17 potential active ingredients were screened through pharmacokinetics and drug-likeness evaluation using SwissADME. It was shown that key targets might include TNF, AKT1, SRC, IL2, CASP9, and CASP3 in our herb-ingredient-target network, and isovitexin, a flavonoid, tended to participate in the inflammatory response, indoles were more likely to affect the cell proliferation processes, and lignans might have a broader affinity to key targets than the other active ingredients, such as regulating immune system (targeting IL-2) and PI3K-Akt signaling pathway. In vitro, indigo and secoisolariciresinol diglucoside markedly reduced TNF-α expression in Poly (I: C)-incubated cells. Isovitexin significantly inhibited TNF-α expression, and isatin treatment markedly reduced IL-1β expression in LPS-incubated cells.ConclusionAs the pharmacodynamics material basis of Ii, indoles, lignans, and flavonoids are believed to confer beneficial properties through various cellular aspects with multiple signaling pathways involved.

Obesity activates immunomodulating properties of mesenchymal stem cells in adipose tissue with differences between localizations.

Mesenchymal stem cells from healthy adipose tissue are adipocytes progenitors with immunosuppressive potential that are used for years in cell therapy. Whether adipose stem cells (ASC) may prevent inflammation in early obesity is not known. To address this question, we performed a kinetic study of high-fat (HF) diet induced obesity in mice to follow the immune regulating functions of adipose stem cells (ASC) isolated from the subcutaneous (SAT) and the visceral adipose tissue (VAT). Our results show that, early in obesity and before inflammation was detected, HF diet durably and differently activated ASC from SAT and VAT. Subcutaneous ASC from HF-fed mice strongly inhibited the proliferation of activated T lymphocytes, whereas visceral ASC selectively inhibited TNFα expression by macrophages and simultaneously released higher concentrations of IL6. These depot specific differences may contribute to the low-grade inflammation that develops with obesity in VAT while inflammation in SAT is delayed. The mechanisms involved differ from those already described for naïve cells activation with inflammatory cytokines and probably engaged metabolic activation. These results evidence that adipose stem cells are metabolic sensors acquiring an obesity-primed immunocompetent state in answer to depot-specific intrinsic features with overnutrition, placing these cells ahead of inflammation in the local dialog with immune cells.

β-Asarone suppresses TNF-α expression through DNA methylation and c-Jun-mediated transcription modulation in scratch-injured neuronal cells.

This study aimed to investigate the role and possible mechanism of β-asarone in regulating neuronal apoptosis and axonal regeneration. A scratch injury was applied to cell cultures of mouse primary cortical neurons to mimic neuronal injury. The neuronal apoptosis was evaluated by terminal deoxynucleotidyl transferase dUTP nick end labelingstaining and western blot analysis of apoptosis-related proteins. The axonal regeneration was assessed by immunofluorescent staining of β-tubulin III and western blot analysis of axonal markers. In the results, β-asarone inhibited neuronal apoptosis and promoted axonal regeneration by suppressing tumor necrosis factor-α (TNF-α) expression in scratch-injured mouse neuronal cells. Research investigating the molecular mechanisms by which β-asarone inhibited TNF-α expression showed that, on the one hand, β-asarone suppressed the JNK/c-Jun pathway and thus transcriptionally inhibited TNF-α expression; on the other hand, β-asarone induced expression of UHRF1 that recruited DNMT1 to induce TNF-α promoter methylation and subsequently decreased the messenger RNA expression of TNF-α. In conclusion, β-asarone suppresses TNF-α expression through DNA methylation and c-Jun-mediated transcription modulation in scratch-injured neuronal cells.

PTH1-34 inhibited TNF-α expression and antagonized TNF-α-induced MMP13 expression in MIO mice

This study aims to investigate the effects and mechanisms of parathyroid hormone [1–34] (PTH1-34) on TNF-α-stimulated mice chondrocytes, as well as cartilage from a meniscus injury induced osteoarthritis (MIO) mice model. The C57BL/6J mice received medial meniscectomy, and then administrated with PTH1-34. The results showed that PTH1-34 administration decreased secondary allodynia and the pain-related transcripts. The IHC, ELISA, Micro-CT imaging and histopathology analysis revealed the significantly improved subchondral plate thickness and bone porosity, the reduced pro-inflammatory cytokines in serum and joint fluid. In vitro, mice chondrocyte was treated with TNF-α or co-cultured with synovial cells. The results showed that TNF-α markedly upregulated the MMP13 expression, and the ERK1/2, NF-κB or PI3K signaling pathway inhibitors could reverse the induction effect of TNF-α on expression of MMP13 in chondrocytes. PTH1-34 alone has no effect on the expression of MMP13 and NF-κB signaling pathways, but the PTH1-34 could reverse the induction effect of TNF-α on MMP13 expression and NF-κB signaling pathway activation in chondrocytes. In addition, PTH1-34 administration inhibited the expression of TNF-α and MMP13, and chondrocyte viability, while the PKA repressor reversed the effect of PTH1-34 in chondrocytes co-cultured with synovial cells. In conclusion, PTH1-34 has an obvious analgesic and anti-inflammatory effect, inhibits the matrix synthesis and alleviates the progression of osteoarthritis. In vitro, PTH1-34 inhibited TNF-α expression and antagonized TNF-α-induced MMP13 expression via the PKA pathway and the NF-κB signaling pathways, respectively.