Blocking the Function of Inflammatory Cytokines and Mediators by Using IL-10 and TGF-β: A Potential Biological Immunotherapy for Intervertebral Disc Degeneration in a Beagle Model

The debilitating effects of lower back pain are a major health issue worldwide. A variety of factors contribute to this, and oftentimes intervertebral disk degeneration (IDD) is an underlying cause of this disorder. Inflammation contributes to IDD, and inflammatory cytokines such as tumor necrosis factor (TNF)-α and interleukin (IL)-1β, play key roles in the pathology of IDD. Therefore, the development of treatments that inhibit the expression and/or effects of TNF-α and IL-1β in IDD patients should be a promising therapeutic approach to consider. This study characterized the potential to suppress inflammatory cytokine production in degenerative intervertebral disc (NP) cells by treatment with IL-10 and TGF-β in a canine model of IDD. IDD was induced surgically in six male beagles, and degenerative NP cells were isolated and cultured for in vitro studies on cytokine production. Cultured degenerative NP cells were divided into four experimental treatment groups: untreated control, IL-10-treated, TGF-β-treated, and IL-10- plus TGF-β-treated cells. Cultured normal NP cells served as a control group. TNF-α expression was evaluated by fluorescence activated cell sorting (FACS) analysis and enzyme-linked immunosorbent assay (ELISA); moreover, ELISA and real-time PCR were also performed to evaluate the effect of IL-10 and TGF-β on NP cell cytokine expression in vitro. Our results demonstrated that IL-10 and TGF-β treatment suppressed the expression of IL-1β and TNF-α and inhibited the development of inflammatory responses. These data suggest that IL-10 and TGF-β should be evaluated as therapeutic approaches for the treatment of lower back pain mediated by IDD.     

Improved Chemotherapeutic Activity by Morus alba Fruits through Immune Response of Toll-Like Receptor 4

Morus alba L. fruits have long been used in traditional medicine by many cultures. Their medicinal attributes include cardiovascular, hepatoprotective, neuroprotective and immunomodulatory actions. However, their mechanism of macrophage activation and anti-cancer effects remain unclear. The present study investigated the molecular mechanisms of immune stimulation and improved chemotherapeutic effect of M. alba L. fruit extract (MFE). MFE stimulated the production of cytokines, nitric oxide (NO) and tumor necrosis factor-α (TNF-α) and tumoricidal properties of macrophages. MFE activated macrophages through the mitogen-activated protein kinase (MAPKinase) and nuclear factor-κB (NF-κB) signaling pathways downstream from toll-like receptor (TLR) 4. MFE was shown to exhibit cytotoxicity of CT26 cells via the activated macrophages, even though MFE did not directly affect CT26 cells. In a xenograft mouse model, MFE significantly enhanced anti-cancer activity combined with 5-fluorouracil and markedly promoted splenocyte proliferation, natural killer (NK) cell activity, cytotoxic T lymphocyte (CTL) activity and IFN-γ production. Immunoglobulin G (IgG) antibody levels were significantly increased. These results indicate the indirect anti-cancer activity of MFE through improved immune response mediated by TLR4 signaling. M. alba L. fruit extract might be a potential anti-tumor immunomodulatory candidate chemotherapy agent.     

Astragalus membranaceus Inhibits Peritoneal Fibrosis via Monocyte Chemoattractant Protein (MCP)-1 and the Transforming Growth Factor-β1 (TGF-β1) Pathway in Rats Submitted to Peritoneal Dialysis

Inflammation and transforming growth factor-β1 (TGF-β1) contribute to the development of peritoneal fibrosis (PF), which is associated with peritoneal dialysis (PD). Astragalus membranaceus (Astragalus) has anti-inflammatory and anti-fibrotic effects in many diseases. The goal of this study was to determine the anti-fibrotic effects of Astragalus on the PF response to PD. A rat model of PD was induced using standard PD fluid, and PF was verified by HE and Masson’s staining, as well as through the expression of fibroblast surface protein (FSP) and collagen III. The expression levels of monocyte chemoattractant protein (MCP)-1, F4/80 (macrophage/monocyte marker in rat), TGF-β1 and the downstream proteins phospho-SMAD 2/3 in dialyzed peritoneal tissue treated with or without Astragalus was evaluated using immunohistochemistry analysis. Overall correlations between MCP-1 and TGF-β1 staining were analyzed using both the Spearman and Pearson methods. The results showed that Astragalus could inhibit the recruitment and activation of monocytes/macrophages, thereby reducing the production of TGF-β1 in the dialyzed peritoneal membrane. PF was also significantly decreased following treatment with Astragalus. MCP-1 expression had a strong positive correlation with TGF-β1 sensitivity, suggesting that the anti-fibrotic function of Astragalus was mediated by MCP-1 and the TGF-β1 pathway. Our results indicate that Astragalus could be a useful agent against PD-induced PF.     

Cucurbitacin B Down-Regulates TNF Receptor 1 Expression and Inhibits the TNF-α-Dependent Nuclear Factor κB Signaling Pathway in Human Lung Adenocarcinoma A549 Cells

Pro-inflammatory cytokines, such as tumor necrosis factor-α (TNF-α), induce the expression of intracellular adhesion molecule-1 (ICAM-1) by activating the nuclear factor κB (NF-κB) signaling pathway. In the present study, we found that cucurbitacin B decreased the expression of ICAM-1 in human lung adenocarcinoma A549 cells stimulated with TNF-α or interleukin-1α. We further investigated the mechanisms by which cucurbitacin B down-regulates TNF-α-induced ICAM-1 expression. Cucurbitacin B inhibited the nuclear translocation of the NF-κB subunit RelA and the phosphorylation of IκBα in A549 cells stimulated with TNF-α. Cucurbitacin B selectively down-regulated the expression of TNF receptor 1 (TNF-R1) without affecting three adaptor proteins (i.e., TRADD, RIPK1, and TRAF2). The TNF-α-converting enzyme inhibitor suppressed the down-regulation of TNF-R1 expression by cucurbitacin B. Glutathione, N-acetyl-L-cysteine, and, to a lesser extent, L-cysteine attenuated the inhibitory effects of cucurbitacin B on the TNF-α-induced expression of ICAM-1, suggesting that an α,β-unsaturated carbonyl moiety is essential for anti-inflammatory activity. The present results revealed that cucurbitacin B down-regulated the expression of TNF-R1 at the initial step in the TNF-α-dependent NF-κB signaling pathway.     

Nuclear Respiratory Factor-1, a Novel SMAD4 Binding Protein,Represses TGF-β/SMAD4 Signaling by Functioning as a Transcriptional Cofactor

SMAD4, a key regulator of transforming growth factor-β (TGF-β) signaling, plays a major role in cell growth, migration, and apoptosis. In particular, TGF-β/SMAD induces growth arrest, and SMAD4 induces the expression of target genes such as p21WAF1 and p15INK4b through its interaction with several cofactors. Thus, inactivating mutations or the homozygous deletion of SMAD4 could be related to tumorigenesis or malignancy progression. However, in some cancer types, SMAD4 is neither mutated nor deleted. In the current study, we demonstrate that TGF-β signaling with a preserved SMAD4 function can contribute to cancer through associations with negative pathway regulators. We found that nuclear respiratory factor-1 (NRF1) is a novel interaction SMAD4 partner that inhibits TGF-β/SMAD4-induced p15INK4b mRNA expression by binding to SMAD4. Furthermore, we confirmed that NRF1 directly binds to the core region of the SMAD4 promoter, thereby decreasing SMAD4 mRNA expression. On the whole, our data suggest that NRF1 is a negative regulator of SMAD4 and can interfere with TGF-β/SMAD-induced tumor suppression. Our findings provide a novel perception into the molecular basis of TGF-β/SMAD4-signaling suppression in tumorigenesis.     

The Multi-Biomarker Approach for Heart Failure in Patients with Hypertension

We assessed the predictive ability of selected biomarkers using N-terminal pro-brain natriuretic peptide (NT-proBNP) as the benchmark and tried to establish a multi-biomarker approach to heart failure (HF) in hypertensive patients. In 120 hypertensive patients with or without overt heart failure, the incremental predictive value of the following biomarkers was investigated: Collagen III N-terminal propeptide (PIIINP), cystatin C (CysC), lipocalin-2/NGAL, syndecan-4, tumor necrosis factor-α (TNF-α), interleukin 1 receptor type I (IL1R1), galectin-3, cardiotrophin-1 (CT-1), transforming growth factor β (TGF-β) and N-terminal pro-brain natriuretic peptide (NT-proBNP). The highest discriminative value for HF was observed for NT-proBNP (area under the receiver operating characteristic curve (AUC) = 0.873) and TGF-β (AUC = 0.878). On the basis of ROC curve analysis we found that CT-1 > 152 pg/mL, TGF-β < 7.7 ng/mL, syndecan > 2.3 ng/mL, NT-proBNP > 332.5 pg/mL, CysC > 1 mg/L and NGAL > 39.9 ng/mL were significant predictors of overt HF. There was only a small improvement in predictive ability of the multi-biomarker panel including the four biomarkers with the best performance in the detection of HF—NT-proBNP, TGF-β, CT-1, CysC—compared to the panel with NT-proBNP, TGF-β and CT-1 only. Biomarkers with different pathophysiological backgrounds (NT-proBNP, TGF-β, CT-1, CysC) give additive prognostic value for incident HF in hypertensive patients compared to NT-proBNP alone.     

Preparation and Characterization of Tripterygium wilfordii Multi-Glycoside Nanoparticle Using Supercritical Anti-Solvent Process

The aim of this study was to prepare nanosized Tripterygium wilfordii multi-glycoside (GTW) powders by the supercritical antisolvent precipitation process (SAS), and to evaluate the anti-inflammatory effects. Ethanol was used as solvent and carbon dioxide was used as an antisolvent. The effects of process parameters such as precipitation pressure (15–35 MPa), precipitation temperature (45–65 °C), drug solution flow rates (3–7 mL/min) and drug concentrations (10–30 mg/mL) were investigated. The nanospheres obtained with mean diameters ranged from 77.5 to 131.8 nm. The processed and unprocessed GTW were characterized by scanning electron microscopy, X-ray diffraction, Fourier-transform infrared spectroscopy and thermal gravimetric analysis. The present study was designed to investigate the beneficial effect of the GTW nanoparticles on adjuvant-induced arthritis in albino rats. The processed and unprocessed GTW were tested against Freund’s complete adjuvant-induced arthritis in rats. Blood samples were collected for the estimation of interleukins (IL-1α, IL-1β) and tumor necrosis factor-α (TNF-α). It was concluded that physicochemical properties and anti-inflammatory activity of GTW nanoparticles could be improved by physical modification, such as particle size reduction using supercritical antisolvent (SAS) process. Further, SAS process was a powerful methodology for improving the physicochemical properties and anti-inflammatory activity of GTW.     

TGF-β Signaling: From Tissue Fibrosis to Tumor Microenvironment

Transforming growth factor-β (TGF-β) signaling triggers diverse biological actions in inflammatory diseases. In tissue fibrosis, it acts as a key pathogenic regulator for promoting immunoregulation via controlling the activation, proliferation, and apoptosis of immunocytes. In cancer, it plays a critical role in tumor microenvironment (TME) for accelerating invasion, metastasis, angiogenesis, and immunosuppression. Increasing evidence suggest a pleiotropic nature of TGF-β signaling as a critical pathway for generating fibrotic TME, which contains numerous cancer-associated fibroblasts (CAFs), extracellular matrix proteins, and remodeling enzymes. Its pathogenic roles and working mechanisms in tumorigenesis are still largely unclear. Importantly, recent studies successfully demonstrated the clinical implications of fibrotic TME in cancer. This review systematically summarized the latest updates and discoveries of TGF-β signaling in the fibrotic TME.     

Anti-Inflammatory Effect of Allicin Associated with Fibrosis in Pulmonary Arterial Hypertension

Pulmonary arterial hypertension (PAH) is characterized by pulmonary vascular remodeling. Recent evidence supports that inflammation plays a key role in triggering and maintaining pulmonary vascular remodeling. Recent studies have shown that garlic extract has protective effects in PAH, but the precise role of allicin, a compound derived from garlic, is unknown. Thus, we used allicin to evaluate its effects on inflammation and fibrosis in PAH. Male Wistar rats were divided into three groups: control (CON), monocrotaline (60 mg/kg) (MCT), and MCT plus allicin (16 mg/kg/oral gavage) (MCT + A). Right ventricle (RV) hypertrophy and pulmonary arterial medial wall thickness were determined. IL-1β, IL-6, TNF-α, NFκB p65, Iκβ, TGF-β, and α-SMA were determined by Western blot analysis. In addition, TNF-α and TGF-β were determined by immunohistochemistry, and miR-21-5p and mRNA expressions of Cd68, Bmpr2, and Smad5 were determined by RT-qPCR. Results: Allicin prevented increases in vessel wall thickness due to TNF-α, IL-6, IL-1β, and Cd68 in the lung. In addition, TGF-β, α-SMA, and fibrosis were lower in the MCT + A group compared with the MCT group. In the RV, allicin prevented increases in TNF-α, IL-6, and TGF-β. These observations suggest that, through the modulation of proinflammatory and profibrotic markers in the lung and heart, allicin delays the progression of PAH.     

TGF-β Induced CTGF Expression in Human Lung Epithelial Cells through ERK,ADAM17, RSK1, and C/EBPβ Pathways

Background: Lung epithelial cells play critical roles in idiopathic pulmonary fibrosis. Methods: In the present study, we investigated whether transforming growth factor-β (TGF-β)-induced expression of connective tissue growth factor (CTGF) was regulated by the extracellular signal-regulated kinase (ERK)/a disintegrin and metalloproteinase 17 (ADAM17)/ribosomal S6 kinases 1 (RSK1)/CCAAT/enhancer-binding protein β (C/EBPβ) signaling pathway in human lung epithelial cells (A549). Results: Our results revealed that TGF-β-induced CTGF expression was weakened by ADAM17 small interfering RNA (ADAM17 siRNA), TNF-α processing inhibitor-0 (TAPI-0, an ADAM17 inhibitor), U0126 (an ERK inhibitor), RSK1 siRNA, and C/EBPβ siRNA. TGF-β-induced ERK phosphorylation as well as ADAM17 phosphorylation was attenuated by U0126. The TGF-β-induced increase in RSK1 phosphorylation was inhibited by TAPI-0 and U0126. TGF-β-induced C/EBPβ phosphorylation was weakened by U0126, ADAM17 siRNA, and RSK1 siRNA. In addition, TGF-β increased the recruitment of C/EBPβ to the CTGF promoter. Furthermore, TGF-β enhanced fibronectin (FN), an epithelial–mesenchymal transition (EMT) marker, and CTGF mRNA levels and reduced E-cadherin mRNA levels. Moreover, TGF-β-stimulated FN protein expression was reduced by ADAM17 siRNA and CTGF siRNA. Conclusion: The results suggested that TGF-β induces CTGF expression through the ERK/ADAM17/RSK1/C/EBPβ signaling pathway. Moreover, ADAM17 and CTGF participate in TGF-β-induced FN expression in human lung epithelial cells.     

A20 Overexpression Inhibits Lipopolysaccharide-Induced NF-κB Activation,TRAF6 and CD40 Expression in Rat Peritoneal Mesothelial Cells

Zinc finger protein A20 is a key negative regulator of inflammation. However, whether A20 may affect inflammation during peritoneal dialysis (PD)-associated peritonitis is still unclear. This study was aimed to investigate the effect of A20 overexpression on lipopolysaccharide (LPS)-induced inflammatory response in rat peritoneal mesothelial cells (RPMCs). Isolated and cultured RPMCs in vitro. Plasmid pGEM-T easy-A20 was transfected into RPMCs by Lipofectamine™2000. The protein expression of A20, phospho-IκBα, IκBα, TNF receptor-associated factor (TRAF) 6 and CD40 were analyzed by Western blot. The mRNA expression of TRAF6, CD40, interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) were determined by real time-PCR. NF-κB p65 DNA binding activity, IL-6 and TNF-α levels in cells culture supernatant were determined by ELISA. Our results revealed that RPMCs overexpression of A20 lead to significant decrease of LPS-induced IκBα phosphorylation and NF-κB DNA binding activity (all p < 0.01). In addition, A20 also attenuated the expression of TRAF6, CD40, IL-6 and TNF-α as well as levels of IL-6 and TNF-α in cells culture supernatant (all p < 0.05). However, A20 only partly inhibited CD40 expression. Our study indicated that A20 overexpression may depress the inflammatory response induced by LPS in cultured RPMCs through negatively regulated the relevant function of adaptors in LPS signaling pathway.     

The Wnt11 Signaling Pathway in Potential Cellular EMT and Osteochondral Differentiation Progression in Nephrolithiasis Formation

The molecular events leading to nephrolithiasis are extremely complex. Previous studies demonstrated that calcium and transforming growth factor-β1 (TGF-β1) may participate in the pathogenesis of stone formation, but the explicit mechanism has not been defined. Using a self-created genetic hypercalciuric stone-forming (GHS) rat model, we observed that the increased level of serous/uric TGF-β1 and elevated intracellular calcium in primary renal tubular epithelial cells (PRECs) was associated with nephrolithiasis progression in vivo. In the setting of high calcium plus high TGF-β1 in vitro, PRECs showed great potential epithelial to mesenchymal transition (EMT) progression and osteochondral differentiation properties, representing the multifarious increased mesenchymal and osteochondral phenotypes (Zeb1, Snail1, Col2A1, OPN, Sox9, Runx2) and decreased epithelial phenotypes (E-cadherin, CK19) bythe detection of mRNAs and corresponding proteins. Moreover, TGF-β-dependent Wnt11 knockdown and L-type Ca²⁺ channel blocker could greatly reverse EMT progression and osteochondral differentiation in PRECs. TGF-β1 alone could effectively promote EMT, but it had no effect on osteochondral differentiation in NRK cells (Rat kidney epithelial cell line). Stimulation with Ca²⁺ alone did not accelerate differentiation of NRK. Co-incubation of extracellular Ca²⁺ and TGF-β1 synergistically promotes EMT and osteochondral differentiation in NRK control cells. Our data supplied a novel view that the pathogenesis of calcium stone development may be associated with synergic effects of TGF-β1 and Ca²⁺, which promote EMT and osteochondral differentiation via Wnt11 and the L-type calcium channel.     

Role of Human Primary Renal Fibroblast in TGF-β1-Mediated Fibrosis-Mimicking Devices

Renal fibrosis is a progressive chronic kidney disease that ultimately leads to end-stage renal failure. Despite several approaches to combat renal fibrosis, an experimental model to evaluate currently available drugs is not ideal. We developed fibrosis-mimicking models using three-dimensional (3D) co-culture devices designed with three separate layers of tubule interstitium, namely, epithelial, fibroblastic, and endothelial layers. We introduced human renal proximal tubular epithelial cells (HK-2), human umbilical-vein endothelial cells, and patient-derived renal fibroblasts, and evaluated the effects of transforming growth factor-β (TGF-β) and TGF-β inhibitor treatment on this renal fibrosis model. The expression of the fibrosis marker alpha smooth muscle actin upon TGF-β1 treatment was augmented in monolayer-cultured HK-2 cells in a 3D disease model. In the vascular compartment of renal fibrosis models, the density of vessels was increased and decreased in the TGF-β-treated group and TGF-β-inhibitor treatment group, respectively. Multiplex ELISA using supernatants in the TGF-β-stimulating 3D models showed that pro-inflammatory cytokine and growth factor levels including interleukin-1 beta, tumor necrosis factor alpha, basic fibroblast growth factor, and TGF-β1, TGF-β2, and TGF-β3 were increased, which mimicked the fibrotic microenvironments of human kidneys. This study may enable the construction of a human renal fibrosis-mimicking device model beyond traditional culture experiments.     

Heightened Crescentic Glomerulonephritis in Immune Challenged 129sv Mice Is TGF-β/Smad3 Dependent

The 129sv mouse strain is particularly sensitive to experimental immune-mediated nephritis. Previous studies have indicated that transforming growth factor-β (TGF-β) plays a critical role in both immune modulation and tissue fibrogenesis in various diseases and that its biological activities are exerted via the SMAD family. In this study, we aimed to determine whether TGF-β/SMAD signaling is essential for the development of immune-mediated nephritis in 129sv mice. Relative to C57BL/6J control mice with anti-glomeruli basement membrane (GBM) nephritis, 129sv mice with anti-GBM nephritis exhibited increased renal collagen deposition. Additionally, higher mRNA levels of pro-collagen and collagen IV, higher serum levels of active and total TGF-β1, and increased TGF-β1, TGF-βIIR, and phosphorylated SMAD expression were detected in these mice. Deletion of Smad3 in 129sv mice ameliorated anti-GBM induced nephritis, including crescentic glomerulonephritis. Collectively, these findings indicate that the heightened experimental nephritis and fibrotic disease in the 129sv strain of mice are regulated by SMAD3, which could be a potential therapeutic target for immune-mediated nephritis.     

TNF-α Induces Mitophagy in Rheumatoid Arthritis Synovial Fibroblasts,and Mitophagy Inhibition Alleviates Synovitis in Collagen Antibody-Induced Arthritis

Mitophagy is a selective form of autophagy that removes damaged mitochondria. Increasing evidence indicates that dysregulated mitophagy is implicated in numerous autoimmune diseases, but the role of mitophagy in rheumatoid arthritis (RA) has not yet been reported. The aim of the present study was to determine the roles of mitophagy in patient-derived RA synovial fibroblasts (RASFs) and in the collagen antibody-induced arthritis mouse model. We measured the mitophagy marker PTEN-induced putative kinase 1 (PINK1) in RASFs treated with tumor necrosis factor-α (TNF-α) using Western blotting and immunofluorescence. Arthritis was induced in PINK1^−/− mice by intraperitoneal injection of an anti-type II collagen antibody cocktail and lipopolysaccharide. RA severity was assessed by histopathology. PINK1 expression and damaged mitochondria increased in TNF-α treated RASFs via increased intracellular levels of reactive oxygen species. PINK1 knockdown RASFs decreased cellular migration and invasion functions. In addition, PINK1^−/− mice with arthritis exhibited markedly reduced swelling and inflammation relative to wild-type mice with arthritis. Taken together, these findings suggest that regulation of PINK1 expression in RA could represent a potential therapeutic and diagnostic target for RA.     

Different Role of Tumor Necrosis Factor-α Polymorphism in Non-Hodgkin Lymphomas among Caucasian and Asian Populations: A Meta-Analysis

Tumor necrosis factor-α (TNF-α) is an immunoregulatory cytokine involved in B- and T-cell function, and also plays an important role in inflammation and cancer. TNF-α-308G>A has been associated with constitutively elevated TNF-α expression. Several studies have reported the association between the TNF-α-308G>A polymorphism and non-Hodgkin lymphomas (NHL) risk, however, results are still inconsistent. To solve these conflicts, we conducted the first meta-analysis to assess the effect of TNF-α-308G>A polymorphism on the risk of NHL and various subtypes (additive model) including 10,619 cases and 12,977 controls in Caucasian and Asian populations. Our meta-analysis indicated that TNF-α-308G>A polymorphism is not associated with NHL risk when pooling all studies together (OR = 1.06, 95% CI: 0.92–1.23, p = 0.413). In stratified analyses, we found TNF-α-308A allele was significantly associated with higher risk of NHL, B-cell lymphomas (BCL), T-cell lymphomas (TCL) and diffuse large B-cell lymphomas (DLBCL) in Caucasians (OR = 1.22, 95% CI: 1.06–1.40, p = 0.007; OR = 1.18, 95% CI: 1.03–1.34, p = 0.014; OR = 1.20, 95% CI: 1.01–1.42, p = 0.040; OR = 1.21, 95% CI: 1.11–1.32, p < 0.001, respectively). Interestingly, it was associated with decreased risk of NHL, BCL and DLBCL in Asians (OR = 0.75, 95% CI: 0.66–0.86, p < 0.001; OR = 0.70, 95% CI: 0.52–0.94, p = 0.018; OR = 0.70, 95% CI: 0.57–0.86, p = 0.001). These findings also suggest TNF-α might play a distinct role in pathogenesis of NHL in different populations.     

Achillea millefolium L. Essential Oil Inhibits LPS-Induced Oxidative Stress and Nitric Oxide Production in RAW 264.7 Macrophages

Achillea millefolium L. is a member of the Asteraceae family and has been used in folk medicine in many countries. In this study, 19 compounds in A. millefolium essential oil (AM-EO) have been identified; the major components are artemisia ketone (14.92%), camphor (11.64%), linalyl acetate (11.51%) and 1,8-cineole (10.15%). AM-EO can suppress the inflammatory responses of lipopolysaccharides (LPS)-stimulated RAW 264.7 macrophages, including decreased levels of cellular nitric oxide (NO) and superoxide anion production, lipid peroxidation and glutathione (GSH) concentration. This antioxidant activity is not a result of increased superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx) activities, but rather occurs as a result of the down-regulation of inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6) and heme oxygenase-1 (HO-1) expression, thus reducing the inflammatory response. Therefore, AM-EO can be utilized in many applications, including the treatment of inflammatory diseases in the future.     

Tumor Necrosis Factor-Alpha Exacerbates Viral Entry in SARS-CoV2-Infected iPSC-Derived Cardiomyocytes

The coronavirus disease 2019 (COVID-19) pandemic with high infectivity and mortality has caused severe social and economic impacts worldwide. Growing reports of COVID-19 patients with multi-organ damage indicated that severe acute respiratory syndrome coronavirus 2 (SARS-CoV2) may also disturb the cardiovascular system. Herein, we used human induced pluripotent stem cell (iPSC)-derived cardiomyocytes (iCMs) as the in vitro platform to examine the consequence of SARS-CoV2 infection on iCMs. Differentiated iCMs expressed the primary SARS-CoV2 receptor angiotensin-converting enzyme-II (ACE2) and the transmembrane protease serine type 2 (TMPRSS2) receptor suggesting the susceptibility of iCMs to SARS-CoV2. Following the infection of iCMs with SARS-CoV2, the viral nucleocapsid (N) protein was detected in the host cells, demonstrating the successful infection. Bioinformatics analysis revealed that the SARS-CoV2 infection upregulates several inflammation-related genes, including the proinflammatory cytokine tumor necrosis factor-α (TNF-α). The pretreatment of iCMs with TNF-α for 24 h, significantly increased the expression of ACE2 and TMPRSS2, SASR-CoV2 entry receptors. The TNF-α pretreatment enhanced the entry of GFP-expressing SARS-CoV2 pseudovirus into iCMs, and the neutralization of TNF-α ameliorated the TNF-α-enhanced viral entry. Collectively, SARS-CoV2 elevated TNF-α expression, which in turn enhanced the SARS-CoV2 viral entry. Our findings suggest that, TNF-α may participate in the cytokine storm and aggravate the myocardial damage in COVID-19 patients.