Dietary tannic acid attenuates elastase-induced pulmonary inflammation and emphysema in mice.

Emphysema is one of the major components of chronic obstructive pulmonary disease (COPD), which is characterised by the destruction and enlargement of air spaces, leading to airflow limitation and dyspnoea, finally progressing to oxygen dependency. The alveolar wall destruction is due to chronic inflammation, oxidative stress, apoptosis, and proteinase/anti-proteinase imbalance. So far, there has been no effective therapy for patients with COPD. We evaluated the therapeutic efficacy of tannic acid (TA), a naturally occurring plant-derived polyphenol in the murine emphysema model. In C57BL/6 J mice, we established emphysema by intratracheal instillation of elastase (EL). Then, mice were treated with TA and evaluated 1 and 21 days post-EL instillation. After 24 h, TA treatment significantly reduced EL-induced histopathological alterations, infiltrating leukocytes, and gene expression of markers of inflammation and apoptosis. Similarly, after 21 days, TA treatment suppressed the mean linear intercept, gene expression of proteinases, and increased elastic fiber contents in the lungs when compared to the EL-alone group. Furthermore, EL induced the activation of p38 mitogen-activated protein kinase (MAPK) and nuclear factor kappa light chain enhancer of activated B cells (NF-kB) p65 pathways in the lungs was suppressed by TA treatment. In summary, TA has the potential to mitigate EL-induced inflammation, apoptosis, proteinase/anti-proteinase imbalance, and subsequent emphysema in mice.

Bacterial Outer Membrane Vesicles Promote Lung Inflammatory Responses and Macrophage Activation via Multi-Signaling Pathways.

Emerging evidence suggests that Gram-negative bacteria release bacterial outer membrane vesicles (OMVs) and that these play an important role in the pathogenesis of bacterial infection-mediated inflammatory responses and organ damage. Despite the fact that scattered reports have shown that OMVs released from Gram-negative bacteria may function via the TLR2/4-signaling pathway or induce pyroptosis in macrophages, our study reveals a more complex role of OMVs in the development of inflammatory lung responses and macrophage pro-inflammatory activation. We first confirmed that various types of Gram-negative bacteria release similar OMVs which prompt pro-inflammatory activation in both bone marrow-derived macrophages and lung alveolar macrophages. We further demonstrated that mice treated with OMVs via intratracheal instillation developed significant inflammatory lung responses. Using mouse inflammation and autoimmune arrays, we identified multiple altered cytokine/chemokines in both bone marrow-derived macrophages and alveolar macrophages, suggesting that OMVs have a broader spectrum of function compared to LPS. Using TLR4 knock-out cells, we found that OMVs exert more robust effects on activating macrophages compared to LPS. We next examined multiple signaling pathways, including not only cell surface antigens, but also intracellular receptors. Our results confirmed that bacterial OMVs trigger both surface protein-mediated signaling and intracellular signaling pathways, such as the S100-A8 protein-mediated pathway. In summary, our studies confirm that bacterial OMVs strongly induced macrophage pro-inflammatory activation and inflammatory lung responses via multi-signaling pathways. Bacterial OMVs should be viewed as a repertoire of pathogen-associated molecular patterns (PAMPs), exerting more robust effects than Gram-negative bacteria-derived LPS.

Caveolin-1 regulates OMV-induced macrophage pro-inflammatory activation and multiple Toll-like receptors.

Macrophages (MФ), the primary cell of the innate immune system, serves as the first line of defense. During bacterial infection, Gram-negative (G-) bacteria release nanosized outer membrane vesicles (OMVs), facilitating the crosstalk between the microbe and the host. The underlying mechanisms by which OMVs induced pro-inflammatory (M1) activation are still unknown. Our study shows that OMVs caused M1 activation via modulating various toll-like receptor (TLR) expressions as they contain LPS, LTA, bacterial DNAs, and flagellins. Also, we found that caveolin-1 (cav-1), a 21-kDa scaffolding protein of caveolae and lipid rafts, plays a significant role in OMV-induced pro-inflammatory response in regulating various TLR signaling pathways. Specifically, cav-1 deletion increased the expression of OMV-induced TLRs, pro-inflammatory cytokine secretions (TNF-α and IL-1ß), and the reactive oxygen species (ROS) production in MФs. Further, we examined the interaction between Cav-1 and TLR4 by immunoprecipitation, colocalization, and computational models, providing future direction to explore the role of cav-1 in OMV-induced other TLR signaling. Altogether, Cav-1 is a key regulator in OMV-induced multiple TLRs response. This study promotes future research to develop drugs by targeting the specific motif of cav-1 or TLRs against bacterial infection and macrophage-mediated inflammation.

Impact of Storage Conditions on EV Integrity/Surface Markers and Cargos.

Extracellular vesicles (EVs) are small biological particles released into biofluids by every cell. Based on their size, they are classified into small EVs (<100 nm or <200 nm) and medium or large EVs (>200 nm). In recent years, EVs have garnered interest for their potential medical applications, including disease diagnosis, cell-based biotherapies, targeted drug delivery systems, and others. Currently, the long-term and short-term storage temperatures for biofluids and EVs are −80 °C and 4 °C, respectively. The storage capacity of EVs can depend on their number, size, function, temperature, duration, and freeze−thaw cycles. While these parameters are increasingly studied, the effects of preservation and storage conditions of EVs on their integrity remain to be understood. Knowledge gaps in these areas may ultimately impede the widespread applicability of EVs. Therefore, this review summarizes the current knowledge on the effect of storage conditions on EVs and their stability and critically explores prospective ways for improving long-term storage conditions to ensure EV stability.

Direct Detection of Extracellular Vesicle miRNAs Using a Single-Step RT-qPCR Assay.

Extracellular vesicles (EVs) are biological carriers, and EV-associated miRNAs (EV-miRNAs) are considered as a novel biomarker in multiple diseases. Currently, the column-based purification method is used to purify miRNAs from EVs. However, this method of purification is complex, time-consuming, and expensive. Therefore, a simple and cost-effective single-step quantitative reverse transcription-polymerase chain reaction (RT-qPCR) method is required to detect the expression of EV-miRNAs. This chapter describes a protocol for directly analyzing the EV-miRNAs expression from mouse bronchoalveolar lavage fluid (BALF) and serum without going for an RNA isolation and purification step from EVs. It is an efficient method in several terms such as cost-wise, time, low expertise, and accuracy in results. This method may be helpful in diagnostic blood tests used in medical centers or research laboratories.

Anti-asthmatic effects of tannic acid from Chinese natural gall nuts in a mouse model of allergic asthma.

Asthma is a chronic inflammatory disease of the airways, which is characterized by infiltration of inflammatory cells, airway hyperresponsiveness (AHR), and airway remodeling. This study aimed to explore the role and mechanism of tannic acid (TA), a naturally occurring plant-derived polyphenol, in murine asthma model. BALB/c mice were given ovalbumin (OVA) to establish an allergic asthma model. The results revealed that TA treatment significantly decreased OVA-induced AHR, inflammatory cells infiltration, and the expression of various inflammatory mediators (Th2 and Th1 cytokines, eotaxin, and total IgE). Additionally, TA treatment also attenuated increases in mucins (Muc5ac and Muc5b) expression, mucus production in airway goblet cells, mast cells infiltration, and airway remodeling induced by OVA exposure. Furthermore, OVA-induced NF-κB (nuclear factor- kappa B) activation and cell adhesion molecules expression in the lungs was suppressed by TA treatment. In conclusion, TA effectively attenuated AHR, inflammatory response, and airway remodeling in OVA-challenged asthmatic mice. Therefore, TA may be a potential therapeutic option against allergic asthma in clinical settings.

An overview on the role of plant-derived tannins for the treatment of lung cancer.

Lung cancer is the leading cause of cancer-related death globally. Despite many advanced approaches to treat cancer, they are often ineffective due to resistance to classical anti-cancer drugs and distant metastases. Currently, alternative medicinal agents derived from plants are the major interest due to high bioavailability and fewer adverse effects. Tannins are polyphenolic compounds existing as specialized products in a wide variety of vegetables, fruits, and nuts. Many tannins have been found to possess protective properties, such as anti-inflammatory, anti-fibrotic, anti-microbial, anti-diabetic, and so on. This review aims to summarize the current knowledge addressing the anti-cancer effects of dietary tannins and their underlying molecular mechanisms. In vivo and in vitro studies provide evidences that anti-cancer effects of various tannins are predominantly mediated through negative regulation of transcription factors, growth factors, receptor kinases, and many oncogenic molecules. In addition, we also discussed the absorption, distribution, metabolism, excretion, and toxicity (ADMET) properties of tannins, clinical trial results as well as our perspective on future research with tannins.

Therapeutic potential of plant-derived tannins in non-malignant respiratory diseases.

Respiratory diseases are the major cause of human illness and death around the world. Despite advances in detection and treatment, very few classes of safe and effective therapy have been introduced to date. At present, phytochemicals are getting more attention because of their diverse beneficial activities and minimal toxicity. Tannins are polyphenolic secondary metabolites with high molecular weights, which are naturally present in a wide variety of fruits, vegetables, cereals, and leguminous seeds. Many tannins are endowed with well-recognized protective properties, such as anti-cancer, anti-microbial, anti-oxidant, anti-hyperglycemic, and many others. This review summarizes a large body of experimental evidence implicating that tannins are helpful in tackling a wide range of non-malignant respiratory diseases including acute lung injury (ALI), pulmonary fibrosis, asthma, pulmonary hypertension, and chronic obstructive pulmonary disease (COPD). Mechanistic pathways by which various classes of tannins execute their beneficial effects are discussed. In addition, clinical trials and our perspective on future research with tannins are also reviewed.

Tannic acid alleviates experimental pulmonary fibrosis in mice by inhibiting inflammatory response and fibrotic process.

Pulmonary fibrosis (PF) is a chronic and irreversible scarring disease in the lung with limited treatment options. Therefore, it is critical to identify new therapeutic options. This study was undertaken to identify the effects of tannic acid (TA), a naturally occurring dietary polyphenol, in a mouse model of PF. Bleomycin (BLM) was intratracheally administered to induce PF. Administration of TA significantly reduced BLM-induced histological alterations, inflammatory cell infiltration and the levels of various inflammatory mediators (nitric oxide, leukotriene B4 and cytokines). Additionally, treatment with TA also impaired BLM-mediated increases in pro-fibrotic (transforming growth factor-ß1) and fibrotic markers (alpha-smooth muscle actin, vimentin, collagen 1 alpha and fibronectin) expression. Further investigation indicated that BLM-induced phosphorylation of Erk1/2 (extracellular signal-regulated kinases 1 and 2) in lungs was suppressed by TA treatment. Findings of this study suggest that TA has the potential to mitigate PF through inhibiting the inflammatory response and fibrotic process in lungs and that TA might be useful for the treatment of PF in clinical practice.

Tannic acid prevents macrophage-induced pro-fibrotic response in lung epithelial cells via suppressing TLR4-mediated macrophage polarization.

BACKGROUND: Polarized macrophages induce fibrosis through multiple mechanisms, including a process termed epithelial-to-mesenchymal transition (EMT). Mesenchymal cells contribute to the excessive accumulation of fibrous connective tissues, leading to organ failure. This study was aimed to investigate the effect of tannic acid (TA), a natural dietary polyphenol on M1 macrophage-induced EMT and its underlying mechanisms. MATERIALS: First, we induced M1 polarization in macrophage cell lines (RAW 264.7 and THP-1). Then, the conditioned-medium (CM) from these polarized macrophages was used to induce EMT in the human adenocarcinomic alveolar epithelial (A549) cells. We also analysed the role of TA on macrophage polarization. RESULTS: We found that TA pre-treated CM did not induce EMT in epithelial cells. Further, TA pre-treated CM showed diminished activation of MAPK in epithelial cells. Subsequently, TA was shown to inhibit LPS-induced M1 polarization in macrophages by directly targeting toll-like receptor 4 (TLR4), thereby repressing LPS binding to TLR4/MD2 complex and subsequent signal transduction. CONCLUSION: It was concluded that TA prevented M1 macrophage-induced EMT by suppressing the macrophage polarization possibly through inhibiting the formation of LPS-TLR4/MD2 complex and blockage of subsequent downstream signal activation. Further, our findings may provide beneficial information to develop new therapeutic strategies against chronic inflammatory diseases.

Tannic acid protects against experimental acute lung injury through downregulation of TLR4 and MAPK.

Acute lung injury (ALI) and its severe form acute respiratory distress syndrome (ARDS) remain a major cause of morbidity and mortality in critically ill patients, and no specific therapies are still available to control the mortality rate. Thus, we explored the preventive and therapeutic effects of tannic acid (TA), a natural polyphenol in the context of ALI. We used in vivo and in vitro models, respectively, using lipopolysaccharide (LPS) to induce ALI in mice and exposing J774 and BEAS-2B cells to LPS. In both preventive and therapeutic approaches, TA attenuated LPS-induced histopathological alterations, lipid peroxidation, lung permeability, infiltration of inflammatory cells, and the expression of proinflammatory mediators. In addition, in-vitro study showed that TA treatment could reduce the expression of proinflammatory mediators. Further studies revealed that TA-dampened inflammatory responses by downregulating the LPS-induced toll-like receptor 4 (TLR4) expression and inhibiting extracellular-signal-regulated kinase (ERK)1/2 and p38 mitogen-activated protein kinase (MAPK) activation. Furthermore, cells treated with the inhibitors of ERK1/2 (PD98059) and p38 (SB203580) mitigated the expression of cytokines induced by LPS, thus suggesting that ERK1/2 and p38 activity are required for the inflammatory response. In conclusion, TA could attenuate LPS-induced inflammation and may be a potential therapeutic agent for ALI-associated inflammation in clinical settings.

Tannic acid modulates fibroblast proliferation and differentiation in response to pro-fibrotic stimuli.

In response to tissue injury, fibroblasts migrate into the wound, where they undergo proliferation and differentiation. The persistence of these differentiated fibroblasts (myofibroblasts) is associated with excessive scarring in various organs. We aimed to investigate the effects of Tannic acid (TA) on fibroblast proliferation and differentiation, and found that TA inhibited fibroblast differentiation as assessed by reduced expression of α-smooth muscle actin, N-cadherin, and type-1-collagen. TA also prevented the TGF-ß1-induced alteration in the expression of two classes of genes involved in the remodeling of extracellular matrix (ECM) proteins, namely matrix metalloproteinases (Mmp-2 and -9) and tissue inhibitors of metalloproteinases (Timp-1 and -3). Further, TA suppressed TGF-ß1-induced cell proliferation and induced cell cycle arrest at G0/G1 phase via targeting Cyclins expression. Finally, TA exerted its inhibitory effects by decreasing the phosphorylation of Smad and ERK signaling. In sum, our results suggesting that TA may be a potential therapeutic agent for pathological fibrosis.

Tannic acid attenuates TGF-ß1-induced epithelial-to-mesenchymal transition by effectively intervening TGF-ß signaling in lung epithelial cells.

Idiopathic pulmonary fibrosis (IPF) is a chronic, progressive, and an irreversible lung disorder characterized by the accumulation of fibroblasts and myofibroblasts in the extracellular matrix. The transforming growth factor-ß1 (TGF-ß1)-induced epithelial-to-mesenchymal transition (EMT) is thought to be one of the possible sources for a substantial increase in the number of fibroblasts/myofibroblasts in IPF lungs. Tannic acid (TA), a natural dietary polyphenolic compound has been shown to possess diverse pharmacological effects. However, whether TA can inhibit TGF-ß1-mediated EMT in lung epithelial cells remains enigmatic. Both the human adenocarcinomic alveolar epithelial (A549) and normal bronchial epithelial (BEAS-2B) cells were treated with TGF-ß1 with or without TA. Results showed that TA addition, markedly inhibited TGF-ß1-induced EMT as assessed by reduced expression of N-cadherin, type-1-collagen, fibronectin, and vimentin. Furthermore, TA inhibited TGF-ß1-induced cell proliferation through inducing cell cycle arrest at G0/G1 phase. TGF-ß1-induced increase in the phosphorylation of Smad (Smad2 and 3), Akt as well as that of mitogen activated protein kinase (ERK1/2, JNK1/2, and p38) mediators was effectively inhibited by TA. On the other hand, TA reduced the TGF-ß1-induced increase in TGF-ß receptors expression. Using molecular docking approach, FTIR, HPLC and Western blot analyses, we further identified the direct binding of TA to TGF-ß1. Finally, we conclude that TA might directly interact with TGF-ß1, thereby repressing TGF-ß signaling and subsequent EMT process in lung epithelial cells. Further animal studies are needed to clarify its potential therapeutic benefit in pulmonary fibrosis.