Butyrate Prevents the Pathogenic Anemia-Inflammation Circuit by Facilitating Macrophage Iron Export.

Most patients with inflammatory bowel disease (IBD) develop anemia, which is attributed to the dysregulation of iron metabolism. Reciprocally, impaired iron homeostasis also aggravates inflammation. How this iron-mediated, pathogenic anemia-inflammation crosstalk is regulated in the gut remains elusive. Herein, it is for the first time revealed that anemic IBD patients exhibit impaired production of short-chain fatty acids (SCFAs), particularly butyrate. Butyrate supplementation restores iron metabolism in multiple anemia models. Mechanistically, butyrate upregulates ferroportin (FPN) expression in macrophages by reducing the enrichment of histone deacetylase (HDAC) at the Slc40a1 promoter, thereby facilitating iron export. By preventing iron sequestration, butyrate not only mitigates colitis-induced anemia but also reduces TNF-α production in macrophages. Consistently, macrophage-conditional FPN knockout mice exhibit more severe anemia and inflammation. Finally, it is revealed that macrophage iron overload impairs the therapeutic effectiveness of anti-TNF-α antibodies in colitis, which can be reversed by butyrate supplementation. Hence, this study uncovers the pivotal role of butyrate in preventing the pathogenic circuit between anemia and inflammation.

Compatibility principle in the Tanyu Tongzhi Formula revealed by a cell-based analysis.

ETHNOPHARMACOLOGICAL RELEVANCE: The concept of the prescription in Traditional Chinese Medicine (TCM) is usually characterized by the compatibility principle "monarch, minister, assistant, and guide", which means herbs play primary, secondary, auxiliary, or harmonic roles, respectively, to achieve the optimally holistic effect. Following this compatibility principle, the Tanyu Tongzhi Formula (TTF), used for many years to treat cardiovascular diseases, has been proved effective clinically and experimentally. AIM OF THE STUDY: The ancient compatibility principle is based on experiences, but whether its underlying interactions can be explained at the cellular level is unknown. We aimed to explore the mechanisms of activity of the TTF herbs and the interactions between them. MATERIALS AND METHODS: We used a real-time cell analyzer to record the responses of COS-7 cells to the herbs in TTF, both individually and in different combinations. We also used biochemical assays to further characterize the TTF activity. RESULTS: Monarch herb Fructus trichosanthis acts as an inhibitor of the EGF signaling. It's cytotoxicity, derived from inhibition of tubulin polymerization, could be completely neutralized by the combination of the phlegm group, or the whole TTF combination. Meanwhile, the minister, assistant, and guide herbs in the TTF did not affect EGF signaling. CONCLUSION: Our results provide a demonstration, at the cellular level, of the compatibility principle of "monarch, minister, assistant, and guide" in TTF. Under the guidance of this principle, TTF exerts the anti-inflammation and anti-tumor effects through inhibiting EGF signaling, while avoiding the microtubule-disrupting activity of Fructus trichosanthis.

Suppression of Th17 cell differentiation by misshapen/NIK-related kinase MINK1.

T helper type 17 cells (Th17 cells) are major contributors to many autoimmune diseases. In this study, we demonstrate that the germinal center kinase family member MINK1 (misshapen/NIK-related kinase 1) negatively regulates Th17 cell differentiation. The suppressive effect of MINK1 on induction of Th17 cells is mediated by the inhibition of SMAD2 activation through direct phosphorylation of SMAD2 at the T324 residue. The importance of MINK1 to Th17 cell differentiation was strengthened in the animal model of experimental autoimmune encephalomyelitis (EAE). Moreover, we show that the reactive oxygen species (ROS) scavenger N-acetyl cysteine boosts Th17 cell differentiation in a MINK1-dependent manner and exacerbates the severity of EAE. Thus, we have not only established MINK1 as a critical regulator of Th17 cell differentiation, but also clarified that accumulation of ROS may limit the generation of Th17 cells. The contribution of MINK1 to ROS-regulated Th17 cell differentiation may suggest an important mechanism for the development of autoimmune diseases influenced by antioxidant dietary supplements.

Cellular Phenotypic Analysis of Macrophage Activation Unveils Kinetic Responses of Agents Targeting Phosphorylation.

Macrophages are highly plastic cells, which serve as sentinels of the host immune system due to their ability to recognize and respond to microbial products rapidly and dynamically. Appropriate regulation of macrophage activation is essential for pathogen clearance or preventing autoimmune diseases. However, regularly used endpoint assays for analyzing macrophage functions have the limitations of being static and non-high throughput. In this study, we introduced a real-time and convenient method based on changes in cellular impedance that are detected by microelectronic biosensors. This new method can record the time/dose-dependent cell response profiles (TCRPs) of macrophages in real time and generates physiologically relevant data. The TCRPs generated from classically interferon-γ/lipopolysaccharide-activated macrophages showed considerable consistency with the data generated from standard endpoint assays. We further explored this approach by using it for global screening of a library of protein tyrosine kinase/phosphatase (PTK/PTP) inhibitors to investigate their impact on macrophage activation. Collectively, our findings suggest that the cellular impedance-based assay provides a promising approach for dynamically monitoring macrophage functions in a convenient and high-throughput manner.

Cell-based phenotypic screening of mast cell degranulation unveils kinetic perturbations of agents targeting phosphorylation.

Mast cells play an essential role in initiating allergic diseases. The activation of mast cells are controlled by a complicated signal network of reversible phosphorylation, and finding the key regulators involved in this network has been the focus of the pharmaceutical industry. In this work, we used a method named Time-dependent cell responding profile (TCRP) to track the process of mast cell degranulation under various perturbations caused by agents targeting phosphorylation. To test the feasibility of this high-throughput cell-based phenotypic screening method, a variety of biological techniques were used. We further screened 145 inhibitors and clustered them based on the similarities of their TCRPs. Stat3 phosphorylation has been widely reported as a key step in mast cell degranulation. Interestingly, our TCRP results showed that a Stat3 inhibitor JSI124 did not inhibit degranulation like other Stat3 inhibitors, such as Stattic, clearly inhibited degranulation. Regular endpoint assays demonstrated that the distinctive TCRP of JSI124 potentially correlated with the ability to induce apoptosis. Consequently, different agents possibly have disparate functions, which can be conveniently detected by TCRP. From this perspective, our TCRP screening method is reliable and sensitive when it comes to discovering and selecting novel compounds for new drug developments.

The Inhibition of Mast Cell Activation of Radix Paeoniae alba Extraction Identified by TCRP Based and Conventional Cell Function Assay Systems.

Chinese herbs have long been used to treat allergic disease, but recently the development was greatly impeded by the lack of good methods to explore the mechanism of action. Here, we showed the effects of Chinese herb Radix Paeoniae alba were identified and characterized by a mast cell activation assay that involves electronic impedance readouts for dynamic monitoring of cellular responses to produce time-dependent cell responding profiles (TCRPs), and the anti-allergic activities were further confirmed with various conventional molecular and cell biology tools. We found Radix P. alba can dose-dependently inhibit TCPRs, and have anti-allergic function in vitro and in vivo. Radix P. alba suppressed mast cell degranulation not only inhibiting the translocation of granules to the plasma membrane, but also blocking membrane fusion and exocytosis; and that there may be other anti-allergic components in addition to paeoniflorin. Our results suggest that Radix P. alba regulated mast cell activation with multiple targets, and this approach is also suitable for discovering other mast cell degranulation-targeting Chinese herbs and their potential multi-target mechanisms.

Prevention of pulmonary fibrosis with salvianolic acid a by inducing fibroblast cell cycle arrest and promoting apoptosis.

ETHNOPHARMACOLOGICAL RELEVANCE: Danshen (Salvia miltiorrhiza Bunge) is widely used in traditional Chinese medicine (TCM), often in combination with other herbs, to treat a diversity of ailments. More recent studies have focused on its possible roles in the treatment of respiratory diseases (pneumonia and pulmonary fibrosis) and found that it has pharmacological activity that protects pulmonary morphology and function. However, the mechanism underlying this activity has not yet been clarified. MATERIALS AND METHODS: The purpose of this study was to investigate the anti-pulmonary fibrosis effects exerted by salvianolic acid A (SAA), the ingredient responsible for the pharmacological activity of Danshen, and the underlying mechanisms. Bleomycin (BLM)-induced rat pulmonary fibrosis was used to evaluate the antifibrotic role of SAA, and fibroblast cells were used to study the mechanism involved. RESULTS: BLM-treated rats exhibited increased alveolar wall thickness and collagen deposition in lung tissues, but these pathologies were greatly attenuated by daily administration of SAA. We also found that SAA significantly inhibited the proliferation, adhesion and migration of fibroblasts in vitro. This was partly due to a strong induction of cell cycle arrest and apoptosis upon SAA treatment. Consistent with these phenotypes, we observed decreased expression of the cell cycle-related proteins cyclin D1, cyclin E1, and cyclin B1, and increased expression of p53 and p21 in SAA-treated cells. In addition, the anti-apoptotic Bcl-2 protein decreased in a dose-dependent manner, while cleaved caspase-3 protein increased upon SAA treatment. CONCLUSIONS: These results suggest that the alleviation of rat pulmonary fibrosis by SAA is due to the inhibition of fibroblast proliferation and induction of apoptosis, which occurs mainly through p53-dependent growth arrest and apoptosis. We suggest that SAA should be considered as a potential novel therapeutic agent for the treatment of fibrotic lung diseases.

Kinetic cellular phenotypic profiling: prediction, identification, and analysis of bioactive natural products.

Natural products have always been a major source of therapeutic agents; however, the development of traditional herbal products has been currently hampered by the lack of analytic methods suitable for both high-throughput screening and evaluating the mechanism of action. Cellular processes such as proliferation, apoptosis, and toxicity are well-orchestrated in real time. Monitoring these events and their perturbation by natural products can provide high-rich information about cell physiological relevancies being involved. Here, we report a novel cell-based phenotypic profiling strategy that uses electronic impedance readouts for real-time monitoring of cellular responses to traditional Chinese medicines (TCMs). The utility of this approach was used to screen natural herbs that have been historically documented to cure human diseases and that have been classified into seven clusters based on their mechanisms of action. The results suggest that herbal medicines with similar cellular mechanisms produce similar time/dose-dependent cell response profiles (TCRPs). By comparing the TCRPs produced by the Chinese medicinal Cordyceps sinensis with similar TCRPs of chemical compounds, we explored the potential use of herbal TCRPs for predicting cellular mechanisms of action, herbal authentications, and bioactive identification. Additionally, we further compared this novel TCRP technology with high-performance liquid chromatography (HPLC)-based methods for herbal origin-tracing authentication and identification of bioactive ingredients. Together, our findings suggest that using TCRP as an alternative to existing spectroscopic techniques can allow us to analyze natural products in a more convenient and physiologically relevant manner.