Uncovering the key pharmacodynamic material basis and possible molecular mechanism of Xiaoke formulation improve insulin resistant through a comprehensive investigation.

ETHNOPHARMACOLOGICAL RELEVANCE: Xiaoke formulation (XKF) has been utilized in clinical practice for decades in China as a treatment option for mild to moderate type 2 diabetes. However, there is still a need for systematic research to uncover the key pharmacodynamic material basis and mechanism of XKF. AIM OF THE STUDY: Aim of to investigate the distribution and metabolism of XKF in normal and insulin resistant (IR) mice were different, and elucidate its key pharmacodynamic material basis and mechanism of action. MATERIALS AND METHODS: Ultra performance liquid chromatography/time of flight mass spectrometry technology was employed to investigate the differences in XKF absorption, distribution, and metabolism between normal and IR mice across blood, liver, feces, and urine samples. Further, network pharmacology was used to predict target proteins and their associated signaling pathways. Then, molecular docking was utilized to validate the activity of key pharmacodynamic components and targets. Finally, IR HepG2 cells were used to detect the glucose consumption under the action of key pharmacodynamic material basis. In addition, the expression of phosphatidylinositol 3-kinase (PI3K), protein kinase B (AKT) and phospho-protein kinase B (p-AKT) was determined using western blotting. RESULTS: The study demonstrates significant distinctions in plasma and liver number and abundance of alkaloids, organic acids, flavonoids, iridoids and saponins between normal and IR mice when XKF was administered. Further analysis has shown that the representative components of XKF, including berberine, chlorogenic acid, calycosin, swertiamarin and astragaloside IV have significantly different metabolic pathways in plasma and liver. Prototypes and metabolites of these components were rarely detected in the urine and feces of mice. According to the network pharmacological analysis, these differential components are predicted to improve IR by targeting key factors such as SRC, JUN, HRAS, NOS3, FGF2, etc. Additionally, the signaling pathways involved in this process include PI3K-AKT pathway, GnRH signaling pathway, and T cell receptor signaling pathway. In addition, in vitro experiments indicate that berberine and its metabolites (berberine and demethyleneberine), chlorogenic acid and its metabolites (3-O-ferulic quinic acid and 5-O-ferulic quinic acid), calycosin and swertiamarin could improve IR in IR-HepG2 cells by elevating the expression of PI3K and AKT, leading to an increase in glucose consumption. CONCLUSION: The key pharmacodynamic material basis of XKF, such as berberine and its metabolites (berberrubine and demethyleneberberine), chlorogenic acid and its metabolites (3-O-feruloylquinic acid and 5-O-feruloylquinic acid), calycosin and swertiamarin influence the glucose metabolism disorder of IR-HepG2 cells by regulating the PI3K/AKT signalling pathway, leading to an improvement in IR.

ß-Elemene in zedoary turmeric oil injection induces dyspnea by binding to hemoglobin and upregulating HIF-1α.

ETHNOPHARMACOLOGICAL RELEVANCE: Zedoary turmeric oil injection (ZTOI) extracted from the rhizome extract of Curcuma phaeocaulis Valeton, Curcuma wenyujin Y. H. Chen et C. Ling or Curcuma kwangsiensis S. G. Lee et C. F. Liang, is widely used for the treatment of virus-induced upper respiratory tract infections, peptic ulcers, viral pneumonia, etc. However, it has attracted widespread attention because it often causes adverse drug reactions (ADRs), including dyspnea. However, little is known about the mechanism underlying dyspnea caused by ZTOI, which limits its clinical application. AIM OF THE STUDY: To investigate the major pathophysiologic signatures and underlying mechanism of ZTOI-related dyspnea. METHODS: Respiratory function detection was used to explore the pathophysiologic signature of dyspnea induced by ZTOI. UV-vis absorption spectroscopy and isothermal titration calorimetry were applied to test the interaction between ZTOI and hemoglobin (Hb). GC‒MS was used to identify the main components in ZTOI. Molecular docking, surface plasmon resonance, and circular dichroism spectroscopy were employed to test the reaction between ß-elemene and Hb. Western blot was performed to investigate the effect of ß-elemene on the hypoxia signaling pathway. RESULTS: The results showed that ZTOI-induced dyspnea was related to a decreased oxygen carrying capacity of Hb. The molecular interaction between ZTOI and Hb was proven. Notably, ß-elemene in ZTOI exhibited high binding affinity to Hb and altered its secondary structure. Furthermore, it was found that ß-elemene downregulated the expression of prolyl hydroxylase-domain protein 2 and upregulated the expression of hypoxia-inducible factor-1α. CONCLUSIONS: Our study is valuable for better understanding the pathophysiological characteristics and underlying mechanism of ZTOI to ensure its safe clinical application. We also provided a strategy to elucidate the underlying mechanism based on inspiration from clinical ADR phenotypes for investigating other medical products with ADRs in the clinic.

SETD2 deficiency accelerates sphingomyelin accumulation and promotes the development of renal cancer.

Patients with polycystic kidney disease (PKD) encounter a high risk of clear cell renal cell carcinoma (ccRCC), a malignant tumor with dysregulated lipid metabolism. SET domain-containing 2 (SETD2) has been identified as an important tumor suppressor and an immunosuppressor in ccRCC. However, the role of SETD2 in ccRCC generation in PKD remains largely unexplored. Herein, we perform metabolomics, lipidomics, transcriptomics and proteomics within SETD2 loss induced PKD-ccRCC transition mouse model. Our analyses show that SETD2 loss causes extensive metabolic reprogramming events that eventually results in enhanced sphingomyelin biosynthesis and tumorigenesis. Clinical ccRCC patient specimens further confirm the abnormal metabolic reprogramming and sphingomyelin accumulation. Tumor symptom caused by Setd2 knockout is relieved by myriocin, a selective inhibitor of serine-palmitoyl-transferase and sphingomyelin biosynthesis. Our results reveal that SETD2 deficiency promotes large-scale metabolic reprogramming and sphingomyelin biosynthesis during PKD-ccRCC transition. This study introduces high-quality multi-omics resources and uncovers a regulatory mechanism of SETD2 on lipid metabolism during tumorigenesis.

USP47 inhibits m6A-dependent c-Myc translation to maintain regulatory T cell metabolic and functional homeostasis.

The functional integrity of Tregs is interwoven with cellular metabolism; however, the mechanisms governing Treg metabolic programs remain elusive. Here, we identified that the deubiquitinase USP47 inhibited c-Myc translation mediated by the RNA N6-methyladenosine (m6A) reader YTHDF1 to maintain Treg metabolic and functional homeostasis. USP47 positively correlated with the tumor-infiltrating Treg signature in samples from patients with colorectal cancer and gastric cancer. USP47 ablation compromised Treg homeostasis and function in vivo, resulting in the development of inflammatory disorders, and boosted antitumor immune responses. USP47 deficiency in Tregs triggered the accumulation of the c-Myc protein and in turn exacerbated hyperglycolysis. Mechanistically, USP47 prevented YTHDF1 ubiquitination to attenuate the association of YTHDF1 with translation initiation machinery, thereby decreasing m6A-based c-Myc translation efficiency. Our findings reveal that USP47 directs m6A-dependent metabolic programs to orchestrate Treg homeostasis and suggest novel approaches for selective immune modulation in cancer and autoimmune diseases by targeting of USP47.

Superparamagnetic Composite Nanobeads Anchored with Molecular Glues for Ultrasensitive Label-free Proteomics.

Mass spectrometry has emerged as a mainstream technique for label-free proteomics. However, proteomic coverage for trace samples is constrained by adsorption loss during repeated elution at sample pretreatment. Here, we demonstrated superparamagnetic composite nanoparticles functionalized with molecular glues (MGs) to enrich proteins in trace human biofluid. We showed high protein binding (>95 %) and recovery (≈90 %) rates by anchor-nanoparticles. We further proposed a Streamlined Workflow based on Anchor-nanoparticles for Proteomics (SWAP) method that enabled unbiased protein capture, protein digestion and pure peptides elution in one single tube. We demonstrated SWAP to quantify over 2500 protein groups with 100 HEK 293T cells. We adopted SWAP to profile proteomics with trace aqueous humor samples from cataract (n=15) and wet age-related macular degeneration (n=8) patients, and quantified ≈1400 proteins from 5 µL aqueous humor. SWAP simplifies sample preparation steps, minimizes adsorption loss and improves protein coverage for label-free proteomics with previous trace samples.

Polystyrene microplastic exposure induces insulin resistance in mice via dysbacteriosis and pro-inflammation.

Microplastics (MPs) as emerging contaminants have become a global environmental problem. However, studies on the effects of MPs on metabolic diseases remain limited. Here, we evaluated the effects of polystyrene (PS), one of the most prominent types of MPs, on insulin sensitivity in mice fed with normal chow diet (NCD) or high-fat diet (HFD), and explained the underlying mechanisms. Mice fed with NCD or HFD both showed insulin resistance (IR) after PS exposure accompanied by increased plasma lipopolysaccharide and pro-inflammatory cytokines such as tumor necrosis factor-α and interleukin-1ß. Exposure to PS also resulted in a significant decrease in the richness and diversity of gut microbiota, particularly an increase in the relative abundance of Gram-negative bacteria such as Prevotellaceae and Enterobacteriaceae. Additionally, PS with a small particle size (5 µm) accumulated in the liver, kidneys and blood vessels of mice. Further analyses showed inhibition of the insulin signaling pathway in the liver of PS exposed mice, such as inhibition of IRS1 and decreased expression of PI3K. Hence, the mechanism of PS exposure to induce IR in mice might be mediated through regulating gut microbiota and PS accumulation in tissues, stimulating inflammation and inhibiting the insulin signaling pathway. In conclusion, PS might be a potential environmental contaminant that causes metabolic diseases associated with IR.

Sickle-like Inertial Microfluidic System for Online Rare Cell Separation and Tandem Label-Free Quantitative Proteomics (Orcs-Proteomics).

Label-free proteomics with trace clinical samples provides a wealth of actionable insights for personalized medicine. Clinically acquired primary cells, such as circulating tumor cells (CTCs), are usually with low abundance that is prohibitive for conventional label-free proteomics analysis. Here, we present a sickle-like inertial microfluidic system for online rare cell separation and tandem label-free proteomics (namely, Orcs-proteomics). Orcs-proteomics adopts a buffer system with 0.1% N-dodecyl ß-d-maltoside (DDM), 1 mM Tris (2-carboxyethyl) phosphine (TCEP), and 2 mM 2-chloroacetamide (CAA) for cell lysis and reductive alkylation. We demonstrate the application of Orcs-proteomics with 293T cells and manage to identify 913, 1563, 2271, and 2770 protein groups with 4, 13, 68, and 119 cells, respectively. We then spike MCF7 cells with white blood cells (WBCs) to simulate the patient's blood sample. Orcs-proteomics identifies more than 2000 protein groups with an average of 61 MCF7 cells. We further recruit two advanced breast cancer patients and collect 5 and 7 CTCs from each patient through minimally invasive blood drawing. Orcs-proteomics manages to identify 973 and 1135 protein groups for each patient. Therefore, Orcs-proteomics empowers rare cells simultaneously to be separated and counted for proteomics and provides technical support for personalized treatment decision making with rare primary patient samples.

Poly (N-vinylpyrrolidone) modification mitigates plasma protein corona formation on phosphomolybdate-based nanoparticles.

Phosphomolybdate-based nanoparticles (PMo12-based NPs) have been commonly applied in nanomedicine. However, upon contact with biofluids, proteins are quickly adsorbed onto the NPs surface to form a protein corona, which induces the opsonization and facilitates the rapid clearance of the NPs by macrophage uptake. Herein, we introduce a family of structurally homologous PMo12-based NPs (CDS-PMo12@PVPx(x = 0 ~ 1) NPs) capping diverse content of zwitterionic polymer poly (N-vinylpyrrolidone) (PVP) to regulate the protein corona formation on PMo12-based NPs. The fluorescence quenching data indicate that the introduction of PVP effectively reduces the number of binding sites of proteins on PMo12-based NPs. Molecular docking simulations results show that the contact surface area and binding energy of proteins to CDS-PMo12@PVP1 NPs are smaller than the CDS-PMo12@PVP0 NPs. The liquid chromatography-tandem mass spectrometry (LC-MS/MS) is further applied to analyze and quantify the compositions of the human plasma corona formation on CDS-PMo12@PVPx(x = 0 ~ 1) NPs. The number of plasma protein groups adsorption on CDS-PMo12@PVP1 NPs, compared to CDS-PMo12@PVP0 NPs, decreases from 372 to 271. In addition, 76 differentially adsorption proteins are identified between CDS-PMo12@PVP0 and CDS-PMo12@PVP1 NPs, in which apolipoprotein is up-regulated in CDS-PMo12@PVP1 NPs. The apolipoprotein adsorption onto the NPs is proposed to have dysoponic activity and enhance the circulation time of NPs. Our findings demonstrate that PVP grafting on PMo12-based NPs is a promising strategy to improve the anti-biofouling property for PMo12-based nanodrug design.

ZFP91 disturbs metabolic fitness and antitumor activity of tumor-infiltrating T cells.

Proper metabolic activities facilitate T cell expansion and antitumor function; however, the mechanisms underlying disruption of the T cell metabolic program and function in the tumor microenvironment (TME) remain elusive. Here, we show a zinc finger protein 91-governed (ZFP91-governed) mechanism that disrupts the metabolic pathway and antitumor activity of tumor-infiltrating T cells. Single-cell RNA-Seq revealed that impairments in T cell proliferation and activation correlated with ZFP91 in tissue samples from patients with colorectal cancer. T cell-specific deletion of Zfp91 in mice led to enhanced T cell proliferation and potentiated T cell antitumor function. Loss of ZFP91 increased mammalian target of rapamycin complex 1 (mTORC1) activity to drive T cell glycolysis. Mechanistically, T cell antigen receptor-dependent (TCR-dependent) ZFP91 cytosolic translocation promoted protein phosphatase 2A (PP2A) complex assembly, thereby restricting mTORC1-mediated metabolic reprogramming. Our results demonstrate that ZFP91 perturbs T cell metabolic and functional states in the TME and suggest that targeting ZFP91 may improve the efficacy of cancer immunotherapy.

ZFP91 is required for the maintenance of regulatory T cell homeostasis and function.

Autophagy programs the metabolic and functional fitness of regulatory T (T reg) cells to establish immune tolerance, yet the mechanisms governing autophagy initiation in T reg cells remain unclear. Here, we show that the E3 ubiquitin ligase ZFP91 facilitates autophagy activation to sustain T reg cell metabolic programming and functional integrity. T reg cell-specific deletion of Zfp91 caused T reg cell dysfunction and exacerbated colonic inflammation and inflammation-driven colon carcinogenesis. TCR-triggered autophagy induction largely relied on T reg cell-derived ZFP91 to restrict hyperglycolysis, which is required for the maintenance of T reg cell homeostasis. Mechanistically, ZFP91 rapidly translocated from the nucleus to the cytoplasm in response to TCR stimulation and then mediated BECN1 ubiquitination to promote BECN1-PIK3C3 complex formation. Therefore, our results highlight a ZFP91-dependent mechanism promoting TCR-initiated autophagosome maturation to maintain T reg cell homeostasis and function.

Methylation-Induced Silencing of ALDH2 Facilitates Lung Adenocarcinoma Bone Metastasis by Activating the MAPK Pathway.

Bone metastasis (BM) dramatically reduces the quality of life and life expectancy in lung adenocarcinoma (LUAD) patients. There is an urgent need to identify potential biomarkers for application in the treatment of this deadly disease. We compared patient BM, LUAD, and para-LUAD tissues using proteomic analysis and identified aldehyde dehydrogenase 2 (ALDH2), which can detoxify acetaldehyde to acetic acid, as one of the key regulators in lung tumor metastasis. Both the mRNA and protein levels of ALDH2 were significantly lower in tumor tissues than in normal tissues and were lowest in BM tissues with increased migratory capacity. Also, ALDH2 was upregulated following treatment with 5-azacitidine, a DNA methyltransferase inhibitor, in H1299, H460, and HCC827 cells. Further, we identified a potential methylated CpG island 3, with the longest methylated CpG island area in ALDH2, and performed bisulfite genomic sequencing of these sites. An average of 78.18% of the sites may be methylated in CpG island 3. Knockdown of DNA (cytosine-5)-methyltransferase 3A (DNMT3A) and methylated CpG binding protein 4 (MBD4) upregulated ALDH2 expression. ALDH2 functions as a mitogen-activated protein kinase (MAPK) upstream to inhibit cell proliferation and migration, promote cell apoptosis, and alter the epithelial-mesenchymal transition (EMT) by elevating E-cadherin and attenuating vimentin. Cell proliferation and migration were inhibited after the addition of the JNK inhibitor SP600125. In the multivariate analysis, M stage (p = 0.003), ALDH2 (p = 0.008), and phospho-c-Jun N-terminal kinase (p-JNK) (p = 0.027) expression were independent prognostic factors for overall survival in patients with BM. In vivo experiments also showed that ALDH2 expression could suppress tumor formation. In summary, we found that ALDH2 expression is a prognostic factor for BM in LUAD and that DNMT3A and MBD4 repression of ALDH2 via a MAPK-dependent pathway alters the EMT process, indicating that these proteins could act as potential biomarkers or therapeutic targets for LUAD metastasis.

Mouse Double Minute 2 Homolog-Mediated Ubiquitination Facilitates Forkhead Box P3 Stability and Positively Modulates Human Regulatory T Cell Function.

Regulatory T cells (Treg cells) are essential for maintaining immune tolerance, and the dysfunction of Treg cells may cause autoimmune diseases and tumors. Forkhead box P3 (FOXP3) is the key transcription factor controlling Treg cell development and suppressive function. Mouse double minute 2 homolog (MDM2), an E3 ubiquitin ligase, has been identified as an oncoprotein that mediates the ubiquitination and degradation of tumor suppressor p53; however, whether it has functions in Treg cells remains unknown. Here, we demonstrate that MDM2 positively regulates human Treg cell suppressive function via its mediated ubiquitination and stabilization of FOXP3. Knockdown of MDM2 with shRNA in human primary Treg cells leads to the impaired ability of FOXP3 to regulate the expression levels of downstream genes and the attenuated suppressive capacity of Treg cells, due to FOXP3 instability. Consistently, MDM2 overexpression in human Treg cells enhances FOXP3 stability and Treg cell suppressive capacity. Mechanistically, MDM2 interacts with FOXP3, and mainly mediates monoubiquitination and polyubiquitination of FOXP3, thus stabilizing the protein level of FOXP3. We have also found lysine residues in FOXP3 required for MDM2-mediated ubiquitination. In addition, TCR/CD28 signaling upregulates the expression level of MDM2 and its mediated FOXP3 ubiquitination in human Treg cells. Therefore, our findings reveal that MDM2 in Treg cells could be a potential therapeutic target for treating autoimmune diseases and tumors.

Triptonide acts as a novel antiprostate cancer agent mainly through inhibition of mTOR signaling pathway.

BACKGROUND: The increasing incidence of prostate cancer (PCa) indicates an urgent need for the development of new effective drugs in PCa therapy. Triptonide has been reported to have a strong inhibition activity in cancers through screening of Chinese herbal medicine. This study aims to investigate the effects of triptonide on anti-PCa activity and its mechanisms. METHODS: Three human advanced PCa cell lines PC3, DU145, and LNCap, and a human normal prostate epithelial cell line RWPE were treated with a range (0, 1.25, 2.5, 5, 10, 20, 40, 80, 160, and 320 nM) of triptonide concentrations for 72 hours respectively. Then, cell viability was assessed by cell counting kit-8. PCa cells were treated with different doses (0-20 nM) of triptonide for 72 hours. Cell cycle and apoptosis were assessed by flow cytometry assays. Nude mice bearing human PCa xenografts were intraperitoneally injected daily with either triptonide (10 mg/kg/d) or phosphate-buffered saline as a control for 35 days. RNA-sequencing (RNA-seq) was performed by an Illumina Hiseq Sequencing platform and confirmed by a real-time polymerase chain reaction. Gene Ontology, Kyoto Encyclopedia of Genes and Genomes pathway analysis, and ingenuity pathway analysis were used to analyze RNA-seq results. RESULTS: Triptonide effectively inhibits the proliferation of human PCa cells PC3, DU145, and LNCap in vitro with their IC50 values as 11.961, 10.259, and 12.012 nM, respectively. Triptonide (10 mg/kg) potently inhibits the growth of PCa cell xenografts in vivo at an inhibition rate of over 97.95%. Treatment with triptonide (5 nM) significantly promotes cell apoptosis and retaining cell-cycle arrest in the G2/M phase. RNA-seq data revealed that total of 936 genes were upregulated or downregulated in triptonide treated. Moreover, the phosphorylation of mechanistic target of rapamycin (mTOR) and the downstream protein p70S6K were both inhibited, most obviously in PCa cells. CONCLUSIONS: Our findings suggest that triptonide can efficaciously suppress PCa growth in vitro and in vivo via inhibiting the phosphorylation of mTOR and the activities of related downstream signaling pathways.

Regulation of T cell differentiation and function by ubiquitin-specific proteases.

T cells play critical roles in immune responses to pathogens, autoimmunity, and antitumor immunity. During the past few decades, increasing numbers of studies have demonstrated the significance of protein ubiquitination in T cell-mediated immunity. Several E3 ubiquitin ligases and deubiquitinases (DUBs) have been identified as either positive or negative regulators of T cell development and function. In this review, we mainly focus on the roles of DUBs (especially ubiquitin-specific proteases (USPs)) in modulating T cell differentiation and function, as well as the molecular mechanisms. Understanding how T cell development and function is regulated by ubiquitination and deubiquitination will provide novel strategies for treating infection, autoimmune diseases, and cancer.

A Chinese medicine formula (Jinqi Jiangtang Tablet): A review on its chemical constituents, quality control, pharmacokinetics studies, pharmacological properties and clinical applications.

ETHNOPHARMACOLOGICAL RELEVANCE: Diabetes belongs to the category of "Xiao Ke Zheng" in the field of traditional Chinese medicine (TCM) and has been listed as one of the predominant diseases of TCM. Jinqi Jiangtang Tablet (JQJTT), a Chinese medicine formula composed of three herbs (Coptis chinensis, Astragalus membranaceus and Lonicera japonica), is an effective prescription for diabetes proved by randomized, double-blind, placebo-controlled trials. AIM OF THE REVIEW: To analyze systematic and up-to-date classification information on the study of JQJTT, explain the problems existing in the current research of classics formulas, and further propose the solution, providing a reference for future study. MATERIALS AND METHODS: Literatures on JQJTT were collected from a variety of databases, including PubMed, Google Scholar, Science Direct, Wiley, Web of Science, China National Knowledge Infrastructure, and WanFang Data. Information was also collected from books and reports, such as Chinese Pharmacopoeia, Chinese herbal classic books and reports of re-evaluation on post-marketing drugs conducted by companies. RESULTS: There are some problems for JQJTT: the quality control system is not perfect, the pharmacological functional mechanism is not fully explained, and clinical applications need to be reevaluated. A few of research directions for future research are proposed: (i) the chemical quality evaluation combined with bioassay to evaluate quality; (ii) interaction based on gut microbiota in vivo; (iii) the effects of interaction between components of the polypharmacy on pharmacokinetic studies; (iv) interaction mechanism between drugs and endogenous small molecules and biomacromolecules; (v) evidence-based medicine reconfirmation for clinical evaluation. CONCLUSIONS: The recent research status of JQJTT was summarized and analyzed from the aspects of chemical constituents, quality control, pharmacokinetics studies, pharmacological properties and clinical applications. This review takes JQJTT as an example, points out some typical problems and opinions about the TCM formulas, highlights the importance of the secondary development of classical formula, and lays a foundation for the further research.

Preparation and Characterization of Cellulose Grafted with Epoxidized Soybean Oil Aerogels for Oil-Absorbing Materials.

The absorbent materials synthesized from biosources with low cost and high selectivity for oils and organic solvents have attracted increasing attention in the field of oil spillage and discharge of organic chemicals. We developed a convenient surface-grafting method to prepare efficient and recyclable biobased aerogels from epoxidized soybean oil (ESO)-modified cellulose at room temperature. The porous network-like structure of the cellulose aerogel was still fully retained after undergoing hydrophobic modification with ESO. Moreover, the modified aerogels possessed excellent hydrophobicity with a water contact angle of 132.6°. Moreover, the absorbent ability of the hydrophobic cellulose aerogels was systematically assessed. The results showed that modified aerogels could retain more than 90% absorption capacity even after 30 absorption-desorption cycles, indicating that the ESO-grafted cellulose aerogels have practical applications in the oil-water separation from industrial wastewater and oil-leakage removal.

Berberine alleviates insulin resistance by reducing peripheral branched-chain amino acids.

Increased circulating branched-chain amino acids (BCAAs) have been involved in the pathogenesis of obesity and insulin resistance (IR). However, evidence relating berberine (BBR), gut microbiota, BCAAs, and IR is limited. Here, we showed that BBR could effectively rectify steatohepatitis and glucose intolerance in high-fat diet (HFD)-fed mice. BBR reorganized gut microbiota populations under both the normal chow diet (NCD) and HFD. Particularly, BBR noticeably decreased the relative abundance of BCAA-producing bacteria, including order Clostridiales; families Streptococcaceae, Clostridiaceae, and Prevotellaceae; and genera Streptococcus and Prevotella. Compared with the HFD group, predictive metagenomics indicated a reduction in the proportion of gut microbiota genes involved in BCAA biosynthesis but the enrichment genes for BCAA degradation and transport by BBR treatment. Accordingly, the elevated serum BCAAs of HFD group were significantly decreased by BBR. Furthermore, the Western blotting results implied that BBR could promote the BCAA catabolism in the liver and epididymal white adipose tissues of HFD-fed mice by activation of the multienzyme branched-chain α-ketoacid dehydrogenase complex (BCKDC), whereas by inhibition of the phosphorylation state of BCKDHA (E1α subunit) and branched-chain α-ketoacid dehydrogenase kinase (BCKDK). The ex vivo assay further confirmed that BBR could increase BCAA catabolism in both AML12 hepatocytes and 3T3-L1 adipocytes. Finally, data from healthy subjects and diabetics confirmed that BBR could improve glycemic control and modulate circulating BCAAs. Together, our findings clarified BBR improving IR associated not only with gut microbiota alteration in BCAA biosynthesis but also with BCAA catabolism in liver and adipose tissues.

Ring finger protein 31-mediated atypical ubiquitination stabilizes forkhead box P3 and thereby stimulates regulatory T-cell function.

The CD4+CD25+FOXP3+ regulatory T (Treg) cells are critical for maintaining immune tolerance in healthy individuals and are reported to restrict anti-inflammatory responses and thereby promote tumor progression, suggesting them as a target in the development of antitumor immunotherapy. Forkhead box P3 (FOXP3) is a key transcription factor governing Treg lineage differentiation and their immune-suppressive function. Here, using Treg cells, as well as HEK-293T and Jurkat T cells, we report that the stability of FOXP3 is directly and positively regulated by the E3 ubiquitin ligase ring finger protein 31 (RNF31), which catalyzes the conjugation of atypical ubiquitin chains to the FOXP3 protein. We observed that shRNA-mediated RNF31 knockdown in human Treg cells decreases FOXP3 protein levels and increases levels of interferon-γ, resulting in a Th1 helper cell-like phenotype. Human Treg cells that ectopically expressed RNF31 displayed stronger immune-suppressive capacity, suggesting that RNF31 positively regulates both FOXP3 stability and Treg cell function. Moreover, we found that RNF31 is up-regulated in Treg cells that infiltrate human gastric tumor tissues compared with their counterparts residing in peripheral and normal tissue. We also found that elevated RNF31 expression in intratumoral Treg cells is associated with poor survival of gastric cancer patients, suggesting that RNF31 supports the immune-suppressive functions of Treg cells. Our results suggest that RNF31 could be a potential therapeutic target in immunity-based interventions against human gastric cancer.

Adipose Tissue-Resident Regulatory T Cells.

Tissue-resident immune cells play critical roles in regulating tissue function and homeostasis. Obesity-associated visceral adipose tissue inflammation is attributed to the accumulation of M1 macrophages which produce inflammatory cytokines like TNF-α, IL-6, and expansion of effector T cells like Th1 cells, CD8+ cytotoxic T cells which produce interferon-γ to further add to the severity of inflammation in the visceral adipose tissue. Regulatory T cells have been reported to exert key roles in suppressing inflammation, thus maintaining the homeostasis of immune responses, and visceral adipose Tregs exert critical roles in defending against obesity-associated metabolic disorders. They inhibit the infiltration of effector T cells and facilitate the reconstitution of adipose tissue macrophages from M1 to M2 phenotype. What is more, they can take up lipids from the adipocytes through CD36 which is driven by PPARγ. Here we review the recent progress in adipose tissue-resident regulatory T cells (Tregs), a subpopulation of CD4+ T cells which suppress adipose tissue inflammation.

Treating metastatic triple negative breast cancer with CD44/neuropilin dual molecular targets of multifunctional nanoparticles.

Metastasis of cancer makes up the vast majority of cancer-related deaths, and it usually initiates from tumor cells invasiveness and develops through tumor neovasculature. In this work, we have fabricated a CD44/neuropilin dual receptor-targeting nanoparticulate system (tLyP-1-HT NPs) with endogenous or FDA approved components for treating metastatic triple negative breast cancer (TNBC). The enhanced specific targeting of tLyP-1-HT NPs to both metastatic tumor cells and metastasis-supporting tumor neovasculature was contributed by means of CD44/neuropilin dual receptor-mediated interaction. The NPs not only effectively suppress the invasive capability of tumor cells themselves, but also significantly restrain the metastasis incidence via extravasation as well as the eventual colonization in lungs. In all the three types of TNBC-bearing mice models, orthotopic, post-metastasis and metastasis prevention models, the docetaxel-loaded tLyP-1-HT NPs exhibited markedly enhanced anti-tumor and anti-metastasis efficacy. The inhibitory rates of tLyP-1-HT NPs against orthotopic tumor growth and lung metastasis achieved 79.6% and 100%, respectively. The metastasis inhibition rate and life extension rate of the tLyP-1-HT NPs against post-pulmonary metastasis mice reached 85.1% and up to 62.5%, respectively. All the results demonstrated the designed dual receptor-targeting multifunctional NPs hold great potential in treating metastatic TNBC and lung metastasis.