HucMSC-Ex alleviates DSS-induced colitis in mice by decreasing mast cell activation via the IL-33/ST2 axis.

BACKGROUND: Inflammatory bowel disease (IBD) is a chronic inflammatory disease that poses challenges in terms of treatment. The precise mechanism underlying the role of human umbilical cord mesenchymal stem cell-derived exosome (HucMSC-Ex) in the inflammatory repair process of IBD remains elusive. Mucosal mast cells accumulate within the intestinal tract and exert regulatory functions in IBD, thus presenting a novel target for addressing this intestinal disease. METHODS: A mouse model of Dextran Sulfate Sodium (DSS)-induced colitis was established and hucMSC-Ex were administered to investigate their impact on the regulation of intestinal mast cells. An in vitro co-culture model using the human clonal colorectal adenocarcinoma cell line (Caco-2) and human mast cell line (LAD2) was also established for further exploration of the effect of hucMSC-Ex. RESULTS: We observed the accumulation of mast cells in the intestines of patients with IBD as well as mice. In colitis mice, there was an upregulation of mast cell-related tryptase, interleukin-33 (IL-33), and suppression of tumorigenicity 2 receptor (ST2 or IL1RL1), and the function of the intestinal mucosal barrier related to intestinal tight junction protein was weakened. HucMSC-Ex treatment significantly reduced mast cell infiltration and intestinal damage. In the co-culture model, a substantial number of mast cells interact with the epithelial barrier, triggering activation of the IL-33/IL1RL1 (ST2) pathway and subsequent release of inflammatory factors and trypsin. This disruption leads to aberrant expression of tight junction proteins, which can be alleviated by supplementation with hucMSC-Ex. CONCLUSION: Our results suggest that hucMSC-Ex may reduce the release of mast cell mediators via the IL-33/IL1RL1 (ST2) axis, thereby mitigating its detrimental effects on intestinal barrier function.

Short-Chain Fatty Acids Weaken Ox-LDL-Induced Cell Inflammatory Injury by Inhibiting the NLRP3/Caspase-1 Pathway and Affecting Cellular Metabolism in THP-1 Cells.

Short-chain fatty acids (SCFAs) are important anti-inflammatory metabolites of intestinal flora. Oxidized low-density lipoprotein (ox-LDL)-induced macrophage activation is critical for the formation of atherosclerosis plaque. However, the association between SCFAs and ox-LDL-induced macrophage activation with respect to the formation of atherosclerosis plaque has not yet been elucidated. The present study investigated whether SCFAs (sodium acetate, sodium propionate, and sodium butyrate) can affect ox-LDL-induced macrophage activation and potential signaling pathways via regulation of the expression of the NLRP3/Caspase-1 pathway. Using human monocyte-macrophage (THP-1) cells as a model system, it was observed that ox-LDL not only induced cell inflammatory injury but also activated the NLRP3/Caspase-1 pathway. The exogenous supplementation of three SCFAs could significantly inhibit cell inflammatory injury induced by ox-LDL. Moreover, three SCFAs decreased the expression of IL-1ß and TNF-α via the inactivation of the NLRP3/Caspase-1 pathway induced by ox-LDL. Furthermore, three SCFAs affected cellular metabolism in ox-LDL-induced macrophages, as detected by untargeted metabolomics analysis. The results of the present study indicated that three SCFAs inhibited ox-LDL-induced cell inflammatory injury by blocking the NLRP3/Caspase-1 pathway, thereby improving cellular metabolism. These findings may provide novel insights into the role of SCFA intervention in the progression of atherosclerotic plaque formation.

Emerging roles of mesenchymal stem cell-derived exosomes in gastrointestinal cancers.

Gastrointestinal tumours are the most common solid tumours, with a poor prognosis and remain a major challenge in cancer treatment. Mesenchymal stem cells (MSC) are multipotent stromal cells with the potential to differentiate into multiple cell types. Several studies have shown that MSC-derived exosomes have become essential regulators of intercellular communication in a variety of physiological and pathological processes. Notably, MSC-derived exosomes support or inhibit tumour progression in different cancers through the delivery of proteins, RNA, DNA, and bioactive lipids. Herein, we summarise current advances in MSC-derived exosomes in cancer research, with particular reference to their role in gastrointestinal tumour development. MSC-derived exosomes are expected to be a novel potential strategy for the treatment of gastrointestinal cancers.

The role of NOD2 in intestinal immune response and microbiota modulation: A therapeutic target in inflammatory bowel disease.

As an intracellular pattern recognition receptor (PPR), the nucleotide-binding oligomerization domain-containing protein 2 (NOD2) triggers a cascade of immune responses. Previous studies of NOD2 regarding inflammatory bowel disease (IBD) mainly focused on the relevance of NOD2 mutations and loss within the disease onset and progression. With increasing research, more studies are exploring other functional roles and clinical applications of NOD2. In this review, we discuss the role of NOD2 in intestinal immune response and microbiota modulation in IBD and explore its clinical potential as a therapeutic target for IBD.

Farnesoid-X receptor as a therapeutic target for inflammatory bowel disease and colorectal cancer.

Farnesoid-X receptor (FXR), as a nuclear receptor activated by bile acids, is a vital molecule involved in bile acid metabolism. Due to its expression in immune cells, FXR has a significant effect on the function of immune cells and the release of chemokines when immune cells sense changes in bile acids. In addition to its regulation by ligands, FXR is also controlled by post-translational modification (PTM) activities such as acetylation, SUMOylation, and methylation. Due to the high expression of FXR in the liver and intestine, it significantly influences intestinal homeostasis under the action of enterohepatic circulation. Thus, FXR protects the intestinal barrier, resists bacterial infection, reduces oxidative stress, inhibits inflammatory reactions, and also acts as a tumor suppressor to impair the multiplication and invasion of tumor cells. These potentials provide new perspectives on the treatment of intestinal conditions, including inflammatory bowel disease (IBD) and its associated colorectal cancer (CRC). Moreover, FXR agonists on the market have certain organizational heterogeneity and may be used in combination with other drugs to achieve a greater therapeutic effect. This review summarizes current data on the role of FXR in bile acid metabolism, regulation of immune cells, and effects of the PTM of FXR. The functions of FXR in intestinal homeostasis and potential application in the treatment of IBD and CRC are discussed.

The gut metagenomics and metabolomics signature in patients with inflammatory bowel disease.

Inflammatory bowel disease (IBD), a chronic gut immune dysregulation and dysbiosis condition is rapidly increasing in global incidence. Regardless, there is a lack of ideal diagnostic markers, while conventional treatment provides scarce desired results, thus, the exploration for better options. Changes in the gut microbial composition and metabolites either lead to or are caused by the immune dysregulation that characterizes IBD. This study examined the fecal metagenomics and metabolomic changes in IBD patients. A total of 30 fecal samples were collected from 15 IBD patients and 15 healthy controls for 16S rDNA gene sequencing and UHPLC/Q-TOF-MS detection of metabolomics. Results showed that there was a severe perturbation of gut bacteria community composition, diversity, metabolites, and associated functions and metabolic pathways in IBD. This included a significantly decreased abundance of Bacteroidetes and Firmicutes, increased disease-associated phyla such as Proteobacteria and Actinobacteria, and increased Escherichia coli and Klebsiella pneumoniae in IBD. A total of 3146 metabolites were detected out of which 135 were differentially expressed between IBD and controls. Metabolites with high sensitivity and specificity in differentiating IBD from healthy individuals included 6,7,4'-trihydroxyisoflavone and thyroxine 4'-o-.beta.-d-glucuronide (AUC = 0.92), normorphine and salvinorin a (AUC = 0.90), and trichostachine (AUC = 0.91). Moreover, the IBD group had significantly affected pathways including primary bile acid biosynthesis, vitamin digestion and absorption, and carbohydrate metabolism. This study reveals that the combined evaluation of metabolites and fecal microbiome can be useful to discriminate between healthy subjects and IBD patients and consequently serve as therapeutic and diagnostic targets.

[Mycoplasma pneumoniae toxin of community-acquired respiratory distress syndrome (CARDS) promotes autophagy of THP-1 cells and activates NLRP3 inflammasomes].

Objective To study the mechanism of community-acquired respiratory distress syndrome (CARDS) toxin of Mycoplasma pneumoniae (Mp) inducing THP-1 cell autophagy and the activation of pyrin domain containing the nucleotide-binding oligomerization domain-like receptor family 3 (NLRP3). Methods The recombinant CARDS (rCARDS) Mp toxin was obtained by Escherichia coli expression system, and THP-1 cells were treated with the toxin at the concentrations of 5 and 10 µg/mL for 20, 40 minutes, 1, 2 and 3 hours. The expression of autophagy-related proteins beclin-1, LC3II and P62 of THP-1 cells were determined by Western blot; the gene expression of NLRP3, caspase-1 and interleukin 1ß (IL-1ß) were detected by real-time quantitative PCR; and the level of reactive oxygen species (ROS) of THP-1 cells was tested by DCFH-DA staining. Results Compared with the control group, when treated with rCARDS toxin for 1 hour, the expression of beclin-1, LC3 and P62 significant increased. When treated with rCARDS toxin for 2 and 3 hours, the expression of beclin-1, LC3 and P62 significant decreased. When treated with rCARDS toxin for 20 and 40 minutes, the NLRP3 gene expression had no significant difference between the groups treated with the concentration of 5 and 10 µg/mL rCARDS toxin. NLRP3 gene expression in the groups treated with rCARDS toxin was higher than that in the control group in the whole experiment. When treated with rCARDS toxin for 1 hour and 2 hours, the NLRP3 gene expression of the 10 µg/mL group was significant higher than that in the 5 µg/mL group. When treated with rCARDS toxin for 3 hours, the NLRP3 gene expression of the 10 µg/mL group and 5 µg/mL group was lower than that in the groups treated for 2 hours. When treated with rCARDS toxin for 40 minutes, 1 hour and 2 hours, the caspase-1 mRNA expression of rCARDS toxin groups was higher than that in the control group. When treated for 40 minutes, 1, 2 and 3 hours, the caspase-1 gene expression of the 10 µg/mL group was significantly higher than that in the 5 µg/mL group. Compared to the control group, when treated with rCARDS toxin for 20 and 40 minutes, IL-1ß gene expression had no significant difference. When the time prolonged to 1 hour and 3 hours, the levels of IL-1ß mRNA expression and ROS had a significant increase in a dose-dependent manner in all groups. Conclusion CARDS Mp toxin can activate NLRP3 inflammasomes and induce cell autophagy in THP-1 cells.

Simultaneous HPLC determination of ergosterol and 22,23-dihydroergosterol in Flammulina velutipes sterol-loaded microemulsion.

An efficient HPLC method was developed and validated for the simultaneous determination of ergosterol and 22,23-dihydroergosterol in Flammulina velutipes sterol-loaded microemulsions (FVSMs). The different chromatographic conditions for in vitro and in vivo determinations were investigated, with the application examined by tissue distribution. Chromatographic separation was achieved on an Inertsil ODS-SP (250 × 4.6 mm, 5 µm) analytical column using a mobile phase of 98% methanol (in vitro), and 93% methanol for stomach samples and 96% methanol for other samples (in vivo) at 1.0 mL/min. The sterol content was detected at 282 nm. The established in vitro linearity ranges for ergosterol and 22,23-dihydroergosterol were 0.58-72.77 µg/mL (r1 = 0.9999) and 0.59-73.25 µg/mL (r2 = 0.9999), respectively, with the biological (in vivo) samples following the same trend. The accuracy of the method was >99% (in vitro) and between 93%-108% (in vivo). The LOQ was 2.15 µg/L for ergosterol and 2.41 µg/L for 22,23-dihydroergosterol in the in vitro studies. Also, the precisions met the acceptance criterion. These results indicate that the established HPLC method was specific, linear, accurate, precise and sensitive for the separation and simultaneous determination of ergosterol and 22,23-dihydroergosterol.

Enhanced oral bioavailability and tissue distribution of a new potential anticancer agent, Flammulina velutipes sterols, through liposomal encapsulation.

This study innovatively investigated the anticancer effect of Flammulina velutipes sterols (FVSs), the in vivo pharmacokinetics, and the tissue distribution of FVS-loaded liposomes. The FVS consisting of mainly 54.8% ergosterol and 27.9% 22,23-dihydroergosterol exhibited evident in vitro antiproliferative activity (liver HepG-2, IC50 = 9.3 µg mL(-1); lung A549, IC50 = 20.4 µg mL(-1)). To improve the poor solubility of FVS, F. velutipes sterol liposome (FVSL) was originally prepared. The encapsulation efficiency of ergosterol was 71.3 ± 0.1% in FVSL, and the encapsulation efficiency of 22,23-dihydroergosterol was 69.0 ± 0.02% in FVSL. In comparison to its two free sterol counterparts, the relative bioavailability of ergosterol and 22,23-dihydroergosterol in FVSL was 162.9 and 244.2%, respectively. After oral administration in Kunming mice, the results of tissue distribution demonstrated that the liposomal FVS was distributed mostly in liver and spleen. The drug was eliminated rapidly within 4 h. These findings support the fact that FVS, a potential nutraceutical and an effective drug for the treatment of liver cancer, could be encapsulated in liposomes for improved solubility and bioavailability.

Cytotoxic effect of novel Flammulina velutipes sterols and its oral bioavailability via mixed micellar nanoformulation.

The aim of this study was to investigate the anti-tumor effect of sterols initially separated from Flammulina velutipes and the pharmacokinetics and tissue distribution after oral administration of F. velutipes sterol nanomicelles (FVSNs). F. velutipes sterol (FVS) consisted of mainly ergosterol (54.78%), 22,23-dihydroergosterol (27.94%) and ergost-8(14)-ene-3ß-ol (discovered for the first time in F. velutipes). In vitro cytotoxicity assay of FVS against U251 cells and HeLa cells showed that at 72h treatment, the FVS (IC50=23.42µg/mL) exhibited strong inhibitory effect against U251 cells, even overwhelmed the standard anti-tumor drug (5-fluorouracil) to an extent, while the HeLa cells were not significantly susceptible to the FVS. To improve the solubility and bioavailability of FVS, a model for insoluble anti-tumor drugs, FVSNs were prepared. In vitro characterization of FVSNs revealed satisfactory size distribution, loading capacity and encapsulation efficiency. Pharmacokinetic study in SD rats demonstrated that the mixed micellar nanoformulation significantly enhanced the bioavailability of FVS than free drug. Additionly, tissue distribution in mice manifested that the biodistribution of FVSNs as compared to the free FVS suspension were significantly improved. In conclusion, the nanomicelles developed in our study provided a promising delivery system for enhancing the oral bioavailability and selective biodistribution of FVS, a potential anti-tumor agent.

Enhanced oral bioavailability of a sterol-loaded microemulsion formulation of Flammulina velutipes, a potential antitumor drug.

PURPOSE: To investigate the growth inhibition activity of Flammulina velutipes sterol (FVS) against certain human cancer cell lines (gastric SGC and colon LoVo) and to evaluate the optimum microemulsion prescription, as well as the pharmacokinetics of encapsulated FVS. METHODS: Molecules present in the FVS isolate were identified by gas chromatography/mass spectrometry analysis. The cell viability of FVS was assessed with methyl thiazolyl tetrazolium (MTT) bioassay. Based on the solubility study, phase diagram and stability tests, the optimum prescription of F. velutipes sterol microemulsions (FVSMs) were determined, followed by FVSMs characterization, and its in vivo pharmacokinetic study in rats. RESULTS: The chemical composition of FVS was mainly ergosterol (54.8%) and 22,23-dihydroergosterol (27.9%). After 72 hours of treatment, both the FVS (half-maximal inhibitory concentration [IC(50)] = 11.99 µg · mL(-1)) and the standard anticancer drug, 5-fluorouracil (IC(50) = 0.88 µg · mL(-1)) exhibited strong in vitro antiproliferative activity against SGC cells, with IC(50) > 30.0 µg · mL(-1); but the FVS performed poorly against LoVo cells (IC(50) > 40.0 µg · mL(-1)). The optimal FVSMs prescription consisted of 3.0% medium chain triglycerides, 5.0% ethanol, 21.0% Cremophor EL and 71.0% water (w/w) with associated solubility of FVS being 0.680 mg · mL(-1) as compared to free FVS (0.67 µg · mL(-1)). The relative oral bioavailability (area-under-the-curve values of ergosterol and 22,23-dihydroergosterol showed a 2.56-fold and 4.50-fold increase, respectively) of FVSMs (mean diameter ~ 22.9 nm) as against free FVS were greatly enhanced. CONCLUSION: These results indicate that the FVS could be a potential candidate for the development of an anticancer drug and it is readily bioavailable via microemulsion formulations.

[Nanoscale drug carriers for traditional Chinese medicine research and development].

Nanocarriers generally made of natural or artificial polymers ranging in size from about 10-1 000 nm, possess versatile properties suitable for drug delivery, including good biocompatibility and biodegradability, potential capability of targeted delivery and controlled release of incorporated drugs, and have been extensively used in the development of new drug delivery systems (DDS). These types of nano-DDS have considerable potential to traditional Chinese medicine (TCM), and recently have attracted increasing efforts on the TCM research and development. In this review, the recently published literature worldwide is covered to describe the latest advances in the applications as TCM delivery carriers, and to highlight the characteristics and preparation methods of some selected examples of promising nanocarriers such as nanoparticles, lipid nanoparticles, nanoemulsions, nanomicelles and nanoliposomes.