[Protective effects of galacto-oligosaccharide and polydextrose on Caco-2 intestinal cell barrier injury model].

OBJECTIVE: To explore the protective effect of different ratios of galactose oligosaccharide(GOS) and polydextrose(PDX) on intestinal cell barrier damage model of Caco-2. METHODS: The same batch of Caco-2 cells were cultured to form a cell barrier model and randomly divided into damaged model group without calcium, calcium-containing blank control group(1.8 mmol/L Ca~(2+)), low-ratio/low-dose group(1.8 mmol/L Ca~(2+)+2 mg/mL GOS+2 mg/mL PDX) and low-ratio/medium-dose group(1.8 mmol/L Ca~(2+)+4 mg/mL GOS+4 mg/mL PDX), low-ratio/high-dose group(1.8 mmol/L Ca~(2+)+8 mg/mL GOS+8 mg/mL PDX) and high-ratio/low-dose group(1.8 mmol/L Ca~(2+)+0.8 mg/mL GOS+3.2mg/mL PDX), high-ratio/medium-dose group(1.8 mmol/L Ca~(2+)+1.6 mg/mL GOS+6.4 mg/mL PDX), high-ratio/high-dose group(1.8 mmol/L Ca~(2+)+3.2mg/mL GOS+12.8 mg/mL PDX), a total of 8 groups, three parallel groups were performed in each group. The Trans Epithelial Electrical Resistance value and apparent permeability coefficient value of each group were determined after 4 d culture, and the morphology of tight junction proteins ZO-1, Occludin and Claudin-1 were observed by immunofluorescence method, and the expression levels of inflammatory related factors in each group were determined by protein microarray method. RESULTS: Compared with damaged model group, TEER ratio in calcium-containing blank control group was significantly increased(P<0.05), while Papp value was significantly decreased(P<0.05);Compared with calcium-containing blank control group, TEER ratio in low-ratio/medium-dose group and high-ratio/high-dose group was significantly increased(P<0.05) while Papp value was significantly decreased(P<0.05), and they could significantly down-regulate some inflammatory response related cytokines. The cell barrier was intact in all groups except for the compact junction protein structure in the model group. CONCLUSION: Compared with Ca~(2+) alone, the combination of two prebiotics can enhance the density of Caco-2 cell barrier and reduced the permeability of cell bypass. And it can significantly reduce the expression level of some inflammatory cytokines and effectively protect the intestinal cell barrier.

Engineered Toll-like Receptor Nanoagonist Binding to Extracellular Matrix Elicits Safe and Robust Antitumor Immunity.

Cancer immunotherapy, such as the Toll-like receptor (TLR) agonist including CpG oligodeoxynucleotide, has shown potency in clinical settings. However, it is still confronted with multiple challenges, which include the limited efficacy and severe adverse events caused by the rapid clearance and systemic diffusion of CpG. Here we report an improved CpG-based immunotherapy approach composed of a synthetic extracellular matrix (ECM)-anchored DNA/peptide hybrid nanoagonist (EaCpG) via (1) a tailor designed DNA template that encodes tetramer CpG and additional short DNA moieties, (2) generation of elongated multimeric CpG through rolling circle amplification (RCA), (3) self-assembly of densely packaged CpG particles composed of tandem CpG building blocks and magnesium pyrophosphate, and (4) incorporation of multiple copies of ECM binding peptide through hybridization to short DNA moieties. The structurally well-defined EaCpG shows dramatically increased intratumoral retention and marginal systemic dissemination through peritumoral administration, leading to potent antitumor immune response and subsequent tumor elimination, with minimal treatment-related toxicity. Combined with conventional standard-of-care therapies, peritumor administration of EaCpG generates systemic immune responses that lead to a curative abscopal effect on distant untreated tumors in multiple cancer models, which is superior to the unmodified CpG. Taken together, EaCpG provides a facile and generalizable strategy to simultaneously potentiate the potency and safety of CpG for combinational cancer immunotherapies.

Multiple Fingerprints and Spectrum-Effect Relationship of Polysaccharides from Saposhnikoviae Radix.

PMP-HPLC, FT-IR, and HPSEC fingerprints of 10 batches of polysaccharides from Saposhnikoviae Radix with different production areas and harvest times have been prepared, and the chemometrics analysis was performed. The anti-allergic activity of 10 batches of Saposhnikoviae Radix polysaccharide (SP) was evaluated, and the spectrum-effect relationship of the 10 batches of SP was analyzed by gray correlation degree with the chromatographic fingerprint as the independent variable. The results showed that the PMP-HPLC, HPSEC, and FT-IR fingerprints of 10 batches of SP had a high similarity. Two monosaccharides (rhamnose and galactose), the polysaccharide fragment Mn = 8.67 × 106~9.56 × 106 Da, and the FT-IR absorption peak of 892 cm-1 can be used as the quality control markers of SPs. All 10 batches of SP could significantly inhibit the release of ß-HEX in RBL-231 cells, and the polysaccharides harvested from Inner Mongolia in the winter had the best anti-allergic activity. The spectrum-effect relationship model showed that the monosaccharide composition and molecular weight were related to the anti-allergic activity of the SPs. Multiple fingerprints combined with spectrum-effect relationship analysis can evaluate and control the quality of SPs from the aspects of overall quality and efficacy, which has more application value.

Integration of a miniaturized DMMB assay with high-throughput screening for identifying regulators of proteoglycan metabolism.

Defective biosynthesis or function of proteoglycans causes pathological conditions in a variety of tissue systems. Osteoarthritis (OA) is a prevalent degenerative joint disorder characterized by progressive cartilage destruction caused by imbalanced proteoglycan synthesis and degradation. Identifying agents that regulate proteoglycan metabolism may benefit the development of OA-modifying therapeutics. High-throughput screening (HTS) of chemical libraries has paved the way for achieving this goal. However, the implementation and adaptation of HTS assays based on proteoglycan measurement remain underexploited. Using primary porcine chondrocytes as a model, we report a miniaturized dimethyl-methylene blue (DMMB) assay, which is commonly used to quantitatively evaluate sulfated glycosaminoglycan (GAG) content, with an optimized detection range and reproducibility and its integration with HTS. Treatment with TGF-ß1 and IL1-α, known as positive and negative proteoglycan regulators, respectively, supported the assay specificity. A pre-test of chemical screening of 960 compounds identified both stimulators (4.48%) and inhibitors (6.04%) of GAG production. Fluorophore-assisted carbohydrate electrophoresis validated the activity of selected hits on chondroitin sulfate expression in an alginate culture system. Our findings support the implementation of this simple colorimetric assay in HTS to discover modifiers of OA or other diseases related to dysregulated proteoglycan metabolism.