Investigation of cellular uptake and transport capacity of Cordyceps sinensis exopolysaccharide­selenium nanoparticles with different particle sizes in Caco-2 cell monolayer.

Cordyceps sinensis exopolysaccharide­selenium nanoparticles (EPS-SeNPs) were successfully constructed, characterized, and its Se release kinetics and mechanism were also evaluated in our previous studies. However, the intestinal cellular uptake and transport capacities of EPS-SeNPs remain unknown. On the basis of our previous researches, this work was designed to evaluate the uptake and transport capacities of EPS-SeNPs (EPS/Se = 20/1, 3/1, 1/1, and 3/4) in intestinal epithelial (Caco-2) cells. Confocal laser scanning microscopy results indicated that the internalization of coumarin-6 labeled EPS-SeNPs was in a time-dependent process and eventually located in the cytoplasm, not in the nucleus. Endocytosis inhibitors were employed to evaluate the cellular uptake pathway of EPS-SeNPs, relevant results revealed that clathrin-, caveolae-, and energy-mediated pathways were participated in the internalization of EPS-SeNPs by Caco-2 cells. In addition, the transportation of EPS-SeNPs across Caco-2 cell monolayers was in a concentration-dependent manner. Different particle sizes of EPS-SeNPs presented different uptake and transport capacities in Caco-2 cells. Noteworthy, EPS/Se = 3/4 with the highest selenium content possessed the most superior cellular uptake and transport abilities in Caco-2 cells. The present work may contribute to illustrate the internalization and transport mechanism of EPS-SeNPs, thus facilitating its application in food and medical industries.

Fei-Yan-Qing-Hua decoction decreases hyperinflammation by inhibiting HMGB1/RAGE signaling and promotes bacterial phagocytosis in the treatment of sepsis.

ETHNOPHARMACOLOGICAL RELEVANCE: Fei-Yan-Qing-Hua decoction (FYQHD), derived from the renowned formula Ma Xing Shi Gan tang documented in Zhang Zhong Jing's "Treatise on Exogenous Febrile Disease" during the Han Dynasty, has demonstrated notable efficacy in the clinical treatment of pneumonia resulting from bacterial infection. However, its molecular mechanisms underlying the therapeutic effects remains elusive. AIM OF THE STUDY: This study aimed to investigate the protective effects of FYQHD against lipopolysaccharide (LPS) and carbapenem-resistant Klebsiella pneumoniae (CRKP)-induced sepsis in mice and to elucidate its specific mechanism of action. MATERIALS AND METHODS: Sepsis models were established in mice through intraperitoneal injection of LPS or CRKP. FYQHD was administered via gavage at low and high doses. Serum cytokines, bacterial load, and pathological damage were assessed using enzyme-linked immunosorbent assay (ELISA), minimal inhibitory concentration (MIC) detection, and hematoxylin and eosin staining (H&E), respectively. In vitro, the immunoregulatory effects of FYQHD on macrophages were investigated through ELISA, MIC, quantitative real-time PCR (Q-PCR), immunofluorescence, Western blot, and a network pharmacological approach. RESULTS: The application of FYQHD in the treatment of LPS or CRKP-induced septic mouse models revealed significant outcomes. FYQHD increased the survival rate of mice exposed to a lethal dose of LPS to 33.3%, prevented hypothermia (with a rise of 3.58 °C), reduced pro-inflammatory variables (including TNF-α, IL-6, and MCP-1), and mitigated tissue damage in LPS or CRKP-induced septic mice. Additionally, FYQHD decreased bacterial load in CRKP-infected mice. In vitro, FYQHD suppressed the expression of inflammatory cytokines in macrophages activated by LPS or HK-CRKP. Mechanistically, FYQHD inhibited the PI3K/AKT/mTOR/4E-BP1 signaling pathway, thereby suppressing the translational level of inflammatory cytokines. Furthermore, it reduced the expression of HMGB1/RAGE, a positive feedback loop in the inflammatory response. Moreover, FYQHD was found to enhance the phagocytic activity of macrophages by upregulating the expression of phagocytic receptors such as CD169 and SR-A1. CONCLUSION: FYQHD provides protection against bacterial sepsis by concurrently inhibiting the inflammatory response and augmenting the phagocytic ability of immune cells.

Naringin alleviates pneumonia caused by Klebsiella pneumoniae infection by suppressing NLRP3 inflammasome.

Klebsiella pneumoniae (Kpn) is an important pathogen of hospital-acquired pneumonia, which can lead to sepsis and death in severe cases. In this study, we simulated pneumonia induced by Kpn infection in mice to investigate the therapeutic effect of naringin (NAR) on bacterial-induced lung inflammation. Mice infected with Kpn exhibited increases in white blood cells (WBC) and neutrophils in the peripheral blood and pathological severe injury of the lungs. This injury was manifested by increased expression of the inflammatory cytokines interleukin (IL)- 18, IL-1ß, tumor necrosis factor-α (TNF-α) and IL-6, and elevated the expression of NLRP3 protein. NAR treatment could decrease the protein expression of NLRP3, alleviate lung inflammation, and reduce lung injury in mice caused by Kpn. Meanwhile, molecular docking results suggest NAR could bind to NLRP3 and Surface Plasmon Resonance (SPR) analyses also confirm this result. In vitro trials, we found that pretreated with NAR not only inhibited nuclear translocation of nuclear factor (NF)-κB protein P65 but also attenuated the protein interaction of NLRP3, caspase-1 and ASC and inhibited the assembly of NLRP3 inflammasome in mice AMs. Additionally, NAR could reduce intracellular potassium (K+) efflux, inhibiting NLRP3 inflammasome activation. These results indicated that NAR could protect against Kpn-induced pneumonia by inhibiting the overactivation of the NLRP3 inflammasome signaling pathway. The results of this study confirm the efficacy of NAR in treating bacterial pneumonia, refine the mechanism of action of NAR, and provide a theoretical basis for the research and development of NAR as an anti-inflammatory adjuvant.

The cellular uptake of Cordyceps sinensis exopolysaccharide­selenium nanoparticles and their induced apoptosis of HepG2 cells via mitochondria- and death receptor-mediated pathways.

This wok investigated the effects of Cordyceps sinensis exopolysaccharide­selenium nanoparticles (EPS-SeNPs), EPS-Se-1, EPS-Se-2, EPS-Se-3, and EPS-Se-4) with particle sizes (79-124 nm) and Se contents (20.11-40.80 µg/mg) on endocytosis and antitumor activity against human hepatocellular carcinoma (HepG2) cells and revealed the apoptosis-related mechanisms. EPS-SeNPs inhibited HepG2 cells proliferation in a dose and Se content-dependent manner by disrupting cell membrane and mitochondrial integrity, promoting reactive oxygen species production. EPS-SeNPs were endocytosed by HepG2 cells through a clathrin-mediated pathway and followed the quasi-first-order kinetics model, indicating physical adsorption played a dominant role in cellular uptake behavior of EPS-SeNPs. Notably, EPS-Se-3 with the lowest particle size (79 nm) showed the highest antitumor activity and the strongest ability to promote cell apoptosis. Western blotting results revealed that EPS-Se-3 increased expressions of Bax, Cytochrome c, cleaved caspase-9, cleaved caspase-3, Fas, p53, and cleaved caspase-8, while decreased the expressions of Bcl-2 and PARP, as contrast to that of control. Overall, EPS-SeNPs induced cell apoptosis through intrinsic mitochondria-mediated and extrinsic death receptor-mediated pathways.

Isolation, structural features, rheological properties and bioactivities of polysaccharides from Lignosus rhinocerotis: A review.

L. rhinocerotis, an edible and medicinal mushroom, has long been utilized as folk medicine and nutritional food in Southeast Asia and southern China. Polysaccharides are the main bioactive substances of L. rhinocerotis sclerotia, and they have attracted extensive attention of researchers both at home and abroad. In the past few decades, various methods have been applied to extract polysaccharides from L. rhinocerotis (LRPs) and the structural features of LRPs are closely related to the used methods of extraction and purification. Many studies have confirmed that LRPs possess various remarkable bioactivities, including immunomodulatory, prebiotic, antioxidant, anti-inflammatory and anti-tumor activities and intestinal mucosa protective effect. As a natural polysaccharide, LRP has the potential to be a drug and functional material. This paper systematically reviews the recent studies on structural characteristics, modification, rheological properties and bioactivities of LRPs, and provides a theoretical basis for an in-depth study of the structure-activity relationship, and utilization of LRPs as therapeutic agents and functional foods. Additionally, the further research and development of LRPs are also prospected.

Protective effects of Cordyceps sinensis exopolysaccharide­selenium nanoparticles on H2O2-induced oxidative stress in HepG2 cells.

This work investigated the protective effects of Cordyceps sinensis exopolysaccharide­selenium nanoparticles (EPS-SeNPs) at different Se/EPS ratios (1/20, 1/3, 1/1, and 4/3) against 0.4 mM H2O2-induced oxidative stress in human hepatoma (HepG2) cells. Results revealed that EPS-SeNPs (0.1-4.0 µg/mL) promoted the proliferation of HepG2 cells with a cell viability of over 90% during H2O2 stress. Different Se/EPS ratios of EPS-SeNPs provided protective effects against H2O2-induced oxidative stress in HepG2 cells by increasing cell viability, restoring cell and nucleus morphology, as well as reducing lactate dehydrogenase (LDH) levels. Particularly, EPS-SeNPs (Se/EPS = 1/1) with the smallest particle size showed the highest cell viability and the greatest inhibitory effect on LDH level. Besides, EPS-SeNPs also inhibited nitric oxide and reactive oxygen species (ROS) production, and increased mitochondrial membrane potential, superoxide dismutase (SOD), and catalase levels while reducing glutathione (GSH) content. Specially, EPS-SeNPs at Se/EPS ratio of 1/3 with smaller size showed the lowest ROS level and the highest antioxidant activities (SOD and GSH), implying that the ROS generation was inhibited by increasing enzymatic and non-enzymatic antioxidants. The enhanced protective effect of EPS-SeNPs (Se/EPS = 1/1 and 1/3) might be attributed to its smaller particle size.

Selenium release kinetics and mechanism from Cordyceps sinensis exopolysaccharide-selenium composite nanoparticles in simulated gastrointestinal conditions.

This work investigated selenium (Se) release kinetics and mechanism from exopolysaccharide-selenium nanoparticles (EPS-SeNPs, Se/EPS = 1/20, 1/1 and 4/3) in simulated gastric (SGF) and intestinal fluids (SIF) using kinetics models of Zero order, First order, Higuchi, Hixson-Crowell and Korsmeyer-Peppas. EPS-SeNPs showed an increase in size from 80-125 nm to 250-320 nm and more ambiguous boundary after gastrointestinal digestion. Se/EPS ratio and pH had significant influence on Se release. Se release kinetics from EPS-SeNPs (Se/EPS = 1/1 and 4/3 in SGF) followed a classical Fickian diffusion, in contrast to an erosion governed by macromolecular chains relaxation for Se/EPS = 1/20 in SIF. Se release from EPS-SeNPs (Se/EPS = 1/1 and 4/3 in SIF) was well-fitted to Korsmeyer-Peppas model and followed a non-Fickian mechanism controlled by both diffusion and erosion. Additionally, EPS-SeNPs (Se/EPS = 1/20) showed a low Se release after SGF digestion, but a high release after SIF digestion, suggesting its application in controlled release of Se-enriched supplements for Se-deficiency treatment.

Effect of ultrasound on size, morphology, stability and antioxidant activity of selenium nanoparticles dispersed by a hyperbranched polysaccharide from Lignosus rhinocerotis.

The differences between ultrasonic and non-ultrasonic approaches in synthesizing Lignosus rhinocerotis polysaccharide-selenium nanoparticles (LRP-SeNPs) were compared in terms of size, morphology, stability and antioxidant activity by UV-VIS, FT-IR, X-ray diffraction (XRD), dynamic light scattering (DLS), transmission electron microscopy (TEM), and energy dispersive X-ray (EDX) with high resolution TEM. Results indicated that the SeNPs were associated with the LRP macromolecules in a physical adsorption pattern without breaking chemical bonds, and the ultrasonic treatment reduced the size of SeNPs, narrowed the size distribution as well as improved the stability. Due to the LRP compact coil structure loosed under ultrasonic cavitation, the SeNPs could be easily diffused into the LRP internal branches instead of gathering on the LRP surface and were well dispersed and eventually stabilized throughout the extended branches. After ultrasound treatment, the SeNPs had a minimum average diameter of ∼50 nm and the LRP-SeNPs could remain homogeneous and translucent for 16 days within 200 nm size. Furthermore, the ultrasound-treated LRP-SeNPs exhibited higher DPPH and ABTS radical-scavenging abilities than those untreated with ultrasound. This difference may be attributed to the reason that ultrasound can reduce the SeNPs size and increase the specific surface area, which provides sufficient active sites to react with the free radicals and suppress the oxidizing reactions. The integrated results demonstrated that ultrasound played a crucial role in the dispersion, size control, stabilization and antioxidant activity of SeNPs.

Construction of a Cordyceps sinensis exopolysaccharide-conjugated selenium nanoparticles and enhancement of their antioxidant activities.

A Cordyceps sinensis exopolysaccharide (EPS)-conjugated selenium nanoparticles (SeNPs) were successfully constructed through the reduction of SeO32-. The EPS-SeNPs were characterized in terms of formation, morphology, size, Se distribution and phase by UV-vis, FT-IR, transmission electron microscopy (TEM), dynamic light scattering (DLS), energy dispersive X-ray (EDX) and wide angle X-ray diffraction (WAXD) measurements. Results revealed that the SeNPs conjugated to EPS were amorphous and could be well dispersed at a size range of 80-125nm. The interactions between the OH groups of EPS and SeNPs substituted for intermolecular interaction in native EPS to form new CO⋯Se bonds, resulting in good dispersion of SeNPs in the EPS matrix. Besides, the EPS-SeNPs at different Se/P ratios exhibited significant scavenging ability on superoxide anion radical (O2-) and ABTS radical cation (ABTS+) when compared to pure EPS, indicating that the conjugated SeNPs reinforced antioxidant effect of EPS. This work not only provides a simple and efficient way to construct well-dispersed SeNPs in aqueous system, and demonstrates the vital role of the EPS as a biopolymer template for dispersion, stabilization and size control of SeNPs, but also finds the EPS-SeNPs can potentially serve as a good antioxidant towards O2- and ABTS+.

Structure, molecular conformation, and immunomodulatory activity of four polysaccharide fractions from Lignosus rhinocerotis sclerotia.

Four polysaccharide fractions, LRP-1, LRP-2, LRP-3 and LRP-4 were extracted stepwise from Lignosus rhinocerotis sclerotia with distilled water at 25, 95, 120°C and 1.0M NaOH solution at 4°C. Their structure, molecular size and chain conformation were clarified using SEC-MALLS-RI, GC, FT-IR and UV-vis. Furthermore, their immunomodulatory activities were evaluated by the model of cyclophosphamide (Cy)-induced immunosuppression. The LRP-1 and LRP-2 were polysaccharide-protein complexes (46-68% ß-d-glucan and 27-48% protein), while LRP-3 and LRP-4 were absolutely composed of ß-d-glucose. The LRP-4 with low polydispersity had much higher molecular weight (Mw, 5.86×106g/mol) and intrinsic viscosity ([η], 202.6ml/g) than other LRP fractions. Based on Mw, radius of gyration (z1/2) and [η] data with the exponent ß of z1/2-Mw and its U-shaped curve, all four LRP fractions were highly branched macromolecules and LRP-3 showed a more compact sphere-like conformation than LRP-2 in aqueous solution. Additionally, all four LRP fractions exhibited protective effects against Cy-induced immunosuppression in mice by improving immune organs as well as stimulating the release of major cytokines TNF-α and INF-γ. This work provides a theoretical basis for the application of polysaccharides and their protein complexes from Lignosus rhinocerotis sclerotia in food- or drug-based therapies.