Chitosan Oligosaccharide Attenuates Lipopolysaccharide-Induced Intestinal Barrier Dysfunction through Suppressing the Inflammatory Response and Oxidative Stress in Mice.

This study was conducted to investigate the protective effect of chitosan oligosaccharide (COS) against lipopolysaccharide (LPS)-induced intestinal injury. The results demonstrated that COS improved the mucosal morphology of the jejunum and colon in LPS-challenged mice. COS alleviated the LPS-induced down-regulation of tight junction protein expressions and reduction of goblet cells number and mucin expression. The mRNA expressions of anti-microbial peptides secreted by the intestinal cells were also up-regulated by COS. Additionally, COS decreased pro-inflammatory cytokine production and neutrophil recruitment in the jejunum and colon of LPS-treated mice. COS ameliorated intestinal oxidative stress through up-regulating the mRNA expressions of nuclear factor E2-related factor 2 and downstream antioxidant enzymes genes. Correlation analysis indicated that the beneficial effects of COS on intestinal barrier function were associated with its anti-inflammatory activities and antioxidant capacity. Our study provides evidence for the application of COS to the prevention of intestinal barrier dysfunction caused by the stress of a LPS challenge.

A Potential Probiotic for Diarrhea: Clostridium tyrobutyricum Protects Against LPS-Induced Epithelial Dysfunction via IL-22 Produced By Th17 Cells in the Ileum.

Probiotics are clinically used for diarrhea and inflammatory bowel diseases in both humans and animals. Previous studies have shown that Clostridium tyrobutyricum (Ct) protects against intestinal dysfunction, while its regulatory function in the gut needs further investigation and the related mechanisms are still not fully elucidated. This study aims to further verify the protective function of Ct and reveal its underlying mechanisms in alleviating diarrhea and intestinal inflammation. Ct inhibited LPS-induced diarrhea and intestinal inflammation in the ileum. IL-22 was identified and the protective role of Ct in the ileum presented an IL-22-dependent manner according to the transcriptomic analysis and in vivo interference mice experiments. The flow cytometric analysis of immune cells in the ileum showed that Ct enhanced the proportions of Th17 cells in response to LPS. The results of in situ hybridization further verified that Ct triggered Th17 cells to produce IL-22, which combined with IL-22RA1 expressed in the epithelial cells. Moreover, Ct was unable to enhance the levels of short-chain fatty acids (SCFAs) in the ileum, suggesting that the protective role of Ct in the ileum was independent of SCFAs. This study uncovered the role of Ct in alleviating diarrhea and inflammation with the mechanism of stimulating Th17 cells in the lamina propria to produce IL-22, highlighting its potential application as a probiotic for diarrhea and inflammation in the ileum.

Optimization and comparison of the production of galactooligosaccharides using free or immobilized Aspergillus oryzae ß-galactosidase, followed by purification using silica gel.

The aim of this study was to optimize and compare the production of galactooligosaccharides (GOSs) by free and cotton cloth-immobilized Aspergillus oryzae ß-galactosidase, and perform economical evaluation of production of GOSs (100%) between them. Using the response surface method, the optimal reaction time (3.9 h), initial lactose concentration (57.13%), and enzyme to lactose ratio (44.81 U/g) were obtained for the free enzyme, which provided a GOSs yield of 32.62%. For the immobilized enzyme, the optimal yield of GOSs (32.48%) was obtained under reaction time (3.09 h), initial lactose concentration (52.74%), and temperature (50.0 ℃). And it showed desirable reusability during five successive enzymatic reactions. The recovery rate of GOSs (100%) is 65% using silica gel filtration chromatography. The economical evaluation showed almost no difference in the manufacturing cost for the GOSs (100%) between these two systems, and that the recovery rate had a great impact on the cost.

A Potential Prophylactic Probiotic for Inflammatory Bowel Disease: The Overall Investigation of Clostridium tyrobutyricum ATCC25755 Attenuates LPS-Induced Inflammation via Regulating Intestinal Immune Cells.

SCOPE: This study aims to roundly investigate whether Clostridium tyrobutyricum (Ct) alleviates inflammation via regulating immune cells in the small intestines. METHODS AND RESULTS: Mice are pre-treated with different concentrations of Ct follow by LPS stimulation. Ct maintains the mice body weight under inflammation. In response to LPS, 107 CFU mL-1 Ct decreases the mRNA expression of inflammatory cytokines in the duodenum, while 108 CFU mL-1 Ct reduces inflammatory cytokines expression in both duodenum and ileum and protected intestinal morphology. The small intestinal immune cells are analyzed using flow cytometry. Ct decreases the numbers of macrophages and mast cells in the intestines in response to LPS. In the duodenum, Ct enhances dentritic cells (DCs), regulatory T cells (Tregs), and T helper cell 17 (Th17) proportions. Ct decreases DCs and Tregs proportions, while enhances Th17 numbers in the ileum. The underlying mechanism of Ct in preventing inflammation may rely on the physiological immune cell composition of the intestines. In response to LPS, Ct may mainly stimulate Tregs via activating DCs in the duodenum while trigger Th17 cells in the ileum, thereby maintaining the intestinal homeostasis. CONCLUSION: Ct alleviates the LPS-induce inflammation via regulating different immune cell types in the small intestines, highlighting that Ct is a potential prophylactic probiotic in intestinal diseases.

Galactooligosaccharide pretreatment alleviates damage of the intestinal barrier and inflammatory responses in LPS-challenged mice.

Galactooligosaccharides (GOS) have been identified as beneficial prebiotics for animals and human beings. Most studies have focused on the effect of GOS on the hindgut populated with abundant microbes. However, few research studies have been conducted on the small intestine, and many results are inconsistent due to the purity of GOS, commonly mixed with monosaccharides or lactose. Therefore, pure GOS with definite structures were prepared and used in the present study to evaluate their effects on intestinal barrier function, inflammatory responses and short-chain fatty acids (SCFAs) produced in the colon of mice challenged with lipopolysaccharide (LPS). The results of 1H and 13C nuclear magnetic resonance spectral analyses indicated that the main structures of GOS with a degree of polymerization of 3 (trisaccharide) and 4 (tetrasaccharide) are [ß-Gal-(1 → 6)-ß-Gal(1 → 4)-ß-Glc] and [ß-Gal-(1 → 6)-ß-Gal-(1 → 6)-ß-Gal-(1 → 4)-ß-Glc], respectively. The results of an in vivo study in mice showed that intragastric administration of 0.5 g per kg BW GOS attenuated intestinal barrier damage and inflammatory responses induced by LPS in the jejunum and ileum, as indicated by increasing villus height and villus-to-crypt ratio, up-regulated intestinal tight junction (ZO-1, occludin, and claudin-1) gene expression, and down-regulated pro-inflammatory cytokines such as IL-1ß, IL-6, IFN-γ, and TNF-α gene expression. Nevertheless, the protective effects of GOS on the intestinal barrier are independent of glucagon-like peptide 2. In addition, 0.5 g per kg BW GOS administration promoted the recovery of colonic acetate, propionate, butyrate, and total SCFA production reduced by LPS challenge. The obtained results provide practical evidence that pure GOS can act as protective agents for intestinal health.

Clostridium tyrobutyricum Protects against LPS-Induced Colonic Inflammation via IL-22 Signaling in Mice.

This study aimed to investigate the effects of Clostridium tyrobutyricum (C. tyrobutyricum) on colonic immunity and the role of IL-22 in the protective function of C. tyrobutyricum. Mice were supplemented with 108 CFU/mL C. tyrobutyricum daily for 20 days, followed by injecting with LPS for 24 h. In vivo interference of IL-22 via injecting with an adeno-associated virus was conducted to elucidate the role of IL-22 in C. tyrobutyricum attenuating colonic inflammation. The results showed that C. tyrobutyricum decreased the mRNA expression of IL-6 and IL-1ß. C. tyrobutyricum enhanced the mRNA expression of IL-22 and the expression of MUC2 in the colon. The in vivo interference results showed that C. tyrobutyricum enhanced the mRNA expression of IL-6 and IL-1ß while decreased the expression of MUC2 after knocking down IL-22. The flow cytometric analysis showed that C. tyrobutyricum decreased the proportions of macrophages, DCs, and mast cells and effectively regulated the proportion of Th17 cells, indicating that C. tyrobutyricum may stimulate the expression of IL-22 via regulating Th17 cells. Our study concluded that C. tyrobutyricum protected against LPS-induced colonic barrier dysfunction and inflammation via IL-22 signaling, suggesting that C. tyrobutyricum could be a potential probiotic in regulating colonic health.

Effects of Supranutritional Selenium Nanoparticles on Immune and Antioxidant Capacity in Sprague-Dawley Rats.

The present study was conducted to investigate the effects of supranutritional selenium nanoparticles (SeNPs) on immune and antioxidant capacity in rats. Forty male Sprague-Dawley (SD) rats were randomly divided into four groups and given intragastric administration of SeNPs at doses of 0, 0.2, 0.4, and 0.8 mg Se/kg BW, respectively, for 2 weeks. Serum immune parameters, serum and organic tissues (liver, heart, kidney) antioxidant indices, and liver mRNA expression of glutathione peroxidase 1 (GPx1) and glutathione peroxidase 4 (GPx4) were examined. The results showed that supranutritional doses of 0.4 and 0.8 mg Se/kg BW SeNPs promoted the immune responses in serum. SeNPs administration improved antioxidant capacity in the liver and kidney, and the best improvement on antioxidant capacity was found in the kidney. Furthermore, intragastric administration of SeNPs upregulated mRNA expression of GPx1 and GPx4 in the liver. The results obtained indicated that SeNPs administration at supranutritional levels had beneficial effects on immune and antioxidant capacity and supplemental SeNPs at dose of 0.4 mg Se/kg BW exhibited the best response in SD rats.

Prooxidation and Cytotoxicity of Selenium Nanoparticles at Nonlethal Level in Sprague-Dawley Rats and Buffalo Rat Liver Cells.

The effects of selenium nanoparticles (SeNPs) on the antioxidant capacity in Sprague-Dawley (SD) rats were investigated. The rats were given intragastric administration of an SeNP suspension at doses of 0, 2, 4, and 8 mg Se/kg BW for two weeks. The antioxidant capacity in serum and organic tissues (liver, heart, and kidney) and the gene expression levels of glutathione peroxidase 1 (GPX1) and glutathione peroxidase 4 (GPX4) in the liver were measured. Buffalo rat liver (BRL) cell lines were further constructed to explore the cytotoxicity mechanism induced by SeNPs through the determination of antioxidant capacity; cell activity; apoptosis; and Caspase-3, Caspase-8, and Caspase-9 family activities. The results showed that SeNP administration over 4.0 mg Se/kg BW decreased the antioxidant capacities in the serum, liver, and heart and downregulated mRNA expression of GPX1 and GPX4 in the liver. The BRL cell line experiments showed that treatment with over 24 µM SeNPs decreased the viability of the cells and damaged the antioxidant capacity. Flow cytometry analysis showed that decreased cell viability induced by SeNPs is mainly due to apoptosis, rather than cell necrosis. Caspase-3 and Caspase-8 activities were also increased when BRL cells were treated with 24 µM and 48 µM SeNPs. Taken together, a nonlethal level of SeNPs could impair the antioxidant capacity in serum and organic tissues of rats, and the liver is the most sensitive to the toxicity of SeNPs. A pharmacological dose of SeNPs could lead to cytotoxicity and induce cell death through apoptosis and extrinsic pathways contributing to SeNP-induced apoptosis in BRL cells.

Effects of inorganic trace minerals replaced by complexed glycinates on reproductive performance, blood profiles, and antioxidant status in broiler breeders.

This study was conducted to investigate the effects of replacing inorganic trace minerals (ITM) with organic trace minerals (OTM; complexed glycinates) on reproductive performance, blood profiles, and antioxidant status in broiler breeders. A total of 648, 23-week-old healthy broiler breeders (ZhenNing), with similar body weight (1.40 ± 0.002 kg), were randomly divided into 4 groups with 6 replicates in each group (27 hens/replicate) and fed the respective experimental diets for 14 wk (including 2 wk for adaptation). The experimental treatments consisted of T1: Cont., commercially recommended levels of ITM (Cu, Zn, Fe, and Mn sulfates); T2: Mix, half trace minerals (TM) were provided from ITM and half from OTM (glycinates); T3: M-OTM, TM were provided from glycinates and reduced to 70% of T1; T4: L-OTM, TM were provided from glycinates and reduced to 50% of T1. The results showed that commercial level of inorganic trace minerals replaced by low-dose complexed glycinates (T3 and T4) exhibited no significant effects on laying performance, 50% ITM replaced by complexed glycinates (T2) numerically improved laying rate by 1.23% than cont. treatment (T1). Broiler breeders fed complexed glycinates tended to produce more qualified eggs (P = 0.05) in T3, with better yolk color (P < 0.01) and eggshell thickness (P = 0.05) in T2 treatment. Replacement of low-dose complexed glycinates reduced fertilization rate (P < 0.01), while it did not affect hatchability. There were no significant differences in serum reproductive hormones such as estrogen and progesterone among the treatments. Serum total protein, albumin, and phosphorus were increased respectively with the replacement of ITM by low-dose OTM from complexed glycinates (P < 0.05). Total liver antioxidant capacity in M-OTM and L-OTM treatment was higher than that of Cont. and Mix treatments (P < 0.01). In conclusion, replacement of high levels of ITM by lower levels of OTM in the form of complexed glycinates is beneficial for egg quality and liver antioxidant status in broiler breeders during the peak laying period.

Effects of Replacing Inorganic with Respective Complexed Glycinate Minerals on Apparent Mineral Bioavailability and Deposition Rate in Tissues of Broiler Breeders.

The study was conducted to evaluate the effects of replacing inorganic trace minerals (ITMs) with respective low-dose complexed glycinate minerals (CGMs) on their bioavailability and retention during peak laying period of broiler breeders. In this experiment, 648 ZhenNing broiler breeders (23 weeks old) were randomly allocated to four treatments with six replicates (27 birds/replicate) and fed for 14 weeks including 2 weeks adaptation period. The treatments were T1-ITM, commercially recommended levels of ITMs (Cu, Zn, Fe, and Mn sulphates); T2-MIX, half of the minerals were supplemented with ITMs and half with CGMs; T3-L-CGMs, minerals were supplemented with CGMs (50% level of T1); and T4-M-CGMs, minerals were supplemented with CGMs (70% level of T1). The results showed that birds fed on ITM had lower bioavailability of Fe, Mn, and Zn (P < 0.05) than those fed on L-CGMs, but the highest (P < 0.01) bioavailability of Cu was found in those fed on MIX. Mineral retention (in serum, muscle, bone, and tissues) was not affected by reducing supplementation levels of trace minerals up to 50% of ITMs, but Zn (in serum, liver, kidney, heart, and albumen) and Fe (in serum and the yolk) retention was negatively affected (P < 0.05). In conclusion, replacing dietary ITMs with low-dose complexed glycinate minerals increases the apparent bioavailability of Fe, Mn, and Zn without compromising the mineral retention rates in most of the tissues tested.

Optimization for galactooligosaccharides synthesis: A potential alternative for gut health and immunity.

AIMS: Present study was conducted to optimize the processing parameters for galactooligosaccharides (GOS) synthesis from whey powder followed by exploring its prebiotic efficiency. MAIN METHODS: All factors (initial lactose concentration, pH, reaction time, temperature and enzyme to substrate ratio; E/S) were analyzed by single factor analysis and optimization for GOS yield was done following the orthogonal experimental design. For in vivo analysis, 60 mice were equally divided into four groups (normal control, NC; low, medium, and high dose of GOS, LG, MG and HG) and fed varying levels (0, 0.25, 0.5 and 1.0 g/kg bw per day) of GOS, for 30 days and sampling was done at the end of experiment regarding gut health, immunity, cecal microbiota and metabolites. KEY FINDINGS: Optimum yield of GOS (25.1%) was obtained at reaction time 25 min, temperature 50 °C, pH 4.5 and the enzyme to substrate ratio (E/S) of 20 U/g. In vivo experiment, shallower crypt and greater villus to crypt ratio (V/C) were found in the duodenum of LG treatment compared to NC mice (P < .05). The GOS promotes thymus development and improve immunity. Intervention with GOS increased the population of bifidobacterium and lactobacillus in MG and bifidobacterium in LG mice (P < .05), and was accompanied by decreased proliferation of desulfovibrio. Correlation analysis also supported the above findings. SIGNIFICANCE: This study optimized the processing parameters for GOS preparation and provided data encouraging to suggest that GOS can be a potential option to improve the gut health and immunity.

Chitosan Oligosaccharide Attenuates Nonalcoholic Fatty Liver Disease Induced by High Fat Diet through Reducing Lipid Accumulation, Inflammation and Oxidative Stress in C57BL/6 Mice.

Nonalcoholic fatty liver disease (NAFLD) has become the most common chronic liver disease closely associated with metabolic syndrome, but there are no validated pharmacological therapies. The aim of this study was to investigate the effect of chitosan oligosaccharide (COS) on NAFLD. Mice were fed either a control diet or a high-fat diet (HFD) with or without COS (200 or 400 mg/kg body weight (BW)) by oral gavage for seven weeks. Administration with COS significantly lowered serum lipid levels in the HFD-fed mice. The hepatic lipid accumulation was significantly decreased by COS, which was attributed to decreased expressions of lipogenic genes and increased expressions of fatty ß-oxidation-related genes. Moreover, pro-inflammatory cytokines, neutrophils infiltration, and macrophage polarization were decreased by COS in the liver. Furthermore, COS ameliorated hepatic oxidative stress by activating the nuclear factor E2-related factor 2 (Nrf2) pathway and upregulating gene expressions of antioxidant enzymes. These beneficial effects were mediated by the activation of the adenosine monophosphate-activated protein kinase (AMPK) signaling pathway. Therefore, COS might be a potent dietary supplement to ameliorate NAFLD.

Gender-related differences in the association between concomitant amplification of AIB1 and HER2 and clinical outcomes in glioma patients.

BACKGROUND: Previous studies demonstrated that AIB1 or HER2 copy number gain (CNG), respectively, were independent predictors for poor prognosis of glioma patients, especially in females. We hypothesize that there are some connections between the two genes and sex-specific characteristics, thus this study aimed to analyze gender-related differences in the prognosis of glioma patients. METHODS: Using Real-Time Quantitative Reverse Transcription PCR (RT-qPCR) method, we examined AIB1 and HER2 CNG in gliomas samples (n = 114), and inspected the correlation of various genotypes with patients outcomes. RESULTS: Concomitant AIB1 and HER2 amplification were closely related to shorter survival time and radiotherapy resistance in female gliomas patients (P < 0.01), which also served as an independent risk factor. No significant prognostic value was found with AIB1 and HER2 CNG in male patients. However, linear regression analysis showed a positive relationship between the copy number of AIB1 and HER2 (P < 0.01) in male patients, rather than female patients. CONCLUSION: In this study, we reveal a gender difference in the prognostic value of concomitant AIB1 and HER2 CNG in glioma patients which were barely noticed before. These observations indicated that genetic alterations synergistic with essential respects of sex determination influence glioma biology and patients outcomes.

[Comparative investigation of the components in rats plasma before and after orally administrating Shuanghuanglian injection].

OBJECTIVE: To investigate the main constituents absorbed in the blood after orally administrating Shuanghuanglian injection extracts of its composed crude drug and provide the foundation for optimizing the prescription of Shuanghuanglian powder for injection. METHOD: Through analysing HPLC fingerprint established based on shuanghuanglian powder for injection and extracts of it composed crude drug, the active investigation were carried out among the peaks of control plasma and plasma containing drug in rats, shuanghuanglian powder for injection and extract of its composed crude drug. And the analysis was performed about relative content of constituents in each sample. Furthermore their original drugs and the metabolites in plasma containing drug were ascertained. RESULT: Eight compounds were detected in plasma after oral administration of Shuanghuanglian, four of them were the original compounds contained in Shuanghuanglian powder for injection and three of them were relative metabolites. It has also found that contents of original compounds such as baicalin and chlorogenic acid fell off gradually, but their metabolites increased in rat plasma in vivo. With increasing time the main components of forsythia have not been detected obviously in rats plasma. CONCLUSION: In some extent, the compounds absorbed into blood from Shuanghuanglian powder for injection and their metabolites are the effective constituents, and its plasma pharmacochemistry should be subject to illuminate the pharmacological effects and the active mechanism.

[Influence on rat intestinal absorption of huanglian alkalis after match-pair of huanglian-wuzhuyu].

OBJECTIVE: To research effect of Wuzhuyu on Huanglian intestinal absorption and to provide some basic principle of match-pair of huanglian-rougui. METHOD: The rat everted gut sac method as well as non everted gut sac method was applied to investigate the influence of Wuzhuyu on Huanglian intestinal absorption. RESULT: The content of each alkalis in Huanglian was decreasing with the comptibale ration increase, and the intestinal absorption had been improved in some degree. The intestinal absorption of Huanglian alkalis reached the highest level when match-pair ration of huang Lian-rougui was 6: 1. CONCLUSION: The chemical reaction in the progress of extraction and the intestinal absorption of huanglian alkalis were the key factors of match-pair of huanglian-wuzhuyu.

[Study on stability of baicalin and baicalein in Rat plasma by HPLC-ECD].

OBJECTIVE: To compare the stability of baicalin and baicalein in buffered aqueous solutions and mouse plasma in order to discuss the degradation mechanism and the method of disposing samples. METHOD: HPLC-ECD was used to determine baicalin and baicalein in different conditions, and the disposing samples methods were selected to stabilize them. RESULT: Baicalin and baicalein in the buffered aqueous solution at pH 7.4 was more stable than that in the mouse plasma. After adding Vit C in the buffered aqueous solution, the stability of baicalin and baicalein could be improved obviously and their remained percents were 102% and 100%. The stability of baicalin and baicalein could be enhanced distinctly in the mouse plasma only when adding Vit C and 1 mol x L(-1) HCl, and furthermore their remained percentage was up to 98% and 103%. CONCLUSION: The stability of baicalin and baicalein is related to the oxidation and enzyme degradation and it can be improved by adding Vit C and 1 mol x L(-1) HCl in the solution.

[Influence of match-pair of huanglian-rougui on rat intestinal absorption of total huanglian alkali].

OBJECTIVE: To research the principle of match-pair of huanglian-rougui. METHOD: A rat intestinal model in vitro had been applied to investigate the absorption and transport characteristics of total huanglian alkali across intestinal mucosa, as well as the influence of match-pair of huanglian-rougui on its intestinal absorption. The identification of alkalis in primitive solution, serosal solution and intestinal homogenate were analyzed by TLC. RESULT: The transport and uptake characteristic of total alkali in huanglian solution was similar to berberine. The uptake and transport of total alkali across intestinal mucosa was concentration-and time-dependent. Transport directions had strong effects on the transport of total alkali. The amount of total alkali transport from serosal side to mucosal side was much more than that from mucosal side to serosal side. After matching with rougui as some ration, the intestinal absorption of total alkali increased significantly comparing with huanglian alone, and there was an optimal ration among match-pair of huanglian-rougui. CONCLUSION: Chemical reaction in vitro and intestinal absorption were the key factors of match-pair of huanglian-rougui.

[Degradation of recombinant hirudin-2 in nasal mucosa of rabbits in vitro].

AIM: To investigate the degradation of recombinant hirudin-2 (rHV2) in nasal mucosa of rabbit. METHODS: The specific and accurate HPLC method was developed for analyzing rHV2; The degrading ratios of rHV2 at different concentrations and at pH conditions in rabbit nasal mucosa homogenate were determined; The results in nasal mucosa homogenate were compared with that in small intestinal mucosa homogenate of rabbits. The stability of rHV2 in the enzyme extract of nasal mucosa surface and the effect of proteolysis inhibitor bacitracin on the degradation of rHV2 in nasal mucosa homogenate were also estimated. RESULTS: The degradation of rHV2 in rabbit nasal mucosa homogenate showed concentration- and pH-dependence; rHV2 in nasal mucosa homogenate was more stable than that in intestinal mucosa homogenate. Also rHV2 was more stable in the enzyme extracts of nasal serosal surface than that of mucosa surface. Addition of bacitracin was able to inhibit the degradation to some degree. CONCLUSION: Comparing with oral administration, rHV2 nasal delivery was a more tolerant route.