An investigation of long-term outcome of rabbit anti-thymocyte globulin and cyclosporine therapy for pediatric severe aplastic anemia.

Children with severe aplastic anemia (SAA) face heterogeneous prognoses after immunosuppressive therapy (IST). There are few models that can predict the long-term outcomes of IST for these patients. The objective of this paper is to develop a more effective prediction model for SAA prognosis based on clinical electronic medical records from 203 children with newly diagnosed SAA. In the early stage, a novel model for long-term outcomes of SAA patients with IST was developed using machine-learning techniques. Among the indicators related to long-term efficacy, white blood cell count, lymphocyte count, absolute reticulocyte count, lymphocyte ratio in bone-marrow smears, C-reactive protein, and the level of IL-6, IL-8 and vitamin B12 in the early stage are strongly correlated with long-term efficacy (P < .05). Taken together, we analyzed the long-term outcomes of rabbit anti-thymocyte globulin and cyclosporine therapy for children with SAA through machine-learning techniques, which may shorten the observation period of therapeutic effects and reduce treatment costs and time.

Compound polysaccharides ameliorate experimental colitis by modulating gut microbiota composition and function.

BACKGROUND AND AIM: Inflammatory bowel disease results from a dysregulated immune response to intestinal microbial flora in individuals with genetic predisposition(s). This study aimed to determine the effects of compound polysaccharides (CP) containing yam polysaccharide and inulin on the rat model of colitis induced by 2,4,6-trinitrobenzenesulfonic acid (TNBS) and to explain the mechanism in terms of gut microbiota composition and function. METHODS: Male SD rats were divided into three groups: the control group, the model group, and the CP group. Disease activity index, serum myeloperoxidase level, and the composition and function of gut microbiota were analyzed. RESULTS: The data in the study showed CP reduced inflammation in the rat model of colitis induced by TNBS and ameliorated the experimental colitis. The results also indicated that CP not only reversed TNBS-induced gut dysbiosis-indexed by increased short-chain fatty acids (SCFAs)-producing bacteria, lactic acid-producing bacteria, and decreased Bacteroides, Proteobacteria as well as sulfate-reducing bacteria, but also restored the dysregulated microbiota function of colitic rats into a normal condition, including an improvement on basic metabolism and a reduction on oxidative stress, cell motility, signal transduction, xenobiotics biodegradation, and metabolism as well as pathogenesis processes. CONCLUSIONS: Compound polysaccharides ameliorated the experimental colitis of rats induced by TNBS by modulating the gut microbiota composition and function profiles, which makes it possible to be used as prebiotic agents to treat gut dysbiosis in colitis individuals.

Age-related changes in hepatic expression and activity of drug metabolizing enzymes in male wild-type and breast cancer resistance protein knockout mice.

This study aimed to reveal age-related changes in the expression and activity of seven hepatic drug metabolizing enzymes (DMEs) in male wild-type and breast cancer resistance protein knockout (Bcrp1-/- ) FVB mice. The protein expression of four cytochrome P450 (Cyps) (Cyp3a11, 2d22, 2e1, and 1a2), and three UDP-glucuronosyltransferases (Ugts) (Ugt1a1, 1a6a, and 1a9) in liver microsomes of wild-type and Bcrp1-/- FVB mice at different ages were determined using a validated ultra high performance liquid chromatography with tandem mass spectrometry (UHPLC-MS/MS) method. The activities and mRNA levels of these DMEs were measured using the probe substrates method and real-time PCR, respectively. In the liver of wild-type FVB mice, Cyp3a11, 2d22, 2e1, 1a2, Ugt1a1, and 1a6a displayed maximum protein levels at 6-9 weeks of age. Cyp1a2, Ugt1a1, 1a6a, and 1a9 showed maximum activities at 6-9 weeks of age, whereas Cyp3a11, 2d22, and 2e1 showed maximum activities in 1-3-week-old mice. Additionally, most of the DMEs showed maximum mRNA levels in 17-week-old mice liver. Compared with wild-type FVB mice, the protein levels of these DMEs showed no significant changes in Bcrp1-/- FVB mice liver. However, the activity of Cyp2e1 was increased and that of Cyp2d22 was decreased. In conclusion, the seven hepatic DMEs in FVB mice liver showed significant alterations in an isoform-specific manner with increased age. Although the protein levels of these DMEs showed no significant changes, the activities of Cyp2e1 and 2d22 were changed in Bcrp1-/- mice.

The effect of probiotics and polysaccharides on the gut microbiota composition and function of weaned rats.

Weaning is an essential and important event for infants and rodent animals, and the dietary supplementation plays a crucial role in regulating the composition and function of gut microbiota in the initial period after weaning. In this study, we investigated the potential effects of two probiotics along with three polysaccharides (PP) in weaned rats. Male SD rats, 21 days of age, were divided into two groups: one group was administered with PP for four weeks and the other was not. Body weight, food intake, gut epithelial barrier function, digestive enzyme activities, and the composition and function of gut microbiota were analyzed. The dietary PP increased the body weight and food intake, promoted gut epithelial barrier integrity, and elevated the activities of digestive enzymes. Moreover, the microbial community structure was different between the two groups. At the genus level, Bifidobacterium, Lactobacillus, and Allobaculum were significantly increased, whereas Anaerostipes, Enterococcus, and Parabacteroides were observably reduced. Furthermore, PP significantly increased amino acid metabolism, energy metabolism, and SCFA-related metabolism. This study shows that synbiotics containing probiotics (L. acidophilus NCFM and B. lactis Bi-07) in combination with polysaccharides (LBP, PCPs, and Lentinan) can modulate the composition of gut microbiota, stimulate the maturation of gut microbiota biological function, and promote the growth performance in weaned rats.

Metabolomics insights into the modulatory effects of long-term compound polysaccharide intake in high-fat diet-induced obese rats.

BACKGROUND: Polysaccharides can alleviate obesity in mammals; however, studies on mechanism of this alleviation are limited. A few studies have indicated that polysaccharides improve obesity by regulating the metabolism of the body. Therefore, a metabolomics approach, consisting of high resolution nuclear magnetic resonance (NMR) spectroscopy and a multivariate statistical technique, was applied to explore the mechanism of the protective effects of lentinan and Flos Lonicera polysaccharides (LF) on high-fat diet (HFD) induced obesity. METHODS: In this study, rats were randomly divided into three groups: control diet (CD), HFD, and HFD supplemented with a mixture of lentinan and Flos Lonicera polysaccharide. Histopathological and clinical biochemical assessments were also conducted. A combination of a NMR metabolomics study and a multivariable statistical analysis method to distinguish urinary and fecal metabolites was applied. RESULTS: Significant obesity symptoms appeared in HFD rats (for example, significant weight gain, epididymal adipose accumulation and lipid deposition in hepatocytes), which was attenuated in the LF group. Additionally, the HFD induced a reduction of choline, citrate, pyruvate and glycerol and increased the levels of trimethylamine oxide (TMAO) and taurine. Of note, these metabolic disorders were reversed by LF intervention mainly through pathways of energy metabolism, choline metabolism and gut microbiota metabolism. CONCLUSIONS: LF supplementation had a re-balancing effect on the disturbed metabolic pathways in the obese body. The results of this study validate the therapeutic effect of the compound polysaccharide--LF in obesity and described the biochemical and metabolic mechanisms involved.

Metabonomic strategy for the detection of metabolic effects of probiotics combined with prebiotic supplementation in weaned rats.

The purpose of this study is to investigate the effects of probiotics combined with prebiotics (PP) supplementation on weaned rat metabolism. A metabonomic strategy employing 1H-NMR spectroscopy and multivariate data analysis was used to examine weaned rat biological responses to PP supplementation. Male Sprague-Dawley rats (post-natal day 21, PD 21) received probiotics (Lactobacillus acidophilus NCFM (L-NCFM) and Bifidobacterium lactis Bi-07 (B-LBi07), 1 : 1, 1.0 × 1011 cfu kg-1) and prebiotics (Lycium barbarum polysaccharides (LBP), Poria cocos polysaccharides (PCPs) and Lentinan, 1 : 1 : 1, 24 g kg-1) via intragastric administration for 28 consecutive days. Urine and feces were collected for analysis. Significant topographical metabolic variations were present in urine and feces. Urinary metabolites upregulated by PP treatment included alanine, N-acetylglycine, glutamine, dimethylamine, phosphorylcholine, ethylene glycol, mannitol, phenylacetylglycine and glycoate, which were related to alanine, aspartate and glutamate metabolism, and choline metabolism. Feces-derived metabolites, including caproate, valerate, butyrate, propionate, lactate, acetate, succinate, methanol, threonine and methionine, were significantly increased, which were related to short-chain fatty acid (SCFA) metabolism and TCA cycle metabolism. These results indicate that dietary PP supplementation can regulate common systemic metabolic processes, including energy metabolism, amino acid metabolism, lipid metabolism, nucleic acid metabolism, and gut microbiota-related metabolism. This study also illuminates the vital role of PP supplementation in regulating the metabolism of weaned rats.

Modulation of Gut Microbiome Composition and Function in Experimental Colitis Treated with Sulfasalazine.

Inflammatory bowel disease (IBD) results from alterations in intestinal flora and the immune system. Sulfasalazine (SASP) is a sulfa antimicrobial used to treat IBD in clinic for years. However, how SASP affects gut microbes and its potential functions remains unclear. To investigate the relationships of SASP, IBD, and gut microbiome, we used 2,4,6-trinitrobenzenesulfonic acid (TNBS) to induce experimental colitis in rats, and analyzed the microbiota in the fecal samples, which come from the control group (treated with ethanol + saline), the model group (treated with TNBS-ethanol + saline) and the SASP group (treated with TNBS-ethanol + SASP), with 16S gene sequencing and followed up a subset sample using shotgun sequencing. The study found that SASP treatment could not only restore the TNBS-induced gut dysbiosis, which was proved by the increasing amount of SCFAs-producing bacteria and lactic acid-producing bacteria as well as the decreasing amount of Proteobacteria, but also modulate the dysregulated function of the TNBS-induced colitis to resemble that of the control group, including an increased capacity for basic metabolism (carbohydrate metabolism, citrate cycle) and a decrease in the oxidative stress (riboflavin, sulfur, cysteine) as well as bacterial pathogenesis (cell motility and secretion, bacterial motility proteins, flagellar assembly). Moreover, a higher proportion of Mycoplasma was observed in the SASP group, which may associate with infertility. In all, the study provides insight into specific microbial clades and pathways linked with SASP treatment to elaborate the mechanism for treatment of IBD.

Profiles and Gender-Specifics of UDP-Glucuronosyltransferases and Sulfotransferases Expressions in the Major Metabolic Organs of Wild-Type and Efflux Transporter Knockout FVB Mice.

Hepatic and extrahepatic tissues participate in xenobiotic detoxication, carcinogen activation, prodrug processing, and estrogen regulation through UDP-glucuronosyltransferases (UGTs/Ugts) and sulfotransferases (SULTs/Sults). Wild-type (WT) and efflux transporter knockout (KO) FVB mice have been commonly used to perform the studies of pharmacokinetics, metabolism, and toxicity. We employed the developed UHPLC-MS/MS approach to gain systematic insight on gender-specific of Ugts and Sults in major metabolic organs. Results showed that the liver was the most abundant with Ugts/Sults, followed by the small intestine and the kidney. In the liver, Ugt2b5, Ugt2b1, Ugt1a6a, Ugt1a1, Sult1a1, and Sult1d1 were the major isoforms. The protein amounts of Ugt1a9 were significantly higher in male efflux transporter KO mice than in WT mice, whereas Ugt1a5 and Sult1a1 severely decreased in female efflux transporter KO mice. In WT and efflux transporter KO mice, the expression levels of Ugt1a1, Ugt1a5, Sult1a1, Sult1d1, and Sult3a1 were female-specific, whereas those of Ugt2b1, Ugt2b5, and Ugt2b36 were male-specific. In the small intestine, Ugt1a1, Sult1b1, and Sult2b1 were the major isoforms. The protein levels and gender differences of Ugts/Sults were obviously affected when KO of Mdr1a, and Bcrp1, Mrp1, Mrp2, and Mdr1a, respectively. The KO of efflux transporter affected the protein amounts of Ugts/Sults in the kidney, heart, and spleen. Therefore, a better understanding of the expression profiles and gender-specific of Ugts and Sults in major metabolic organs of WT and efflux transporter KO mice is useful for the evaluation of potential efficacy, and toxicity of corresponding substrates.

Sulfotransferases and Breast Cancer Resistance Protein Determine the Disposition of Calycosin in Vitro and in Vivo.

Sulfation is a key process of drug disposition that generally regulates drug effectiveness and toxicity. Calycosin derived from the dry root extract of Radix Astragali exhibits a variety of biological effects that easily undergo extensive phase II metabolism. However, the sulfation pathway of calycosin lacks information. We investigated the disposition mechanisms of calycosin sulfate in vitro and in vivo. We characterized the sulfation metabolism and excretion of calycosin using bidirectional transport studies. We confirmed that sulfate conjugate is breast cancer resistance protein (BCRP) substrate using the intestinal perfusion model and pharmacokinetics studies in Bcrp1-/- mice. Results showed that calycosin is rapidly and extensively metabolized to calycosin-3'-sulfate (C-3'-S) in the intestine and liver. The overexpression of BCRP led to a substantial increase (approximately 14-fold, p < 0.01) of excreted C-3'-S in the BCRP overexpressed Madin-Darby canine kidney II (MDCK II/BCRP) cells. The chemical inhibition of BCRP caused reduction (about 2-fold, p < 0.01) in C-3'-S apical excretion. Furthermore, in intestinal perfusion studies, the deletion of Bcrp1 significantly decreased C-3'-S excretion in the small intestine (82.6-90.6%, p < 0.01) and colon (97.6-98.2%, p < 0.01). In contrast, plasma level of C-3'-S was increased to 40-fold (p < 0.01) in Bcrp1-/- mice. In conclusion, calycosin undergoes an extensive sulfation metabolism and BCRP is a critical determinant to the disposition of C-3'-S.

Breast Cancer Resistance Protein and Multidrug Resistance Protein 2 Regulate the Disposition of Acacetin Glucuronides.

PURPOSE: To determine the mechanism responsible for acacetin glucuronide transport and the bioavailability of acacetin. METHODS: Area under the curve (AUC), clearance (CL), half-life (T1/2) and other pharmacokinetic parameters were determined by the pharmacokinetic model. The excretion of acacetin glucuronides was evaluated by the mouse intestinal perfusion model and the Caco-2 cell model. RESULTS: In pharmacokinetic studies, the bioavailability of acacetin in FVB mice was 1.3%. Acacetin was mostly exposed as acacetin glucuronides in plasma. AUC of acacetin-7-glucuronide (Aca-7-Glu) was 2-fold and 6-fold higher in Bcrp1 (-/-) mice and Mrp2 (-/-) mice, respectively. AUC of acacetin-5-glucuronide (Aca-5-Glu) was 2-fold higher in Bcrp1 (-/-) mice. In mouse intestinal perfusion, the excretion of Aca-7-Glu was decreased by 1-fold and 2-fold in Bcrp1 (-/-) and Mrp2 (-/-) mice, respectively. In Caco-2 cells, the efflux rates of Aca-7-Glu and Aca-5-Glu were significantly decreased by breast cancer resistance protein (BCRP) inhibitor Ko143 and multidrug resistance protein 2 (MRP2) inhibitor LTC4. The use of these inhibitors markedly increased the intracellular acacetin glucuronide content. CONCLUSIONS: BCRP and MRP2 regulated the in vivo disposition of acacetin glucuronides. The coupling of glucuronidation and efflux transport was probably the primary reason for the low bioavailability of acacetin.

High-Throughput and Reliable Isotope Label-free Approach for Profiling 24 Metabolic Enzymes in FVB Mice and Sex Differences.

FVB mice are extensively used in transgenic and pharmacokinetic research. In this study, a validated isotope label-free method was constructed using ultrahigh-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) to quantify 24 drug-metabolizing enzymes (DMEs) in FVB mice. The DMEs include cytochrome P450s (CYP450s/Cyp450s), UDP-glucuronsyltransferases (UGTs/Ugts), and sulfotransferases (SULTs/Sults), which catalyze a variety of reactions to detoxify xenobiotics and endobiotics. The proposed UHPLC-MS/MS method exhibited good range and high sensitivity for signature peptides, as well as acceptable accuracy, precision, and recovery. The protein expression profiles of the DMEs were determined in male and female mice. Overall, the major Cyps, Ugts, and Sults were expressed in male mice followed the rank order: Cyp2c29 > 2e1 > 3a11 > 1a2 > 2d22 > 27a1 > 2c39; Ugt2b5 > 2b1 > 1a6a > 1a9 > 1a1 > 2a3 > 1a2 > 1a5; and Sult1a1 > 3a > 1d1. In contrast, the rank order in female mice was Cyp2c29 > 2e1 > 2c39 > 2d22 > 3a11 > 1a2 > 27a1; Ugt1a6a > 2b5 > 1a1 > 2b1 > 2a3 > 1a9 > 1a5 > 1a2; and Sult1a1 > 3a1 > 1d1. Cyp2c29, Cyp1a2, Cyp27a1, Ugt2b1, Ugt2b5 and Ugt2b36 were male predominant, whereas Cyp2c39, Cyp2d22, Cyp7a1, Ugt1a1, Ugt1a5, Sult1a1, Sult3a1, and Sult1d1 were female predominant. This work could serve as a useful reference for the metabolic study of new drugs and for elucidating the effectiveness and toxicity of drugs. The method is stable, simple, and rapid for determining the expression of DMEs in animals.

In Vitro Study of UGT Metabolism and Permeability of Orientin and Isoorientin, Two Active flavonoid C-glycosides.

C-glycosides are important flavonoids with significant pharmacological activities implicated in anticancer and antioxidative effects. However, their characteristics of metabolism and transportation have been rarely investigated. This research aimed to examine the metabolic characteristics of two active C-glycosides, namely, orientin and isoorientin, in human liver microsomes (HLMs) and rat liver microsomes (RLMs) and to confirm the specific uridine 5'-diphospho glucuronosyltransferase (UGT) isoforms involved in glucuronidation by HLMs. Furthermore, the permeability of orientin and isoorientin was also determined by using Caco-2 cell monolayers. Results revealed that orientin and isoorientin could generate two metabolites, which were identified as monoglucuronides. HLM- and RLM-mediated glucuronide formations were in accordance with typical Michaelis-Menten kinetics. Conversely, RLM initially metabolized orientin to its corresponding metabolite, and this process was consistent with biphasic kinetics. Among the UGT isoform, UGT1A1, 1A8, 1A9 and 1A10 exhibited the highest enzyme activity. Passive diffusion was the predominant orientin and isoorientin transportation mechanism in Caco-2 cell monolayers, and their apparent permeability further confirmed that orientin and isoorientin were well absorbed. Therefore, orientin and isoorientin can be metabolized by UGT isoforms and microsomes; these flavonoids can also be transported via passive diffusion in Caco-2 cells, which are relatively permeable.

SGLT-1 Transport and Deglycosylation inside Intestinal Cells Are Key Steps in the Absorption and Disposition of Calycosin-7-O-ß-d-Glucoside in Rats.

Hydrolysis by lactase-phloridzin hydrolase (LPH) is the first and critical step in the absorption of isoflavonoid glucosides. However, the absorption characteristics of calycosin-7-O-ß-d-glucoside (CG) slightly differ from other isoflavonoid glucosides. In this study, we used the rat intestinal perfusion model and performed pharmacokinetic studies and in vitro experiments to determine the factors influencing CG absorption and disposition. After oral administration of isoflavonoid glucosides, LPH was found to play minimal or no role on the hydrolysis of CG, in contrast to that of daidzin. CG was mainly transported into the small intestinal cells by sodium-dependent glucose transporter 1 (SGLT-1) as intact. This pathway could be the main mechanism underlying the high permeability of CG in the small intestine. CG was likely to be hydrolyzed in enterocytes to its aglycone calycosin by broad-specific ß-glucuronides (BSßG) and glucocerebrosidase or rapidly metabolized. Calycosin was also rapidly and extensively metabolized to 3'-glucuronide in the enterocytes and liver, and the glucuronidation rates of calycosin and CG were much higher in the former. The metabolites were also transported into lumen by breast cancer resistance protein and multidrug resistance-associated protein 2. In conclusion, the enterocytes could be an important site for CG absorption, deglycosylation, and metabolism in rats. This study could contribute to the theoretical foundation and mechanism of absorption and disposition of flavonoid compounds.