Prediction of teicoplanin plasma concentration in critically ill patients: a combination of machine learning and population pharmacokinetics.

BACKGROUND: Teicoplanin has been widely used in patients with infections caused by Staphylococcus aureus, especially for critically ill patients. The pharmacokinetics (PK) of teicoplanin vary between individuals and within the same individual. We aim to establish a prediction model via a combination of machine learning and population PK (PPK) to support personalized medication decisions for critically ill patients. METHODS: A retrospective study was performed incorporating 33 variables, including PPK parameters (clearance and volume of distribution). Multiple algorithms and Shapley additive explanations were employed for feature selection of variables to determine the strongest driving factors. RESULTS: The performance of each algorithm with PPK parameters was superior to that without PPK parameters. The composition of support vector regression, categorical boosting and a backpropagation neural network (7:2:1) with the highest R2 (0.809) was determined as the final ensemble model. The model included 15 variables after feature selection, of which the predictive performance was superior to that of models considering all variables or using only PPK. The R2, mean absolute error, mean squared error, absolute accuracy (±5 mg/L) and relative accuracy (±30%) of external validation were 0.649, 3.913, 28.347, 76.12% and 76.12%, respectively. CONCLUSIONS: Our study offers a non-invasive, fast and cost-effective prediction model of teicoplanin plasma concentration in critically ill patients. The model serves as a fundamental tool for clinicians to determine the effective plasma concentration range of teicoplanin and formulate individualized dosing regimens accordingly.

Cyclosporine-A induced cytotoxicity within HepG2 cells by inhibiting PXR mediated CYP3A4/CYP3A5/MRP2 pathway.

Cyclosporine-A (CsA) is currently used to treat immune rejection after organ transplantation as a commonly used immunosuppressant. Liver injury is one of the most common adverse effects of CsA, whose precise mechanism has not been fully elucidated. Pregnane X receptor (PXR) plays a critical role in mediating drug-induced liver injury as a key regulator of drug and xenobiotic clearance. As a nuclear receptor, PXR transcriptionally upregulates the expression of drug-metabolizing enzymes and drug transporters, including cytochrome P4503A (CPY3A) and multidrug resistance-associated protein 2 (MRP2). Our study established CsA-induced cytotoxic hepatocytes in an in vitro model, demonstrating that CsA dose-dependently increased the aspartate aminotransferase (AST) and lactate dehydrogenase (LDH) level secreted in the HepG2 cell supernatant, as well as viability and oxidative stress of HepG2 cells. CsA also dose-dependently decreased the PXR, CYP3A4, CPY3A5, and MRP2 levels of HepG2 cells. Mechanistically, altering the expression of PXR, CYP3A4, CYP3A5, and MRP2 affected the impact of CsA on AST and LDH levels. Moreover, altering the expression of PXR also changed the level of CYP3A4, CPY3A5, and MRP2 of HepG2 cells treated by CsA. Our presented findings provide experimental evidence that CsA-induced liver injury is PXR tightly related. We suggest that PXR represents an attractive target for therapy of liver injury due to its central role in the regulation of the metabolizing enzymes CYP3A and MRP2-mediated bile acid transport and detoxification.

Vitamin D receptor (VDR) on the cell membrane of mouse macrophages participates in the formation of lipopolysaccharide tolerance: mVDR is related to the effect of artesunate to reverse LPS tolerance.

It is unclear whether membrane vitamin D receptor (mVDR) exists on the macrophage membrane or whether mVDR is associated with lipopolysaccharide (LPS) tolerance. Herein, we report that interfering with caveolae and caveolae-dependent lipid rafts inhibited the formation of LPS tolerance. VDR was detected as co-localized with membrane molecular markers. VDR was detected on the cell membrane and its level was higher in LPS-tolerant cells than that in only LPS treatment cells. Anti-VDR antibodies could abolish the effect of artesunate (AS) to reverse LPS tolerance, and the wild-type peptides (H397 and H305) of VDR, but not the mutant peptide (H397D and H305A), led to the loss of AS's effect. AS decreased the mVDR level in LPS-tolerant cells. In vivo, AS significantly reduced VDR level in the lung tissue of LPS-tolerant mice. In summary, mVDR exists on the cell membrane of macrophages and is closely associated with the formation of LPS tolerance and the effects of AS. Video Abstract.

Effect of CYP2C19 polymorphism on the plasma voriconazole concentration and voriconazole-to-voriconazole-N-oxide concentration ratio in elderly patients.

BACKGROUND: Effects of CYP2C19 polymorphism on voriconazole concentration (C0 ), dose-adjusted trough concentrations (C0 /dose) and voriconazole-to-voriconazole-N-oxide concentration ratio (C0 /CN ) have not been fully investigated. OBJECTIVES: To investigate correlations of CYP2C19 polymorphisms with plasma concentrations of voriconazole and the major metabolite voriconazole-N-oxide in elderly patients. METHODS: A prospective, multi-centre, non-intervention, open clinical study was conducted within Southwestern Chinese patients clinically diagnosed with invasive fungal infections, to investigate the associations of CYP2C19∗2 (681G > A), CYP2C19∗3 (636G > A) and CYP2C19∗17 (-806C > T) genetic polymorphisms with voriconazole C0 , C0 /dose and C0 /CN . RESULTS: The study included 131 adult patients, of which 72 were elderly (≥60 years) and 59 were adults (<60 years). The allele frequencies of CYP2C19∗2, ∗3 and ∗17 in the elderly cohort were 61.1%, 29.9% and 7.6%, respectively, which were similar to those in the adult cohort (66.9%, 29.7% and 2.5%, respectively; P > .05). The median voriconazole C0 (C0 ), C0 /dose and C0 /CN ratio in patients with the CYP2C19∗1/∗2 and CYP2C19∗2/∗2 genotypes were significantly higher than those in patients with the CYP2C19∗1/∗1 genotype in the adult cohort (P < .05). The C0 and C0 /dose in patients with the CYP2C19∗1/∗3 and CYP2C19∗2/∗2 genotypes, and the C0 /CN ratio for patients with the CYP2C19∗1/∗2 genotype were numerically higher than those in patients with the CYP2C19∗1/∗1 genotype in the elderly cohort, but this difference was not statistically significant (P > 0.05). The C0 , C0 /dose and C0 /CN in patients with poor metaboliser phenotypes were higher than in those with normal metaboliser phenotypes and C0 in patients with intermediate metaboliser phenotypes were significantly higher than in those with normal metaboliser phenotypes in the adult cohort (P < .05). However, there were no significant differences in the C0 , C0 /dose and C0 /CN among different CYP2C19-predicted metabolic phenotypes in the elderly cohort. CONCLUSIONS: Voriconazole C0 , C0 /dose and C0 /CN ratio are not significantly affected by the CYP2C19∗2/∗3 polymorphisms in the elderly patients.

Artesunate Attenuates Pro-Inflammatory Cytokine Release from Macrophages by Inhibiting TLR4-Mediated Autophagic Activation via the TRAF6-Beclin1-PI3KC3 Pathway.

BACKGROUND/AIMS: Lipopolysaccharide (LPS) plays a critical role in excessive inflammatory cytokine production during sepsis. Previously, artesunate (AS) was reported to protect septic mice by reducing LPS-induced pro-inflammatory cytokine release. In the present study, the possible mechanism of the anti-inflammatory effect of AS was further investigated. METHODS: An enzyme-linked immunosorbent assay was used to detect TNF-α and IL-6 release from macrophages. Specific small interfering RNAs (siRNAs) were used to knockdown the mRNA expression of target genes. Transmission electron microscopy and laser confocal microscopy were used to observe changes in autophagy. Western blotting was performed to detect the protein levels of tumor necrosis factor receptor-associated factor6 (TRAF6), Beclin1, phosphatidylinositol 3-kinase class III (PI3KC3), autophagy-related protein 5 (ATG5), and sequestosome 1. Immunoprecipitation (IP) and fluorescent co-localization were used to detect the interactions between TRAF6-Beclin1 and Beclin1-PI3KC3, and the ubiquitination of Beclin1. RESULTS: AS inhibited TNF-α and IL-6 release from RAW264.7 cells, mouse bone marrow-derived monocytes (BMDMs) and peritoneal macrophages (PMs) induced by LPS. However, the inhibition by AS of LPS-induced cytokine release decreased when autophagy was inhibited using 3-MA, bafilomycin A1, or a siRNA targeting the Atg5 gene. Notably, AS showed an inhibition of LPS-induced autophagic activation not degradation. Whereas, these effects of AS were lost in macrophages lacking TLR4 and decreased in macrophages with down-regulated TRAF6, indicating that AS inhibited LPS-induced cytokine release and autophagic activation via TLR4-TRAF6 signaling. Western blotting results showed AS could reduce the levels of TRAF6, Beclin1, and PI3KC3. Importantly, the IP results showed AS only inhibited K63-linked ubiquitylation not total ubiquitylation of Beclin1 by acting on TRAF6. This interrupted the TRAF6-Beclin1 interaction and subsequent the formation of Beclin1- PI3KC3 core complex of the PI3K-III complex. CONCLUSION: AS inhibited LPS-induced cytokine release from macrophages by inhibiting autophagic activation. This effect was tightly related to blockade of the TRAF6-Beclin1-PI3KC3 pathway via decreasing K63-linked ubiquitination of Beclin1 and then interrupting the formation of Beclin1-PI3KC3 core complex of the PI3K-III complex. Our findings reveal the mechanism of AS's anti-inflammatory effect and is significant for future targeted investigations of sepsis treatment.

NF-κB potentiates tumor growth by suppressing a novel target LPTS.

BACKGROUND: Chronic inflammation is causally linked to the carcinogenesis and progression of most solid tumors. LPTS is a well-identified tumor suppressor by inhibiting telomerase activity and cancer cell growth. However, whether and how LPTS is regulated by inflammation signaling is still incompletely elucidated. METHODS: Real-time PCR and western blotting were used to determine the expression of p65 and LPTS. Reporter gene assay, electrophoretic mobility shift assay and chromatin immunoprecipitation were performed to decipher the regulatory mechanism between p65 and LPTS. Cell counting kit-8 assays and xenograt models were used to detect p65-LPTS-regulated cancer cell growth in vitro and in vivo, respectively. RESULTS: Here we for the first time demonstrated that NF-κB could inhibit LPTS expression in the mRNA and protein levels in multiple cancer cells (e.g. cervical cancer and colon cancer cells). Mechanistically, NF-κB p65 could bind to two consensus response elements locating at -1143/-1136 and -888/-881 in the promoter region of human LPTS gene according to EMSA and ChIP assays. Mutation of those two binding sites rescued p65-suppressed LPTS promoter activity. Functionally, NF-κB regulated LPTS-dependent cell growth of cervical and colon cancers in vitro and in xenograft models. In translation studies, we verified that increased p65 expression was associated with decreased LPTS level in multiple solid cancers. CONCLUSIONS: Taken together, we revealed that NF-κB p65 potentiated tumor growth via suppressing a novel target LPTS. Modulation of NF-κB-LPTS axis represented a potential strategy for treatment of those inflammation-associated malignancies.

Effect of aspirin on platelet-rich plasma of diabetes mellitus with lower extremity atherosclerosis.

Aim: Platelet-rich plasma (PRP), enriched with multiple growth factors, is a promising adjunctive therapy for diabetic foot ulcers (DFUs). As a classic anti-platelet drug for diabetic patients, the effects of aspirin on the content of growth factors in PRP remains unclear.Methods: Our study enrolled diabetic patients who were currently taking or not taking aspirin as the research subjects, with healthy volunteers as the control. PRP from these individuals was activated with glucose calcium and thrombin. Growth factors levels in PRP activated supernatant (PRP-AS) and wound healing ability of platelet gel (PG) in the full-thickness skin defect diabetic mouse model were compared.Results: We found the level of growth factors in PRP-AS derived from two groups of diabetic patients were not statistically different, whereas both lower than that from healthy volunteers. Similarly, we found better wound healing ability of PG from healthy volunteers than those from diabetic patients, but no difference between the two groups of diabetic patients in the mouse model.Discussion: Aspirin does not interfere with autologous PRP therapy when using calcium gluconate and thrombin as agonists. However considering the content of growth factors, PRP from healthy volunteers is a preferable option for promoting DFU repair.

Platelet-rich plasma (PRP), enriched with multiple growth factors, effectively promote the healing of refractory diabetic foot ulcer (DFU). We found that aspirin, as a commonly used antiplatelet drug for diabetic patients, did not affect the growth factors content in autologous PRP from diabetic patients or its wound-healing ability in diabetic mouse skin defects. However, PRP from healthy volunteers showed superior performance in both aspects. Our study provide a theoretical basis for personalized PRP treatment plans for DFU patients.

Joint use of population pharmacokinetics and machine learning for prediction of valproic acid plasma concentration in elderly epileptic patients.

BACKGROUND: Valproic acid (VPA) is a commonly used broad-spectrum antiepileptic drug. For elderly epileptic patients, VPA plasma concentrations have a considerable variation. We aim to establish a prediction model via a combination of machine learning and population pharmacokinetics (PPK) for VPA plasma concentration. METHODS: A retrospective study was performed incorporating 43 variables, including PPK parameters. Recursive Feature Elimination with Cross-Validation was used for feature selection. Multiple algorithms were employed for ensemble model, and the model was interpreted by Shapley Additive exPlanations. RESULTS: The inclusion of PPK parameters significantly enhances the performance of individual algorithm model. The composition of categorical boosting, light gradient boosting machine, and random forest (7:2:1) with the highest R2 (0.74) was determined as the ensemble model. The model included 11 variables after feature selection, of which the predictive performance was comparable to the model that incorporated all variables. CONCLUSIONS: Our model was specifically tailored for elderly epileptic patients, providing an efficient and cost-effective approach to predict VPA plasma concentration. The model combined classical PPK with machine learning, and underwent optimization through feature selection and algorithm integration. Our model can serve as a fundamental tool for clinicians in determining VPA plasma concentration and individualized dosing regimens accordingly.

A 47-Year-Old Man with Hyperphosphatemia Due to Chronic Renal Failure Treated with Lanthanum Carbonate Tablets Presenting Acutely with Partial Large Bowel Obstruction.

BACKGROUND Hyperphosphatemia is a complication of chronic renal failure (CRF) due to reduction in the glomerular filtration rate. Lanthanum carbonate is a commonly used phosphate binder for patients with CRF and hyperphosphatemia, but has adverse effects if patients are not monitored. This report is of a 47-year-old man with hyperphosphatemia due to CRF treated with lanthanum carbonate tablets who presented acutely with partial large bowel obstruction. The incidence of lanthanum carbonate causing intestinal obstruction is rare, and few cases in the literature have described the course of the disease in detail. CASE REPORT A 47-year-old man diagnosed with diabetic nephropathy underwent hemodialysis treatment and was prescribed 0.5 g/day of chewable lanthanum carbonate tablets. After taking lanthanum carbonate for 5 months, the patient experienced symptoms of decreased bowel movements and exhaustion, which progressively worsened. Abdominal computed tomography (CT) revealed multiple hyperdensities in the large bowel, indicating the presence of lanthanum deposition. Lanthanum carbonate was promptly discontinued. After undergoing enema and catharsis treatment, the large bowel obstruction was relieved, and the hyperdensities in the abdominal CT disappeared. The colonoscopy and histologic examination revealed ulcerations and inflammatory changes in the large bowel mucosa. CONCLUSIONS This report highlights the rare association between the use of lanthanum carbonate tablets and intestinal obstruction. Healthcare providers should enhance their vigilance regarding lanthanum carbonate-induced serious gastrointestinal adverse reactions and actively seek to detect lanthanum deposition by abdominal CT or radiography (X-ray). After the occurrence of lanthanum deposition, drug withdrawal and promotion of defecation are primary treatment methods.

Pharmacokinetic/pharmacodynamic comparison between generic and brand-name levofloxacin based on Monte Carlo simulation.

OBJECTIVE: Generic medications are widely used because of their low cost. However, some generic medications show lower quality and clinical efficacy compared with brand-name medications, especially for antimicrobial drugs. Levofloxacin is a fluoroquinolone antimicrobial drug with excellent antimicrobial activity and wide antimicrobial spectrum, while it is susceptible to drug resistance. Our study aims to evaluate the bioequivalence of generic and brand-name levofloxacin. METHODS: The pharmacokinetic (PK) parameters (Cmax, AUC0∼24, Tmax, and t1/2), pharmacodynamic (PD) parameters (in vitro antibacterial activity and the inhibition of resistant mutation), and PK/PD analysis (the probability of target attainment; the cumulative fraction of response) calculated by Monte Carlo simulation were investigated. RESULTS: Our results demonstrated that compared with generics, brand-name levofloxacin not only had higher drug content, it also showed higher antimicrobial susceptibility, higher resistance to mutation ability, and higher percentage of each dosage interval wherein plasma concentration of antimicrobial agents exceeded the MPC90 (mutant prevention concentration to prevent the mutation of 90% strains) against various clinical isolates. Although the differences in AUC0∼24 between brand-name levofloxacin and generics were not statistically significant (P > 0.05, F test), Monte Carlo simulation results showed cumulative fraction of response values for PK/PD of brand-name medications were higher than generics. CONCLUSION: Our results indicated that PK or PD equivalence did not imply therapeutic equivalence; thus, we suggest including PK/PD analysis in the bioequivalence evaluation system, which benefits prediction of clinical outcome with high application value.

Construction and Interpretation of Prediction Model of Teicoplanin Trough Concentration via Machine Learning.

Objective: To establish an optimal model to predict the teicoplanin trough concentrations by machine learning, and explain the feature importance in the prediction model using the SHapley Additive exPlanation (SHAP) method. Methods: A retrospective study was performed on 279 therapeutic drug monitoring (TDM) measurements obtained from 192 patients who were treated with teicoplanin intravenously at the First Affiliated Hospital of Army Medical University from November 2017 to July 2021. This study included 27 variables, and the teicoplanin trough concentrations were considered as the target variable. The whole dataset was divided into a training group and testing group at the ratio of 8:2, and predictive performance was compared among six different algorithms. Algorithms with higher model performance (top 3) were selected to establish the ensemble prediction model and SHAP was employed to interpret the model. Results: Three algorithms (SVR, GBRT, and RF) with high R 2 scores (0.676, 0.670, and 0.656, respectively) were selected to construct the ensemble model at the ratio of 6:3:1. The model with R 2 = 0.720, MAE = 3.628, MSE = 22.571, absolute accuracy of 83.93%, and relative accuracy of 60.71% was obtained, which performed better in model fitting and had better prediction accuracy than any single algorithm. The feature importance and direction of each variable were visually demonstrated by SHAP values, in which teicoplanin administration and renal function were the most important factors. Conclusion: We firstly adopted a machine learning approach to predict the teicoplanin trough concentration, and interpreted the prediction model by the SHAP method, which is of great significance and value for the clinical medication guidance.

Artesunate interacts with the vitamin D receptor to reverse sepsis-induced immunosuppression in a mouse model via enhancing autophagy.

BACKGROUND AND PURPOSE: Immunosuppression is the predominant cause of mortality for sepsis because of failure to eradicate pathogens. No effective and specific drugs capable of reversing immunosuppression are clinically available. Evidences implicate the involvement of the vitamin D receptor (NR1I1) in sepsis-induced immunosuppression. The anti-malarial artesunate was investigated to determine action on sepsis-induced immunosuppression. EXPERIMENTAL APPROACH: The effect of artesunate on sepsis-induced immunosuppression was investigated in mice and human and mice cell lines. Bioinformatics predicted vitamin D receptor as a candidate target for artesunate, which was then identified using PCR and immunoblotting. Vdr, Atg16l1 and NF-κB p65 were modified to investigate artesunate 's effect on pro-inflammatory cytokines release, bacterial clearance and autophagy activities in sepsis-induced immunosuppression. KEY RESULTS: Artesunate significantly reduced the mortality of caecal ligation and puncture (CLP)-induced sepsis immunosuppression mice challenged with Pseudomonas aeruginosa and enhanced pro-inflammatory cytokine release and bacterial clearance to reverse sepsis-induced immunosuppression in vivo and in vitro. Mechanistically, artesunate interacted with vitamin D receptor, inhibiting its nuclear translocation, which influenced ATG16L1 transcription and subsequent autophagy activity. Artesunate inhibited the physical interaction between vitamin D receptor and NF-κB p65 in LPS-tolerant macrophages and then promoted the nuclear translocation of NF-κB p65, which activated the transcription of NF-κB p65 target genes such as pro-inflammatory cytokines. CONCLUSION AND IMPLICATIONS: Our findings provide evidence that artesunate interacted with vitamin D receptor to reverse sepsis-induced immunosuppression in an autophagy and NF-κB-dependent manner, highlighting a novel approach for sepsis treatment and drug repurposing of artesunate has a bidirectional immunomodulator.

Artesunate reverses LPS tolerance by promoting ULK1-mediated autophagy through interference with the CaMKII-IP3R-CaMKKß pathway.

The progress of sepsis is increasingly recognized by the transition from early hyperinflammation to long term immunosuppression, which is characterized in innate immune cells by diminished responsiveness termed as lipopolysaccharide (LPS) tolerance. In this study, we investigated the ability of the antimalarial drug artesunate to reverse LPS tolerance and explored the underlying mechanisms. Initially, we detected a dramatic decline in autophagy accompanied by decreased cytokine production and impaired bacterial clearance by LPS tolerant macrophages. Then we demonstrated that artesunate restored cytokine production and enhanced bacterial clearance by inducing autophagy. Moreover, artesunate caused greater suppression of inhibitory phosphorylation than of activating phosphorylation of Unc-51 like autophagy activating kinase 1 (ULK1), a kinase that is essential for initiating autophagy through the inhibition of excessive AMP-activated protein kinase (AMPK) activation. This effect was shown to be achieved by suppression of Ca2+/calmodulin-dependent protein kinase II (CaMKII) phosphorylation, resulting in reduction of the inositol 1,4,5-triphate receptor (IP3R) dependent Ca2+ release from the endoplasmic reticulum (ER) and inhibiting the overactive CaMKKß-AMPK cascade. Administration of artesunate also upregulated autophagy and reversed the tolerant status in LPS tolerant mice. In summary, our findings reveal a novel immunopharmacological action of artesunate to reverse LPS tolerance by restoring autophagy. Our results may also indicate the significance of autophagy induction for treating immunosuppression in sepsis.

Macrophage ABHD5 Suppresses NFκB-Dependent Matrix Metalloproteinase Expression and Cancer Metastasis.

Metabolic reprogramming in tumor-associated macrophages (TAM) is associated with cancer development, however, the role of macrophage triglyceride metabolism in cancer metastasis is unclear. Here, we showed that TAMs exhibited heterogeneous expression of abhydrolase domain containing 5 (ABHD5), an activator of triglyceride hydrolysis, with migratory TAMs expressing lower levels of ABHD5 compared with the nonmigratory TAMs. ABHD5 expression in macrophages inhibited cancer cell migration in vitro in xenograft models and in genetic cancer models. The effects of macrophage ABHD5 on cancer cell migration were dissociated from its metabolic function as neither triglycerides nor ABHD5-regulated metabolites from macrophages affected cancer cell migration. Instead, ABHD5 deficiency in migrating macrophages promoted NFκB p65-dependent production of matrix metalloproteinases (MMP). ABHD5 expression negatively correlated with MMP expression in TAMs and was associated with better survival in patients with colorectal cancer. Taken together, our findings show that macrophage ABHD5 suppresses NFκB-dependent MMP production and cancer metastasis and may serve as a prognostic marker in colorectal cancer. SIGNIFICANCE: These findings highlight the mechanism by which reduced expression of the metabolic enzyme ABHD5 in macrophages promotes cancer metastasis.Graphical Abstract: http://cancerres.aacrjournals.org/content/canres/79/21/5513/F1.large.jpg.

Monoacylglycerol lipase regulates cannabinoid receptor 2-dependent macrophage activation and cancer progression.

Metabolic reprogramming greatly contributes to the regulation of macrophage activation. However, the mechanism of lipid accumulation and the corresponding function in tumor-associated macrophages (TAMs) remain unclear. With primary investigation in colon cancer and confirmation in other cancer models, here we determine that deficiency of monoacylglycerol lipase (MGLL) results in lipid overload in TAMs. Functionally, macrophage MGLL inhibits CB2 cannabinoid receptor-dependent tumor progression in inoculated and genetic cancer models. Mechanistically, MGLL deficiency promotes CB2/TLR4-dependent macrophage activation, which further suppresses the function of tumor-associated CD8+ T cells. Treatment with CB2 antagonists delays tumor progression in inoculated and genetic cancer models. Finally, we verify that expression of macrophage MGLL is decreased in cancer tissues and positively correlated with the survival of cancer patients. Taken together, our findings identify MGLL as a switch for CB2/TLR4-dependent macrophage activation and provide potential targets for cancer therapy.

Systematic investigation on the turning point of over-inflammation to immunosuppression in CLP mice model and their characteristics.

Immunosuppression is involved in refractory innate and adaptive immune responses and is considered to be the predominant driving force for mortality in sepsis. The cecal ligation and puncture (CLP) model is regarded as a golden standard model for sepsis study, but the turning point of over-inflammation to immunosuppression was reported differently. Herein, systematic investigation on the turning point of over-inflammation to immunosuppression in CLP mice model was carried out. The results showed only the mortality of mice challenged with of Pseudomonas aeruginosa on Day 1 not other days after the surgery was higher than that of other mice with Sham surgery, suggesting Day 1 after the CLP surgery might be the turning point. There was very low mortality even without death in Sham mice but the mortality was 80% after mice were challenged with 2.5×107, 5.0×106 and 1.0×106CFU/10g of Pseudomonas aeruginosa, further demonstrating Day 1 after the CLP surgery was the turning point. And, CLP mice presented low levels of pro-inflammatory and anti-inflammatory cytokines, and high bacterial loads on Day 1. Additionally, the amounts and proportion of blood cells and monocytes significantly changed, too. In conclusion, Day 1 after the CLP surgery was the turning point of over-inflammation to immunosuppression, and low levels of cytokines and high bacterial loads were the characteristics of this model on Day 1, which is significant for pharmacological investigation on sepsis.

Quantification of neomangiferin in rat plasma by liquid chromatography-tandem mass spectrometry and its application to bioavailability study.

Neomangiferin, a natural C-glucosyl xanthone, has recently received a great deal of attention due to its multiple biological activities. In this study, a rapid and sensitive ultra-high performance liquid chromatography tandem mass spectrometry (UHPLC-MS/MS) method for the quantification of neomangiferin in rat plasma was developed. Using chloramphenicol as an internal standard (IS), plasma samples were subjected to a direct protein precipitation process using methanol (containing 0.05% formic acid). Quantification was performed by multiple reactions monitoring (MRM) method, with the transitions of the parent ions to the product ions of m/z 583.1→330.9 for NG and m/z 321.1→151.9 for IS. The assay was shown to be linear over the range of 0.2-400 ng/mL, with a lower limit of quantification of 0.2 ng/mL. Mean recovery of neomangiferin in plasma was in the range of 97.76%-101.94%. Relative standard deviations (RSDs) of intra-day and inter-day precision were both <10%. The accuracy of the method ranged from 94.20% to 108.72%. This method was successfully applied to pharmacokinetic study of neomangiferin after intravenous (2 mg/kg) and intragastric (10 mg/kg) administration for the first time. The oral absolute bioavailability of neomangiferin was estimated to be 0.53%±0.08% with an elimination half-life (t1/2) value of 2.74±0.92 h, indicating its poor absorption and/or strong metabolism in vivo.