Population pharmacokinetics of intravenous colistin sulfate and dosage optimization in critically ill patients.

Aims: To explore the population pharmacokinetics of colistin sulfate and to optimize the dosing strategy for critically ill patients. Methods: The study enrolled critically ill adult patients who received colistin sulfate intravenously for more than 72 h with at least one measurement of plasma concentration. Colistin concentrations in plasma or urine samples were measured by ultraperformance liquid chromatography tandem mass spectrometry (LC-MS/MS). The population pharmacokinetics (PPK) model for colistin sulfate was developed using the Phoenix NLME program. Monte Carlo simulation was conducted to evaluate the probability of target attainment (PTA) for optimizing dosing regimens. Results: A total of 98 plasma concentrations from 20 patients were recorded for PPK modeling. The data were adequately described by a two-compartment model with linear elimination. During modeling, creatinine clearance (CrCL) and alanine aminotransferase (ALT) were identified as covariates of the clearance (CL) and volume of peripheral compartment distribution (V2), respectively. In addition, colistin sulfate was predominantly cleared by the nonrenal pathway with a median urinary recovery of 10.05% with large inter-individual variability. Monte Carlo simulations revealed a greater creatinine clearance associated with a higher risk of sub-therapeutic exposure to colistin sulfate. The target PTA (≥90%) of dosage regimens recommended by the label sheet was achievable only in patients infected by pathogens with MIC ≤0.5 mg/L or with renal impairments. Conclusion: Our study showed that the dose of intravenous colistin sulfate was best adjusted by CrCL and ALT. Importantly, the recommended dosing regimen of 1.0-1.5 million units daily was insufficient for patients with normal renal functions (CrCL ≥80 ml/min) or those infected by pathogens with MIC ≥1.0 mg/L. The dosage of colistin sulfate should be adjusted according to renal function and drug exposure.

Naringin improves sepsis-induced intestinal injury by modulating macrophage polarization via PPARγ/miR-21 axis.

Naringin exhibited various pharmacological activities. However, its biological function and underlying mechanism in regulating macrophage polarization remain elusive. This study aimed to investigate the regulatory network between naringin and macrophage polarization in sepsis-induced intestinal injury. Cecal ligation and puncture (CLP) was used to establish the animal model of sepsis. Chromatin immunoprecipitation and a luciferase reporter assay were used to determine the interplay between peroxisome proliferator-activated receptor γ (PPARγ) and miR-21 promoter, as well as miR-21 and its target genes. Naringin enhanced the overall survival of septic mice and alleviated the CLP-induced inflammatory response and intestinal damage. This was accompanied by the increased expression of PPARγ in the intestines and the stimulation of ileal macrophages toward the M2 phenotype. Furthermore, in lipopolysaccharide-stimulated bone marrow-derived macrophages, naringin stimulated M2 polarization. Mechanistically, PPARγ inhibition attenuated the promotion of M2 polarization caused by naringin, and the naringin/PPARγ regulatory work was compromised by miR-21 inhibition. The present study suggested that naringin promoted M2 polarization via the PPARγ/miR-21 axis, thus relieving sepsis-induced intestinal injury. This study provides novel insights into the mechanism by which naringin alleviated sepsis-induced intestinal injury through regulation of macrophage polarization.

MiR-20b-5p modulates inflammation, apoptosis and angiogenesis in severe acute pancreatitis through autophagy by targeting AKT3.

BACKGROUND: Severe acute pancreatitis (SAP) is a common acute abdominal disease with high morbidity and mortality. However, the mechanism underlying SAP is still unclear. METHODS: Cerulean and LPS (Cer-LPS) was used to establish a rat model and an in vitro model of SAP. qRT-PCR, western blot and IHC were determined to analyse the expression of mRNA and proteins. IL-1ß, TNF-α and IL-6 levels were measured applying ELISA. H&E staining was determined to observe the pathological changes. Apoptosis was tested by AV-PI staining using flow cytometry. CCK8 assay was taken to detect cell viability. Cell migration was assessed by transwell assay. Tube formation assay was conducted to evaluate angiogenesis. Luciferase assay was used to detect relationship of miR-20b-5p and AKT3. RESULTS: MiR-20b-5p was lowly expressed in SAP models both in vivo and in vitro. Overexpression of miR-20b-5p restrained inflammation and apoptosis in Cer-LPS treated pancreatic acinar cells. Furthermore, miR-20b-5p promoted the angiogenesis of vascular endothelial cells, since the viability, migration and the capability of tube formation were increased by miR-20b-5p. Mechanically, miR-20b-5p directly targeted AKT3 to promote autophagy. Furthermore, miR-20b-5p could prevent the inflammation, apoptosis and enhance angiogenesis via enhancing autophagy, which was verified in vivo. CONCLUSION: This study demonstrated miR-20b-5p attenuates SAP through directly targeting AKT3 to regulate autophagy, subsequently inhibit inflammation and apoptosis, and promote angiogenesis. Our findings suggested a novel target of miR-20b-5p for the therapy of SAP.

Long Non-coding RNA FENDRR Modulates Autophagy Through Epigenetic Suppression of ATG7 via Binding PRC2 in Acute Pancreatitis.

Acute pancreatitis (AP) is an inflammatory, complicated pancreatic disease, carrying significant morbidity and mortality. However, the molecular and cellular mechanisms involved in AP pathogenesis remain to be elucidated. Here, we explore the role of FOXF1 adjacent non-coding developmental regulatory RNA (FENDRR) in AP progression. Caerulein with or without LPS- induced or taurolithocholic acid 3-sulfate (TLC-S)-induced AP mouse models and cell models were performed for the validation of FENDRR expression in vivo and in vitro, respectively. Histopathological examinations of pancreatic tissues were performed to evaluate the severity of AP. Transmission electron microscopy was utilized to visualize the autophagic vacuoles. siRNA specifically targeting FENDRR was further applied. Flow cytometry was employed to assess cell apoptosis. ELISA, immunoflureoscence, and western blotting analysis were also performed to determine the levels of inflammatory cytokines and autophagy activity. RNA immunoprecipitation (RIP) and chromatin immunoprecipitation (ChIP) assays were carried out to reveal the epigenetic regulation of FENDRR on ATG7. Additionally, silencing FENDRR was also verified in AP mouse models. Higher FENDRR and impaired autophagy were displayed in both AP mouse models and cell models. FENDRR knockdown dramatically attenuated caerulein- or TLC-S-induced AR42J cells apoptosis and autophagy suppression. Further mechanistic experiments implied that the action of FENDRR is moderately attributable to its repression of ATG7 via direct interaction with the epigenetic repressor PRC2. Moreover, the silencing of FENDRR significantly induced the promotion of ATG7, thus alleviating the development of AP in vivo. Our study highlights FENDRR as a novel target that may contribute to AP progression, suggesting a therapeutic target for AP treatment.

Sesamin attenuates intestinal injury in sepsis via the HMGB1/TLR4/IL-33 signalling pathway.

CONTEXT: Sepsis is currently one of the leading causes of death in intensive care units (ICUs). Sesamin was previously reported to inhibit inflammation. However, no studies have revealed the impact of sesamin on sepsis. OBJECTIVE: We studied the mechanism underlying the effect of sesamin on the pathophysiology of sepsis through the HMGB1/TLR4/IL-33 signalling pathway. MATERIALS AND METHODS: Fifty male BALB/c mice (n = 10 per group) were used to establish a caecal ligation and puncture (CLP) mouse model, and given daily injections of sesamin at a low, middle, or high concentration (25, 50, or 100 µM) during the seven-day study period; survival curves were generated by the Kaplan-Meier method. H&E staining and TUNEL staining were performed to assess changes in intestinal morphology intestinal damage in the mouse intestinal epithelium. Molecules related to the HMGB1/TLR4/IL-33 pathway were assessed by RT-qPCR and Western blotting. RESULTS: We found mice in the sepsis group survived for only 4 days, while those treated with sesamin survived for 6-7 days. In addition, sesamin significantly relieved the increase in the levels of MPO (21%, 33.3%), MDA (40.5% and 31.6%), DAO (1.24-fold and 2.31-fold), and pro-inflammatory cytokines such as TNF-α (75% and 79%) and IL-6 (1-fold and 1.67-fold) 24 and 48 h after sepsis induction and downregulated the expression of HMGB1, TLR4, and IL-33 while upregulating the expression of ZO-1 and occludin. DISCUSSION AND CONCLUSIONS: Sesamin improved the 7-day survival rate of septic mice, suppressed the inflammatory response in sepsis through the HMGB-1/TLR4/IL-33 signalling pathway, and further alleviated intestinal injury.

miR-375 Inhibits Autophagy and Further Promotes Inflammation and Apoptosis of Acinar Cells by Targeting ATG7.

OBJECTIVES: MicroRNAs have been considered to be closely related with the development of severe acute pancreatitis (SAP), and microRNA-375 (miR-375) was believed to be a marker of SAP. We aim to investigate the role of miR-375 in regulating SP. METHODS: Cerulein and lipopolysaccharide were used to establish the models of SAP. AR42J cell line was chosen for study in vitro. Flow cytometry was applied for assessing apoptosis. The contents of inflammatory factors were detected with related enzyme-linked immunosorbent assay and quantitative real-time polymerase chain reaction assays. Hematoxylin and eosin staining was applied to observe the pathological changes of pancreatic tissues. Immunohistochemistry analysis was conducted for investigating the expression of light chain 3. RESULTS: The level of miR-375 in pancreatitis tissues and cell lines was upregulated. Overexpression of miR-375 promoted inflammation and the apoptosis of acinar cells through inhibiting autophagy. The binding site between miR-375 and ATG7 was identified, and miR-375 could directly regulate the ATG7. microRNA-375 suppressed autophagy and promoted inflammation and the apoptosis of acinar cells via targeting ATG7. CONCLUSIONS: We proved that miR-375 could inhibit autophagy and promote inflammation and the apoptosis of acinar cells through regulating ATG7. This study first proves that miR-375 modulates the development of SAP through targeting ATG7.

[Clone of hepatitis B virus X gene and its protein expression].

OBJECTIVE: To construct the hepatitis B virus (HBV) X gene recombinant and to induce the expression of X protein. METHODS: HBV DNA was extracted from the serum of patient with hepatitis B. The X gene was amplified by PCR using the primers with EcoRI and HindIII digestion sites, and then cloned into pronucleus expression vector pMAL-C2X, which was detected by EcoRI and HindIII digestion and sequence. Finally, the recombinant was induced by IPTG to express X protein in JM109. RESULTS: The band similar to X gene was amplified by PCR. There were fragments similar to X gene when the recombinant was digested by the enzyme digestion. It was tested by DNA sequence that the correct and entire opening reading frame of HBV X gene was inserted. The X protein was expressed by the IPTG induction. CONCLUSION: Pronucleus expression recombinant pMAL-C2X-HBV-X is constructed successfully and with the IPTG induction, the recombinant pMAL-C2X-HBV-X can express the X protein in E. coli JM109, which lays the foundation for the HBV X protein purification and its biological study.