Integrative multi-omics analysis unravels the host response landscape and reveals a serum protein panel for early prognosis prediction for ARDS.

BACKGROUND: The multidimensional biological mechanisms underpinning acute respiratory distress syndrome (ARDS) continue to be elucidated, and early biomarkers for predicting ARDS prognosis are yet to be identified. METHODS: We conducted a multicenter observational study, profiling the 4D-DIA proteomics and global metabolomics of serum samples collected from patients at the initial stage of ARDS, alongside samples from both disease control and healthy control groups. We identified 28-day prognosis biomarkers of ARDS in the discovery cohort using the LASSO method, fold change analysis, and the Boruta algorithm. The candidate biomarkers were validated through parallel reaction monitoring (PRM) targeted mass spectrometry in an external validation cohort. Machine learning models were applied to explore the biomarkers of ARDS prognosis. RESULTS: In the discovery cohort, comprising 130 adult ARDS patients (mean age 72.5, 74.6% male), 33 disease controls, and 33 healthy controls, distinct proteomic and metabolic signatures were identified to differentiate ARDS from both control groups. Pathway analysis highlighted the upregulated sphingolipid signaling pathway as a key contributor to the pathological mechanisms underlying ARDS. MAP2K1 emerged as the hub protein, facilitating interactions with various biological functions within this pathway. Additionally, the metabolite sphingosine 1-phosphate (S1P) was closely associated with ARDS and its prognosis. Our research further highlights essential pathways contributing to the deceased ARDS, such as the downregulation of hematopoietic cell lineage and calcium signaling pathways, contrasted with the upregulation of the unfolded protein response and glycolysis. In particular, GAPDH and ENO1, critical enzymes in glycolysis, showed the highest interaction degree in the protein-protein interaction network of ARDS. In the discovery cohort, a panel of 36 proteins was identified as candidate biomarkers, with 8 proteins (VCAM1, LDHB, MSN, FLG2, TAGLN2, LMNA, MBL2, and LBP) demonstrating significant consistency in an independent validation cohort of 183 patients (mean age 72.6 years, 73.2% male), confirmed by PRM assay. The protein-based model exhibited superior predictive accuracy compared to the clinical model in both the discovery cohort (AUC: 0.893 vs. 0.784; Delong test, P < 0.001) and the validation cohort (AUC: 0.802 vs. 0.738; Delong test, P = 0.008). INTERPRETATION: Our multi-omics study demonstrated the potential biological mechanism and therapy targets in ARDS. This study unveiled several novel predictive biomarkers and established a validated prediction model for the poor prognosis of ARDS, offering valuable insights into the prognosis of individuals with ARDS.

Consecutive baicalin treatment relieves its accumulation in rats with intrahepatic cholestasis by increasing MRP2 expression.

Baicalin, an important flavonoid isolated from Scutellaria baicalensis Georgi, is a Chinese herb widely used in clinical practice. We previously reported the in vivo accumulation of baicalin in rats with intrahepatic cholestasis (IHC) after a single dose. However, the effects of the long-term administration of baicalin on its pharmacokinetics are unknown. Thus, we investigated the disposition of baicalin in normal rats and those with IHC after single and multiple consecutive administrations. In addition, we further investigated the effect of baicalin on multidrug resistance protein 2 (MRP2) in vivo to explore the underlying mechanism. In our study, the liquid chromatography-mass spectrometry (LC-MS) method established to determine baicalin concentrations in rat blood was simple, specific, and with linearity (R2 = 0.9980) in the range of 1.01-506.00 µg/mL. The relative standard deviations (RSD) for intra-day and inter-day precision were not more than 10.55%, and the intra-day and inter-day accuracies were 94.94%-109.13%. The recovery rate and stability were in line with the requirements of the quantitative analysis of biological samples as stated in the Chinese Pharmacopoeia (2020 Edition). Compared with that in normal rats, the Cmax and t1/2 increased significantly in EE-induced rats with IHC, whereas the clearance (CL) decreased after a single administration of baicalin. However, the area under the curve decreased, CL increased, and the t1/2 was shortened after the continuous administration of baicalin in the IHC rat model compared with the single administration of baicalin, and the pharmacokinetic characteristics were similar to those in normal rats. Moreover, MRP2 expression increased in rats with IHC with the continuous administration of baicalin. Continuous baicalin intervention could effectively reduce its accumulation in rats with IHC, and the mechanism may be attributed to its enhancement of MRP2 expression.

Evaluation on the Metabolic Activity of Two Carboxylesterase Isozymes in Mouse Liver Microsomes by a LC-MS/MS Method.

An applicable method for the precise measurement of major carboxylesterase (CESs) activity in liver still limited. Clopidogrel and irinotecan are specific substrates for CES1 and CES2, respectively. Clopidogrel is metabolized to the inactive metabolite clopidogrel carboxylate (CCAM) by CES1. Irinotecan is metabolized to the active metabolite 7-ethyl-10-hydroxycamptothecin (SN-38) by CES2. In the present study, the LC-MS/MS method for the determination of CCAM and SN-38 were separately developed to characterize the metabolic activities of CES1 and CES2 in mouse liver microsomal. CCAM was separated on a Ecosil ODS column with an isocratic mobile phase consisted of 5 mmol/L ammonium formate and 0.1% formic acid in water and acetonitrile (15:85, V:V) at a flow rate of 0.4mL/min. SN-38 was separated on a Waters symmetry C18 column with an gradient mobile phase consisted of 5 mmol/L ammonium formate and 0.1% formic acid in water and acetonitrile at a flow rate of 0.3 mL/min. Calibration curves were linear within the concentration range of 100-20,000 ng/mL for CCAM and 1-200 ng/mL for SN-38. The results of method showed excellent accuracy and precision. The recovery rate, matrix effect and stability inspection results were within the acceptance criteria. The optimized incubation conditions were as follows: protein concentration of microsomes were all 0.1 mg/mL, incubation time was 60 min for clopidogrel and 30 min for irinotecan, respectively. This method was sensitive and applicable for the determination of the activity of CESs in the mouse liver microsomes.

IL-6 downregulates hepatic carboxylesterases via NF-κB activation in dextran sulfate sodium-induced colitis.

Ulcerative colitis (UC) is associated with increased levels of inflammatory factors, which is attributed to the abnormal expression and activity of enzymes and transporters in the liver, affecting drug disposition in vivo. This study aimed to examine the impact of intestinal inflammation on the expression of hepatic carboxylesterases (CESs) in a mouse model of dextran sulfate sodium (DSS)-induced colitis. Two major CESs isoforms, CES1 and CES2, were down-regulated, accompanied by decreases in hepatic microsomal metabolism of clopidogrel and irinotecan. Meanwhile, IL-6 levels significantly increased compared with other inflammatory factors in the livers of UC mice. In contrast, using IL-6 antibody simultaneously reversed the down-regulation of CES1, CES2, pregnane X receptor (PXR), and constitutive androstane receptor (CAR), as well as the nuclear translocation of NF-κB in the liver. We further confirmed that treatment with NF-κB inhibitor abolished IL-6-induced down-regulation of CES1, CES2, PXR, and CAR in vitro. Thus, it was concluded that IL-6 represses hepatic CESs via the NF-κB pathway in DSS-induced colitis. These findings indicate that caution should be exercised concerning the proper and safe use of therapeutic drugs in patients with UC.

Estrogen cholestasis induces gut and liver injury in rats involving in activating PI3K/Akt and MAPK signaling pathways.

BACKGROUNDS: Estrogen and its metabolites often lead to intrahepatic cholestasis in susceptible women with pregnancy, administration of oral contraceptives and postmenopausal hormone replacement therapy. Recently, dysfunction of the gut-liver axis has been suggested to play a pivotal role in the progression of cholestasis, but details about estrogen cholestasis (EC)-induced gut and liver injury are still largely unknown. This study aims to gain insight into EC-induced gut and liver injury and cell signaling implicated. METHODS: Male rats were exposed to 5 and 10 mg/kg of 17α-ethinylestradiol via subcutaneous injection for 5 successive days to simulate human EC. RESULTS: By detection of these estrogen cholestatic rats, we found that EC induced inflammation in the liver but not in the intestine through activating NF-κB signaling pathway. EC strongly induced oxidative stress in both the liver and intestine, and activated the hepatic Nrf2/Gclm/Gclc pathway and the intestinal Nrf2/Ho-1 pathway, respectively, for adaptively regulating oxidative stress. EC increased cell apoptosis in both the liver and intestine. Additionally, EC elevated phosphorylation of Akt, ERK1/2, and p38 in the liver and increased phosphorylation of p38 in the intestine. CONCLUSIONS: EC induces liver inflammation, both gut and liver oxidative stress and apoptosis, involving in activating PI3K/Akt and MAPK signaling pathways. Investigation of EC-induced gut and liver injury contributes to the development of new potential therapeutic strategies.

Detection techniques of carboxylesterase activity: An update review.

Mammalian carboxylesterases (CESs) are essential members of serine esterase hydrolase superfamily, which are widely distributed in many tissues including liver, intestine, lung and kidney. CESs play an important role in the metabolism of various xenobiotics including ester drugs and environmental toxicants, and also participate in lipid homeostasis, so the development of CESs activity detection techniques are of great significance for drug discovery and biomedical research. With the rapid development of separated and detection technologies such as chromatography, capillary electrophoresis, fluorescent probe-based detection technology, bioluminescent sensor and colorimetric sensor in recent decade, the research of physiological functions of CESs have make huge breakthrough. This review summarizes the development and application of CESs activity detection techniques, as well as comparatively analyzes the characteristics of various detection techniques. The information and knowledge represented here will help the researchers carry out various biochemical studies for understanding activation mechanism and role of CESs in drug metabolism.

Baicalin Protects Against 17α-Ethinylestradiol-Induced Cholestasis via the Sirtuin 1/Hepatic Nuclear Receptor-1α/Farnesoid X Receptor Pathway.

Estrogen-induced cholestasis (EIC) is characterized by impairment of bile flow and accumulated bile acids (BAs) in the liver, always along with the liver damage. Baicalin is a major flavonoid component of Scutellaria baicalensis, and has been used in the treatment of liver diseases for many years. However, the role of baicalin in EIC remains to be elucidated. In this study, we demonstrated that baicalin showed obvious hepatoprotective effects in EIC rats by reducing serum biomarkers and increasing the bile flow rate, as well as by alleviating liver histology and restoring the abnormal composition of hepatic BAs. In addition, baicalin protected against estrogen-induced liver injury by up-regulation of the expression of hepatic efflux transporters and down-regulation of hepatic uptake transporters. Furthermore, baicalin increased the expression of hepatic BA synthase (CYP27A1) and metabolic enzymes (Bal, Baat, Sult2a1) in EIC rats. We showed that baicalin significantly inhibited hepatic inflammatory responses in EIC rats through reducing elevated levels of TNF-α, IL-1ß, IL-6, and NF-κB. Finally, we confirmed that baicalin maintains hepatic BA homeostasis and alleviates inflammation through sirtuin 1 (Sirt1)/hepatic nuclear receptor-1α (HNF-1α)/farnesoid X receptor (FXR) signaling pathway. Thus, baicalin protects against estrogen-induced cholestatic liver injury, and the underlying mechanism involved is related to activation of the Sirt1/HNF-1α/FXR signaling pathway.