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.

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.

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.

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.