Functional metabolomics characterizes the contribution of farnesoid X receptor in pyrrolizidine alkaloid-induced hepatic sinusoidal obstruction syndrome.

Consumption of herbal products containing pyrrolizidine alkaloids (PAs) is one of the major causes for hepatic sinusoidal obstruction syndrome (HSOS), a deadly liver disease. However, the crucial metabolic variation and biomarkers which can reflect these changes remain amphibious and thus to result in a lack of effective prevention, diagnosis and treatments against this disease. The aim of the study was to determine the impact of HSOS caused by PA exposure, and to translate metabolomics-derived biomarkers to the mechanism. In present study, cholic acid species (namely, cholic acid, taurine conjugated-cholic acid, and glycine conjugated-cholic acid) were identified as the candidate biomarkers (area under the ROC curve 0.968 [95% CI 0.908-0.994], sensitivity 83.87%, specificity 96.55%) for PA-HSOS using two independent cohorts of patients with PA-HSOS. The increased primary bile acid biosynthesis and decreased liver expression of farnesoid X receptor (FXR, which is known to inhibit bile acid biosynthesis in hepatocytes) were highlighted in PA-HSOS patients. Furtherly, a murine PA-HSOS model induced by senecionine (50 mg/kg, p.o.), a hepatotoxic PA, showed increased biosynthesis of cholic acid species via inhibition of hepatic FXR-SHP singling and treatment with the FXR agonist obeticholic acid restored the cholic acid species to the normal levels and protected mice from senecionine-induced HSOS. This work elucidates that increased levels of cholic acid species can serve as diagnostic biomarkers in PA-HSOS and targeting FXR may represent a therapeutic strategy for treating PA-HSOS in clinics.

Comprehensive profiling of amino acids and derivatives in biological samples: A robust UHPLC-MS/MS method for investigating acute lung injury.

The severe respiratory dysfunctions associated with acute lung injury (ALI) and its sequelae have a high morbidity and mortality rate, are multifactorial, and lack a viable treatment. Considering the critical function that amino acids and derivatives play in the genesis of illnesses and the regulation of metabolic processes, monitoring the levels of metabolites associated with amino acids in biological matrices is necessary and interesting to study their pathological mechanisms. Exploring the dynamics of amino acids and derivatives level and searching for biomarkers provides improved clinical ideas for the diagnosis and treatment of ALI. Therefore, we developed an ultra-high-performance liquid chromatography-electrospray tandem mass spectrometry (UHPLC-MS/MS) method that can simultaneously determine the amino acid and derivatives metabolic levels to study amino acid profiles in different biological samples to facilitate clinical research of ALI. In this study, 48 amino acids and derivatives, including neurotransmitters, polyamines, purines, and other types, were quantified simultaneously in a fast, high-throughput, sensitive, and reliable manner within a 15-minute run time without derivatization. No relevant studies have been reported to quantify these 48 amino acid metabolites in three biological samples simultaneously. Satisfactory linearity (R > 0.995), inter-day and intra-day accuracy (85.17-112.67 % and 85.29-111.60 %, respectively), inter-day and intra-day precision (RSD < 13.80 % and RSD < 12.01 %, respectively), matrix effects (81.00 %-118.00 %), recovery (85.09 %-114.65 %) and stability (RSD < 14.72 %) were all demonstrated by the optimized method's successful validation for all analytes. In addition, the suggested method was effectively implemented in plasma, urine, and lung tissue from normal mice and mice with ALI, with the aim of finding potential biomarkers associated with ALI. Potential biomarkers were screened through multivariate statistical analysis and volcanic map analysis, and the changes of markers in ALI were again identified through heat map analysis and correlation analysis with biochemical indicators, which provided ideas and references for subsequent mechanism studies. Here, the technique created in this work offers a quick and dependable way to perform an integrated analysis of amino acids in a variety of biological materials, which can provide research ideas for understanding the physiopathological state of various diseases.

[Comparison on anti-inflammatory activity of Gynostemma pentaphyllum processed with different methods].

The aim of this study is to investigate the effects of Gynostemma pentaphyllum dried with two different methods(air drying and heating) on inflammation in acute lung injury(ALI) mice in vivo and in vitro. Lipopolysaccharide(LPS) was sprayed into the airway of wild type C57BL/6J male mice to establish the model, and the drug was injected into the tail vein 24 h after modeling. Lung function, lung tissue wet/dry weight(W/D) ratio, the total protein concentration, interleukin 6(IL-6), IL-1ß, and tumor necrosis factor-α(TNF-α) in the bronchoalveolar lavage fluid(BALF), and pathological changes of the lung tissue were used to evaluate the effects of different gypenosides on ALI mice. The results showed that total gypenosides(YGGPs) and the gypenosides substituted with one or two glycosyl(GPs_(1-2)) in the air-dried sample improved the lung function, significantly lowered the levels of IL-1ß and TNF-α in BALF, and alleviated the lung inflammation of ALI mice. Moreover, GPs_(1-2) had a more significant effect on inhibiting NO release in RAW264.7 cells. This study showed that different drying methods affected the anti-inflammatory activity of G. pentaphyllum, and the rare saponins in the air-dried sample without heating had better anti-inflammatory activity.

Integrative analysis of renal microRNA and mRNA to identify hub genes and pivotal pathways associated with cyclosporine-induced acute kidney injury in mice.

Cyclosporine (CsA) is an immunosuppressive agent that often causes acute kidney injury (AKI) in children. The specific mechanisms underlying CsA-induced AKI are currently unknown. This study used an integrated network analysis of microRNA (miRNA) and mRNA expression profiles, biochemical and pathological analyses to further investigate these potential mechanisms of CsA-induced AKI. Small RNA sequence analysis identified 25 differentially expressed miRNAs, RNA sequencing analysis identified 4,109 differentially expressed mRNAs. We obtained a total of 4,367 target genes from the 25 differentially expressed miRNAs based on three algorithms, including the Mirdb, Mirtarbase, and TargetScan. 971 target genes overlapped between the 4,367 target genes and 4,109 differentially expressed mRNAs were identified for further bioinformatics analysis. Finally, 30 hub genes and two main modules were recognized. Functional enrichment analysis of 30 hub genes indicated that inflammation and epithelial-mesenchymal transition (EMT) related genes were mainly concentrated together. Pathway analysis revealed that the PI3K-Akt signaling pathway plays an integral role in CsA-induced AKI. Network analysis identified 3 important miRNAs, mmu-miR-17b-5p, mmu-miR-19b-3p, and mmu-mir-423-5p that may further promote the development of inflammatory responses and EMT by mediating a complex network of factors. Our research provides a clearer understanding the molecular mechanism of this specific drug-induced AKI by CsA use, which is useful for discovering potential targets for gene therapies, and drug development in CsA-induced AKI.

Hyperoside attenuates pyrrolizidine alkaloids-induced liver injury by ameliorating TFEB-mediated mitochondrial dysfunction.

Pyrrolizidine alkaloids (PAs) are potent hepatotoxins that can cause liver damage. Hyperoside (Hyp), a natural flavonoid, can be extracted from medicinal plants. Hyp displays hepatoprotective activity in various liver diseases. However, the potential effect and mechanism of action of Hyp in ameliorating PA-induced liver injury remain obscure. This study aimed to explore the protective effect of Hyp against PA-induced hepatotoxicity and its underlying mechanism. We established an in vitro model of PAs in mouse primary hepatocytes and developed a mouse model of acute PA toxicity to investigate the protective effect of Hyp. We found that Hyp notably attenuated PA-induced hepatotoxicity. RNA-sequencing showed that the beneficial effect of Hyp against PA-induced hepatotoxicity was associated with the transcription factor EB (TFEB)-peroxisome proliferator-activated receptor-γ coactivator-1-α (PGC1α) pathway. Our results confirmed that both the autophagy-lysosomal pathway and mitochondrial biogenesis were induced by Hyp through TFEB nuclear translocation in PA-induced liver injury. Furthermore, we demonstrated that activation of the mechanistic target of rapamycin complex 1 (mTORC1) by MHY 1485 decreased TFEB nuclear translocation and abrogated the protective effect of Hyp against PA-induced liver injury in mice. In contrast, inhibition of mTORC1 activity increased the level of TFEB and reduced hepatotoxicity induced by PAs in mouse livers. Likewise, Hyp-induced TFEB activation was validated in vitro. In conclusion, Hyp can activate the TFEB-mediated autophagy-lysosomal pathway and mitochondrial biogenesis through inhibition of mTORC1 activity, alleviating the liver injury induced by PAs, thus suggesting the potential value of Hyp in the treatment of PA-induced hepatotoxicity.

Red ginseng extracts ameliorate high-fat diet-induced obesity and insulin resistance by activating the intestinal TGR5-mediated bile acids signaling pathway.

BACKGROUND: Obesity has emerged as a worldwide metabolic disease, given its rapid growth in global prevalence. Red ginseng extracts (RGS), one of the traditional processed products of ginseng, show the potential to improve the metabolic phenotype of obesity. However, the RGS mechanism for regulating obesity and late insulin resistance remains to be clarified. PURPOSE: This study aimed to emphasize the potential use of RGS in treatment of obesity and insulin resistance (IR) and explore the underlying mechanism affecting glucose and lipid metabolism improvements. METHODS: The role of RGS was evaluated in a high-fat diet (HFD) rodent model. Glucose tolerance test (GTT) and insulin tolerance test (ITT) were performed to characterize the glucose metabolism level. The expression of lipolysis proteins and uncoupling protein-1 (UCP-1) were investigated by western blot. Glucagon-like peptide-1 (GLP-1) and apical sodium-dependent bile acid transporter (ASBT) protein expression in the intestine were determined via immunofluorescence. UPLC-Q-TOF-MS were used to detect the alterations in bile acids (BAs) levels in serum, ileum, and inguinal white adipose tissue (iWAT). In addition, intestine-specific Tgr5 knockout mice were employed to verify the efficacy of RGS in improving obesity. RESULTS: RGS treatment alleviated dietary-induced dyslipidemia and IR in obese mice in a dose-dependent manner and improved glucose and insulin tolerance, and energy expenditure. RGS treatment significantly reduced lipid deposition and induced GLP-1 secretion in the intestine of wild-type mice but not in Tgr5ΔIN obese mice. Furthermore, RGS intervention increased BA levels in serum, ileum, and iWAT. The increase of circulating BAs in mice was related to the activation of ileal TGR5 and the promotion of ASBT translocation to the plasma membrane, thus affecting BA transport. Next, the increased level of circulating BAs entered the periphery, which might facilitate lipolysis and energy consumption by activating TGR5 in iWAT. CONCLUSION: Our results demonstrated that RGS significantly alleviated HFD-induced obesity and insulin resistance in mice. RGS intervention improved glucose metabolism, promoted lipolysis, and energy metabolism by activating TGR5 in the intestine. In addition, we found that activating intestinal TGR5 facilitated the localization of ASBT to the plasma membrane, which ultimately promoted the transport of BAs to regulate metabolic phenotype.

A TMT-based shotgun proteomics uncovers overexpression of thrombospondin 1 as a contributor in pyrrolizidine alkaloid-induced hepatic sinusoidal obstruction syndrome.

Hepatic sinusoidal obstruction disease (HSOS) is a rare but life-threatening vascular liver disease. However, its underlying mechanism and molecular changes in HSOS are largely unknown, thus greatly hindering the development of its effective treatment. Hepatic sinusoidal endothelial cells (HSECs) are the primary and essential target for HSOS. A tandem mass tag-based shotgun proteomics study was performed using primary cultured HSECs from mice with HSOS induced by senecionine, a representative toxic pyrrolizidine alkaloid (PA). Dynamic changes in proteome were found at the initial period of damage and the essential role of thrombospondin 1 (TSP1) was highlighted in PA-induced HSOS. TSP1 over-expression was further confirmed in human HSECs and liver samples from patients with PA-induced HSOS. LSKL peptide, a known TSP1 inhibitor, protected mice from senecionine-induced HSOS. In addition, TSP1 was found to be covalently modified by dehydropyrrolizidine alkaloids in human HSECs and mouse livers upon senecionine treatment, thus to form the pyrrole-protein adduct. These findings provide useful information on early changes in HSECs upon PA treatment and uncover TSP1 overexpression as a contributor in PA-induced HSOS.

Notoginsenoside Ft1 acts as a TGR5 agonist but FXR antagonist to alleviate high fat diet-induced obesity and insulin resistance in mice.

Obesity and its associated complications are highly related to a current public health crisis around the world. A growing body of evidence has indicated that G-protein coupled bile acid (BA) receptor TGR5 (also known as Gpbar-1) is a potential drug target to treat obesity and associated metabolic disorders. We have identified notoginsenoside Ft1 (Ft1) from Panax notoginseng as an agonist of TGR5 in vitro. However, the pharmacological effects of Ft1 on diet-induced obese (DIO) mice and the underlying mechanisms are still elusive. Here we show that Ft1 (100 mg/100 diet) increased adipose lipolysis, promoted fat browning in inguinal adipose tissue and induced glucagon-like peptide-1 (GLP-1) secretion in the ileum of wild type but not Tgr5 -/- obese mice. In addition, Ft1 elevated serum free and taurine-conjugated bile acids (BAs) by antagonizing Fxr transcriptional activities in the ileum to activate Tgr5 in the adipose tissues. The metabolic benefits of Ft1 were abolished in Cyp27a1 -/- mice which have much lower BA levels. These results identify Ft1 as a single compound with opposite activities on two key BA receptors to alleviate high fat diet-induced obesity and insulin resistance in mice.

Validation of the Nanjing Criteria for Diagnosing Pyrrolizidine Alkaloids-induced Hepatic Sinusoidal Obstruction Syndrome.

BACKGROUND AND AIMS: Hepatic sinusoidal obstruction syndrome (HSOS) is caused by toxic injury to sinusoidal endothelial cells in the liver. The intake of pyrrolizidine alkaloids (PAs) in some Chinese herbal remedies/plants remains the major etiology for HSOS in China. Recently, new diagnostic criteria for PA-induced HSOS (i.e. PA-HSOS) have been developed; however, the efficacy has not been clinically validated. This study aimed to assess the performance of the Nanjing criteria for PA-HSOS. METHODS: Data obtained from consecutive patients in multiple hospitals, which included 86 PA-HSOS patients and 327 patients with other liver diseases, were retrospectively analyzed. Then, the diagnostic performance of the Nanjing criteria and simplified Nanjing criteria were evaluated and validated. The study is registered in www.chictr.org.cn (ID: ChiCTR1900020784). RESULTS: The Nanjing criteria have a sensitivity and specificity of 95.35% and 100%, respectively, while the simplified Nanjing criteria have a sensitivity and specificity of 96.51% and 96.33%, respectively, for the diagnosis of PA-HSOS. Notably, a proportion of patients with Budd-Chiari syndrome (11/49) was misdiagnosed as PA-HSOS on the basis of the simplified Nanjing criteria, and this was mainly due to the overlapping features in the enhanced computed tomography/magnetic resonance imaging examinations. Furthermore, most of these patients (10/11) had occlusion or thrombosis of the hepatic vein, and communicating vessels in the liver were found in 8/11 patients, which were absent in PA-HSOS patients. CONCLUSIONS: The Nanjing criteria and simplified Nanjing criteria exhibit excellent performance in diagnosing PA-HSOS. Thus, both could be valuable diagnostic tools in clinical practice.

Blood microRNA Signatures Serve as Potential Diagnostic Biomarkers for Hepatic Sinusoidal Obstruction Syndrome Caused by Gynura japonica Containing Pyrrolizidine Alkaloids.

Background and Aims: The Gynura japonica-induced hepatic sinusoidal obstruction syndrome (HSOS) is closely related to pyrrolizidine alkaloids (PAs), and its prevalence has been increasing worldwide in recent years. However, no effective therapy for PA-induced HSOS in clinics is available, partially due to the failure of quick diagnosis. This study aims to identify blood microRNA (miRNA) signatures as potential biomarkers for PA-induced HSOS in clinics. Methods: The microarray-based miRNA profiling was performed on blood samples of the discovery cohort, which consisted of nine patients with HSOS and nine healthy donors. Differentially expressed miRNAs were further confirmed using a validation cohort, which consisted of 20 independent patients with HSOS. In addition, the rat model was established through the oral administration of the total alkaloid extract from G. japonica to investigate the association of miRNA biomarkers with the progression of HSOS. Bioinformatic analyses, including GO and KEGG enrichment, receiver operating characteristics curve, and correlation analyses were conducted to evaluate the accuracy of the potential miRNA biomarkers. Results: Three miRNAs, namely miR-148a-3p, miR-362-5p, and miR-194-5p, were overexpressed in patients and rats with PA-induced HSOS. These miRNAs were positively related to the severity of liver injury and displayed considerable diagnostic accuracy for patients with HSOS with areas under the curve over 0.87. Conclusion: In summary, this study demonstrated that three miRNAs, hsa-miR-148a-3p, hsa-miR-362-5p, and hsa-miR-194-5p, might serve as potential biomarkers for PA-induced HSOS in clinics.