Multi-omics analysis reveals mechanism of Schisandra chinensis lignans and acteoside on EMT in hepatoma cells via ERK1/2 pathway.

BACKGROUND: Hepatocellular carcinoma (HCC), a globally common cancer, often presents late and shows high resistance to chemotherapy, resulting in suboptimal treatment efficacy. Components from traditional Chinese medicines have been recognized for their anti-cancer properties. OBJECTIVE: Exploring the mechanism of Schisandra chinensis lignans and acteoside in suppressing Epithelial-Mesenchymal Transition (EMT) in hepatoma cells through the Extracellular signal-Regulated Kinases (ERK)1/2 pathway and identifying biomarkers, molecular subtypes, and targets via multi-omics for precision oncology. METHODS: Proliferation was assessed using cell counting kit-8 (CCK-8) assays, with scratch and transwell assays for evaluating invasion and migration. Flow cytometry quantified apoptosis rates. Expression levels of CCL20, p-ERK1/2, c-Myc, Vimentin, and E-cadherin/N-cadherin were analyzed by real-time PCR and Western blot. Tumor volume was calculated with a specific formula, and growth. RESULTS: The Schisandra chinensis lignans and acteoside combination decreased CCL20 expression, inhibited hepatoma proliferation and migration, and enhanced apoptosis in a dose- and time-dependent manner. Molecular analysis revealed increased E-cadherin and decreased N-cadherin, p-ERK1/2, c-Myc, and Vimentin expression, indicating ERK1/2 pathway modulation. In vivo, treated nude mice showed significantly reduced tumor growth and volume. CONCLUSION: Schisandra chinensis lignans and acteoside potentially counteract CCL20-induced EMT, invasion, and migration in hepatocellular carcinoma cells via the ERK1/2 pathway, enhancing apoptosis. Multi-omics analysis further aids in pinpointing novel biomarkers for precision cancer therapy.

A systematic comparison of anti-angiogenesis efficacy and cardiotoxicity of receptor tyrosine kinase inhibitors in zebrafish model.

Pathological angiogenesis is fundamental to progression of cancerous tumors and blinding eye diseases. Anti-angiogenic receptor tyrosine kinase inhibitors (TKIs) are in broad use for the treatment of these diseases. With more and more TKIs available, it is a challenge to make an optimal choice. It remains unclear whether TKIs demonstrate similar anti-angiogenesis activities in different tissues. Many TKIs have shown varying degrees of toxic effects that should also be considered in clinical use. This study investigates the anti-angiogenic effects of 13 FDA-approved TKIs on the intersegmental vessels (ISVs), subintestinal vessels (SIVs) and retinal vasculature in zebrafish embryos. The results show that vascular endothelial growth factor receptor TKIs (VEGFR-TKIs) exhibit anti-angiogenic abilities similarly on ISVs and SIVs, and their efficacy is consistent with their IC50 values against VEGFR2. In addition, VEGFR-TKIs selectively induces the apoptosis of endothelial cells in immature vessels. Among all TKIs tested, axitinib demonstrates a strong inhibition on retinal neovascularization at a low dose that do not strongly affect ISVs and SIVs, supporting its potential application for retinal diseases. Zebrafish embryos demonstrate cardiotoxicity after VEGFR-TKIs treatment, and ponatinib and sorafenib show a narrow therapeutic window, suggesting that these two drugs may need to be dosed more carefully in patients. We propose that zebrafish is an ideal model for studying in vivo antiangiogenic efficacy and cardiotoxicity of TKIs.

The Role and Mechanism of Hyperoside against Depression-like Behavior in Mice via the NLRP1 Inflammasome.

BACKGROUND AND OBJECTIVES: Hypericum perforatum (HP) is widely used for depressive therapy. Nevertheless, the antidepressant effect and potential mechanism of hyperoside (Hyp), the main active component of HP, have not been determined. MATERIALS AND METHODS: We performed ultra-performance liquid chromatography-quadrupole-time-of-flight-tandem mass spectrometry (UPLC-Q-TOF-MS/MS) technology to analyze the components in HP. Using data mining and network pharmacology methods, combined with Cytoscape v3.7.1 and other software, the active components, drug-disease targets, and key pathways of HP in the treatment of depression were evaluated. Finally, the antidepressant effects of Hyp and the mechanism involved were verified in chronic-stress-induced mice. RESULTS: We identified 12 compounds from HP. Hyp, isoquercetin, and quercetin are the main active components of HP. The Traditional Chinese Medicine Systems Pharmacology Database (TCMSP), the Analysis Platform, DrugBank, and other databases were analyzed using data mining, and the results show that the active components of HP and depression are linked to targets such as TNF-, IL-2, TLR4, and so on. A potential signaling pathway that was most relevant to the antidepressant effects of Hyp is the C-type lectin receptor signaling pathway. Furthermore, the antidepressant effects of Hyp were examined, and it is verified for the first time that Hyp significantly alleviated depressive-like behaviors in chronic-stress-induced mice, which may be mediated by inhibiting the NLRP1 inflammasome through the CXCL1/CXCR2/BDNF signaling pathway. CONCLUSION: Hyp is one of the main active components of HP, and Hyp has antidepressant effects through the NLRP1 inflammasome, which may be connected with the CXCL1/CXCR2/BDNF signaling pathway.

[Sorting Nexin 16:Structure,Function,and Role in Diseases].

Sorting nexin 16(SNX16),a member of the SNX family,contains a phoxhomology domain that is prone to bind with phosphatidylinositol-3-phosphate domain and a C-terminal coiled-coil domain. SNX16 participates in diverse cellular processes such as endocytosis,protein sorting,and signal transduction. The dysfunctions of SNX16 are demonstrated to be involved in the occurrence of several diseases.Here,we review the structural characteristics and biological functions of SNX16 and discuss the regulatory role of SNX16 in diseases,surveying how SNX16 can be applied to the prevention and treatment of related disorders.

Field-amplified sample injection in capillary zone electrophoresis for the pharmacokinetic research of trace risperidone and its major metabolite 9-hydroxyrisperidone in beagle dogs.

This article describes a method for the simultaneous quantitation of risperidone and its major metabolite, 9-hydroxyrisperidone, in beagle dog plasma by field-amplified sample injection in capillary zone electrophoresis. The separation was carried out at 25°C in a 48 cm × 75 µm fused-silica capillary with an applied voltage of 20 kV using 60 mM NaH2 PO4 buffer (pH 3.6). The detection wavelength was 280 nm. Clean-up and preconcentration of plasma samples were conducted by 96-well formatted liquid-liquid extraction. In this study, this stacking technique provided a sensitivity enhancement of approximately 158 to 188 fold compared with the same sample without stacking. The method was suitably validated with respect to stability, specificity, linearity, lower limit of quantitation, accuracy, precision, and extraction recovery. Calibration curves exhibited good linearity (r2  > 0.995) over a wide concentration range of 2.5 to 200 ng/mL for both risperidone and 9-hydroxyrisperidone. The intra- and interday precisions at the three quality control levels were less than 11.40%. The intra- and interday accuracies ranged from 87.90 to 107.17% for risperidone and from 88.43 to 105.92% for 9-hydroxyrisperidone. All validation data were within the required limits. In conclusion, the method developed was successfully applied to pharmacokinetic studies of risperidone and 9-hydroxyrisperidone in beagle dogs.

High-Throughput Determination of Sodium Danshensu in Beagle Dogs by the LCMS/MS Method, Employing Liquid-Liquid Extraction Based on 96-Well Format Plates.

Sodium Danshensu (sodium d-(+)-ß-(3,4-dihydroxyphenyl) lactate), one of the water-soluble ingredients in Salvia miltiorrhiza, exhibits potent relaxation of the coronary artery and anticoagulation effection. A high-throughput, rapid, and sensitive method combining liquid chromatography with electrospray ionization tandem mass spectrometry to determine the sodium danshensu in beagle dog plasma was developed and validated, using gallic acid as an internal standard (IS). Acidified plasma samples were extracted using 96-well liquid-liquid extraction, and were eluted on a CNW Athena C18 column (3 µm, 2.1 × 100 mm) by using a gradient mobile phase system of methanol and water (containing 0.2% formic acid). The mass spectrometric detection was achieved using negative ion electrospray ionization mode and monitoring the precursor→production combinations of m/z 197→135 for sodium danshensu and 169→125 for IS, in multiple reaction monitoring modes. Good linearity was achieved, and the linear range was 10-1000 ng/mL (R² > 0.996) with a quantification limit of 10 ng/mL for sodium danshensu in beagle dog plasma. The intra- and inter-day precision (RSD) ranged from 2.1% to 9.0%. The accuracy (RE) was between -8.6% and 5.7% at all quality control levels. The validated method was successfully applied to the pharmacokinetics study of sodium danshensu in beagle dog plasma after intravenous injection and oral administration of sodium danshensu.

Determination of Vancomycin in Human Serum by Cyclodextrin-Micellar Electrokinetic Capillary Chromatography (CD-MEKC) and Application for PDAP Patients.

A simple and sensitive cyclodextrin-micellar electrokinetic capillary chromatography (CD-MEKC) method with UV detection was developed and validated for the determination of vancomycin (VCM) in serum. The separation was achieved in 14 min at 25 °C with a fused-silica capillary column of 40.2 cm × 50 mm i.d. (effective length 30.2 cm) and a run buffer containing 25 mM borate buffer with 50 mM sodium dodecylsulfonate (SDS) (pH 9.5) and 2% sulfobutyl-ß-cyclodextrin (sulfobutyl-ß-CD). Under optimal conditions for biological samples, good separations with high efficiency and short analysis time were achieved. Several parameters affecting the drug separation from biological matrices were studied, including buffer types, concentrations, and pHs. The methods were validated over the range of 0.9998-99.98 µg/mL. Calibration curves of VCM also showed good linearity (r² > 0.999). Intra- and interday precisions (relative standard deviation, RSD) were less than 5.80% and 7.38%, and lower limit of quantification (LLOQ) were lower than 1.0 µg/mL. The mean recoveries ranged between 84.03% and 91.69%. The method was successfully applied for monitoring VCM concentrations in serum of patients with peritoneal dialysis-associated peritonitis (PDAP). The assay should be applicable to pharmacokinetic studies and routine therapeutic drug monitoring of this drug in serum.

Association of endothelin-1 gene single-nucleotide polymorphisms and haplotypes with risk of hormone refractory prostate cancer.

Androgen deprivation is often the treatment of choice for patients with a new diagnosis of metastatic or locally advanced prostate cancer (CaP). However, most CaP patients showing a first response to androgen deprivation will progress to a hormone refractory phase of the disease (HRPC) with a much poorer prognosis. Accumulating evidence suggests that endothelin-1 (ET-1) plays an important role in CaP progression. Singlenucleotide polymorphisms (SNPs) of the ET-1 gene reportedly have been associated with cancer progression and chemoresistance. In the present study, we explored the association of SNPs and haplotypes of the ET-1 gene with the risk of HRPC. We genotyped three SNPs (rs1800541, rs2070699 and rs5370) in the ET-1 gene in a case-control study; 234 CaP patients who developed HRPC within six years after androgen deprivation therapy was used as HRPC cases, and 234 age- and primary therapy-matched CaP patients who had not developed HRPC within six years after androgen deprivation therapy were used as non-HRPC controls. Our results revealed that the G allele at rs1800541 and the G allele at rs2070699 were respectively associated with reduced and increased risk of HRPC at borderline statistical significance (p=0.047 and p=0.058, respectively). With adjustment for potential confounders including body mass index, initial Gleason score at diagnosis of CaP, and post-treatment nadir serum PSA level, we found that rs1800541-rs2070699 TG haplotype was significantly associated with increased risk of HRPC (p=0.033; adjusted OR, 2.10; 95% CI, 1.37-5.04). In conclusion, this study provides the first evidence that a 2-SNP haplotype of the ET-1 gene is associated with increased risk of HRPC, which adds new insights into early identification of CaP patients who are likely to develop HRPC in a later stage of the disease.

Identification of 3',4'-Dimethoxy Flavonol-3-ß-d-Glucopyranoside Metabolites in Rats by Liquid Chromatography-Electrospray Ionization Ion Trap Mass Spectrometry.

A method using liquid chromatography-electrospray ionization ion trap mass spectrometry was established for the identification of metabolites in feces, urine and bile in rats after oral administration of 3',4'-dimethoxy flavonol-3-ß-d-glucopyranoside (abbreviated DF3G). Seven metabolites in rat feces, urine and bile were firstly identified on the basis of their MS fragmentation behaviors. Three metabolites were identified in the feces, 6 in the urine and 2 in the bile, which suggested that demethylation, deglycosylation and deglycosylation followed by glucuronide conjugation were the major metabolic pathways for DF3G in vivo. Hydrolyzation might be the first step in the absorption and metabolism of DF3G. The possible metabolic pathway was proposed for the first time. The established method was simple, reliable and sensitive, revealing that it could be used to rapidly screen and identify the structures of metabolites of DF3G to better understand its metabolism in vivo.

Nucleotidyl transferase TUT1 inhibits lipogenesis in osteosarcoma cells through regulation of microRNA-24 and microRNA-29a.

Osteosarcoma is the most common type of bone cancer. In the present study, by way of PCR-based microarrays, we found that TUT1, a nucleotidyl transferase, was significantly downregulated in osteosarcoma, compared with adjacent normal tissues. In the current study, we performed PCR-based microarrays using the cDNA prepared from osteosarcoma and adjacent normal tissues. The enforced expression of TUT1 was able to inhibit cell proliferation in U2OS and MG63 cells, while its knockdown using small interfering RNA (siRNA) oligos promoted cell proliferation. At the molecular level, we found that TUT1 could inhibit the expression levels of PPARgamma and SREBP-1c, two key regulators in lipogenesis, through upregulation of microRNA-24 and microRNA-29a. Therefore, our results suggest that TUT1 may act as a tumor suppressor for osteosarcoma, which might provide a novel mechanism for the tumor development.

Antidepressant-like effects of hyperoside on chronic stress-induced depressive-like behaviors in mice: Gut microbiota and short-chain fatty acids.

BACKGROUND: The antidepressant effect of hyperoside (HYP), which is the main component of Hypericum perforatum, is not established. This study aimed to determine the effects of HYP on depression. METHODS: The antidepressant-like effect of HYP was studied in mice induced by chronic restraint stress (CRS). The effects of HYP on behavior, inflammation, neurotransmitters, gut microbiota, and short-chain fatty acids (SCFAs) were studied in CRS mice. RESULTS: HYP improved depressive-like behavior in mice induced by CRS. Nissl staining analysis showed that HYP improved neuronal damage in CRS mice. Western blot (WB) analysis showed that HYP increased the expression levels of BDNF and PSD95 in the hippocampus of CRS mice. The results of ELISA showed that HYP down-regulated the expression levels of IL-6, IL-1ß, TNF-α, and CORT in the hippocampus, blood, and intestinal tissues of mice and up-regulated the expression levels of 5-HT and BDNF. Hematoxylin and eosin (HE) staining results indicate that HYP can improve the intestinal histopathological injury of CRS mice. The results of 16S rRNA demonstrated that HYP attenuated the dysbiosis of the gut microbiota of depressed mice, along with altering the concentration of SCFAs. LIMITATIONS: In the present study, direct evidence that HYP improves depressive behaviors via gut microbiota and SCFAs is lacking, and only female mice were evaluated, which limits the understanding of the effects of HYP on both sexes. CONCLUSIONS: HYP can improve CRS-induced depressive-like behaviors in mice, which is associated with regulating the gut microbiota and SCFAs concentration.

Multi-technology integrated network pharmacology-based study on phytochemicals, active metabolites, and molecular mechanism of Psoraleae Fructus to promote melanogenesis.

ETHNOPHARMACOLOGICAL RELEVANCE: According to the Compendium of Materia Medica (Shizhen Li, Ming dynasty) and Welfare Pharmacy (Song dynasty), Psoraleae Fructus (PF), a traditional Chinese medicine (TCM) has a bitter taste and warm nature, which has the effect of treating spleen and kidney deficiency and skin disease. Although PF has been widely used since ancient times and has shown satisfactory efficacy in treating vitiligo, the active substances and the mechanism of PF in promoting melanogenesis remain unclear. AIM OF THE STUDY: To explore the active substances and action mechanisms of PF in promoting melanogenesis. MATERIALS AND METHODS: Firstly, UPLC-UV-Q-TOF/MS was used to characterize the components in PF extract and identify the absorption components and metabolites of PF after oral administration at usual doses in rats. Secondly, the active substances and related targets and pathways were predicted by network pharmacology and molecular docking. Finally, pharmacodynamic and molecular biology experiments were used to verify the prediction results. RESULTS: The experimental results showed that 15 compounds were identified in PF extract, and 44 compounds, consisting of 8 prototype components and 36 metabolites (including isomers) were identified in rats' plasma. Promising action targets (MAPK1, MAPK8, MAPK14) and signaling pathways (MAPK signaling pathway) were screened and refined to elucidate the mechanism of PF against vitiligo based on network pharmacology. Bergaptol and xanthotol (the main metabolites of PF), psoralen (prototype drug), and PF extract significantly increased melanin production in zebrafish embryos. Furthermore, bergaptol could promote the pigmentation of zebrafish embryos more than psoralen and PF extract. Bergaptol significantly increased the protein expression levels of p-P38 and decreased ERK phosphorylation in B16F10 cells, which was also supported by the corresponding inhibitor/activator combination study. Moreover, bergaptol increased the mRNA expression levels of the downstream microphthalmia-associated transcription factor (MITF) and tyrosinase in B16F10 cells. Our data elucidate that bergaptol may promote melanogenesis by regulating the p-P38 and p-ERK signaling pathway. CONCLUSIONS: This study will lay a foundation for discovering potential new drugs for treating vitiligo and provide feasible ideas for exploring the mechanism of traditional Chinese medicine.

A comprehensive quality control evaluation for standard decoction of Smilax glabra Roxb based on HPLC-MS-UV/CAD methods combined with chemometrics analysis and network pharmacology.

An effective, sensitive, and rapid method was developed for the quality control evaluation of the standard decoction of Smilax glabra Roxb (SGR). SGR is a primary ingredient of the traditional functional foods of turtle jelly and SGR tea. Chemometrics, Network Pharmacology, and molecular docking were used to screen for six quality markers. Multiple extraction parameters were optimized. HPLC-UV/CAD-QAMS was used to rapidly quantify the six quality markers (neoastilbin, astilbin, neoisoastilbin, isoastilbin, quercitrin, and isoengeletin) in 10 batches of the standard decoction of SGR samples. The relative correction factor (RCF) values of the five compounds were close to 1, demonstrating that the charged aerosol detection (CAD) showed a consistent response to compounds with similar parent nucleus structures. This method can serve as a guide for rapid quantitative analysis of the multi-components of the SGR standard decoction and all the traditional functional foods of turtle jelly with the homology of medicine.

A Systematic Study of Yiqi Qubai Standard Decoction for Treating Vitiligo Based on UPLC-Q-TOF/MS Combined with Chemometrics, Molecular Docking, and Cellular and Zebrafish Assays.

The Yiqi Qubai (YQ) formula is a hospital preparation for treating vitiligo in China that has had reliable efficacy for decades. The formula consists of four herbs; however, the extraction process to produce the formula is obsolete and the active ingredients and mechanisms remain unknown. Therefore, in this paper, fingerprints were combined with the chemometrics method to screen high-quality herbs for the preparation of the YQ standard decoction (YQD). Then, the YQD preparation procedure was optimized using response surface methodology. A total of 44 chemical constituents, as well as 36 absorption components (in rat plasma) of YQD, were identified via UPLC-Q-TOF/MS. Based on the ingredients, the quality control system of YQD was optimized by establishing the SPE-UPLC-Q-TOF/MS identification method and the HPLC quantification method. Network pharmacological analysis and molecular docking showed that carasinaurone, calycosin-7-O-ß-d-glucoside, methylnissolin-3-O-glucoside, genkwanin, akebia saponin D, formononetin, akebia saponin B, and apigenin may be the key active components for treating vitiligo; the core targets associated with them were AKT1, MAPK1, and mTOR, whereas the related pathways were the PI3K-Akt, MAPK, and FoxO signaling pathways. Cellular assays showed that YQD could promote melanogenesis and tyrosinase activity, as well as the transcription and expression of tyrosinase-associated proteins (i.e., TRP-1) in B16F10 cells. In addition, YQD also increased extracellular tyrosinase activity. Further efficacy validation showed that YQD significantly promotes melanin production in zebrafish. These may be the mechanisms by which YQD improves the symptoms of vitiligo. This is the first systematic study of the YQ formula that has optimized the standard decoction preparation method and investigated the active ingredients, quality control, efficacy, and mechanisms of YQD. The results of this study lay the foundations for the clinical application and further development of the YQ formula.

Safety Profile of Eltrombopag in Different Age Groups: An Analysis of Real-World Pharmacovigilance and Randomized Clinical Trials.

Eltrombopag is clinically approved for use in immune thrombocytopenia (ITP), chronic hepatitis C-related thrombocytopenia, and aplastic anemia and suitable for children; however, data on its overall safety profile are scarce. This study aimed to explore the clinical features of adverse drug events (ADEs) associated with eltrombopag in different age groups using individual case safety reports (ICSRs) from the World Health Organization database VigiBase and the US Food and Drug Administration Adverse Event Reporting System database from 2008 to 2022 in combination with a meta-analysis of data from randomized clinical trials in the literature from inception to July 28, 2022. We conducted disproportionality analyses by grouping patients into the following age groups: 0-17 (0-23 months, 2-11 years, and 12-17 years), 18-64, and ≥ 65 years. The ADEs about hepatobiliary disorders, thrombosis, skin and subcutaneous tissue disorders, infections, and so on were observed more differently in each age group. Meta-analysis results showed differences in the four system organ classes between adults and children with ITP: infections and infestations, general disorders and administration site conditions, skin and subcutaneous tissue disorders, and investigations. The adverse drug reactions in the latest version of instructions were searched in the databases to analyze their postmarketing safety signal strength. We observed signals of elevated alanine aminotransferase, aspartate aminotransferase, and blood bilirubin levels in all age groups. For children, urinary tract infection and back pain showed signals. Due to the inherent limitations of pharmacovigilance studies, more experiments are needed to assess the risks of eltrombopag in different ages.

Simultaneous online SPE-HPLC-MS/MS quantification of gefitinib, osimertinib and icotinib in dried plasma spots: Application to therapeutic drug monitoring in patients with non-small cell lung cancer.

Gefitinib, osimertinib and icotinib are the most commonly used tyrosine kinase inhibitors (TKIs) in non-small cell lung cancer (NSCLC) with EGFR mutation. Therapeutic drug monitoring (TDM) for these TKIs has become a standard and essential procedure. Dried plasma spots (DPS) was choosen for microsampling strategies for TDM, allowing easy and cost-effective logistics in many settings. This study developd and validated an assay for the simultaneous quantitative determination of gefitinib, osimertinib and icotinib in DPS by online solid-phase extraction-liquid chromatography-tandem mass spectrometry (online SPE-LC-MS) system. The TKIs were extracted from DPS with methanol and enriched on a Welch Polar-RP SPE column (30 × 4.6 mm, 5 µm), followed by separation on Waters X Bridge C18 analytical column(4.6 × 100 mm, 3.5 µm). The method achieved LLOQ of 2 ng mL-1 for gefitinib and osimertinib (4 ng mL-1 for icotinib), respectively (r2 > 0.99). Precision (within-run 1.54-7.41 % RSD; between-run 3.03-12.84 % RSD), accuracy (range from 81.47 % to 105.08 %; between-run bias 87.87-104.13 %). Osimertinib and icotinib were stable in DPS stored at - 40 °C for 30 days, 4 °C, 42 °C and 60 °C for 5 days and well-sealed 37 °C,75 % humidity (except gefitinib). Lastly, the assay was applied to TDM of TKIs in 46 patients and the results were compared to SALLE assisted LC-MS analysis, it could be confirmed that the developed method achieves similarly good results as the already established one and no bias could be detected. It implies that this method capable of supporting clinical follow-up TDM of TKIs in DPS from poor medical environment.

Identification of bicyclol metabolites in rat plasma, urine and feces by UPLC-Q-TOF-MS/MS and evaluation of the efficacy and safety of these metabolites based on network pharmacology and molecular docking combined with toxicity prediction.

Bicyclol (BIC) has been widely used to treat drug-induced liver injury (DILI), however, it still has the problems of low solubility and bioavailability. Besides, the metabolic characteristics of BIC remain unclear. In the current study, we identified the metabolite of BIC in rat plasma, urine and feces, and evaluated the efficacy and safety of these metabolites. Based on the fragmentation behavior, we totally identified 11 metabolites and 7 metabolites in plasma, 8 metabolites in urine and 8 metabolites in feces. Notably, M1-M3, M6, M7, M10 and M11 were identified for the first time. M7 was the most abundant metabolite in the rat plasma. The metabolic pathways mainly involved demethylation, dealkylation, hydrolysis, methylation, oxidation and glucuronidation. In addition, the efficacy and safety of BIC's metabolites were evaluated by network pharmacology and molecular docking combined with toxicity prediction. The analysis of network pharmacology indicated that BIC's metabolites against DILI through the MAPK signaling pathway and Hepatitis B pathway. The molecular docking results showed that the binding energy of 5 compounds that docked with "7nuw" and 10 compounds that docked with "4tjz" was lower than BIC. 11 compounds possessed higher solubility and lower toxicity than BIC in prediction. Thus, the identification and evaluation of BIC's metabolites contributed to a better understanding of pharmacological mechanism of BIC and the high-value metabolites of high efficacy, safety and solubility provided a basis for drug development.

Schisandrol A, the main active ingredient of Schisandrae Chinensis Fructus, inhibits pulmonary fibrosis through suppression of the TGF-ß signaling pathway as revealed by UPLC-Q-TOF/MS, network pharmacology and experimental verification.

ETHNOPHARMACOLOGICAL RELEVANCE: Schisandra chinensis decoction derived from the book of Waitai Miyao (Tao Wang, Tang dynasty) is often used in the treatment of idiopathic pulmonary fibrosis (IPF), which is included in the Grand Ceremony of Chinese formulae (Huairen Peng, 1994). Schisandrae Chinensis Fructus (Sch) is one of the most important herbs in this formula. According to the "Shennong's Herbal Classicherbal" of the Han Dynasty, Sch has sour taste, warm nature, which has the effect of tonifying qi and curing cough. In addition, according to the "Compendium of Materia Medica" of the Ming Dynasty, Sch is used to treat cough and asthma, which has the effect of moistening the lung and tonifying the kidney. However, the active ingredients of Sch absorption into the plasma and its pharmacological mechanism of treatment for IPF still remained unclear. AIM OF THE STUDY: Our research aimed at identifying the absorbed active ingredients and metabolized of Sch in rat plasma and the mechanism of anti-IPF based on serum pharmacochemistry. MATERIALS AND METHODS: First, the rats were divided into control group and Sch group. Sch sample was orally administrated to the rats for seven days. The blood samples were drawn into an Eppendorf tube after the last dosing. The ultrahigh performance liquid chromatography coupled with quadrupole-time of flight mass spectrometry (UPLC-Q-TOF/MS) was applied to identify the absorption components and metabolites of Sch in rat plasma. Second, the network pharmacology combined with molecular docking analysis was further investigated to illuminate its potential mechanism of treatment for IPF by the biological targets regulating related pathways. Finally, the mechanism of action was verified by experimental in vitro and in vivo. RESULTS: A total of 78 compounds, consist of 13 prototype lignans and 65 metabolites (including isomers) were identified. Network pharmacology study and molecular docking analysis indicated that schisandrol A (L1) play an anti-fibrosis role by regulating the TGF-ß signaling pathway. Experimental in vitro and in vivo verified that the schisandrol A could inhibiting pulmonary fibrosis through TGF-ß signaling pathway. The effect and mechanism of schisandrol A inhibiting pulmonary fibrosis were reported for the first time. CONCLUSIONS: In this study, the absorption active ingredients of Sch in rat plasma were combined with the network pharmacology investigation and experimental in vitro and in vivo to elucidate its biological mechanism of treatment for IPF. The results provided a theoretical support for understanding the bioactive compounds and the pharmacological mechanism of Sch.

Network Pharmacology-Based Investigation and Experimental Exploration of the Antiapoptotic Mechanism of Colchicine on Myocardial Ischemia Reperfusion Injury.

Background: The beneficial effects of colchicine on cardiovascular disease have been widely reported in recent studies. Previous research demonstrated that colchicine has a certain protective effect on ischemic myocardium and has the potential to treat myocardial ischemia reperfusion injury (MIRI). However, the potential targets and pharmacological mechanism of colchicine to treat MIRI has not been reported. Methods: In this study, we used network pharmacology and experimental verification to investigate the pharmacological mechanisms of colchicine for the treatment of MIRI. Potential targets of colchicine and MIRI related genes were screened from public databases. The mechanism of colchicine in the treatment of MIRI was determined by protein-protein interaction (PPI), gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis. Additionally, we evaluated the effect of colchicine on H9C2 cell activity using CCK-8 assays, observed the effect of colchicine on H9C2 cell apoptosis via flow cytometry, and further verified the expression of key targets after colchicine treated by Western blot. Results: A total of 626 target genes for colchicine and 1549 MIRI disease targets were obtained. 138 overlapping genes were determined as potential targets of colchicine in treating MIRI. the PPI network analysis demonstrated that the targets linked to MIRI were ALB, TNF, ACTB, AKT1, IL6, TP53, IL1B, CASP3 and these targets showed nice affinity with colchicine in molecular docking experiments. The results of GO analysis and KEGG pathway enrichment demonstrated that the anti-MIRI effect of colchicine involves in apoptotic signaling pathway. Further tests suggested that colchicine can protect H9C2 cell from Hypoxia/Reoxygenation (H/R) injury through anti-apoptotic effects. Western blot results demonstrated that colchicine can inhibited MIRI induced apoptosis of H9C2 cell by enhancing the decreased levels of Caspase-3 in myocardial injure model induced by H/R and activating the PI3K/AKT/eNOS pathway. Conclusions: we performed network pharmacology and experimental evaluation to reveal the pharmacological mechanism of colchicine against MIRI. The results from this study could provide a theoretical basis for the development and clinical application of colchicine.

Simultaneous and rapid determination of 12 tyrosine kinase inhibitors by LC-MS/MS in human plasma: Application to therapeutic drug monitoring in patients with non-small cell lung cancer.

In recent years, more than 50 tyrosine kinase inhibitors (TKIs) was indicated against numerous cancers, especially outstanding advantages in the treatment of non-small cell lung cancer (NSCLC), and several studies have shown that therapeutic drug monitoring (TDM) of TKIs can improve treatment efficacy and safety. The present study aimed to develop and validate a LC-MS/MS method for the TDM of 12 TKIs (gefitinib, erlotinib, afatinib, dacomitinib, icotinib, osimertinib, crizotinib, ceritinib, alectinib, dabrafenib, trametinib, anlotinib) in patients with NSCLC. The analytes of interest and internal standard were extracted from human plasma. Salting-out assisted liquid-liquid extraction (SALLE) with 5 M ammonium acetate solution was optimized for method validation and compared to simple protein precipitation (PPT). Chromatographic separation was conducted on Waters X bridge C18 column (100 × 4.6 mm, 3.5 µm) using a gradient elution of acetonitrile/5mM ammonium acetate in pure water with 0.1% (v/v) formic acid at 40 °C within 6 min. The total flow was maintained at 1 mL/min, 30% of the post column flow was split into the mass spectrometer and the rest to waste via a 3-way tee. The mass analysis was performed by positive ion electrospray ionization (ESI) in multiple-reaction monitoring (MRM) mode. The assay was validated based on the guidelines on bioanalytical methods by FDA. This quantification method was proved to be satisfactory in selectivity, accuracy, precision, linearity (r2 > 0.995), recovery, matrix effect and stability and the accuracy was further assessed in plasma with a degree of hemolysis of 4%. The described method to simultaneously quantify the 12 selected anticancer drugs in human plasma was successfully validated and applied to routine TDM of gefitinib, erlotinib, icotinib, osimertinib, crizotinib and anlotinib in cancer patients. TKIs plasma monitoring helps to individualize dose adjustment and manage adverse effects in NSCLC patients.