Functional evolution and diversification of the CYP82D subfamily members shape flavonoid diversification in the genus Scutellaria.

Flavonoids, the largest class of polyphenols, exhibit substantial structural and functional diversity, yet their evolutionary diversification and specialized functions remain largely unexplored. The genus Scutellaria is notable for its rich flavonoid diversity, particularly the 6/8-hydroxylated variants biosynthesized by the cytochrome P450 subfamily CYP82D. Our study analyzes metabolic differences between Scutellaria baicalensis and Scutellaria barbata, suggesting that CYP82Ds have acquired a broad range of catalytic functions over their evolution. By integrating analyses of metabolic networks and gene evolution across 22 Scutellaria species, we rapid identified 261 flavonoids and delineated five clades associated with various catalytic functions of CYP82Ds. This approach uncovered a unique catalytic mode for 6/8-hydroxylated function under flavanone substrates and the first instance of 7-O-demethylation of flavonoid substrates catalyzed by cytochrome P450. Ancestral sequence reconstruction and functional validation demonstrated that gradual neofunctionalization of CYP82Ds has driven the chemical diversity of flavonoids in Scutellaria throughout its evolutionary history. Our study enhances the understanding of flavonoid diversity, elucidates the intricate roles of CYP82Ds in Scutellaria plants, and underscores the extensive catalytic versatility of cytochrome P450 members within plant taxa.

Disturbance of bile acids profile aggravates the diarrhea induced by capecitabine through inhibiting the Wnt/ß-catenin pathway.

INTRODUCTION: Diarrhea is the primary dose-limiting side effect of capecitabine(Cap) hindering its clinical application, but the mechanism is unclear. Clarifying this mechanism may enhance the patient compliance and improve the treatment outcome. OBJECTIVES: To assess if the endogenous metabolic profile could prodict the diarrhea induced by Cap and explore and validate underlying mechanisms. METHODS: Untargeted and targeted bile acids(BAs) metabolomics were performed to analyzed the metabolic profile of baseline samples from colorectal cancer(CRC) patients and the association with the diarrhea induced by Cap was assessed. The toxicity of BAs and Cap and its metabolites alone or their combinations to the human normal intestinal epithelial cell(HIEC) was assessed, and the key genes that mediated the BAs-enhanced toxicity of Cap were discovered by RNA-seq and then validated. A mouse model with high exposure levels of BAs was constructed and then treated with Cap to verify the Cap-induced diarrhea enhanced by BAs. RESULTS: The baseline endogenous metabolic profile showed obviously difference between diarrhea and non-diarrhea CRC patients, and the differential metabolites mainly enriched in BAs metabolism; the deoxycholic acid(DCA) and lithocholic acid(LCA) were selected to be the key BAs that enhanced the toxicity of Cap metabolite 5-FU to the HIEC cell; the DCA and LCA could inhibit the Wnt/ß-catenin signaling pathway, which then suppressed the P-glycoprotein and increased the exposure level of 5-FU in the HIEC cell. The results of animal experiment verified that the excessive DCA and LCA could aggravate the Cap-induced diarrhea through inhibiting Wnt/ß-catenin-P-glycoprotein pathway. CONCLUSIONS: The disordered BAs metabolic profile showed close relationship with diarrhea induced by Cap, and excessive DCA and LCA were proved to be the key BAs, which could aggravate the Cap-induced diarrhea through inhibiting Wnt/ß-catenin-P-glycoprotein pathway.

Targeting 5-Hydroxytryptamine Receptor 1A in the Portal Vein to Decrease Portal Hypertension.

BACKGROUND & AIMS: Portal hypertension (PH) is one of the most frequent complications of chronic liver disease. The peripheral 5-hydroxytryptamine (5-HT) level was increased in cirrhotic patients. We aimed to elucidate the function and mechanism of 5-HT receptor 1A (HTR1A) in the portal vein (PV) on PH. METHODS: PH models were induced by thioacetamide injection, bile duct ligation, or partial PV ligation. HTR1A expression was detected using real-time polymerase chain reaction, in situ hybridization, and immunofluorescence staining. In situ intraportal infusion was used to assess the effects of 5-HT, the HTR1A agonist 8-OH-DPAT, and the HTR1A antagonist WAY-100635 on portal pressure (PP). Htr1a-knockout (Htr1a-/-) rats and vascular smooth muscle cell (VSMC)-specific Htr1a-knockout (Htr1aΔVSMC) mice were used to confirm the regulatory role of HTR1A on PP. RESULTS: HTR1A expression was significantly increased in the hypertensive PV of PH model rats and cirrhotic patients. Additionally, 8-OH-DPAT increased, but WAY-100635 decreased, the PP in rats without affecting liver fibrosis and systemic hemodynamics. Furthermore, 5-HT or 8-OH-DPAT directly induced the contraction of isolated PVs. Genetic deletion of Htr1a in rats and VSMC-specific Htr1a knockout in mice prevented the development of PH. Moreover, 5-HT triggered adenosine 3',5'-cyclic monophosphate pathway-mediated PV smooth muscle cell contraction via HTR1A in the PV. We also confirmed alverine as an HTR1A antagonist and demonstrated its capacity to decrease PP in rats with thioacetamide-, bile duct ligation-, and partial PV ligation-induced PH. CONCLUSIONS: Our findings reveal that 5-HT promotes PH by inducing the contraction of the PV and identify HTR1A as a promising therapeutic target for attenuating PH. As an HTR1A antagonist, alverine is expected to become a candidate for clinical PH treatment.

Association between preoperative anxiety states and postoperative complications in patients with esophageal cancer and COPD: a retrospective cohort study.

BACKGROUND: Esophageal cancer brings emotional changes, especially anxiety to patients. Co-existing anxiety makes the surgery difficult and may cause complications. This study aims to evaluate effects of anxiety in postoperative complications of esophageal cancer patients with chronic obstructive pulmonary disease (COPD). METHODS: Patients with esophageal cancer and co-existing COPD underwent tumor excision. Anxiety was measured using Hospital Anxiety and Depression Scale (HAD) before surgery. Clavien-Dindo criteria were used to grade surgical complications. A multiple regression model was used to analyze the relationship between anxiety and postoperative complications. The chi-square test was used to compare the differences in various types of complications between the anxiety group and the non-anxiety group. A multinomial logistic regression model was used to analyze the influencing factors of mild and severe complications. RESULTS: This study included a total of 270 eligible patients, of which 20.7% had anxiety symptoms and 56.6% experienced postoperative complications. After evaluation by univariate analysis and multivariate logistic regression models, the risk of developing complications in anxious patients was 4.1 times than non-anxious patients. Anxious patients were more likely to develop pneumonia, pyloric obstruction, and arrhythmia. The presence of anxiety, surgical method, higher body mass index (BMI), and lower preoperative oxygen pressure may increase the incidence of minor complications. The use of surgical methods, higher COPD assessment test (CAT) scores, and higher BMI may increase the incidence of major complications, while anxiety does not affect the occurrence of major complications (P = 0.054). CONCLUSION: Preoperative anxiety is associated with postoperative complications in esophageal cancer patients with co-existing COPD. Anxiety may increase the incidence of postoperative complications, especially minor complications in patient with COPD and esophageal cancer.

Effects of Natural Products through Inhibiting Endoplasmic Reticulum Stress on Attenuation of Idiopathic Pulmonary Fibrosis.

With ever-increasing intensive studies of idiopathic pulmonary fibrosis (IPF), significant progresses have been made. Endoplasmic reticulum stress (ERS)/unfolded protein reaction (UPR) is associated with the development and progression of IPF, and targeting ERS/UPR may be beneficial in the treatment of IPF. Natural product is a tremendous source of new drug discovery, and accumulating studies have reported that many natural products show potential therapeutic effects for IPF via modulating one or more branches of the ERS signaling pathway. Therefore, this review focuses on critical roles of ERS in IPF development, and summarizes herbal preparations and bioactive compounds which protect against IPF through regulating ERS.

Tyrosine kinase inhibitor-induced hypothyroidism: mechanism and clinical implications.

INTRODUCTION: Since the first experimentally proven tyrosine kinase inhibitor (TKI) imatinib was introduced in the clinical setting, TKIs have attracted widespread attention because of their remarkable therapeutic effects and improvement of survival rates. TKIs are small-molecule, multi-target, anti-cancer agents that target different tyrosine kinases and block downstream signaling. ADVERSE REACTIONS AND CONCERNS: However, with in-depth research on TKI drugs, the adverse reactions-for example, thyroid dysfunction-have become a concern and thus have attracted the attention of numerous researchers. Thyroid dysfunction, especially hypothyroidism, that occurs in high incidence during TKI therapy has a close relationship with treatment efficacy, but the mechanism of TKI-induced thyroid dysfunction is obscure. DISCUSSION: This review discusses the epidemiology, possible mechanisms, and clinical significance of hypothyroidism in cancer patients treated with TKI.

Versatile CYP98A enzymes catalyse meta-hydroxylation reveals diversity of salvianolic acids biosynthesis.

Salvianolic acids (SA), such as rosmarinic acid (RA), danshensu (DSS), and their derivative salvianolic acid B (SAB), etc. widely existed in Lamiaceae and Boraginaceae families, are of interest due to medicinal properties in the pharmaceutical industries. Hundreds of studies in past decades described that 4-coumaroyl-CoA and 4-hydroxyphenyllactic acid (4-HPL) are common substrates to biosynthesize SA with participation of rosmarinic acid synthase (RAS) and cytochrome P450 98A (CYP98A) subfamily enzymes in different plants. However, in our recent study, several acyl donors and acceptors included DSS as well as their ester-forming products all were determined in SA-rich plants, which indicated that previous recognition to SA biosynthesis is insufficient. Here, we used Salvia miltiorrhiza, a representative important medicinal plant rich in SA, to elucidate the diversity of SA biosynthesis. Various acyl donors as well as acceptors are catalysed by SmRAS to form precursors of RA and two SmCYP98A family members, SmCYP98A14 and SmCYP98A75, are responsible for different positions' meta-hydroxylation of these precursors. SmCYP98A75 preferentially catalyses C-3' hydroxylation, and SmCYP98A14 preferentially catalyses C-3 hydroxylation in RA generation. In addition, relative to C-3' hydroxylation of the acyl acceptor moiety in RA biosynthesis, SmCYP98A75 has been verified as the first enzyme that participates in DSS formation. Furthermore, SmCYP98A enzymes knockout resulted in the decrease and overexpression leaded to dramatic increase of SA accumlation. Our study provides new insights into SA biosynthesis diversity in SA-abundant species and versatility of CYP98A enzymes catalytic preference in meta-hydroxylation reactions. Moreover, CYP98A enzymes are ideal metabolic engineering targets to elevate SA content.

External Evaluation of Population Pharmacokinetic Models for High-Dose Methotrexate in Adult Patients with Hematological Tumors.

Currently, numerous population pharmacokinetic (popPK) models for methotrexate (MTX) have been published for estimating PK parameters and variability. However, it is unclear whether the accuracy of these models is sufficient for clinical application. The aim of this study is to evaluate published models and assess their predictive performance according to the standards of scientific research. A total of 237 samples from 74 adult patients who underwent high-dose MTX (HDMTX) treatment at Shanghai Changzheng Hospital were collected. The software package NONMEM was used to perform an external evaluation for each model, including prediction-based diagnosis, simulation-based diagnosis, and Bayesian forecasting. The simulation-based diagnosis includes normalized prediction distribution error (NPDE) and visual predictive check (VPC). Following screening, 7 candidate models suitable for external validation were identified for comparison. However, none of these models exhibited excellent predictive performance. Bayesian simulation results indicated that the prediction precision and accuracy of all models significantly improved when incorporating prior concentration information. The published popPK models for MTX exhibit significant differences in their predictive performance, and none of the models were able to accurately predict MTX concentrations in our data set. Therefore, before adopting any model in clinical practice, extensive evaluation should be conducted.

Profiling the metabolic disorder and detection of colorectal cancer based on targeted amino acids metabolomics.

BACKGROUND: The morbidity of cancer keeps growing worldwide, and among that, the colorectal cancer (CRC) has jumped to third. Existing early screening tests for CRC are limited. The aim of this study was to develop a diagnostic strategy for CRC by plasma metabolomics. METHODS: A targeted amino acids metabolomics method was developed to quantify 32 plasma amino acids in 130 CRC patients and 216 healthy volunteers, to identify potential biomarkers for CRC, and an independent sample cohort comprising 116 CRC subjects, 33 precancerosiss patients and 195 healthy volunteers was further used to validate the diagnostic model. Amino acids-related genes were retrieved from Gene Expression Omnibus and Molecular Signatures Database and analyzed. RESULTS: Three were chosen out of the 32 plasma amino acids examined. The tryptophan / sarcosine / glutamic acid -based receiver operating characteristic (ROC) curve showed the area under the curve (AUC) of 0.955 (specificity 83.3% and sensitivity 96.8%) for all participants, and the logistic regression model were used to distinguish between early stage (I and II) of CRC and precancerosiss patients, which showed superiority to the commonly used carcinoembryonic antigen. The GO and KEGG enrichment analysis proved many alterations in amino acids metabolic pathways in tumorigenesis. CONCLUSION: This altered plasma amino acid profile could effectively distinguish CRC patients from precancerosiss patients and healthy volunteers with high accuracy. Prognostic tests based on the tryptophan/sarcosine/glutamic acid biomarkers in the large population could assess the clinical significance of CRC early detection and intervention.

Separation and quantification of Azvudine in plasma of patients with COVID-19 using LC-MS/MS.

Azvudine (FNC) is a new drug conditionally approved in 2022 for the treatment of coronavirus disease 2019 (COVID-19) in China. However, the exposure level of FNC in COVID-19 patients in clinical practice is still obscure, and there is no liquid chromatography-tandem mass spectrometry (LC-MS/MS) or LC method reported for quantifying the FNC. In this study, a simple, fast, and reliable LC-MS/MS method using L-phenylalanine-D5 (Phe-D5) as the internal standard (IS) was developed for the quantification of FNC in plasma from COVID-19 patients. After simple protein precipitation with methanol, the analyte in the supernatant was separated on Waters Atlantis® T3 (2.1 ×100 mm, 3.0 µm) column with the mobile phase consisting of acetonitrile (ACN) - aqueous solution (containing 0.03% heptafluorobutyric acid and 0.2% formic acid). The mobile phase was delivered at 0.3 mL/min in an isocratic elution program (15:85, V: V). The linear relationship of FNC was good within the calibration range of 2.0 - 2000.0 ng/mL, with the recovery of FNC ranging from 81.37% to 103.31% and the matrix effect was 94.77%- 109.83%. The short-term, long-term, and freeze-thaw stability of the FNC assessed in method was acceptable, and all other items met the requirements of validation of the biological analytical method. Finally, the method was applied to detect the exposure level of FNC in plasma samples from patients diagnosed with COVID-19, and the results, which are within the linear range of the method, showed huge inter-individual variation, supporting the significance of therapeutic drug monitoring of FNC.

AaWRKY6 contributes to artemisinin accumulation during growth in Artemisia annua.

Artemisinin, which is extracted from the plant Artemisia annua L., is a crucial drug for curing malaria and has potential applications for treating cancer, diabetes, pulmonary tuberculosis, and other conditions. Demand for artemisinin is therefore high, and enhancing its yield is important. Artemisinin dynamics change during the growth cycle of A. annua; however, the regulatory networks underlying these changes are poorly understood. Here, we collected A. annua leaves at different growth stages and identified target genes from transcriptome data. We determined that WRKY6 binds to the promoters of the artemisinin biosynthesis gene artemisinic aldehyde Δ11(13) reductase (DBR2). In agreement, overexpression of WRKY6 in A. annua resulted in higher expression levels of genes in the artemisinin biosynthesis pathway and greater artemisinin contents than in the wild type. When expression of WRKY6 was down-regulated, artemisinin biosynthesis pathway genes were also down-regulated and the content of artemisinin was lower. WRKY6 mediates the transcriptional activation of artemisinin biosynthesis by binding to the promoter of DBR2, making it a key regulator for modulating the dynamics of artemisinin changes during the A. annua growth cycle.

A LC-MS/MS Method for Quantifying the Schisandrin B and Exploring Its Intracellular Exposure Correlating Antitumor Effect.

Schisandrin B (Sch.B) shows antineoplastic activity in colorectal cancer, but the mechanism is still obscure. The intracellular spatial distribution may be helpful in elucidating the mechanism. To investigate the intracellular drug distribution of Sch.B in cancer cells, a simple, rapid, and sensitive ultra-highperformance liquid chromatography tandem mass spectrometry (UHPLC-MS/MS) method was established for the determination of Sch.B in colorectal cancer cells. Warfarin was utilized as an internal standard. The sample pretreatment was carried out with protein precipitation using methanol. The analyte was separated on an Atlantis T3-C18 column (3 µm, 2.1∗100 mm) using gradient elution with a mobile phase comprised of methanol and 0.2% formic acid in water. The flow rate was 0.4 mL/min. The linear range of Sch.B was 20.0-1000.0 ng/mL with a correlation coefficient (R) more than 0.99. The matrix effect and recovery ranged from 88.01% to 94.59% and from 85.25% to 91.71%; the interday and intraday precision and accuracy, stability, specificity, carryover, matrix effect, and recovery all conformed to the requirements of pharmacopoeia. Cell viability and apoptosis assays demonstrated that Sch.B has an inhibitory effect in a dose-dependent way on HCT116 proliferation and achieved significant suppression at 75 µM (IC50). It was found that in HCT116 cell, nucleus, and mitochondria, exposure levels of Sch.B peaked at 36 h and then decreased, and mitochondria possessed more Sch.B than nucleus. These results may help to elucidate the antitumor effect of Sch.B.

The transcription factors SbMYB45 and SbMYB86.1 regulate flavone biosynthesis in Scutellaria baicalensis.

Scutellaria baicalensis Georgi is an important Chinese medicinal plant that is rich in the flavones baicalin, wogonoside, and wogonin, providing it with anti-cancer, anti-inflammatory, and antibacterial properties. However, although the biosynthetic pathways of baicalin and its derivates have been elucidated, the regulation of flavone biosynthesis in S. baicalensis is poorly understood. Here, we found that the contents of baicalin and its derivates increased and that baicalin biosynthetic pathway genes were induced in response to light, and baicalin and baicalein are not exclusively produced in the roots of S. baicalensis. Based on the fact that MYB transcription factors are known to play important roles in flavone biosynthesis, we identified SbMYB45 and SbMYB86.1 in S. baicalensis and determined that they bind to the promoter of the flavone biosynthesis gene SbCHI to enhance its transcription. Moreover, overexpressing SbMYB45 and SbMYB86.1 enhanced the accumulation of baicalin in S. baicalensis leaves. We demonstrate that SbMYB45 and SbMYB86.1 bind to the cis-acting element MBSII in the promoter of CHI to redundantly induce its expression upon light exposure. These findings indicate that SbMYB45 and SbMYB86.1 transcriptionally activate SbCHI in response to light and enhance flavone contents in S. baicalensis.

Ameliorative effects of mangiferin derivative TPX on insulin resistance via PI3K/AKT and AMPK signaling pathways in human HepG2 and HL-7702 hepatocytes.

BACKGROUND: As a multifaceted metabolic disorder, insulin resistance is accompanied by the preceding onset of type 2 diabetes mellitus, hyperinsulinemia, metabolic dysfunction-associated fatty liver disease (MAFLD) and other metabolic syndromes. Currently, the number of existing drugs and mechanism-based strategies is limited to alleviate insulin resistance in clinics. As a natural polyphenol product derivative, 1,3,6,7-tetrapropylene acyloxy-ketone (TPX) showed a significant hypoglycemic effect in our previous studies. However, whether TPX could improve hepatic insulin sensitivity was unknown. PURPOSE: To explore whether insulin sensitivity can be improved by the treatment with TPX and further investigate its mechanism(s) of activity. METHODS: To mimic hyperglycemia and insulin resistance in vitro, human HepG2 and HL-7702 hepatocytes were exposed to high glucose. Cellular glucose uptake, glucose consumption, glycogen synthesis, and glucose production were quantified after TPX treatment. The effects of TPX on AMP-activated protein kinase (AMPK) phosphorylation, glucose metabolism, and insulin signal transduction were evaluated by western blotting and network pharmacology analysis. The eGFP-membrane of glucose transporter type 4 (GLUT4) lentivirus transfected cells were constructed to investigate the effects of TPX on GLUT4 mobilization. Reactive oxygen species activity in high glucose-induced insulin-resistant cells was measured by DCFH-DA to show oxidative stress. RESULTS: Treatment with TPX improved glycogen synthesis and inhibited gluconeogenesis by regulating GSK3ß, G6Pase, and PEPCK. Furthermore, high glucose-induced inhibition of glucose consumption, glucose uptake, and GLUT4-mediated membrane translocation were reverted by TPX. Accordingly, mechanistic investigations revealed that TPX interacted with AMPK protein and activated the phosphorylation of AKT, thereby improving energy homeostasis and further ameliorating hepatic insulin resistance. Network pharmacology analysis and molecular docking further confirmed AMPK as an active target of TPX. Concordantly, the pharmacological activity of TPX was reversed by the AMPK inhibitor compound C when hepatocytes were exposed to high glucose stimulation. CONCLUSION: In summary, our study confirmed TPX contributions to insulin resistance improvements by targeting AMPK and PI3K/AKT to restore the insulin signaling pathway, which may be an important potential treatment strategy for insulin-resistance-related diseases, including MAFLD and diabetes.

In vitro and in vivo analyses on anti-NSCLC activity of apatinib: rediscovery of a new drug target V600E mutation.

BACKGROUND: Apatinib (YN968D1) is the first small-molecule-targeting drug with anti-tumor activity created in China for the treatment of advanced gastric cancer (GC) and hepatocellular carcinoma (HCC). It showed significant variation in the efficacy for treating cancers, including advanced non-squamous non-small-cell lung cancer (NSCLC). Whether its efficacy could be optimized by subgrouping patients with certain genetic variation remains elusive. METHODS: Here, we firstly used kinase screening to identify any possible target of apatinib against 138 kinases. The effects of apatinib on proliferation rates, cell cycle, cell apoptosis, and cell migration on cancer cell lines were analyzed; the in vitro potential pathways of apatinib on cancer cell lines were screened. The effect of apatinib on mouse cancer models in vivo was also analyzed. RESULTS: Based on HCC364 cells with BRAF V600E mutation, we have shown that apatinib could inhibit their growth, migration, cell cycle, and induce their apoptosis. Based on mice with transplanted HCC364 cells, we have also shown that apatinib could inhibit the tumor growth. Based on immunohistochemistry, we have demonstrated that apatinib could suppress the phosphorylation of mitogen-activated protein kinase/extracellular signal-regulated kinase and extracellular regulated protein kinases. This may account at least part of the apatinib's inhibitory effect on HCC364 cancer cells. CONCLUSIONS: BRAF V600E protein kinase is a target of apatinib by kinase screening. We have demonstrated that apatinib can effectively inhibit tumor cells with BRAF V600E mutation by in vitro and in vivo experiments. Our results have demonstrated that targeting BRAF V600E mutation, apatinib appears to be effective and safe for treating NSCLC and possibly other cancers with the same mutation.

Tanreqing injection protects against bleomycin-induced pulmonary fibrosis via inhibiting STING-mediated endoplasmic reticulum stress signaling pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: Idiopathic pulmonary fibrosis (IPF), characterized by excessive collagen deposition, is a progressive and typically fatal lung disease without effective therapeutic methods. Tanreqing injection (TRQ), a Traditional Chinese Patent Medicine, has been widely used to treat inflammatory respiratory diseases clinically. AIM OF THE STUDY: The present work aims to elucidate the therapeutic effects and the possible mechanism of TRQ against pulmonary fibrosis. METHODS: The pulmonary fibrosis murine model were constructed by the intratracheal injection of bleomycin (BLM). 7 days later, TRQ-L (2.6 ml/kg) and TRQ-H (5.2 ml/kg) were administered via intraperitoneal injection respectively for 21 days. The efficacy and underlying molecular mechanism of TRQ were investigated. RESULTS: Here, we showed that TRQ significantly inhibited BLM-induced lung edema and pulmonary function. TRQ markedly reduced BLM-promoted inflammatory cell infiltration in BALF and inflammatory cytokines release (TNF-α, IL-6, and IL-1ß) in serum and lung tissues. Meanwhile, TRQ also alleviated BLM-induced collagen synthesis and deposition. Simultaneously, TRQ attenuated BLM-induced pulmonary fibrosis through regulating the expression of fibrotic hallmarks, manifested by down-regulated α-SMA and up-regulated E-cadherin. Moreover, we found that TRQ significantly prevented STING, p-P65, BIP, p-PERK, p-eIF2α, and ATF4 expression in lung fibrosis mice. CONCLUSIONS: Taken together, our results indicated that TRQ positively affects inflammatory responses and lung fibrosis by regulating STING-mediated endoplasmic reticulum stress (ERS) signal pathway.

Pharmacokinetic study of the main components of Tanreqing capsules and Tanreqing injections in beagles by liquid chromatography-tandem mass spectrometry.

BACKGROUND: Tanreqing capsules (TRQCs) and Tanreqing injections (TRQIs) are widely used in the treatment of respiratory diseases. In this study, a simple, rapid, and sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed for simultaneous quantification of the main components of Tanreqing, which include chlorogenic acid, ursodeoxycholic acid, chenodeoxycholic acid, and baicalin, in beagle dog plasma to compare their pharmacokinetic parameters. METHODS: Plasma samples were pretreated with protein precipitation. Chromatographic separation was performed on Waters Acquity UPLC HSS T3 (2.1 mm × 100 mm, 1.8 µm) column using a gradient elution with (A) 0.1% (v/v) formic acid aqueous solution and (B) acetonitrile. Six healthy beagles were divided into two groups, and a crossover, comparative pharmacokinetic study of TRQC (0.09 g/kg) and TRQI (0.5 mL/kg) after a single-dose administration or daily doses over 7 days was carried out. One group was administrated a single dose of TRQC and followed continuously for 7 days, whereas the other group was treated with TRQI in the same way. RESULTS: The calibration curves were linear over the ranges of 2.00-1000.00 ng/mL for baicalin, 10.00-5000.00 ng/mL for ursodeoxycholic acid, 1.00-500.00 ng/mLfor chenodeoxycholic acid and chlorogenic acid, respectively. The relative standard deviation of both intra-day and inter-day accuracy is less than 11.23%. The average extraction recovery of all compounds was greater than 82.21%. The major pharmacokinetic parameters of the four compounds were not significantly different between the two formulations (P > 0.05). CONCLUSIONS: The measured levels of the four major components of TRQCs and TRQIs were comparable in these dogs, providing a reference for the clinical application of TRQCs instead of TRQIs.

A rapid and sensitive LC-MS/MS method for determination of the active component K6 in serum of patients with depression.

Depression is a mental health disorder characterized by chronic negative mood, and depression has become a major threat to human health and quality of life. Anyupeibo capsule, a fifth-class new Chinese medicine, was prepared with extracts of Piper laetispicum C.DC. (Piperaceae), and the alkaloid K6 (5'-methoxy-3',4'-methyl-enedioxycinnamic-acidisob-utylamide-isobutylamide) was found to be the main active component. Using high-performance liquid chromatography coupled with triple-quadrupole tandem mass spectrometry (LC-MS/MS), we developed a method to quantify the concentration of K6 in serum samples from patients with depression. Pretreatment of samples was completed based on solid-phase extraction, and the mobile phase for subsequent LC analysis consisted of aqueous ammonium acetate (0.1 mmol/L, phase A) and acetonitrile (phase B) with isocratic elution at 60% B. Chromatographic separation of K6 was achieved within 3 min with an Agilent ZORBAX SB-C18 column (2.1 × 150 mm, 3.5 µm) at a flow rate of 0.3 mL/min. A linear regression equation for K6 yielded correlation coefficients of r2 > 0.99 within a linear range 0.0503-100.5000 ng/mL. Extraction recovery ranged from 85.33% to 101.18%, and the matrix effect ranged from 87.15% to 100.28%. The inter-day and intra-day precision values-expressed as relative standard deviation-were less than 15%, and the corresponding accuracy values were within ±15%. All validation results for stability, specificity, and carry-over met the requirements of Pharmacopoeia. The LC-MS/MS method was applied to determine the K6 concentration in serum samples from patients with depression in a phase III clinical trial of Anyupeibo capsule.

LC-MS/MS method for quantifying aescinate A and B and assessing their relationship with phlebitis.

The purpose of this study is to establish and validate a sensitive, robust and rapid liquid chromatography-tandem mass spectrometry method for quantifying the aescinate A and aescinate B in human plasma and assessing the association of phlebitis and aescinate A and aescinate B in vivo exposure. The chromatographic separation was completed on Agilent ZORBAX SB-C18 (2.1 mm × 100 mm, 3.5 µm, Agilent, USA) column with isocratic elution. The flow rate was 0.3 mL/min and the total run time was optimized within 5 min. The protein precipitation was applied to pretreat plasma sample using methanol as precipitant. The data acquisition was achieved with positive electrospray ionization in multi-reaction monitoring mode for both aescinate A and aescinate B. The calibration range of aescinate A and aescinate B are constructed in 100-2000 ng/mL, and their correlation coefficients are both >0.990. The intra-day and inter-day precision and accuracy of this method are less than 9.04% and within -13.75% and -0.93%. This analytical method has been successfully applied for the determination of plasma aescinate A and aescinate B concentrations in patients with cerebral infarction, and the results showed that the incidence and grade of phlebitis were not associated with the in vivo exposure of aescinate A and aescinate B.

Choline deficiency-related multi-omics characteristics are susceptible factors for chemotherapy-induced thrombocytopenia.

The XELOX chemotherapy protocol that includes capecitabine and oxaliplatin is the routine treatment for colorectal cancer (CRC), but it can cause chemotherapy-related adverse events such as thrombocytopenia (TCP). To identify predictive biomarkers and clarify the mechanism of TCP susceptibility, we conducted integrative analysis using normal colorectal tissue (CRT), plasma, and urine samples collected before CRC patients received adjuvant XELOX chemotherapy. RNA-sequencing and DNA methylation arrays were performed on CRT samples, while liquid chromatography-mass spectrometry was performed on CRT, plasma, and urine samples. Differentially expressed features (DEFs) from each uni-omics analysis were then subjected to integrative analysis using Multi-Omics Factor Analysis (MOFA). Choline-deficiency in plasma and CRT was found as the most critical TCP-related feature. Based on bioinformatic analysis and literature research, we further concluded that choline-deficiency was the possible reason for most of the other TCP-related multi-omics DEFs, including metabolites representing reduced sphingolipid de novo synthesis and elevated solute carrier-mediated transmembrane transportation in CRT and plasma, DNA hypermethylation and elevated expression of genes involved in neuronal system genes. In terms of thrombocytopoiesis, these TCP-related DEFs may cause atypical maintenance and differentiation of megakaryocyte, resulting a suppressed ability of thrombocytopoiesis, making patients more susceptible to chemotherapy-induced TCP. At last, prediction models were developed and validated with reasonably good discrimination. The area under curves (AUCs) of training sets were all > 0.9, while validation sets had AUCs between 0.778 and 0.926. In conclusion, our results produced reliable marker systems for predicting TCP and promising target for developing precision treatment to prevent TCP.