OBJECTIVE: To investigate the susceptibility of venetoclax-resistant acute myeloid leukemia (AML) cell lines to ferroptosis and to uncover the underlying molecular mechanisms using transcriptomic and metabolomic analysis methods. METHODS: Venetoclax-resistant AML cell lines were constructed using a low-dose concentration escalation method. The sensitivity of cells to chemotherapeutic drugs was detected by CCK-8 assay. The susceptibility of drug-resistant cell lines to ferroptosis was assessed using transcriptomic and metabolomic analysis methods. The expression of cellular GPX4 and SLC7A11 protein was detected by Western blot, and cell death and lipid peroxidation levels were measured by flow cytometry. Depmap database and TCGA cohort were applied to explore the effect of ferroptosis-related genes expression on prognosis. RESULTS: Venetoclax-resistant cell lines exhibited sensitivity to ferroptosis inducers RSL3, APR246, and sorafenib. The ferroptosis inhibitor Fer-1 partially inhibited cell death induced by these inducers. Compared with the parental cells, significant changes in metabolites and gene expression levels related to ferroptosis were observed in the resistant cell lines. In particular, deregulated expression of SLC7A11 and GPX4 may play critical role in ferroptosis susceptibility. Besides, GPX4 was identified as more important for AML cell survival and higher GPX4 expression may predict shortened overall survival, NPM1 mutant and IDH1 R132 mutation positive patients may prone to possess higher GPX4 expression. CONCLUSION Venetoclax-resistant AML cell lines remain susceptible to ferroptosis, higher GPX4 expression maybe a critical marker for poor prognosis. Regulating the expression of ferroptosis-related genes and metabolites may enhance the efficacy of venetoclax and provide new treatment options for AML patients.
Humans ; Ferroptosis ; Leukemia, Myeloid, Acute/metabolism* ; Sulfonamides/pharmacology* ; Bridged Bicyclo Compounds, Heterocyclic/pharmacology* ; Drug Resistance, Neoplasm ; Metabolomics ; Cell Line, Tumor ; Phospholipid Hydroperoxide Glutathione Peroxidase ; Amino Acid Transport System y+/metabolism* ; Transcriptome

OBJECTIVE: To investigate the effect of Arsenic trioxide (ATO) combined with Norcantharidin (NCTD) on the proliferation and apoptosis of Jurkat cells, and to evaluate its effect on the proliferation and apoptosis of acute T-lymphoblastic leukemia (T-ALL) based on the Wnt/β-catenin signaling pathway. METHODS: Jurkat cell lines were used as the study subjects and treated with different concentrations of ATO (0, 2, 4, 8, 16 μmol/L) and NCTD (0, 10, 25, 50, 100 μmol/L) for 72 hours, and the cell proliferation was detected by CCK-8. Meanwhile, flow cytometry was used to detect the apoptosis rate, EdU staining to detect cell proliferation viability, cell clone formation assay to assess cell cloning ability, Transwell assay to assess cell invasion ability, and Western blot to detect apoptosis and the expression of Wnt/β-catenin signaling pathway-related proteins. RESULTS: Compared with the control group, both ATO and NCTD effectively inhibited Jurkat cell proliferation when used alone, and the inhibition effect was more significant when used in combination ( P < 0.05). The combination significantly increased the apoptosis rate of Jurkat cells ( P < 0.05). Meanwhile, the combination significantly decreased the proliferation vitality and clone formation ability of the cells ( P < 0.05), and inhibited the invasion ability of Jurkat cells ( P < 0.05). Western blot analysis showed that the combination of ATO and NCTD significantly up-regulated the expression of pro-apoptotic proteins Bax and E-cadherin, and down-regulated the expression of anti-apoptotic proteins Bcl-2, c-myc and Cyclin D1 ( P < 0.05). CONCLUSION The combination of ATO and NCTD had a synergistic effect in inhibiting proliferation and promoting apoptosis in Jurkat cells, which may be related to the inhibition of Wnt/β-catenin signaling pathway.
Humans ; Apoptosis/drug effects* ; Jurkat Cells ; Cell Proliferation/drug effects* ; Arsenic Trioxide ; Wnt Signaling Pathway/drug effects* ; Bridged Bicyclo Compounds, Heterocyclic/pharmacology* ; beta Catenin/metabolism* ; Arsenicals/pharmacology* ; Oxides/pharmacology*

OBJECTIVE: To establish venetoclax-resistant acute myeloid leukemia (AML) cell lines, assess the sensitivity of venetoclax-resistant cell lines to the BCL-2 protein family, and investigate their resistance mechanisms. METHODS: CCK-8 method was used to screen AML cell lines (MV4-11, MOLM13, OCI-AML2) that were relatively sensitive to venetoclax. Low concentrations of venetoclax continuously induced drug-resistance development in the cell lines. Changes in cell viability and apoptosis rate before and after resistance development were measured using the CCK-8 method and flow cytometry. BH3 profiling assay was performed to anayze the transform of mitochondrion-dependent apoptosis pathway as well as the sensitivity of resistant cell lines to BCL-2 family proteins and small molecule inhibitors. Real-time fluorescence quantitative PCR (RT-qPCR) was utilized to examine changes in the expression levels of BCL-2 protein family members in both venetoclax-resistant cell lines and multidrug-resistant patients. RESULTS: Venetoclax-resistant cell lines of MV4-11, MOLM13, and OCI-AML2 were successfully established, with IC₅₀ values exceeding 10-fold. Under the same concentration of venetoclax, the apoptosis rate of resistant cells decreased significantly (P < 0.05). BH3 profiling assay revealed that the drug-resistant cell lines showed increased sensitivity to many pro-apoptotic proteins (such as BIM,BID and NOXA). RT-qPCR showed significantly upregulated MCL1 and downregulated NOXA1 were detected in drug-resistant cell lines. Expression changes in MCL1 and NOXA1 in venetoclax-resistant patients were consistent with our established drug-resistant cell line results. CONCLUSION The venetoclax-resistant AML cell lines were successfully established through continuous induction with low concentrations of venetoclax. The venetoclax resistance resulted in alterations in the mitochondrial apoptosis pathway of the cells and an increased sensitivity of cells to pro-apoptotic proteins BIM, BID, and NOXA, which may be associated with the upregulation of MCL1 expression and downregulation of NOXA1 expression in the drug-resistant cells.
Humans ; Sulfonamides/pharmacology* ; Drug Resistance, Neoplasm ; Bridged Bicyclo Compounds, Heterocyclic/pharmacology* ; Leukemia, Myeloid, Acute/pathology* ; Proto-Oncogene Proteins c-bcl-2/metabolism* ; Cell Line, Tumor ; Apoptosis ; Antineoplastic Agents/pharmacology*

This study aims to establish a rapid and sensitive UPLC-MS/MS method for simultaneously determining the content of strychnine and paeoniflorin in plasma and brain tissue of rats, and compare the pharmacokinetic behavior and brain tissue distribution of paeoniflorin combined with normal and toxic doses of strychnine in rats after percutaneous administration. Compared with those in the toxic-dose strychnine group, the AUC_(0-t), AUC_(0-∞), and C_(max) of strychnine decreased by 51.51%, 45.68%, and 46.03%, respectively(P<0.01), and the corresponding values of paeoniflorin increased by 91.41%, 102.31%, and 169.32%, respectively(P<0.01), in the compatibility group. Compared with the normal-dose strychnine group, the compatibility group showed insignificantly decreased C_(max), AUC_(0-t), and AUC_(0-∞) of strychnine, increased C_(max) and T_(max) of paeoniflorin(P<0.01), 66.88% increase in AUC_(0-t), and 70.55% increase in AUC_(0-∞) of paeoniflorin. In addition, the brain tissue concentration of strychnine decreased and that of paeoniflorin increased after compatibility. The combination of paeoniflorin with normal dose and toxic dose of strychnine can inhibit the percutaneous absorption of strychnine, and greatly promote the percutaneous penetration of paeoniflorin, whereas the interaction mechanism remains to be explored. The UPLC-MS/MS method established in this study is easy to operate and has good precision. It is suitable for in vivo study of pharmacokinetic behavior and brain tissue distribution of paeoniflorin and strychnine after percutaneous administration in rats, which provides reference for the safe and rational clinical use of strychnine and the combined use of drugs, and lays a solid foundation for the development of external preparations containing Strychni Semen.
Administration, Cutaneous ; Animals ; Brain ; Bridged-Ring Compounds/pharmacology* ; Chromatography, Liquid/methods* ; Glucosides ; Monoterpenes ; Rats ; Rats, Sprague-Dawley ; Strychnine ; Tandem Mass Spectrometry/methods* ; Tissue Distribution

Animals ; Apoptosis ; Bridged Bicyclo Compounds, Heterocyclic/pharmacology* ; Cell Line ; Hypoxia ; MicroRNAs/genetics* ; Myocardial Reperfusion Injury/drug therapy* ; Myocytes, Cardiac ; Oxidative Stress ; Rats ; Reperfusion Injury ; Tetrahydronaphthalenes/pharmacology*

In order to find the endogenous potential biomarkers of in vitro hepatic injury caused by NCTD-Na and elucidate the mechanism of hepatic injury of NCTD-Na,ultra-high performance liquid chromatography coupled quadrupole time-of-flight mass spectrometry(UPLC-Q-TOF-MS/MS) was used for lipidomics detection.Multivariate statistical analysis was used to study the endogenous lipid metabolic changes of human normal liver cells LO2 injury after the treatment with sodium norcantharidate(NCTD-Na).The results showed that the half maximal inhibitory concentration(IC50) of NCTD-Na was 0.034 mmol·L-1.A total of 280 differential metabolites were found between the control group and the low-dose group,with VIP > 2.0 and P<0.05.At the same time,a total of 273 differential metabolites were found between the control group and the high-dose group,with VIP > 2.0 and P<0.05.Cell metabolite profiles showed clear separation among control group,the low-dose group and the high-dose group,and 111 differential metabolites were found,with VIP > 2.0,P<0.05,RSD<30% and in a dose-dependent manner.It was found that most of the above differential metabolites were lipid metabolites after the analysis of simple preparnation methods and database search.A total of 32 potential biomarkers were identified,including 3 phosphatidylcholine(PC),5 lysophosphatidylcholine(Lyso PC),3 ceramide(Cer),1 sphingomyelin(SM),1 phosphatidylethanolamine(PE),10 lysophosphatidylethanolamine(LysoPE),4 diacylglycerol(DG),1 Phosphatidic acid(PA),1 lysophosphatidic acid(Lyso PA),1 phosphatidyl glycerol(PG),1 fatty acid hydroxy fatty acid(FAHFA) and 1 phosphatidylserine(PS).The changes of PCs,Cers,SM,PE and DGs were closely related liver protection,DNA methylation and self-repair in hepatocytes,apoptosis,methylation and detoxification of carcinogens,as well as lipid peroxides production process.Also,they had impact on the proliferation of hepatocytes,differentiation and gene transcription disorders.Cells stimulated by NCTD-Na could promote the production of PA as well as the synthesis and catabolism of FAHFA in a variety of ways.The levels of Lyso PCs,LysoPEs and Lyso PA were correlated with PCs,PE and PA;PE and PS might have valgus during apoptosis,triggering phagocytosis.
Bridged Bicyclo Compounds, Heterocyclic ; pharmacology ; Cells, Cultured ; Hepatocytes ; drug effects ; metabolism ; Humans ; Lipid Metabolism ; Lipids ; analysis ; Tandem Mass Spectrometry

OBJECTIVETo observe the effect of norcantharidin (NCTD) on collagen-induced arthritis (CIA) rats.

METHODSSixty Sprague-Dawley(SD) rats were randomly divided into 6 groups (n=10): normal group, CIA model group(model group), NCTD low-dose group [1.35 mg/(kg•d)], NCTD middle-dose group [2.7 mg/(kg•d)], NCTD high-dose group [5.4 mg/(kg•d)] and methotrexate (MTX) group [1.8 mg/(kg/w)]. Anesthetized rats were sacrificed by luxation of cervical vertebra after 4 weeks of administration. The arthritis scores were evaluated twice a week. The pathological changes in the ankle joints of rats were observed by hematoxylin-eosin (H&E) staining. The serum levels of interleukin (IL) 1β, IL-6, tumor necrosis factor (TNF)-α, vascular endothelial growth factor (VEGF), IL-17 and transform growth factor (TGF) β were detected by enzyme linked immunosorbent assay (ELISA). The mRNA expression of retinoid-related orphan nuclear receptorγt (RORγt) and forkhead box P3 (Foxp3) in peripheral blood lymphocytes were confirmed by real-time polymerase chain reaction.

RESULTSMTX and high-dose NCTD not only decreased the arthritis scores but also alleviated the pathological changes in CIA rats' ankle joints compared with the model group (P<0.05 or P<0.01). All doses of NCTD significantly inhibited the serum levels of IL-6, IL-17 and TNF-α in CIA rats (P<0.05). Only middle- and high-dose of NCTD prominently decreased serum IL-1β and TGF-β levels of CIA rats (P<0.05). However, NCTD has no effect on vascular endothelial growth factor (VEGF) level in CIA rats. The Foxp3 mRNA expression in all NCTD groups were increased significantly than in the model group (P<0.05). The mRNA expression of RORγt in NCTD high-dose group was decreased apparently in comparison with the model group (P<0.05).

CONCLUSIONSNCTD showed therapeutic effect on CIA rats by inhibition of cytokines and regulation of Th17/Treg cells.

Animals ; Arthritis, Experimental ; blood ; drug therapy ; pathology ; Bridged Bicyclo Compounds, Heterocyclic ; pharmacology ; therapeutic use ; Cytokines ; blood ; Forkhead Transcription Factors ; metabolism ; Joints ; drug effects ; pathology ; Male ; Nuclear Receptor Subfamily 1, Group F, Member 3 ; metabolism ; RNA, Messenger ; genetics ; metabolism ; Rats, Sprague-Dawley

Mammalian pancreatic β-cells play a pivotal role in development and glucose homeostasis through the production and secretion of insulin. Functional failure or decrease in β-cell number leads to type 2 diabetes (T2D). Despite the physiological importance of β-cells, the viability of β-cells is often challenged mainly due to its poor ability to adapt to their changing microenvironment. One of the factors that negatively affect β-cell viability is high concentration of free fatty acids (FFAs) such as palmitate. In this work, we demonstrated that Yes-associated protein (Yap1) is activated when β-cells are treated with palmitate. Our loss- and gain-of-function analyses using rodent insulinoma cell lines revealed that Yap1 suppresses palmitate-induced apoptosis in β-cells without regulating their proliferation. We also found that upon palmitate treatment, re-arrangement of F-actin mediates Yap1 activation. Palmitate treatment increases expression of one of the Yap1 target genes, connective tissue growth factor (CTGF). Our gain-of-function analysis with CTGF suggests CTGF may be the downstream factor of Yap1 in the protective mechanism against FFA-induced apoptosis.
Actins ; metabolism ; Adaptor Proteins, Signal Transducing ; antagonists & inhibitors ; genetics ; metabolism ; Animals ; Apoptosis ; drug effects ; physiology ; Bridged Bicyclo Compounds, Heterocyclic ; pharmacology ; Cell Line, Tumor ; Connective Tissue Growth Factor ; genetics ; metabolism ; pharmacology ; Cytochalasin D ; pharmacology ; Fatty Acids, Nonesterified ; pharmacology ; HEK293 Cells ; Humans ; Immunohistochemistry ; Insulin-Secreting Cells ; cytology ; drug effects ; metabolism ; Mice ; Microscopy, Fluorescence ; Palmitic Acid ; pharmacology ; Phosphoproteins ; antagonists & inhibitors ; genetics ; metabolism ; RNA Interference ; RNA, Small Interfering ; metabolism ; Rats ; Recombinant Proteins ; genetics ; metabolism ; pharmacology ; Thiazolidines ; pharmacology

OBJECTIVETo compare the effects and mechanism of blood enriching on mouse model of blood deficiency syndrome induced by cyclophosphamide of albiflorin and paeoniflorin.

METHODAlbiflorin and paeoniflorin were determined by using animal models of blood deficiency syndrome induced by cyclophosphamide. The amount of WBC, RBC, HGB, index of thymus gland and spleen, and the changes of GM-CSF, IL-3 and TNF-α in serum were detected after the treatment.

RESULTCompared with the model group, the amount of WBC in the group of 30 mg x kg(-1) albiflorin and 30 mg x kg(-1) paeoniflorin were increased obviously (P < 0.01). The amount of RBC in the group of 30 mg x kg(-1) albiflorin and 30 mg x kg(-1) paeoniflorin were increased obviously (P < 0.01, P < 0.001), which did not had a significant difference compared with the same dose. The index of thymus gland in the group of 30 mg x kg(-1) albiflorin was superior to the model group (P < 0.01), the difference was significant compared with the same dose of paeoniflorin (P < 0.05). The GM-CSF in serum in all groups of 30 mg x kg(-1) albiflorin, 15 mg x kg(-1) albiflorin, 30 mg x kg(-1) paeoniflorin and 15 mg x kg(-1) paeoniflorin increased obviously (P < 0.01, P < 0.05, P < 0.01, P < 0.05); The IL-3 in serum in both group of 30 mg x kg(-1) albiflorin and 30 mg x kg(-1) paeoniflorin also increased (P < 0.001). The content of TNF-α in group of 30 mg x kg(-1) albiflorin and 30 mg x kg(-1) paeoniflorin were reduced (P < 0.01), which showed the obvious difference compared with the same dose group (P < 0.01).

CONCLUSIONAlbiflorin had the effect of blood enriching by regulating the immune function, same with the paeoniflorin. The probable mechanism of nourishing blood and liver of Paeoniae Radix Alba was not only the better effect of adjusting the content of TNF-α, but also might act synergistically with paeoniflorin.

Animals ; Blood Cells ; drug effects ; Bridged-Ring Compounds ; pharmacology ; Cyclophosphamide ; toxicity ; Glucosides ; pharmacology ; Granulocyte-Macrophage Colony-Stimulating Factor ; blood ; Hematopoiesis ; drug effects ; Interleukin-3 ; blood ; Male ; Mice ; Monoterpenes ; pharmacology ; Tumor Necrosis Factor-alpha ; blood

The present study aims to predict the action targets of antidepressant active ingredients of Xiaoyaosan to understand the "multi-components, multi-targets and multi-pathways" mechanism. Using network pharmacology, the reported antidepressant active ingredients in Xiaoyaosan (saikosaponin A, saikosaponin C, saikosaponin D, ferulic acid, Z-ligustilide, atractylenolide I, atractylenolide II, atractylenolide III, paeoniflorin, albiflorin, liquiritin, glycyrrhizic acid and pachymic acid), were used to predict the targets of main active ingredients of Xiaoyaosan according to reversed pharmacophore matching method. The prediction was made via screening of the antidepressive drug targets approved by FDA in the DrugBank database and annotating the information of targets with the aid of MAS 3.0 biological molecular function software. The Cytoscape software was used to construct the Xiaoyaosan ingredients-targets-pathways network. The network analysis indicates that the active ingredients in Xiaoyaosan involve 25 targets in the energy metabolism-immune-signal transmutation relevant biological processes. The antidepressant effect of Xiaoyaosan reflects the features of traditional Chinese medicine in multi-components, multi-targets and multi-pathways. This research provides a scientific basis for elucidation of the antidepressant pharmacological mechanism of Xiaoyaosan.
Antidepressive Agents ; pharmacology ; Benzoates ; Bridged-Ring Compounds ; Coumaric Acids ; Drug Evaluation, Preclinical ; Drugs, Chinese Herbal ; pharmacology ; Flavanones ; Glucosides ; Glycyrrhizic Acid ; Lactones ; Medicine, Chinese Traditional ; Monoterpenes ; Sesquiterpenes ; Software