Sucrose synthase plays a crucial role in the plant sugar metabolism pathway by catalyzing the production of uridine diphosphate (UDP)-glucose, which serves as a bioactive glycosyl donor for various metabolic processes. In this study, a sucrose synthase gene named CtSus was cloned from Cistanche tubulosa, a traditional Chinese medicine known for its rich content of diverse glycosides, based on transcriptome analysis results. The open reading frame of CtSus was 2 418 bp long encoding 805 amino acids. Sequence analysis revealed conserved domains associated with the plant sucrose synthase family at both the N-terminal and C-terminal regions of CtSus protein. Phylogenetic analysis demonstrated that CtSus shares a close evolutionary relationship with sucrose synthases from other plants within the same order. Functional identification of CtSus was performed through whole-cell catalysis coupling with UGT71BD1, a characterized glycosyltransferase enzyme. The results showed that the introduction of CtSus significantly enhanced the conversion rate of glycosylation catalyzed by UGT71BD1. The soluble expression of CtSus in Escherichia coli was further achieved using the pCold^TM TF expression vector. In vitro enzymatic assay indicated the activity of CtSus to catalyze the formation of UDP-glucose in the presence of sucrose and UDP. Real-time quantitative-polymerase chain reaction results showed that the expression patterns of CtSus gene in different parts of C. tubulosa and the suspension cell cultures of C. tubulosa under drought stress correlated with the accumulation patterns observed for phenylethanol glycosides, respectively. Furthermore, key amino acids and their interactions between enzyme and substrate were explored based on protein structure prediction and molecular docking results. Overall, our findings identify a sucrose synthase CtSus responsible for the supply of the active glycosyl donor UDP-glucose during the biosynthesis of glycoside products in C. tubulosa and have also provided a gene element that can be utilized in engineering strain construction for glycoside products production.

Aim To investigate the effect of microinjection of EX527, a selective SIRT1 antagonist, into the ventrolateral orbital cortex (VLO) on morphine-induced conditioned place preference (CPP), and to explore the role of CREB/BDNF in it. Methods The cannulas were implanted bilaterally in the VLO of rats by brain stereotaxis surgery, and the model of morphine-induced CPP was established. The behavioral experiment consisted of four stages:habituation (d 1), pre-test (d 2-4), conditioning training (d 5-14) and test (d 15). At the stage of conditioning training, EX527 (1 μL, 5 g·L

OBJECTIVE: The leukemia cells from patients with T-cell acute lymphoblastic leukemia (T-ALL) were inoculated into NCG mice to establish a stable human T-ALL leukemia animal model. METHODS: Leukemia cells from bone marrow of newly diagnosed T-ALL patients were isolated, and the leukemia cells were inoculated into NCG mice via tail vein. The proportion of hCD45 positive cells in peripheral blood of the mice was detected regularly by flow cytometry, and the infiltration of leukemia cells in bone marrow, liver, spleen and other organs of the mice was detected by pathology and immunohistochemistry. After the first generation mice model was successfully established, the spleen cells from the first generation mice were inoculated into the second generation mice, and after the second generation mice model was successfully established, the spleen cells from the second generation mice were further inoculated into the third generation mice, and the growth of leukemia cells in peripheral blood of the mice in each group was monitored by regular flow cytometry to evaluate the stability of this T-ALL leukemia animal model. RESULTS: On the 10th day after inoculation, hCD45⁺ leukemia cells could be successfully detected in the peripheral blood of the first generation mice, and the proportion of these cells was gradually increased. On average, the mice appeared listless 6 or 7 weeks after inoculation, and a large number of T lymphocyte leukemia cells were found in the peripheral blood and bone marrow smear of the mice. The spleen of the mice was obviously enlarged, and immunohistochemical examination showed that hCD3⁺ leukemia cells infiltrated into bone marrow, liver and spleen extensively. The second and third generation mice could stably develop leukemia, and the average survival time was 4-5 weeks. CONCLUSION Inoculating leukemia cells from bone marrow of patients with T-ALL into NCG mice via tail vein can successfully construct a patient-derived tumor xenografts (PDTX) model.
Humans ; Animals ; Mice ; Precursor T-Cell Lymphoblastic Leukemia-Lymphoma ; Heterografts ; Bone Marrow ; Disease Models, Animal ; T-Lymphocytes ; Mice, SCID

The present study investigated the anti-oxidative and anti-apoptotic effects and molecular mechanisms of catalpol on the H_2O_2-induced pancreatic β-cells(INS-1 cells).The oxidative damage model of INS-1 cells was induced and optimized by the stimulation of H_2O_2 of different concentrations for different time.CCK-8 assay was used to detect cell viability after catalpol intervention(1, 5, 10, 20, 40, 80, and 160 μmol·L~(-1)) for 24 h.Intracellular reactive oxygen species(ROS), superoxide dismutase(SOD), and lipid peroxide malondialdehyde(MDA) were measured by DCFH-DA fluorescent probe, WST-1, and TBA respectively.Moreover, the apo-ptotic effect was detected by AO-EB and Annexin V-FITC/PI staining.In addition, the protein expression levels were detected by Wes-tern blot, and intracellular insulin concentration was measured by ELISA.The results showed that the oxidative damage model of INS-1 cells was stably induced by 50 μmol·L~(-1) H_2O_2 treatment for 2 h, and catalpol at 1-80 μmol·L~(-1) did not affect cell viability of INS-1 cells.Compared with the conditions in the model group, 1, 5, and 10 μmol·L~(-1) catalpol intervention for 2 h could protect INS-1 cells from oxidative damage(P<0.001), reduce ROS and MDA, increase SOD, and inhibit excessive cell apoptosis.Moreover, 1, 5, and 10 μmol·L~(-1) catalpol could also up-regulate the phosphorylation of nuclear transcription factor NF-E2 related factors, negatively regulate Kelch-like ECH-associated protein 1(Keap1), phosphorylation of extracellular signal-regulated kinase(ERK), and heme oxyge-nase 1(HO-1), and promote the protein expression of pancreatic-duodenal homeobox factor-1(PDX-1) and glucose transporter 2(GLUT2).In addition, 1, 5, and 10 μmol·L~(-1) catalpol increased insulin secretion of INS-1 cells under oxidative damage in the high-glucose culture medium, indicating function recovery of pancreatic β cells.PDX-1 is a key nuclear transcription factor of pancreatic β cell function that directly regulates GLUT2 and insulin synthesis, and affects glucose homeostasis.In conclusion, catalpol can reduce the oxidative damage and apoptosis of INS-1 cells, activate antioxidant pathway, protect the function of pancreatic β cells, and improve insulin synthesis and secretion.
Apoptosis ; Glucose/metabolism* ; Insulin/metabolism* ; Insulin-Secreting Cells/metabolism* ; Iridoid Glucosides ; Kelch-Like ECH-Associated Protein 1/metabolism* ; NF-E2-Related Factor 2/metabolism* ; Oxidative Stress ; Reactive Oxygen Species/metabolism* ; Superoxide Dismutase/metabolism*

OBJECTIVE: To investigate the effect of celastrol on the proliferation and apoptosis of human multiple myeloma (MM) cell lines, reveal the relationship between IRAK4/ERK/p38 signaling pathway and celastrol regulating the proliferation and apoptosis of H929 and ARP-1 cells, and explore whether celastrol combined with bortezomib has synergistic effect. METHODS: CCK-8 method was used to detect the viability of MM cell lines H929 and ARP-1 treated by different concentrations of celastrol, bortezomib, and their combination, and the synergistic effect was determined by Kim's formula. The apoptosis rate of H929 cells and necrosis rate of ARP-1 were detected by Annexin V/PI method. The expression of key proteins and apoptosis proteins in IRAK4/ERK/p38 signaling pathway were detected by Western blot. RESULTS: Celastrol could significantly inhibit the proliferation of H929 and ARP-1 cells (r=0.9018, r=0.9244) and induce apoptosis in a time-dependent manner. Compared with the control group, celastrol could significantly up-regulate the expression of PARP and cleaved caspase-3 while down-regulate the expression of p-IRAK4, p-ERK, and p-p38 in H929 and ARP-1 cells. Celastrol and bortezomib alone inhibited the proliferation of H929 and ARP-1 cells. Compared with celastrol and bortezomib alone, their combination had lower cell survival rate and higher apoptosis rate (P<0.05). CONCLUSION Celastrol can inhibit the proliferation and promote the apoptosis of H929 and ARP-1 cells, which may be related to inhibiting the phosphorylation of IRAK4 and blocking the activation of IRAK4/ERK/p38 signaling pathway. Celastrol combined with bortezomib has synergistic effect, which can more effectively inhibit the proliferation and induce apoptosis of H929 and ARP-1 cells.
Apoptosis ; Bortezomib/pharmacology* ; Cell Line, Tumor ; Cell Proliferation ; Humans ; Interleukin-1 Receptor-Associated Kinases ; Multiple Myeloma ; Pentacyclic Triterpenes ; Signal Transduction

A new method of MS/MS~(ALL) was designed to sequentially record a MS~2 spectrum at each unit mass window through gas phase fractionation concept, so as to offer an opportunity for universal MS~2 spectral recording with direct infusion(DI). As a proof-of-concept, DI-MS/MS~(ALL) was applied for rapid chemome profiling of a famous herbal medicine named Lonicerae Japonicae Flos. After each MS~2 spectrum was correlated to its precursor ion, the structural annotation was conducted by applying well-defined mass cracking rules, matching the mass spectral data with literatures and referring to those accessible databases. As a result, a total of 54 components were identified from Lonicerae Japonicae Flos extract, including 21 phenolic acids, 13 flavonoids, 12 iridoids, 4 triterpenoids and 4 other compounds. Therefore, DI-MS/MS~(ALL) is a powerful tool for comprehensive, rapid qualitative analysis of chemical profiles of traditional Chinese medicine and other chemical components of complex systems.
Chromatography, High Pressure Liquid ; Drugs, Chinese Herbal ; Lonicera ; Plant Extracts ; Tandem Mass Spectrometry

Rhus chinensis is an important resource plant. The aqueous extract of R. chinensis roots or stems was to produce Shuguantong Syrup, which is mainly used for the treatment of coronary heart disease and angina pectoris with definite curative effect. On this basis, the crude phenolic part of R. chinensis prepared by macroporous resin was evaluated for the cardio protective effect against myocardial ischemia in mice. The results showed that the phenolic part group with oral administration at the dosages of 190.8-381.6 mg·kg~(-1), compared with the model group, reduced the values of left ventricular end systolic diameter(LVEDs) and the left ventricular end diastolic diameter(LVEDd), and increased the cardiac ejection fraction(EF) and left ventricular fractional shortening(FS) rate, which could effectively improve cardiac function and exert its anti-myocardial ischemia effect, and reduce the rising levels of creatine kinase isoenzyme(CK-MB) and lactate dehydrogenase(LDH) in serum. HE staining showed that the phenolic part group reduced the infiltration of myocardial inflammatory cells and alleviated the degree of myocardial fibrosis and collagen deposition. TUNEL staining showed that the blue-green fluorescence of the phenolic part group decreased successively, and the degree of myocardial cell apoptosis was reduced. Immunohistochemical staining suggested that it could reduce the number of positive cells for p53 protein expression and significantly improve myocardial cell damage. All above data suggested that the phenolic part group had an anti-mycardial ischemis effect. Related mechanism studies revealed that the crude phenolic part could regulate the expressions of the p53 gene(p53), Bcl-2-associated X protein(Bax), B lymphoma-2 gene(Bcl-2), and caspase-3 protein(caspase-3) in myocardial tissue, suggesting that it could reduce cardiac remodeling and myocardial ischemic damage, and improve cardiac function by inhibiting myocardial apoptosis.This research laid a foundation for the elucidation of the pharmacological ingredients R. chinensis.
Animals ; Apoptosis ; Mice ; Myocardial Ischemia/drug therapy* ; Myocardium ; Myocytes, Cardiac ; Plant Extracts/pharmacology* ; Rhus ; bcl-2-Associated X Protein

China ; Humans ; Risk Factors ; Stroke/prevention & control*

The biochemical integrity of the brain is necessary to maintain normal function. Oxidative damage is one of the mortal important reasons leading to the destruction of this integrity. The nervous system is enriched in phospholipid and polyunsaturated fatty acids (PUFAs). Due to the nature of high oxygen-consumption and rich lipids, brain is particularly vulnerable to oxidative damages. Phospholipid peroxidation is one of the results of imbalance in oxidation-antioxidant system. Once the antioxidant system is insufficient to resist oxidative damage, membrane phospholipids will be prone to free radical attack. Phospholipid peroxidation leads to a variety of toxic oxidation products, including membrane damage, mitochondrial dysfunction, rapid accumulation of amyloid, etc. Multiple proteins and nucleic acids can be covalently modified by peroxidation products, resulting in the loss of the protein functions, which eventually triggers programmed cell death and general neuroinflammation in brain, and ends up with an increased susceptibility to neurodegenerative diseases. Based on the knowledge of mechanisms of phospholipid peroxidation, this review focuses on the characteristics of phospholipid peroxidation as a key factor in the development of neurodegenerative diseases, in order to provide theoretical basis for targeted intervention of phospholipid peroxidation as a potential strategy to prevent neurodegenerative diseases.