In this study, an ultra-performance liquid chromatography-quadrupole time-of-flight high resolution mass spectrometer(UPLC-Q-TOF-HRMS) was used to investigate the effects of the active ingredients in Periploca forrestii compound on spleen metabolism in rats with collagen-induced arthritis(CIA), and its potential anti-inflammatory mechanism was analyzed by network pharmacology. After the model of CIA was successfully established, the spleen tissues of rats were taken 28 days after administration. UPLC-Q-TOF-HRMS chromatograms were collected and analyzed by principal component analysis(PCA), orthogonal partial least squares discriminant analysis(OPLS-DA), and MetPA. The results showed that as compared with the blank control group, 22 biomarkers in the spleen tissues such as inosine, citicoline, hypoxanthine, and taurine in the model group increased, while 9 biomarkers such as CDP-ethanolamine and phosphorylcholine decreased. As compared with the model group, 21 biomarkers such as inosine, citicoline, CDP-ethanolamine, and phosphorylcholine were reregulated by the active ingredients in P. forrestii. Seventeen metabolic pathways were significantly enriched, including purine metabolism, taurine and hypotaurine metabolism, glycerophospholipid metabolism, and cysteine and methionine metabolism. Network pharmacology analysis found that purine metabolism, glycerophospholipid metabolism, and cysteine and methionine metabolism played important roles in the pathological process of rheumatoid arthritis. This study suggests that active ingredients in P. forrestii compound can delay the occurrence and development of inflammatory reaction by improving the spleen metabolic disorder of rats with CIA. The P. forrestii compound has multi-target and multi-pathway anti-inflammatory mechanism. This study is expected to provide a new explanation for the mechanism of active ingredients in P. forrestii compound against rheumatoid arthritis.
Rats ; Animals ; Periploca ; Cysteine ; Cytidine Diphosphate Choline ; Network Pharmacology ; Phosphorylcholine ; Metabolomics ; Arthritis, Rheumatoid/drug therapy* ; Biomarkers ; Glycerophospholipids ; Methionine ; Purines ; Chromatography, High Pressure Liquid

OBJECTIVE: To investigate the effects of methionine restriction on proliferation, cell cycle and apoptosis of human acute leukemia cells. METHODS: Cell Counting Kit-8 (CCK-8) assay was used to detect the effect of methionine restriction on HL-60 and Jurkat cells proliferation. The effect of methionine restriction on cell cycle of HL-60 and Jurkat cells was examined by PI staining. Annexin V-FITC / PI double staining was applied to detect apoptosis of HL-60 and Jurkat cells following methionine restriction. The expression of cell cycle-related proteins cyclin B1, CDC2 and apoptosis-related protein Bcl-2 was evaluated by Western blot assay. RESULTS: Methionine restriction significantly inhibited the proliferation of HL-60 and Jurkat cells in a time-dependent manner (HL-60: r =0.7773, Jurkat: r =0.8725), arrested the cells at G2/M phase (P < 0.001), and significantly induced apoptosis of HL-60 and Jurkat cells (HL-60: P < 0.001; Jurkat: P < 0.05). Furthermore, Western blot analysis demonstrated that methionine restriction significantly reduced the proteins expression of Cyclin B1 (P < 0.05), CDC2 (P < 0.01) and Bcl-2 (P < 0.001) in HL-60 and Jurkat cells. CONCLUSION Acute leukemia cells HL-60 and Jurkat exhibit methionine dependence. Methionine restriction can significantly inhibit the proliferation, promote cell cycle arrest and induce apoptosis of HL-60 and Jurkat cells, which suggests that methionine restriction may be a potential therapeutic strategy for acute leukemia.
Humans ; Cyclin B1/pharmacology* ; Cell Proliferation ; Methionine/pharmacology* ; Cell Cycle ; Apoptosis ; Leukemia, Myeloid, Acute ; Cell Division ; Cell Cycle Proteins ; Jurkat Cells ; Proto-Oncogene Proteins c-bcl-2/metabolism* ; HL-60 Cells

Animals ; Cadmium/toxicity* ; Dysbiosis/metabolism* ; Gastrointestinal Microbiome ; Glutamine/pharmacology* ; Methionine ; Mice ; Mice, Inbred C57BL ; Ostreidae ; Protein Hydrolysates ; Tyrosine

The aim of this study was to investigate the effects of D-methionine (D-met) on the hematopoietic system injury in irradiated mice. C57BL/6 mice were divided into control group, irradiated group, 300 mg/kg D-met plus irradiation group and 1000 mg/kg D-met plus irradiation group. The control mice received sham irradiation, and the mice in remainder groups were exposed to 7.5 Gy; 1,4,8 Gy and 1 Gy of (137)Cs γ-ray respectively, were used to detect the survival rate, survival rate of bone marrow cells, WBC and its differential counts as well the colony formation ability in irradiated mice, respectively. The D-met was intraperitoneally injected to mice at 30 min before irradiation. The results showed that 300 and 1000 mg/kd D-met did not obviously enhance the survival rate of mice exposed to 7.5 Gy; the 10(-2),10(-3),10(-4) mol/L D-met significantly increased the survival rate of bone marrow cells in mice exposed to 1,4,8 Gy; 300 and 1000 mg/kg D-met even so increased the WBC count of peripheral blood in mice exposed to 1 Gy, but there was no statistical difference as compared with irradiated alone mice, moreover 300 and 1000 mg/kg D-met could obviously promote the colony formation ability of bone marrow cells in irradiated mice, the CFU-GM count was higher than that in 1 Gy irradiated mice (P < 0.05). It is concluded that the D-met can effectively mitigate the marrow cell injury resulted from irradiation, enhance the survival rate of bone marrow cells in irradiated mice, promote the recovery of hematopoietic function from radiation injury in mice.
Animals ; Bone Marrow Cells ; drug effects ; radiation effects ; Hematopoietic System ; drug effects ; radiation effects ; Leukocyte Count ; Methionine ; pharmacology ; Mice ; Mice, Inbred C57BL ; Radiation Injuries ; prevention & control

AIM: The potential of Trifolium pratense (red clover) extract in the prevention of lipid disorder has attracted increasing attention in recent years. In this study, the aim was to determine whether and how red clover extract affected the development of murine diet-induced non-alcoholic steatohepatitis. METHODS: Non-alcoholic steatohepatitis was induced in C57BL/6 mice by feeding mice with a methionine-choline-deficient (MCD) diet. Hematoxylin and eosin staining was used for histological analyses. Real-time PCR was used to analyze the mRNA expression levels. RESULTS: Hepatic steatosis and necroinflammation was observed in MCD diet-fed mice, and this diet-induced steatosis was significantly attenuated, whereas liver inflammation was not significantly attenuated, by red clover extract treatment. Consistent with the results of H&E staining, the MCD diet-induced increase of liver triglycerides and cholesterol levels were significantly reduced by red clover extract treatment. However, with the improvement in hepatic steatosis, mRNA levels of acetyl CoA oxidase, carnitine palmitoyl transferase-1, and liver fatty acid-binding protein, three genes regulated by peroxisome proliferator-activated receptor (PPAR) α, were unaffected. CONCLUSION Red clover extract alleviated MCD diet-induced hepatic steatosis, but did not ameliorate liver inflammation in C57BL/6 mice, and the improvement in hepatic steatosis was not through activating PPARα.
Animals ; Cholesterol ; metabolism ; Choline Deficiency ; complications ; Diet ; adverse effects ; Disease Models, Animal ; Inflammation ; drug therapy ; metabolism ; Liver ; drug effects ; metabolism ; Male ; Methionine ; deficiency ; Mice ; Mice, Inbred C57BL ; Non-alcoholic Fatty Liver Disease ; drug therapy ; etiology ; metabolism ; PPAR gamma ; metabolism ; Phytotherapy ; Plant Extracts ; pharmacology ; therapeutic use ; RNA, Messenger ; metabolism ; Trifolium ; Triglycerides ; metabolism

OBJECTIVETo study the effects of methionine and choline on the expression levels of CaMKII and CREB mRNA and proteins in hippocampus of rats exposed to lead.

METHODSMale SD rats were divided into five groups. (1) control group, (2) group exposed to lead+2 by drinking water with 0.40 g/L lead acetate, (3) group exposed to methionine and choline (1:1, 400 mg/kg), (4) group exposed to 0.40 g/L lead acetate plus methionine and choline (1:1, 100 mg/kg), (5) group exposed to 0.40 g/L lead acetate plus methionine and choline (1:1, 400 mg/kg). In 8 weeks after exposure, all rats were killed. Then CREB mRNA and CaMK II mRNA expression levels in hippocampus were detected by real-time PCR, CREB and CaMK II protein expression levels in hippocampus were measured by western blot assay.

RESULTSThe expression levels (0.743 ± 0.185 and 0.729 ± 0.199) of CaMKII mRNA and CREB mRNA in the hippocampus of lead group were significantly lower than those (0.950 ± 0.238 and 0.901 ± 0.232) of control group (P < 0.05), also the expression levels (0.271 ± 0.045 and 0.212 ± 0.058) of CREB protein and pCREB protein in the hippocampus of lead group were significantly lower than those (0.319 ± 0.058 and 0.506 ± 0.125) of control group (P < 0.05). The expression levels (1.014 ± 0.210 and 1.126 ± 0.379) of CaMKII mRNA and the expression levels (1.029 ± 0.335 and 0.932 ± 0.251) of CREB mRNA in the hippocampus of 2 groups exposed to lead acetate plus methionine and choline were significantly higher than those of lead group (P < 0.05). The expression levels (0.407 ± 0.951 and 0.563 ± 0.178) of CREB protein and pCREB protein in the hippocampus of group exposed to lead acetate plus 400 mg/kg methionine and choline were significantly higher than those of lead group (P < 0.05).

CONCLUSIONMethionine and choline could decrease the inhibition effects of lead on the expression of CaMKII and CREB mRNA or CREB and pCREB proteins in the hippocampus of rats.

Animals ; Calcium-Calmodulin-Dependent Protein Kinase Type 2 ; metabolism ; Choline ; pharmacology ; Cyclic AMP Response Element-Binding Protein ; metabolism ; Hippocampus ; drug effects ; metabolism ; Lead ; toxicity ; Male ; Methionine ; pharmacology ; RNA, Messenger ; genetics ; Rats ; Rats, Sprague-Dawley

Effective drug to manage constipation has been unsatisfactory. We sought to determine whether methionine has effect on the human colon. Human colon tissues were obtained from the specimens of colon resection. Microelectrode recording was performed and contractile activity of muscle strips and the propagation of the contractions in the colon segment were measured. At 10 microM, methionine depolarized the resting membrane potential (RMP) of circular muscle (CM) cells. In the CM strip, methionine increased the amplitude and area under the curve (AUC) of contractions. In the whole segment of colon, methionine increased the amplitude and AUC of the high amplitude contractions in the CM. These effects on contraction were maximal at 10 microM and were not observed in longitudinal muscles in both the strip and the colon segment. Methionine reversed the effects of pretreatment with sodium nitroprusside, tetrodotoxin and Nw-oxide-L-arginine, resulting in depolarization of the RMP, and increased amplitude and AUC of contractions in the muscle strip. Methionine treatment affected the wave pattern of the colon segment by evoking small sized amplitude contractions superimposed on preexisting wave patterns. Our results indicate that a compound mimicking methionine may provide prokinetic functions in the human colon.
Area Under Curve ; Arginine/pharmacology ; Colon/drug effects/physiology ; Humans ; Membrane Potentials/drug effects ; Methionine/*pharmacology ; Microelectrodes ; Muscle Contraction/*drug effects ; Muscle, Smooth/drug effects/*physiology ; Nitroprusside/pharmacology ; Tetrodotoxin/pharmacology

OBJECTIVETo study the protective impact of tea polyphenols (TP) on the injury of fibrinolytic functions induced by high-methionine dietary in rats.

METHODS50 male Wistar rats were divided by stratified based on body weight into 5 groups with 10 in each group: namely control group, model group, low-dose TP group, medium-dose TP group and high-dose TP group. The rats in model group and TP groups were fed with 3% methionine dietary, control group rats with routine diet. In addition, rats in low-dose, medium-dose and high-dose TP groups were treated with TP at 50, 100 and 200 mg/kg dosage respectively by gavages every day, control group and model group rats were given with same amount distilled water. The animals were sacrificed after 8 weeks. The levels of tissue-type plasminogen activator (t-PA) and type-1 plasminogen activator inhibitor (PAI-1) in plasma were determined by ELISA assays, mRNA levels of t-PA and PAI-1 in aortic arch were detected by RT-PCR, t-PA and PAI-1 expression in aortic arch were detected by immunohistochemistry strept-avidin-biotin complex (SABC).

RESULTSAfter experiment, the t-PA expression of aortic arch in control group, model group, low-dose TP group, medium-dose TP group and high-dose TP group were 133.03 ± 10.14, 95.46 ± 11.08, 111.97 ± 11.91, 130.23 ± 10.80, 139.39 ± 9.41 (F = 14.15, P < 0.01), respectively, and the PAI-1 expression were 90.91 ± 8.67, 166.76 ± 12.18, 139.63 ± 12.71, 134.66 ± 13.19, 109.49 ± 10.82 (F = 31.44, P < 0.01). The t-PA concentration of plasma were (10.69 ± 1.26), (6.13 ± 0.92), (8.56 ± 1.19), (9.69 ± 0.92), (11.97 ± 1.08) ng/ml, respectively (F = 41.98, P < 0.01), and the PAI-1 concentration of plasma were (6.31 ± 0.81), (16.98 ± 1.27), (11.39 ± 0.82), (8.46 ± 0.67), (8.08 ± 0.91) ng/ml, respectively (F = 207.74, P < 0.01). The mRNA levels of t-PA in aortic arch were 1.12 ± 0.02, 0.75 ± 0.14, 1.01 ± 0.09, 0.95 ± 0.08, 1.05 ± 0.13 (F = 5.77, P < 0.05), and the mRNA levels of PAI-1 in aortic arch were 1.25 ± 0.11, 1.74 ± 0.06, 1.23 ± 0.05, 1.09 ± 0.14, 1.23 ± 0.04 (F = 23.56, P < 0.01).

CONCLUSIONThe results indicate that TP seems to have regulatory function on transcription and protein levels of t-PA and PAI-1, in addition to maintaining the balance between PAI-1 and t-PA and healing the injury of fibrinolytic functions in rats induced by high-methionine dietary.

Animals ; Diet ; Fibrinolysis ; drug effects ; Male ; Methionine ; adverse effects ; Plasminogen Activator Inhibitor 1 ; blood ; Polyphenols ; pharmacology ; Rats ; Rats, Wistar ; Tea ; chemistry ; Tissue Plasminogen Activator ; blood

The present study investigated the effects of intrathecal (i.t.) application of bovine adrenal medulla 22 (BAM22), an endogenous opioid peptide potently activating opioid receptors and sensory neuron-specific receptor (SNSR), on a model of complete Freund's adjuvant (CFA)-induced inflammatory pain. Unilateral, but not bilateral, inflammatory pain was induced by intraplantar (i.pl.) injection of CFA in one side, as indicated by the shortened paw withdrawal latency and the increased edema of paw. Paw withdrawal latency test, paw edema determination and immunohistochemistry were used in CFA-induced inflammatory pain model after i.t. administration of BAM22 or saline. It was found that administration of BAM22 dose-dependently attenuated CFA-induced hyperalgesia and edema, and resumed antinociceptive effects against thermal stimulation in behavioral test. In 10 nmol BAM22 group, paw withdrawal latency was resumed to 83.2% of normal, and edema increased only by 60% of normal at 48 h. The potency of BAM22 was 33.5% of maximal possible effect (MPE) at 24 h, and the antinociception persisted for at least 1 h. Furthermore, i.t. treatment of 10 nmol BAM22 evidently decreased the expressions of CFA-evoked neuronal nitric oxide synthase (nNOS)-positive cells and calcitonin gene-related peptide (CGRP)-immunoreactivity positive nerve fibers by 25.6% (P<0.01) and 25.2% (P<0.001) compared with saline group, respectively, at L3-L5 segments of the spinal cord. Small and medium CGRP-positive cells were 57.4% and 35.2% in dorsal root ganglion (DRG) in 10 nmol BAM22 group, respectively, which were remarkably lower than those in saline group (P<0.001). The present study suggests that BAM22 relieves CFA-induced thermal hyperalgesia in the early phase and resumes antinociceptive effects through down-regulation of nNOS and CGRP expressions in DRG and spinal cord, which is possibly mediated via SNSR.
Animals ; Calcitonin Gene-Related Peptide ; metabolism ; Enkephalin, Methionine ; analogs & derivatives ; pharmacology ; Freund's Adjuvant ; Hyperalgesia ; etiology ; physiopathology ; Inflammation ; chemically induced ; complications ; Male ; Nitric Oxide Synthase Type I ; metabolism ; Pain ; etiology ; physiopathology ; Pain Measurement ; drug effects ; Protein Precursors ; pharmacology ; Rats ; Rats, Sprague-Dawley ; Receptors, G-Protein-Coupled ; physiology

OBJECTIVEThe present study is to observe in vitro the proliferation ability of the muscle cells from permanent myopathy (PM) patients of nomokalaemic periodic paralysis (normKPP), which is caused by mutations of Met1592Val in the skeletal muscle voltage gated sodium channel (SCN4A) gene on chromosome 17q23.1. We also evaluate the possible effect of the foreign basic fibroblast growth factor (bFGF) in preventing and curing PM.

METHODSThe gastrocnemius muscle cells were taken from two male patients with PM of the same Chinese family with Met1592Val mutation of SCN4A, determined by gene screening. Four male patients suffering from the skeletal injury without PM were taken as control. All preparations were protogenerationally cultured in vitro. Proliferation of the cultured preparations was measured by MTT. Activities of the lactic dehydrogenase (LDH), creatine kinase (CK), and protein content in these cells were also detected. The effects of bFGF with different doses (10 ng/mL, 20 ng/mL, 40 ng/mL, 80 ng/mL, 120 ng/mL and 160 ng/mL) on the above mentioned parameters were also evaluated.

RESULTSCells from both PM and control subjects were successfully cultured in vitro. The cultivation of the muscle cells from PM patients in vitro was not yet seen. Results indicated the obvious stimulation of bFGF on cell proliferation, activities of LDH and CK, protein synthesis, in a dose dependent manner. The optimal dose of bFGF was 120 ng/mL (P<0.05), beyond which greater dose caused a less effect. The effect of bFGF on 160 ng /mL was stronger than that on 80 ng/mL, but there was no significant difference (P>0.05).

CONCLUSIONMyoblastic cells from patients with PM had a weaker ability of developing into the myotubules, thus they were unable to perform effective regeneration, which resulted in a progressive necrosis. The exogenous bFGF could promote the division and proliferation of the muscle cells in vitro. These results shield a light on bFGFos potential role in preventing and treating PM.

Adult ; Cell Proliferation ; drug effects ; Cells, Cultured ; Creatine Kinase ; metabolism ; Dose-Response Relationship, Drug ; Fibroblast Growth Factor 2 ; pharmacology ; Humans ; L-Lactate Dehydrogenase ; metabolism ; Male ; Methionine ; genetics ; Middle Aged ; Muscle Development ; genetics ; physiology ; Muscular Diseases ; genetics ; pathology ; Mutation ; genetics ; Myoblasts ; drug effects ; NAV1.4 Voltage-Gated Sodium Channel ; Sodium Channels ; genetics ; Valine ; genetics