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Objective To investigate the effects of 5-hydroxy-1-methylhydantoin (HMH) on kidney injury induced by paraquat (PQ). Methods Fifteen SPF healthy Kunming mice were randomly divided into normal saline (NS) control group, PQ poisoning model group and HMH intervention group, with 5 mice in each group. PQ poisoning model was challenged by one-time gavage of 30 mg/kg PQ solution. The NS group received the same amount of NS by gavage. The HMH group was given 100 mg/kg of HMH immediately after the model was made and continued to be gavaged. Mice in each group were sacrificed 1 day after HMH gavage and heart blood and renal tissue were harvested for examination. The morphological changes of renal tissue were observed under light microscope by hematoxylin-eosin (HE) staining. The content of malondialdehyde (MDA) and the activity of superoxide dismutase (SOD) in renal tissue were detected according to the instructions of the kit. The expression of heme oxygenase-1 (HO-1) and interleukin-1β (IL-1β) in renal tissues were detected by Western Blot. The serum metabolites were detected by gas chromatography time-of-flight mass spectrometry (GC-TOF-MS), the overall distribution of each sample was observed by principal component analysis (PCA), the accuracy of the model was evaluated by multidimensional analysis orthogonal partial least squares-discriminant analysis (OPLS-DA), and the difference metabolites were screened by variable importance in the projection (VIP) value > 1. Results Light microscopic observation showed that: glomerular structure in NS group was clear, there was no hyperemia and inflammatory cell infiltration in renal interstitium and blood vessels. In PQ group, some glomeruli atrophy and necrosis, capillary congestion in glomeruli, infiltration of inflammatory cells around glomeruli, swelling of renal tubular epithelial cells, slight stenosis of lumen, and occasional necrosis and exfoliation of epithelial cells occurred. The degree of kidney injury in HMH group was significantly less than that in PQ group. Compared with the NS group, the content of MDA in the PQ group was significantly increased (nmol/g: 6.70±0.84 vs. 2.70±0.43, P < 0.01) and the activity of SOD was significantly decreased (kU/L: 33.30±4.66 vs. 50.20±3.23, P < 0.05), the protein expression of HO-1 and IL-1β were significantly increased (HO-1/β-actin: 1.11±0.12 vs. 0.61±0.13, IL-1β/β-actin: 0.93±0.13 vs. 0.32±0.06, both P < 0.05). Compared with the PQ group, the content of MDA in the HMH group was significantly decreased (nmol/g: 5.10±0.93 vs. 6.70±0.84, P < 0.05) and the activity of SOD was significantly increased (kU/L:61.00±9.02 vs. 33.30±4.66, P < 0.05), the protein expression of HO-1 was significantly decreased (HO-1/β-actin:0.77±0.07 vs. 1.11±0.12, P < 0.05), however, there was no significant difference in the protein expression of IL-1β (IL-1β/β-actin: 0.87±0.13 vs. 0.93±0.13, P > 0.05). Metabolite detection results showed that: compared with NS group, the levels of creatinine, glycine, succinic acid, fumaric acid and citric acid were significantly increased in the PQ group (VIP value was 1.50, 1.58, 1.64, 1.74 and 1.95 respectively, all P < 0.05), while the levels of palmitic acid, α-tocopherol and 6-phosphogluconic acid were significantly decreased (VIP value was 1.10, 1.55 and 1.56 respectively, all P < 0.05). Compared with the PQ group, the levels of creatinine and citric acid were significantly decreased in the HMH group (VIP value was 1.50 and 1.86, both P < 0.05), while trans-4-hydroxy-proline, D-glyceric acid, 2, 6-fructose phosphate, 6-phosphate gluconic acid and aminomalonic acid were significantly increased (VIP value was 1.36, 1.55, 1.63, 1.68 and 1.76 respectively, all P < 0.05). Conclusions HMH protects kidney injury caused by PQ poisoning by correcting tricarboxylic acids cycle disturbance, lipid peroxidation and energy metabolism disturbance, and its mechanism is related to the regulation of HO-1 protein expression through Nrf2 pathway.
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Objective To establish a gas chromatographic-mass spectrometric (GC-MS) analysis method for quantifying 1-methylhydantoin concentration in whole blood. To provide technical support to forensic identification related cases of 1-methylhydantoin. Methods As an internal standard, 500 ng SKF525A was added to 0.5 mL blood sample, and then 2 mL 0.01 mol/L dilute hydrochloric acid and 0.5 g ammonium carbonate were added in order to buffer the pH value to 9, and following 2 mL ethyl acetate. The organic solvent layer was obtained after centrifuge and then analysed by GC-MS after drying. Results Good linear relationship of 1-methylhydantoin in blood was obtained in the range of 0.5-50 ng/mL. The equation of linear regression was y=0.01551 x+0.00726(R2=0.9997) with 0.1 ng/mL detection limit, and the recovery was 93.02％-108.12％. The intra-day and inter-day precision were less than 6.07％ and 13.37％, respec-tively. Conclusion The results gotten by this method is accurate and reproducible, which can be used for the determination of 1-methylhydantoin concentration in blood samples.
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Aim Toexplorewhether1-methylhydan-toin(MH)could inhibit the basal secretion of growth hormone (GH ) and suppress the promoting effect of growth hormone-releasing hormone (GHRH ) in rab-bits.Methods Thirty-sixrabbitswererandomlydi-vided into six experimental groups according to the kind of dosing drugs,namely normal saline group(A), MH group (B ),octreotide group (C ),GHRH group (D),GHRH +MH group(E),GHRH +octreotide group(F),with 6 rabbits in each group.Blood was sampled (1. 0 mL each time)from each rabbit before injecting drugs and 5,15,30,45,60 min after drug administration.Clotting spontaneously,rabbits blood samples were centrifugated for 20 minutes at approxi-mately 1000 ×g and the supernatant was collected. Serum GH concentrations were determined by enzyme linked immunosorbent assay kit(ELISA Kit).Mean-while,the behavior of rabbits in each group after injec-tingdrugswascloselyobserved.Results TheGH level of rabbits in group A at each time point had no significant differences(P>0. 05 ).Group B and group C rabbit GH levels were significantly lower than those of group A (P<0. 05 ),while GH levels in group D were obviously higher than those of group A (P <0. 05 ).Compared with group D,rabbit GH levels in group E and group F decreased markedly(P<0. 05 ). No obvious toxic and side effects had been observed within one week after the experimental rabbits were ad-ministered corresponding drugs by intravenous injec-tion.Conclusions 1-methylhydantoincouldinhibit the basal secretion of GH in rabbits.1-methylhydan-toin could suppress the promoting effect of GHRH in rabbits.
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ObjectiveTo investigate the protective effects of 5-hydroxy-1-methylhyantoin (HMH) on paraquat (PQ)-induced nephrotoxicity in rat and its possible mechanism.Methods Twenty-four male Sprague-Dawley (SD) rats were randomly divided into four groups: namely control, PQ, vitamin C and HMH groups, with 6 rats in each group. The rats in control group were given an injection of 2 mg/kg of normal saline intraperitoneally. The rats in PQ group were given an injection of 50 mg/kg of PQ intraperitoneally. The rats in vitamin C and HMH groups were given 1 mmol/kg of vitamin C or HMH through gastric tube right after PQ injection. The hydroxyl free radical scavenging ability of HMH and vitamin C was determined by Fenton method. Blood sample was collected after 24 hours of PQ treatment, then the animals were sacrificed and renal tissues were harvested. Blood urea nitrogen (BUN), serum creatinine (SCr), protein content of renal cortex, blood malondialdehyde (MDA), reduced glutathione (GSH) and superoxide dismutase (SOD) activity were determined.Results Both vitamin C and HMH showed a very good ability to scavenge hydroxyl radicals, and the 50% inhibiting concentration (IC50) was both 4.02 mg/mL. Compared with control group, serum BUN, SCr and MDA in renal tissue were significantly increased in PQ group, and the protein, GSH contents and SOD activity were significantly decreased [BUN (mmol/L): 40.80±2.49 vs. 13.67±1.58, SCr (μmol/L): 163.46±8.67 vs. 51.80±4.37, MDA (nmol/g): 7.51±0.23 vs. 4.52±0.33, protein (μmol/L): 0.94±0.14 vs. 1.35±0.10, GSH (mg/g): 1.08±0.48 vs. 3.30±0.44, SOD (kU/L): 70.74±6.42 vs. 112.89±8.72, allP 0.05).Conclusion HMH can protect the kidney against PQ-induced nephrotoxicity, and the mechanism of which maybe attributed to its anti-oxidation property and ability to scavenge hydroxyl radical.
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Objective To investigate the effect of 1-methylhydantoin (1-MH)on asthma and cough animal model, and to clarify its mechanism preliminarily.Methods 50 Wistar rats with ovalbumin-induced asthma were randomly divided into model group, 1-MH 20, 40, and 80 mg · kg-1 groups, positive control group (aminophylline, 60 mg·kg-1 ),another 10 Wistar rats served as control group.The levels of interleukin-5 (IL-5 ),eosinophil chemotactic factor (Eotaxin)and eosinophil (EOS)count in bronchoalveolar lavage fluid (BALF)were measured in various groups. 40 guinea pigs of asthma were randomly divided into model group, 1-MH 15, 30, and 60 mg·kg-1 groups, positive control group (aminophylline, 50 mg · kg-1 );the asthma incubation period of guinea pig was measured in various groups. The bronchus of 10 guinea pigs were selected and put in Krebs solution,and the antispasmodic percentages against histamine phosphate of 1-MH 0.25,0.50,and 1.00 g ·L-1 were recorded and calculated. 50 mice of cough were randomly divided into model group,1-MH 25,50,and 100 mg·kg-1 groups, positive control group (codeine, 50 mg · kg-1 );the cough incubation period and the number of cough in the mice were measured in various groups.40 guinea pigs of cough were randomly divided into model group,1-MH 15,30,and 60 mg·kg-1 groups,positive control group (codeine,20 mg·kg-1);the cough incubation period and the number of cough of the guinea pigs in various groups were measured. Results Compared with sensitized model control group,the levels of IL-5,Eotaxin and EOS count in BALF of the rats in 1-MH 40 mg·kg-1 and 80 mg·kg-1 groups were reduced (P<0.05 or P<0.01 );compared with acetylcholine-induced asthma model control group, the asthma incubation period of guinea pig in 1-MH 30 mg·kg-1 and 60 mg·kg-1 groups was prolonged(P<0.05 or P<0.01);compared with blank control group, the antispasmodic percentages against histamine phosphate in 1-MH 0.50 g · L-1 and 1.00 g · L-1 groups were increased(P<0.01);compared with mouse cough model control group,the cough incubation period of the mice was prolonged,the cough number of the mice was decreased in 50 mg·kg-1 and 100 mg·kg-1 groups(P<0.05 or P<0.01);compared with guinea pig cough model control group,the cough incubation period of the guinea pig was prolonged,the cough number of the guinea pig was decreased in 1-MH 30 mg·kg-1 and 60 mg·kg-1 groups (P<0.05 or P<0.01).Conclusion 1-MH has good antiasthmatic and antitussive effects,which may be related to inhibition of airway inflammation and relaxation of bronchial smooth muscle directly .