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ObjectiveTo investigate the effect of NOD-like receptor family pyrin domain containing 3 (NLRP3) knockdown on a mouse model of nonalcoholic steatohepatitis (NASH) induced by high-fat high-carbohydrate (HFHC) diet. MethodsA total of 44 mice were randomly divided into normal diet group (CON group) with 20 mice and HFHC group with 24 mice. At the end of week 14 of modeling, 4 mice were randomly selected from the HFHC group for the pre-experiment of adeno-associated virus (AAV) by tail vein injection, and NLRP3 knockdown was verified after 4 weeks. After NLRP3 knockdown was verified at the end of week 18, the remaining 40 mice were given a single tail vein injection of AAV, and then they were divided into CON+NLRP3 knockdown negative control group (CON+NLRP3-NC group), CON+NLRP3 knockdown group (CON+NLRP3-KD group), HFHC+NLRP3-NC group, and HFHC+NLRP3-KD group, with 10 mice in each group. At the end of week 24, the activation of NLRP3 inflammasome was observed; related indicators were measured, including body weight, liver weight, liver index, and glucose metabolism (fasting blood glucose, fasting insulin, and Homeostasis Model Assessment of Insulin Resistance [HOMA-IR] index); the indicators of liver lipid content (liver triglyceride [TG] and oil red O staining), liver inflammation (serum alanine aminotransferase [ALT] activity, HE staining, and inflammation-related genes), and liver fibrosis (Sirius Red staining and fibrosis-related genes) were measured. A one-way analysis of variance was used for comparison of continuous data between multiple groups, and the least significant difference t-test was used for further comparison between two groups. ResultsCompared with the CON+NLRP3-NC group based on the results of Western Blot, the HFHC+NLRP3-NC group had significant increases in the protein expression levels of NLRP3, pro-Caspase1, Caspase1, ASC, and IL-1β, while the HFHC+NLRP3-KD group had significant reductions in these levels (all P<0.05). The HFHC+NLRP3-NC group showed varying degrees of increase in body weight, liver weight, liver index, and glucose metabolism indicators, while the HFHC+NLRP3-KD group showed significant improvements in these indicators (all P<0.05). As for hepatic fat deposition, compared with the CON+NLRP3-NC group, the HFHC+NLRP3-NC group had a significant increase in liver TG, with a large number of red lipid droplets shown by oil red O staining, and the HFHC+NLRP3-KD group had significant reductions in liver TG and the number of lipid droplets in the liver (all P<0.01). In terms of liver inflammation, compared with the CON+NLRP3-NC group, the HFHC+NLRP3-NC group had significant increases in serum ALT, NAFLD activity score, and inflammation-related genes, while the HFHC+NLRP3-KD group had significant reductions in these indicators (all P<0.01). As for liver fibrosis, compared with the CON+NLRP3-NC group, the HFHC+NLRP3-NC group had significant increases in collagen fiber area and fibrosis-related genes, and the HFHC+NLRP3-KD group had significant reductions in fibrosis-related genes (all P<0.05) and a tendency of reduction in collagen fiber area (P>0.05). ConclusionNLRP3 knockdown can significantly improve hepatic fat deposition and inflammation in a mouse model of HFHC-induced NASH.
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Objective:To detect the expression and clinical significance of microRNA-128-3p (miR-128-3p) in the plasma of patients with severe pancreatitis (SAP) .Methods:A total of 175 patients with acute pancreatitis who were treated in our hospital from Jun. 2017 to Dec. 2020 were selected as observation objects. According to the severity of the patients, the patients were divided into 78 cases in mild acute pancreatitis (MAP) group, 50 cases in moderate to severe acute pancreatitis (MSAP) group and 47 cases in severe acute pancreatitis (SAP) group; according to the patient’s prognosis, the SAP group was divided into 28 cases in the survival group and 19 cases in the death group. The level of miR-128-3p was detected by qRT-PCR method, and patients were evaluated with Acute Physiology and Chronic Health Evaluation Ⅱ (APACHE Ⅱ) score and Ranson score, the correlation between plasma miR-128-3p level and APACHEⅡ, Ranson score was analyzed by Pearson, the predictive value of plasma miR-128-3p for poor prognosis of SAP patients was analyzed by ROC curve.Results:There were statistically significant differences in APACHE II[ (3.41±1.56) , (5.63±1.78) , (6.57±1.83) points], Ranson [ (2.58±1.34) , (4.95±1.47) , (6.06±1.92) points] and plasma CRP [ (39.73) ±12.31) , (70.25± 24.38) , (142.51±40.55) mg/L], serum calcium [ (1.95±0.31) , (13.26±5.24) , (14.21±6.32) mmol/L], among the MAP group, MSAP group and SAP group ( P<0.05) ; Compared with the MAP group (1.05±0.12) , the plasma miR-128-3p expression levels in the MSAP group (0.83±0.08) and the SAP group (0.57±0.05) were significantly decreased ( P<0.05) ; compared with the MSAP group (0.83±0.08) Compared with the SAP group (0.57±0.05) , the plasma miR-128-3p expression level was significantly decreased ( P<0.05) ; Compared with the survival group [ (0.65±0.08) , (5.91±1.64) points, (5.45±1.25) points, (120.43±30.56) mg/L], the plasma miR-128-3p of SAP patients in the death group was (0.43±0.05) expression levels were significantly reduced, APACHE II [ (7.43±2.21) points], Ranson score [ (7.22±1.59) points] and plasma CRP level [ (171.52±42.38) mg/L] were significantly increased ( P<0.05) ; the results of correlation analysis showed that plasma miR-128-3p level was negatively correlated with APACHEⅡ and Ranson scores ( r=-0.531, -0.609, P<0.05) ; The diagnostic efficacy of plasma miR-128-3p in predicting poor prognosis of SAP patients is better than APACHEⅡ, Ranson score, and CRP. Conclusion:Plasma miR-128-3p level is elevated in patients with severe pancreatitis, which is of certain value for the diagnosis and prognostic evaluation of SAP.
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Objective To investigate the changes of metabolites in serum of paroxysmal patients with atrial fibrillation based on metabonomics.Methods The metabolites of serum in 15 patients with atrial fibrillation (case group) and 20 healthy subjects (control group) were measured by gas chromatography-mass spectrometry (GC-MS) group.The metabolic profile difference in the two groups was analyzed and compared by orthogonal design method (OPLS).Results The metabolic profile of the control and case groups was significantly distinguished,and 29 statistically significant differences metabolites were identified using commercially available metabolite libraries (such as the Wiley and NIST mass pools) and the standard metabolite library established in our laboratory,The areas under the ROC curve of 29 metabolites were calculated.The area under the curve of glycerol was 0.937,the area under the serine curve was 0.853,the area under the curve of aspartic acid was 0.933,the area of threonine was 0.823,the area of tryptophan was 1.Conclusion There exist differences in serum of sma] l molecular metabolites between patients with atrial fibrillation and normal healthy people.Glycerol,serine,aspartic acid,threonine and tryptophan of serum in patients with atrial fibrillation may be biomarkers of diagnosis of atrial fibrillation.
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Objective To study the effect of methylphenidate on urinary endogenous metabolites in children with ADHD, and to explore the mechanism of methylphenidate. Methods The metabolites of urine in 37 children with ADHD treated with methylphenidate before intervention (case 0 week group), intervention for 6 weeks (6 weeks group), intervention 12 weeks (12 weeks group, and 40 healthy subjects (control group) were measured by gas chromatography-mass spectrometry (GC-MS), and analyzed by orthogonal partial least squares (OPLS) to find the significant differences biomarkers. Results The urine metabolic profile of the control group, the 0 week group and the 12 week group showed obvious changes. The metabolic profile of the 0 week group and the control group was differentiated, and the 12 week group was away from the 0 week group, and closed to the control group. 24 significant differences metabolites were identified using commercially available metabolite libraries (such as the Wiley and NIST mass pools) and the standard metabolite library established in our laboratory. The levels of these metabolites were reversed to normal level due to methylphenidate intervention. Conclusion This study shows that methylphenidate has a good curative effect on children with ADHD. Its mechanism is related to the metabolic abnormalities of pyruvate metabolism, tryptophan metabolism, phenylalanine metabolism, glycine, serine and threonine metabolism. The possible mechanism of methylphenidate in the treatment of children with ADHD was revealed from the overall metabolic point of the body. Metabolomics reveals possible therapeutic mechanism of methylphenidate for children with ADHD from overall metabolism of the body.
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OBJECTIVE:To establish a method for simultaneous determination of 6 residual organic solvents in aprepitant raw material as methanol,ethanol,acetone,isopropyl alcohol,methyl tert-butyl ether and tetrahydrofuran.METHODS:Headspace capillary gas chromatography was adopted.The determination was performed on DB-624 capillary column using temperature programming.The temperature of injector port was 180 ℃,and flame ionization detector was used with temperature of 260 ℃.Nitrogen was used as carrier gas with flow rate 3.0 mL/min.The spilt ratio was 5 ∶ 1,and head-space injection volume was 1.0 mL.The head-space equilibrium temperature was set at 80 ℃,and equilibrium time was 40 min.RESULTS:The linear ranges of methanol,ethanol,acetone,isopropyl alcohol,methyl tert-butyl ether,tetrahydrofuran were 6.052-605.232 μ g/mL (r=0.999 9),9.987-998.718 μg/mL(r=0.999 9),9.998-999.768 μg/mL(r=0.999 8),9.986-998.634 μg/mL(r=0.999 9),9.991-999.090 μg/mL (r=0.999 7),1.461-146.133 μg/mL(r=0.999 5),respectively.The limits of quantitation were 1.782 1,2.079 0,0.749 8,1.777 8,0.223 1,0.607 0 μg/mL;the limits of detection were 0.594 0,0.693 0,0.249 9,0.592 6,0.074 4,0.202 3 μg/mL,respectively.RSD of precision test was lower than 2.0%.Only acetone and isopropyl alcohol were detected in stability test and reproducibility tests,RSD<2.0%.Their recoveries were 99.34-100.75% (RSD=0.52%,n=9),98.20%-100.24% (RSD=0.69%,n=9),98.07%-100.07% (RSD=0.84%,n=9),99.86%-101.32% (RSD=0.58%,n=9),97.87%-104.02% (RSD=2.13%,n=9),98.26 %-100.58 % (RSD =0.75 %,n =9),respectively.CONCLUSIONS:The established method is simple,accurate and reproducible,and can be used for simultaneous determination of 6 residual organic solvents in aprepitant raw material.
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This study was aimed to reveal the material basis on different diseases of the same syndrome damp-heat syndrome from the level of metabonomics.The typical damp-heat syndrome patients diagnosed as chronic viral hepatitis B,non-alcoholic fatty liver disease,or chronic glomerulonephritis were included,with 30 cases in each disease.There were 30 healthy volunteers in the control group.The serum samples were detected by UPLC-QTOFMS and GC-TOFMS.And then,the results were analyzed by variance analysis in order to find out the generality and specificity of metabolic material in three different diseases with damp-heat syndrome.The results showed that through comparisons of different diseases with damp-heat syndrome,as well as the healthy group as control,it was revealed that inosine,uridine,aspartic acid,oleic acid glyceride and lactate were the same substances of three diseases of damp-heat syndrome.It was concluded that based on metabonomics,as for three different diseases with damp-heat syndrome,there were different substances among different diseases,but common substances related to damp-heat syndrome.Thus,it provided objective evidences for the theory of different diseases of the same syndrome in traditional Chinese medicine (TCM) from the level of metabonomics.
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OBJECTIVE:To provide reference for improving the cognition of medical staff for adverse drug reactions(ADR). METHODS:A questionnaire survey was conducted on the spot among the physicians,nurses and pharmacists with different profes-sional titles in our hospital to investigate their cognition about ADR concept,their judgment for ADR and ADR reporting,and the results were analyzed by statistical methods. RESULTS:Totally 452 questionnaires were sent out,and 439 were effectively re-ceived,with effective rate of 97.12%. In terms of correct cognition rate about ADR basic concept and the main reasons,the sur-veyed pharmacists were higher than physicians and nurses,the senior professional titles of surveyed pharmacists were higher than the intermediate and junior,the pharmacists with senior professional titles were the highest and the junior were the lowest,the dif-ferences were statistically significant(P<0.05);in terms of the proportion of certain for ADR judgment,the surveyed nurses were lower than pharmacists and physicians,the surveyed physicians with senior and intermediate professional titles were higher the ju-nior,the surveyed nurses with intermediate titles were higher than the junior,the surveyed pharmacists with senior professional ti-tles were higher than the intermediate and the junior,the differences were statistically significant(P<0.05);in terms of cognition about ADR reporting,the surveyed pharmacists were the highest and the nurses were the lowest,the differences were statistically significant (P<0.05),while in terms of the proportion of knowing ADR reporting timing limit,reporting procedures and depart-ments,the surveyed pharmacists and physicians with junior professional titles were lower than the senior and intermediate,the dif-ferences were statistically significant(P<0.05);in terms of reasons for missed ADR reporting,the proportion of consideration of the risk of causing medical disputes for doctors of the surveyed physicians was higher than pharmacists and nurses,while the sur-veyed nurses showed higher proportion for doing not understand reporting procedures,doing not reporting,thinking ADR reporting was dispensable and uncertain for ADR identification,the differences were statistically significant (P<0.05). CONCLUSIONS:Medical staff with different professional titles and occupations have different cognition about ADR basic concept,judgment and re-porting,the pharmacist are relatively good,followed by the physician and nurse,especially the nurse,and the medical staff with junior professional titles are relatively poor. It is necessary to strengthen ADR education and training for them,play professional ad-vantages from the pharmacist and optimize ADR reporting procedures to improve the reporting rate and quality of ADR.
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Faldaprevir analogue molecule (FAM) has been reported to effectively inhibit the catalytic activity of HCV NS3/4A protease, making it a potential lead compound against HCV. A series of HCV NS3/4A protease crystal structures were analyzed by bioinformatics methods, and the FAM-HCV NS3/4A protease crystal structure was chosen for this study. A 20.4 ns molecular dynamics simulation of the complex consists of HCV NS3/4A protease and FAM was conducted. The key amino acid residues for interaction and the binding driving force for the molecular recognition between the protease and FAM were identified from the hydrogen bonds and binding free energy analyses. With the driving force of hydrogen bonds and van der Waals, FAM specifically bind to the active pocket of HCV NS3/4A protease, including V130-S137, F152-D166, D77-D79 and V55, which agreed with the experimental data. The effect of R155K, D168E/V and V170T site-directed mutagenesis on FAM molecular recognition was analyzed for their effect on drug resistance, which provided the possible molecular explanation of FAM resistance. Finally, the system conformational change was explored by using free energy landscape and conformational cluster. The result showed four kinds of dominant conformation, which provides theoretical basis for subsequent design of Faldaprevir analogue inhibitors based on the structure of HCV NS3/4A protease.