Detection of Carbamazepine and Its Metabolites in Blood Samples by LC-MS/MS / 法医学杂志

OBJECTIVES@#To establish a method for the detection of carbamazepine and its metabolites 10,11-dihydro-10,11-epoxycarbamazepine and 10,11-dihydro-10-hydroxycarbamazepine in blood samples by liquid chromatography-tandem mass spectrometry (LC-MS/MS).@*METHODS@#The blood samples were treated with 1-butyl-3-methylimidazolium hexafluorophosphate as an extraction solvent. The samples were extracted by ultrasound-assisted extraction and separated by ZORBAX Eclipse Plus C18, 95Å column. The mobile phase A aqueous solution containing 0.1% formic acid and 10 mmol/L ammonium acetate, and mobile phase B mixed organic solvent containing acetonitrile/methanol (Vacetonitrile∶Vmethanol=2∶3) were used for gradient elution at the flow rate of 1.00 mL/min. An electrospray ion source in positive mode was used for detection in the multiple reaction monitoring.@*RESULTS@#The linearities of carbamazepine and its metabolites 10,11-dihydro-10,11-epoxycarbamazepine and 10,11-dihydro-10-hydroxycarbamazepine in blood samples were good within the corresponding range, with correlation coefficients (r) greater than 0.995 6. The limits of detection were 3.00, 0.40 and 1.30 ng/mL, respectively. The limit of quantitation were 8.00, 1.00 and 5.00 ng/mL, respectively. The extraction recoveries ranged from 76.00% to 106.44%. The relative standard deviations of the intra-day and inter-day precisions were less than 16%. Carbamazepine and its main metabolite 10,11-dihydro-10,11-epoxycarbamazepine were detected in blood samples of death cases with a mass concentration of 2.71 μg/mL and 252.14 ng/mL, respectively.@*CONCLUSIONS@#This method has high sensitivity and good selectivity, which is suitable for the detection of carbamazepine and its metabolites in blood samples, and can be used for carbamazepine-related forensic identifications.

Hypoglycemic Effect of Total Flavonoids from Potentillae Discoloris Herba in Type 2 Diabetic db/db Mice / 中国实验方剂学杂志

Objective:The hypoglycemic effects and mechanisms of total flavonoids from Potentillae Discoloris Herba（TFE） on insulin resistance through the phosphatidylinositol-3 kinase （PI3K）/protein kinase B （Akt） signaling pathway in db/db mice were investigated. Method:The 24 db/db mice were randomly divided into four groups， model group， metformin group and TFE 100，400 mg·kg^-1 group respectively. The 6 db/m mice as normal control group. After 4 weeks treatment， the mice were processed and the levels of fasting blood glucose（FBG）， glycated serum protein（GSP），fasting blood insulin（FINS），triglyceride（TG）， total cholesterol（TC）， low density lipoprotein cholesterol （LDL-C），high density lipoprotein cholesterol （HDL-C） in serum were detected. Homeostatic model assessments of insulin resistance（HOMA-IR）were quantified. Hematoxylin-eosin（HE）staining of liver and pancreatic tissues were examined. The expression of IR<italic>β</italic>， IRS-1，PI3K，phosphorylation-PI3K （p-PI3K）， Akt， phosphorylation-Akt（p-Akt） and glucose transporter 4（GLUT4） in livers were assessed by Western blot. Result:Compared with normal group， model group showed liver and pancreas injury. FBG， GSP， TC， TG， LDL-C， FINS and MDA levels in serum were significantly increased （<italic>P</italic><0.01）， HDL-C and SOD levels in serum were significantly decreased （<italic>P</italic><0.05）， liver glycogen content was significantly decreased （<italic>P</italic><0.01）， as well as expression of IR， IRS-1， p-PI3K/PI3K， p-Akt/Akt and GLUT4 protein in liver tissues were significantly decreased （<italic>P</italic><0.01）. Compared with model group， TFE was able to relieve liver and pancreas injury，while the levels of FBG， GSP， TC， TG， FINS and MDA in serum were significantly decreased （<italic>P</italic><0.05）， HDL-C and SOD levels liver were significantly increased （<italic>P</italic><0.05）， liver glycogen content was significantly increased （<italic>P</italic><0.01）， and the expressions of IRS-1， p-PI3K/PI3K， p-Akt/Akt and GLUT4 protein in liver tissues were significantly up-regulated （<italic>P</italic><0.05）. Conclusion:These findings indicate that TFE has the potential to reduce blood sugar and alleviates insulin resistance through the PI3K/Akt signaling pathway in the livers of db/db mice.

Determination of Escitalopram in Biological Samples by Dispersive Liquid-Liquid Microextraction Combined with GC-MS/MS / 法医学杂志

Objective To establish a method for determination of escitalopram in biological samples by ultrasound-assisted ionic liquid-dispersive liquid-liquid microextraction combined with gas chromatography-tandem mass spectrometry （GC-MS/MS） and provide evidences for forensic determination of cases related to escitalopram. Methods The 1-hexyl-3-methylimidazolium hexafluorophosphate （[C6MIM][PF6]） was selected as an extract solvent to process biological samples. Ultrasound-assisted extraction was used on the samples. Then the samples were detected by GC-MS/MS. Results The linear range of escitalopram in blood and liver were 5.56-1 111.10 ng/mL and 0.025-5.00 mg/g, respectively. The correlation coefficient （r） were greater than 0.999, limit of detection （LOD） were 4.00 ng/mL and 2.00 μg/g, limit of quantitation （LOQ） were 14.00 ng/mL and 6.00 μg/g, respectively. The extraction recovery rates were all greater than 50%, the interday and intraday precision were less than 20%. Escitalopram was detected in blood and liver samples from the actual poisoning case by this method with a content of 1.26 μg/mL and 0.44 mg/g, respectively. Conclusion The ultrasound-assisted ionic liquid-dispersive liquid-liquid microextraction combined with GC-MS/MS is environment friendly, rapid, has good enriching effect and consumes less organic solvent and can be used for forensic determination of escitalopram related cases.

Determination of Endosulfan Concentrations in Biological Samples by GC-MS/MS / 法医学杂志

OBJECTIVES@#To establish an analytical method of the endosulfan concentrations （α-endosulfan and β-endosulfan） in biological samples by GC-MS/MS. To observe the distribution of endosulfan in aquatic animals and provide experimental evidence for forensic identification of relevant cases.@*METHODS@#Acetonitrile was added to the blood and muscle samples for precipitating the protein. The endosulfan concentrations were determined by GC-MS/MS in multiple reaction monitoring mode. Qualitative analysis was performed according to the retention time and ion rate, and quantitative analysis was performed by external standard working curve method.@*RESULTS@#In blood samples, the calibration curves of α-endosulfan and β-endosulfan ranging from 0.062 5 to 10 μg/mL had good linear relationship, the correlation coefficients （r） of which were >0.99. The limits of detection （LOD） were 1 ng/mL and 2 ng/mL and the limits of quantification （LOQ） were 4 ng/mL and 8 ng/mL, respectively. In muscle samples, the calibration curves of α-endosulfan and β-endosulfan ranging from 0.062 5 to 10 μg/g, the r of which were >0.98. The LOD were 1 ng/g and 4 ng/g and the LOQ were 4 ng/g and 16 ng/g, respectively. The accuracy of α-endosulfan and β-endosulfan was 90.76%-108.91% both in blood and muscle samples, the interday and intraday precision were 2.35%-8.71% and 5.44%-10.29%, respectively. In poisoning cases, endosulfan were detected in all parts of fish and crab and the content difference was statistically significant.@*CONCLUSIONS@#The endosulfan detection method based on GC-MS/MS established in the present study is rapid, sensitive and accurate, which can be applied to the endosulfan detection in traces biological samples. The distribution of endosulfan in fish and crab was different, which can provide evidence to the sample collection and analysis for toxicological analysis in relevant forensic identification.

The polysaccharide isolated from Pleurotus nebrodensis (PN-S) shows immune-stimulating activity in RAW264.7 macrophages / 中国天然药物

A novel Pleurotus nebrodensis polysaccharide (PN-S) was purified and characterized, and its immune-stimulating activity was evaluated in RAW264.7 macrophages. PN-S induced the proliferation of RAW264.7 cells in a dose-dependent manner, as determined by the MTT assay. After exposure to PN-S, the phagocytosis of the macrophages was significantly improved, with remarkable changes in morphology being observed. Flow cytometric analysis demonstrated that PN-S promoted RAW264.7 cells to progress through S and G2/M phases. PN-S treatment enhanced the productions of interleukin-6 (IL-6), nitric oxide (NO), interferon gamma (INF-γ), and tumor necrosis factor-α (TNF-α) in the macrophages, with up-regulation of mRNA expressions of interleukin-6 (IL-6), inducible nitric oxide synthase (iNOS), interferon gamma(INF-γ) and tumor necrosis factor-α (TNF-α) being observed in a dose-dependent manner, as measured by qRT-PCR. In conclusion, these results suggest that the purified PN-S can improve immunity by activating macrophages.

Effect of PKF118-310 on Cell Cycle and Proliferation of K562 Cells and Its Mechanism / 中国实验血液学杂志

<p><b>UNLABELLED</b>Objetive: To investigate the effects of PKF118-310 on cell cycle and proliferation of K562 cell lines and its mechanism.</p><p><b>METHODS</b>After treatment of PKF118-310 with different concentration, the proliferation inhibition on K562 cell lines was detected by MTT, the existance of β-catenin and TCF-4 in the cells was observed by immunohistochemistry. The change of the cell cycle was detected by flow cytometry. The expressions of caspase-3, β-catenin, TCF and BCL-9 were detected by Western blot.</p><p><b>RESULTS</b>PKF118-310 can inhibit the proliferation of K562 cell line by S phase blocking. The β-catenin and TCF in the cells were observed by immunohistochemistry. After treating this cell line with PKF118-310 of different concentrations for 72 h, the expression level of caspase-3 increased, the expression levels of β-catenin, TCF and BCL-9 significantly decreased.</p><p><b>CONCLUSION</b>PKF118-310 induces cycle arest of K562 cells at the S phase and inhibits the proliferation of these cells through decreasing β-catenin/TCF/BCL-9 thrascriptional activity.</p>

Pleurotus nebrodensis polysaccharide (PN-S) enhances the immunity of immunosuppressed mice / 中国天然药物

In the present study, the effects of Pleurotus nebrodensis polysaccharide (PN-S) on the immune functions of immunosuppressed mice were determined. The immunosuppressed mouse model was established by treating the mice with cyclophosphamide (40 mg/kg/2d, CY) through intraperitoneal injection. The results showed that PN-S administration significantly reversed the CY-induced weight loss, increased the thymic and splenic indices, and promoted proliferation of T lymphocyte, B lymphocyte, and macrophages. PN-S also enhanced the activity of natural killer cells and increased the immunoglobulin M (IgM) and immunoglobulin G (IgG) levels in the serum. In addition, PN-S treatment significantly increased the phagocytic activity of mouse peritoneal macrophages. PN-S also increased the levels of interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), interferon-γ (INF-γ), and nitric oxide (NOS) in splenocytes. qRT-PCR results also indicated that PN-S increased the mRNA expression of IL-6, TNF-α, INF-γ, and nitric oxide synthase (iNOS) in the splenocytes. These results suggest that PN-S treatment enhances the immune function of immunosuppressed mice. This study may provide a basis for the application of this fungus in adjacent immunopotentiating therapy against cancer and in the treatment of chemotherapy-induced immunosuppression.

Study on the correlation between polymorphisms of genes with susceptibility to tuberculosis and drug-resistant tuberculosis in Chinese Han population / 中华流行病学杂志

Objective To investigate the distribution of polymorphisms of SLC11A1 gene,VDR gene,MBL gene and IFNG gene with susceptibility to tuberculosis (TB) in Chinese Han population suffering from drug-sensitive TB and drug-resistant TB so as to identify the correlation between gene polymorphisms and the development of drug-resistant TB.Methods Single nucleotide polymorphisms (SNP) of VDR gene,SLC11A1 gene,MBL gene,IFNG gene were typed and analyzed by pyrosequencing,Real-time Probe and SNaPshot among 229 patients with drug-sensitive TB and 230 patients with drug-resistant TB.Results The polymorphic foci of VDR gene from the drug-sensitive TB group and the drug-resistant TB group showed no significant difference (P＞0.05).The genotype of INT4 site and allelic frequency of SLC11A1 gene for drug-sensitive TB group were significantly different from those for drug-resistant TB group(P=0.031,0.046).If recessive inheritance was assumed,the genotypes of INT4 site from the two groups were significantly different (0R=5.756,95% CI:1.261-26.269,P=0.011).Considering the relationship between OR values under various combination,our findings confirmed that the genetic mode of INT4 site was in accordance with recessive inheritance.The genotypes of Q/P site and allelic frequencies of MBL gene from drug-sensitive and drug-resistant groups were significantly different (P=0.029,0.033).The difference still existed under the hypothesis of recessive inheritance (OR=9.290,95% CI:1.167-73.949,P=0.011).The polymorphic foci of IFNG gene from the two groups showed no significant difference.Conclusion INT4 sites on SLC11A1 gene and Q/P site on MBL gene were probably associated with the development of drug-resistant TB in Chinese Han population.Further study on this issue would be helpful in locating the population at high risk of drug-resistant TB and exploring the effective intervention to decrease the incidence of this disease.

Mechanism of cinnamic aldehyde-inducing apoptosis of chronic myeloid leukemic cells in vitro / 中国实验血液学杂志

The aim of this study was to investigate the apoptosis-inducing effect of cinnamic aldehyde (CA) on chronic myeloid leukemic (CML) cells and its mechanism. K562 cells and primary bone marrow mononuclear cells (MNC) from patients with CML were treated by various concentrations of CA. Flow cytometry was employed to measure the apoptosis of K562 cells and primary CML bone marrow MNC. Western blot was used to determine the expression of C-MYC and the phosphorylation of CrkL in K562 cells, and real-time polymerase chain reaction (real-time PCR) was used to quantify the expression of BCR-ABL mRNA in K562 cells. The results indicated that CA induced the apoptosis of K562 cells in a time- and dose-dependent manner. CA induced apoptosis of CML MNC dose-dependently. CA inhibited the expression of BCR-ABL mRNA and C-MYC, reduced CrkL phosphorylation levels in K562 cells. It is concluded that CA induces apoptosis of CML cells in vitro. Down-regulation of the expression and function of BCR-ABL may be one of its most important anti-leukemia mechanisms.

Relationship between germ cell apoptosis and Sertoli cell vimentin in prepubertal rats induced by local exposure to heat / 中华男科学杂志

<p><b>OBJECTIVE</b>To explore the relationship between germ cell apoptosis and the expression as well as the distribution of Sertoli cell vimentin induced by local exposure to heat.</p><p><b>METHODS</b>Local short-term exposure of prepubertal male rats testis to heat (43 degrees C for 15 min). Histochemical method was used to observe morphological characteristics of seminiferous tubule. The distribution and expression of Sertoli cell cytoskeletons were analyzed by immunohistochemistry and germ cell apoptosis was evaluated by TUNEL technique at different hour-intervals.</p><p><b>RESULTS</b>After 2 h and 4 h heat exposure, the disattachment phenomenon between Sertoli cell and spermatogonia occurred. Spermatogonia arranged in disorder and displaced away from the basement membrane of seminiferous tubules. Immunohistochemical staining showed that vimentin positive staining was seen radiating from the Sertoli cell perinuclear region with apical "spoke-like" pattern in controls. There was an intense vimentin immunoreactivity surrounding Sertoli cell nuclei along with the collapse of the apical extensions in 2 h group, but no significant difference compared with the controls. The expressions of vimentin in 12 h and 24 h groups were higher than those of the controls (P <0.01), respectively. TUNEL showed that incidence of apoptosis was observed to increases markedly in 12 h and 24 h groups, but it was found that the incidences of apoptotic events were decreased in these two groups compared with the controls.</p><p><b>CONCLUSION</b>The changes of expression and distribution of Sertoli cell vimentin filaments correlate with the increased germ cell apoptosis. Local heat may disrupt spermatogenesis by injuring Sertoli cell directly.</p>