[Study on the effect of diallyl sulfide on peripheral nerve injury in n-hexane intoxicated rats].

Objective: To investigate the antagonistic effect of diallyl sulfide (DAS) against peripheral nerve injury induced by n-hexane in rats. Methods: A total of 68 adult male Wistar rats were selected, among which 50 were randomly selected and divided into blank control group, DAS control group (100 mg/kg·bw) , n-hexane model group, low-dose DAS intervention group (50 mg/kg·bw) , and high-dose DAS intervention group (100 mg/kg·bw) . A rat model of peripheral nerve injury was established by n-hexane exposure, and the rats were treated with DAS at different doses. The changes in pyrrole adducts and behavior were observed, a metabolic analysis was performed for serum pyrrole adducts, and the intervention effect was evaluated. The remaining 18 rats were randomly assigned to the n-hexane model group, the low-dose DAS intervention group, and the high-dose DAS intervention group, with 6 rats in each group, as satellite groups used for the toxicokinetic analysis of serum pyrrole adducts. Results: Compared with the blank control group, the n-hexane model group and low-and high-dose DAS intervention groups had a significant reduction in body weight since week 2 (P<0.01) . Compared with the n-hexane model group at the end of the experiment at week 7, the high-dose DAS intervention group had a significantly higher body weight (P<0.05) , while there was no significant difference in body weight between the n-hexane model group and the low-dose DAS intervention group (P>0.05) . The n-hexane model group developed gait abnormality at week 2 of poisoning, while the low-and high-dose DAS intervention groups developed gait abnormality at weeks 3 and 5 of poisoning, respectively. At the end of the experiment, the n-hexane model group and the low-and high-dose DAS intervention groups had a significantly higher gait score than the blank control group (P<0.01) . At the end of the experiment, the n-hexane model group and the low-dose DAS intervention group had significantly shorter latency in rotarod test than the blank control group (P<0.01) , while there was no significant difference in latency between the DAS control group and the high-dose DAS intervention group (P>0.05) . Compared with the n-hexane model group, the low-and high-dose DAS intervention groups had a significant increase in latency in rotarod test (P<0.01) . Compared with blank control group, the n-hexane model group and the low-dose DAS intervention group had a significant increase in mean nerve conduction velocity (P<0.01) , while there was no significant difference between the blank control group and the DAS control group or high-dose DAS intervention group (P>0.05) , and compared with the n-hexane model group, the low-and high-dose DAS intervention groups had a significant increase in nerve conduction velocity (P<0.01) . Compared with the blank control group at the end of the experiment at week 7, the n-hexane model group and the low-and high-dose DAS intervention groups had significant increases in the concentration of pyrrole adducts in serum, urine, and hair (P<0.01) , while there was no significant difference between the blank control group and the DAS control group (P>0.05) , and the high-dose DAS intervention group had a significantly lower concentration of pyrrole adducts in serum, urine, and hair than the low-dose DAS intervention group (P<0.05) . Serum pyrrole adducts reached the peak level at 9-12 hours and then started to decrease. Compared with the n-hexane model group, the high-and low-dose DAS intervention groups had a significantly shorter half-life period of serum pyrrole adducts (P<0.01) . Compared with the n-hexane model group, the high-and low-dose DAS intervention groups had a significant reduction in the area under the curve of serum pyrrole adducts (P<0.05) . Conclusion: DAS can antagonize peripheral nerve injury induced by n-hexane.

[The protective effects of diallyl sulfide (DAS) on benzene-induced leukopenia in mice].

Objective: To study the protective effects of diallyl sulfide (DAS) on leukopenia induced by benzene. Methods: 90 Healthy male ICR mice, adaptive feeding 5 days later, 15 were randomly divided into blank control group、model group、low、middle、high dose DAS intervention groups and DAS control group. Mice in intervention groups and DAS control group were orally given DAS at 40, 80, 160, 160 mg/kg·bw, while mice in the other two groups received an equal volume of corn oil. After 2 hours, model group and the other three intervention groups were given benzene, corn oil suspension (1.3 g/kg·bw) , the two control groups treated with the same volume of corn oil, Benzene and DAS are dissolved in corn oil. one time for each day. 4 weeks later, Anesthesia at 14/29, make blood routine examination and count organ index and observe pathological examinations of spleen and thymus. Results: On day 14, the counts of peripheral blood white blood cells (WBC) , lymphocytes, monocytes in the model group decreased to 68.99%, 71.72%, 53.19% (P<0.05) ; On day 29, the counts of peripheral blood lymphocytes, monocytes, neutrophils in the model group decreased to 83.00%, 81.03%, 89.37%, 20.84%, 19.25% (P<0.05) ; spleen weight, spleen index, white pulp area ratio of spleen, thymus weight, thymus index, thymic cortex area ratio of mice in the model group decreased (P<0.05) . On day 14, the counts of peripheral blood monocytes and lymphocytes in the DAS high dose intervention group increased by 136.36%, 260.00% (P<0.05) ; On day 29, the counts of White blood cells, lymphocytes, red blood cells, platelets, hemoglobin in the DAS low, middle and high dose intervention groups increased (P<0.05) ; spleen weight, spleen index, white pulp area ratio of spleen, thymus weight, thymus index, thymic cortex area ratio of mice in the DAS high dose intervention groups increased (P<0.05) . Conclusion: DAS can effectively suppress benzene-induced leucopenia in mice.

[Antioxidant mechanism of diallyl sulfide in inhibiting leucopenia in peripheral blood induced by benzene in rats].

Objective: To investigate the antioxidant mechanism of diallyl sulfide (DAS) in antagonizing the reduction in peripheral blood white blood cells (WBC) induced by benzene in rats. Methods: A total of 60 specific pathogen-free adult male Sprague-Dawley rats, with a body weight of 180-220 g, were selected, and after 5 days of adaptive feeding, they were randomly divided into blank control group, DAS control group, benzene model group, benzene+low-dose DAS group, benzene+middle-dose DAS group, and benzene+high-dose DAS group, with 10 rats in each group. The rats in the benzene+low-dose DAS group, the benzene+middle-dose DAS group, the benzene+high-dose DAS group, and the DAS control group were given DAS by gavage at a dose of 40, 80, 160, and 160 mg/kg·bw, respectively, and those in the blank control group and the benzene model group were given an equal volume of corn oil; 2 hours later, the rats in the benzene model group, the benzene+low-dose DAS group, the benzene+middle-dose DAS group, and the benzene+high-dose DAS group were given a mixture of benzene (1.3 g/kg·bw) and corn oil (with a volume fraction of 50%), and those in the blank control group and the DAS control group were given an equal volume of corn oil. The above treatment was given once a day for 4 consecutive weeks. At 1 day before treatment, anticoagulated blood was collected from the jugular vein for peripheral blood cell counting. After anesthesia with intraperitoneally injected pentobarbital (50 mg/kg·bw), blood samples were collected from the abdominal aorta, serum was isolated, and the thymus, the spleen, and the femur were freed at a low temperature to measure oxidative and antioxidant indices. The femur at one side was freed for WBC counting in bone marrow. Results: Compared with the blank control group, the benzene model group had significant reductions in the volume, weight, and organ coefficient of the spleen and the thymus (P<0.05) ; compared with the benzene model group, the benzene+low-dose DAS group, the benzene+middle-dose DAS group, and the benzene+high-dose DAS group had significant increases in the volume of the spleen and the thymus and the weight and organ coefficient of the spleen (P<0.05), and the benzene+middle-dose DAS group and the benzene+high-dose DAS group had significant increases in the weight and organ coefficient of the thymus (P<0.05). Compared with the blank control group, the benzene model group had a significant reduction in WBC count in peripheral blood and bone marrow (P<0.05), and compared with the benzene model group, the benzene+middle-dose DAS group and the benzene+high-dose DAS group had a significant increase in WBC count in peripheral blood and bone marrow (P<0.05). Compared with the blank control group, the benzene model group had a significant increase in the serum level of malondialdehyde (MDA) (P<0.05) and significant reductions in total superoxide dismutase (T-SOD) activity, reduced glutathione (GSH) level, GSH/oxidized glutathione (GSSG) ratio, total antioxidant capacity (T-AOC) (P<0.05) ; compared with the benzene model group, the benzene+high-dose DAS group had a significant reduction in the serum level of MDA and significant increases in T-SOD activity, GSH level, GSH/GSSG ratio, and T-AOC (P<0.05). Compared with the blank control group, the benzene model group had a significant increase in the level of MDA (P<0.05) and significant reductions in GSH level, GSH/GSSG ratio, and T-AOC (P<0.05) in the spleen; compared with the benzene model group, the benzene+low-dose DAS group, the benzene+middle-dose DAS group, and the benzene+high-dose DAS group had a significant reduction in MDA level (P<0.05) and significant increases in GSH level and T-AOC (P<0.05), and the benzene+high-dose DAS group had significant increases in T-SOD activity and GSH/GSSG ratio (P<0.05). Compared with the blank control group, the benzene model group had a significant increase in the level of MDA in bone marrow cells (BMCs) and peripheral blood mononucleated cells (PBMCs) (P<0.05) and a significant reduction in T-AOC in PBMCs (P<0.05) ; compared with the benzene model group, the benzene+low-dose DAS group, the benzene+middle-dose DAS group, and the benzene+high-dose DAS group had a significant reduction in the level of MDA in BMCs and PBMCs (P<0.05), and the benzene+high-dose DAS group had significant increases in GSH level and GSH/GSSG ratio (P<0.05) . Conclusion: DAS can antagonize the benzene-induced reduction in peripheral blood WBC, possibly by exerting an anti-oxidative stress effect.

[Protective effects of N-acetyl-L-cysteine against binge drinking-induced fatty liver in mice].

Objective: To investigate the roles of N-acetyl-L-cysteine (NAC) against binge drinking-induced fatty liver in mice. Methods: SPF male C57BL/6 mice were randomly divided into 3 groups, i.e. control group, model group, and NAC/ethanol group (n=10). Mice in model and NAC/ethanol groups were exposed to 3 doses of ethanol (6 g/kg bw) to induced fatty liver, while mice in control group received equal volume and equal energy of maltodextrin solution. NAC was administered to mice at 1 h before ethanol exposure (100 mg/kg bw, i.p.). The mice were sacrificed at 6 h after the last ethanol exposure. The liver and epididymal adipose tissues were collected. Histopathological examination and biochemical assay kit were used to evaluate the fat accumulation, while Western-blot was performed to detect the protein levels of some key factors involved in fat metabolism in liver and adipose tissues. Results: Compored with control group mice, the liver index and liver weight were significantly increased compared with model group, the liver index and TG level in NAC/ethanol group mice were all significantly decreased (P<0.05). Histological examination showed NAC effectively suppressed binge drinking-induced fat accumulation in mice liver. In addition, NAC had no significant effects on the protein levels of peroxisome proliferator-activated receptor-α (PPAR-α), Acy-CoA oxidase (ACOX), sterol regulatory element binding protein 1 c (SREBP-1c) and fatty acid synthase (FAS). Furthermore, the protein levels of hormone sensitive lipase (HSL) did not significantly differ among 3 groups, whereas NAC prevented binge drinking-induced increase of HSL phosphorylation at ser563 and ser660. Conclusion: NAC could effectively attenuate binge drinking-induced fatty liver, which might be associated with the inhibition of lipid mobilization by suppressing the phosphorylation of HSL.

[Study on the determination of pyrrole adduct content in hair samples].

Objective: To find out a method to determine the pyrrole adducts in the hairs. Methods: Collected the hair from common people and rats, defatted after completely washed, steeped the hair in different concentration of 2, 5-hexandione to build hair model containing pyrrole adducts; dissolved the hair and determined the concentration of pyrrole adducts. Results: (1) . The combination of 0.72 mol/L of sodium hydrate and 2% tyrisin could dissolve the hair, and the digestion liquid could react with the Ehrlich's reagent showing fuchsia color; (2) . The color could maintain longer after adding more ethanol; (3) . More pyrrole adducts would be produced by the increasing the concentration of 2, 5-dihexandione (P<0.01) ; (4) . Concentration of pyrrole adducts in n-hexane treated hair showed no difference compared with control (P>0.05) . Conclusion: the method could be used to determine the concentration of pyrrole adducts in hair exposed to n-hexane.

Cloning and characterization of the UBC gene from lotus (Nelumbo nucifera).

Protein ubiquitination is extensively involved in the regulation of a considerable number of physiological processes in plant cells. E2 (ubiquitin-conjugating enzyme, UBC), one of the essential enzymes of eukaryotic ubiquitination, catalyzes protein ubiquitination together with E1 and E3. In this study, we cloned four full-length cDNA NnUBCs of Nelumbo nucifera. With the same coding sequence length of 459 bp and coding 153 amino acids, these four genes are highly homologous with the AtUBC1 and AtUBC2 of Arabidopsis thaliana. Quantitative fluorescence polymerase chain reaction showed that these four genes exhibited different expression patterns in different tissues of N. nucifera. Overall, the expression of NnUBC3 was the highest in all plant tissues. Tests of different stress treatments showed that NnUBC3 plays an important role in response to heat, salt, and drought stresses in N. nucifera. Moreover, transgenic Arabidopsis plants (Atubc1-1Atubc2-1 mutant) expressing NnUBC3 presented a wild-type phenotype, indicating that NnUBC3 performs the same function as AtUBC1 and AtUBC2.

Identification and characterization of chalcone synthase cDNAs (NnCHS) from Nelumbo nucifera.

Chalcone synthase (CHS) catalyzes the first committed step in flavonoids biosynthetic pathway. In this study, six full-length cDNAs (NnCHS) encoding CHS from Nelumbo nucifera were successfully isolated, using rapid amplification cDNA end (RACE) assay. The obtained cDNAs were 1426 bp in size, containing a 1167 bp open reading frame coding 389 amino acids. Exons-intron architecture of NnCHS gene was illustrated, consisting two exons inserted by a 426 bp intron. The putative NnCHS possessed all the conserved active sites for CHS function as well as the family signature. Phylogenetic analysis revealed that NnCHS shared high homology with CHS from high plants, and the homology-based structural modeling showed that NnCHS had the typical structure of CHS. Moreover, Real-time PCR assays demonstrated that NnCHS mRNAs were expressed in various tissues of N. nucifera, with the highest expression in red flower and lowest level in the leaves. Moreover, patterns of NnCHS expression illustrated short-time wounding or low temperature significantly induced the up-regulation of NnCHS mRNA.

Development and characterization of genic-SSR markers from different Asia lotus (Nelumbo nucifera) types by RNA-seq.

Nelumbo nucifera is an important economic vegetable and traditional medicine, but available genetic resources remain limited. Next generation sequencing has proven to be a rapid and effective means of identifying genic simple sequence repeat (genic-SSR) markers. This study developed genic-SSRs for N. nucifera using Illumina sequencing technology to assess diversity across cultivated and wild lotus. A total of 105,834 uni-contigs were produced with an average read length of 722 bp. Exactly 11,178 genic-SSR loci were identified in 9523 uni-contigs. Di-nucleotide (64.5%) was the most abundant SSR, followed by tri-nucleotide (23%), tetra-nucleotide (8.9%), penta-nucleotide (2.5%), and hexa-nucleotide (1%) repeat types. The most common di- and tri-nucleotide repeat motifs were AG/CT (51%) and AAG/CTT (8%), respectively. Based on these SSRs sequences, 6568 primer pairs were designed, of which 72 primers were randomly selected for synthesis and validation, and 38 in-silico polymorphic primers were obtained using in-house perl scripts. A total of 110 primers were screened in the lotus samples and the results showed that 101 primers yielded amplification products, of which 80 were polymorphs. The number of alleles ranged from 2 to 17 and the PIC (polymorphism information content) ranged from 0.19 to 0.87 with a mean value of 0.55. An Unweighted Pair Group Method with Arithmetic Mean (UPGMA) dendrogram based on Jaccard's similarity coefficients showed that the correlation between geographical source and genotype was low. This study describes the distribution of genic-SSRs in the expressed portion of the lotus genome. These genic-SSRs have an important role to play in molecular mapping, diversity analysis, and marker-assisted selection strategies in Nelumbo.

Comparative analysis of protein expression in differentiated thyroid tumours: a multicentre study.

This study compared clinical features and protein expression profiles in differentiated thyroid tumours to identify protein markers with the potential for indicating malignancy status. Tissue microarrays were constructed using 119 thyroid tumour samples (45 papillary carcinomas, 26 follicular carcinomas, 48 adenomas). Generally, there was overexpression of proliferating cell nuclear antigen (PCNA), p53, matrix metalloproteinase (MMP)-7, Hector Battifora mesothelial-1 (HBME-1), MMP-2, pituitary tumour-transforming gene (PTTG) and human telomerase reverse transcriptase (hTERT) in malignant thyroid carcinomas, and overexpression of fragile histidine triad (FHIT), p16 and E-cadherin in thyroid adenomas. Multiple factor binary logistic regression analysis indicated that MMP-2, HBME-1, p16 and FHIT were independently related to differentiated thyroid tumours. Receiver-operating characteristics for these four factors showed HBME-1 as best for diagnostic accuracy. Sensitivity and specificity were enhanced using an HBME-1 and p16 cluster. HBME-1 expression was not significantly different for papillary and follicular carcinomas, whereas p16 expression was significantly specific.

[Characterization and quantification of dermatan sulfate from donkey skin].

Dermatan sulfate (DS), a recently known antithrombotic glycosaminoglycan, was isolated and purified from donkey skin. Physiochemical characteristics of the glycan, including constituent analysis, electrophoretic behaviour, molecular mass, specific lyase degradations, IR and PMR spectra were described, using porcine skin-origin dermatan sulfate as a standard reference. Contents of DS in donkey skin and its gelatinized preparations (Ejiao) were also measured. Results indicate that the presence of DS may explain the long reputed clinical efficacy of donkey skin and Ejiao in treating serious symptoms associated with what has been called endogenous wind in traditional Chinese medicine.

Decelerated transport and its mechanism of 2,5-hexanedione on middle-molecular-weight neurofilament in rat dorsal root ganglia cells.

Chronic exposure to n-hexane induces peripheral-central axonopathy, mediated by its metabolite 2,5-hexanedione (2,5-HD), in occupational workers and experimental animals, but the underlying mechanism is still unclear. In the current study, we investigated the effects of 2,5-HD on middle-molecular-weight neurofilament (NF-M) axonal transport using live-cell imaging technique in cultured rat dorsal root ganglia (DRG) cells. PA-GFP-NF-M plasmid was transfected into DRG neurons and live-cell imaging was performed to observe the slow axonal transport of NF-M. The levels of cytoskeleton and motor proteins in DRG cells were detected by Western-blot and the concentration of ATP was determined using an ATP Assay Kit. The results showed that 2,5-HD administration resulted in a decrease of NF-M axonal transport and a reduction of three neurofilament subunits levels in DRG cells. Furthermore, 2,5-HD exposure significantly decreased ATP contents and the protein levels of kinesin heavy chain (KHC). These findings indicated that 2,5-HD reduced slow axonal transport, neurofilaments cargoes, motor proteins and ATP energy in rat DRG cells, which may contribute to 2,5-HD-induced neurotoxicity.