Effect of Danzhi Xiaoyaosan on Phosphorylation of Tau Protein in Rats with Alzheimer's Disease Based on PP2A and GSK-3β / 中国实验方剂学杂志

ObjectiveTo investigate the effect of Danzhi Xiaoyaosan on the phosphorylation of tau protein and different sites of glycogen synthase kinase-3β （GSK-3β） and phosphoseryl/suanyl phosphate protein phosphatase 2A （PP2A） in the hippocampus of rats with Alzheimer's disease （AD） and its mechanism. MethodThe rat model of AD was established by injecting okadaic acid into the bilateral hippocampus of 90 male Wistar rats in SPF grades. The rats with successful modeling were selected and randomly divided into model group， aricept group （0.5 mg·kg-1）， and Danzhi Xiaoyaosan high， medium， and low groups （17.55， 8.77， and 4.38 g·kg-1）， and then gavaged for 42 d， once a day. Morris water maze was used to detect the learning and memory ability of rats， Nissl's staining was used to observe the morphological structure of neurons in the hippocampus， and Real-time polymerase chain reaction （Real-time PCR） was used to detect the mRNA expression levels of tau protein， GSK-3β， and PP2A. Western blot was used to determine the protein expression levels of tau protein， GSK-3β， and PP2A. ResultAs compared with the control group， the learning and memory abilities of the rats in the model group were significantly decreased （P<0.01）， and the hippocampal CA3 region cells had abnormal structure， disorderly arrangement， and decreased number. The expression levels of GSK-3β mRNA， GSK-3β， p-GSK-3β-Tyr216， p-PP2A， and p-tau were increased in the model group as compared with the control group （P<0.01）， and those of p-GSK-3β-Ser9 and PP2A decreased significantly （P<0.01）. As compared with the model group， the learning and memory ability of the Aricept group and the Danzhi Xiaoyaosan groups were improved （P<0.05， P<0.01）， and the cell morphology and the number of hippocampal CA3 regions were better. The mRNA expression levels of PP2A and tau in the Aricept group were significantly up-regulated （P<0.05）， the mRNA expression level of GSK-3β was significantly down-regulated （P<0.01）， and the protein expression levels of GSK-3β， p-GSK-3β-Tyr216， and p-PP2A were down-regulated （P<0.05， P<0.01）， and the protein expression level of PP2A was significantly up-regulated （P<0.01）. As compared with the model group， the mRNA expression level of PP2A in the high-dose Danzhi Xiaoyaosan group was significantly up-regulated （P<0.01）， and that of GSK-3β was significantly down-regulated （P<0.01）， whereas the protein expression levels of p-PP2A， p-GSK-3β-Tyr216， and p-tau were down-regulated （P<0.05， P<0.01）， and the protein expression level of PP2A was significantly up-regulated （P<0.01）. As compared with the model group， the mRNA expression level of GSK-3β was significantly down-regulated in the medium-dose Danzhi Xiaoyaosan group （P<0.01）， the protein expression levels of GSK-3β， p-GSK-3β-Tyr216， and p-tau were down-regulated （P<0.05， P<0.01）， and the protein expression level of PP2A was significantly up-regulated （P<0.01）. As compared with the model group， the mRNA expression level of PP2A was significantly up-regulated in the low-dose Danzhi Xiaoyaosan group （P<0.01）， and that of GSK-3β was significantly down-regulated （P<0.01）， whereas the protein expression levels of GSK-3β and p-GSK-3β-Tyr216 were down-regulated （P<0.05， P<0.01）， and those of p-GSK-3β-Ser9 and PP2A were significantly up-regulated （P<0.01）. ConclusionDanzhi Xiaoyaosan can improve the learning and memory ability of rats with AD， and its mechanism may be related to the regulation of the activities of GSK-3β and PP2A protein-related sites and the phosphorylation of tau protein.

Development of a species-specific PCR assay for authentication of Agkistrodon acutus based on mitochondrial cytochrome b gene

BACKGROUND: Agkistrodon acutus, a traditional Chinese medicine, clinically used in the treatment of rheumatism, tumor, and cardiovascular and cerebrovascular diseases. Due to the unique medicinal value and the difficulty of artificial breeding of Agkistrodon acutus, the supply of Agkistrodon acutus on the market exceeds the demand, and a large number of its adulterants are found on the market. In this study, the cytb gene sequences of Agkistrodon acutus and 9 snakes were compared and analyzed, specific primers were designed, and specific PCR methods were established to detect Agkistrodon acutus medicinal samples on the market. RESULTS: This method was successfully applied to distinguish the snake from other adulterated species, and tested 18 Agkistrodon acutus samples randomly purchased from six cities. Twelve samples were counterfeit and six were genuine. The standard reference material of Agkistrodon acutus was cloned by molecular cloning and sequencing, and the gene sequence difference with other species was significant. It shows that the region could be used as the fingerprint region of the target species. CONCLUSIONS: The proposed method can be used as a species-specific marker and can be highly distinguished from other adulterated snake species, which is helpful to effectively avoid the problem of false sale of Agkistrodon acutus.

Study on Identification Methods of Bullwhip by PCR Technology Based on Cyt b Gene / 中国药房

OBJECTIVE： To establish the method for PCR identification of bullwhip， and to identify the authenticity of bullwhip at the molecular level. METHODS： DNA samples of bullwhip and its counterfeits （donkey whip， pig whip， sheep whip） were extracted and their integrity， purity and concentration were detected. Using GenBank related information， using mitochondrial cytochrome b （Cyt b） gene of bullwhip as target gene， Primer-BLAST online software was used to design specific primer. PCR amplification was performed for whips of different species， and electrophoretic analysis was conducted for the product. PCR products of bullwhip samples were cloned and confirmed by DNA sequencing. The specificity and repeatability of the established PCR method were verified. RESULTS： DNA purity of the bullwhip and its counterfeits was high， and there was no protein or RNA pollution. 1.5％ agarose gel electrophoresis showed that there were obvious target gene bands of bullwhip samples at 200-300 bp， while no corresponding bands appeared in other counterfeit products. The results of DNA sequencing showed that the nucleotide sequence of the gene fragment of bullwhip was 100％ similar to that of the bullwhip in GeneBank. Results of methodological validation showed that established method was specific and reproducible. CONCLUSIONS： The established PCR identification method based on Cyt b gene in the study is simple， rapid， accurate， specific and reproducible， and can meet the requirements of analysis and identification of bullwhip and its counterfeits.

The Research Progress of Metformin in Type Ⅱ Diabetes Mellitus with Liver Cancer / 现代生物医学进展

As a safe,cheap and effective diabetes drug,metformin has been used for many years.Diabetes increases the risk of liver cancer and affects its prognosis.In recent years,it is found that metformin reduces the pancreatic cancer risk in the treatment of diabetic patients,a large of experiments also prove that it has anti-cancer and synergistic anticancer effect.This paper focused on the effects of metformin on treatment of Ⅱ type diabetes,discussed the curative effect on liver cancer,suggested the molecular biology mechanism of inhibiting tumor,listed the latest experiment researches,analyzed the existed clinical data,proposed the further study of anticancer mechanism and clinical treatment.Metformin for a future role in prevention of hepatocellular carcinoma in patients with type Ⅱ diabetes are briefly summarized and future prospects,which in type Ⅱ diabetic patients with liver cancer in a prospective study of the effect of treatment.Mefformin for application in other cancer prevention also raises possibilities.

Identification and characterizaton of mitochondrial DNA and cytochrome b patial gene in Martes zibellina L / 吉林大学学报(医学版)

Objective To explore the characterization of mitochondrial DNA and cytochrome b(Cytb) gene from Martes zibellina L..Methods The mtDNA were extracted from fresh heart and liver of Martes zibellina L.by alkaline lysis method respectively,and Cyt b gene segment was amplified by PCR technique and sequenced.Results The complete 16800 bp mtDNA was separated from both samples,and the Cytb gene with 310 bp segments was amplified by PCR and the sequence indicated that the homologous similarity was 99% with sable Martes zibellina L.(GenBank:AB026109.1) and it was 45% with other animal similarity.Conclusion There is complete mtDNA in tissues of Martes zibellina L.and Cytb partial gene from martes zibellina L.may be used as the genetic marker for identification of different species.

Characteristic of class 1 integron-cassette among Shiga toxin-producing E. coli isolates in China / 吉林大学学报(医学版)

Objective To explore the distribution and characteristic of class 1 integrons among Shiga toxin-producing E.coli(STEC) in China,and to elucidate the type of cassettes.Methods Antibiotics susceptibility was tested by the disk diffusion method,class 1 integron was detected by PCR assay and PCR products were sequenced and analyzed in eight isolates.Results Four isolates were multiple-drug resistant whose antibiogram were ampicillin,tetracycline,erythromycin,sulfamethoxazole/trimethoprim and ciprofloxacin,but two isolates conferred resistance of streptomycin and spectinomycin simultaneously.Only the two isolates carried class 1 integrons ranging in 1 000 bp which located on the plasmid of 7 800 bp.The sequenced PCR product demonstrated that the 1 000 bp integron harbored aad A1 gene cassettes conferred the resistance of streptomycin and spectinomycin;and linked with sul 1 and qacE?1 gene conferred the resistance of sulfamethoxazole and quaternary disinfectants.Conclusion The class 1 integrons exists among STEC isolates in China and determines the resistant antibiotics.

Identification and characterization of class 1 integron among E. coli from healthy students' enteric strains / 吉林大学学报(医学版)

Objective To explore the distribution and characterization of class 1 integrons in E.coli from healthy feces,and to elucidate the status of gene-cassettes.Methods Routine method was used to isolate E.coli,antibiotics susceptibility was tested by the disk diffusion method;class 1 integron was detected by PCR assay;PCR products were sequenced and analyzed.Results Of 97 samples,76 isolates were identified,and 25 isolates were multiple-drug resistant.The antibiogram was sulfamethoxazole-trimethoprim,ampicillin,streptomycin,tetracycline,erythromycin.14 of 25 isolates carried class 1 integrons,and the size of integrons differed from 1 800 bp(10 strains) to 750 bp(4 strains).The sequenced PCR product demonstrated that the 1 800 bp integron laboured aadA1-dfrA14-orf gene cassette conferred the resistance to sulfamethoxazole-timethoprim,streptomycin and aminoglycoside;the 750 bp integron laboured dfrA14 gene cassette conferred the resistance to sulfamethoxazole-trimethoprim.Conclusion The different kinds of class 1 integrons exist in E.coli from the healthy students,and determine the multiple-resistant antibiotics.