Study on metabolites derived from Zhideke granules in rats in vivo / 中国药房

OBJECTIVE To analyze the metabolites of Zhideke granules and speculate its metabolic pathway in rats in vivo. METHODS Male SD rats were randomly divided into blank group and administration group （Zhideke granules， 9.45 g/kg）； they were given ultrapure water or relevant medicine， twice a day， every 6-8 h， for 3 consecutive days. Serum， urine and feces samples of rats were collected， and their metabolites were identified by UPLC-Q-Exactive-MS technique after intragastric administration of Zhideke granules； their metabolic pathways were speculated. RESULTS After intragastric administration of Zhideke granules， 16 prototype components （i.g. irisflorentin， baicalin， chlorogenic acid） and 11 metabolites （i.g. hydration products of kaempferol or luteolin， methylation products of chlorogenic acid， and hydroxylation products of baicalin） were identified in serum， urine and feces of rats. Among them， 8 prototype components and 4 metabolites were identified in serum samples； 10 prototype components and 7 metabolites were identified in urine samples； 8 prototype components and 5 metabolites were identified in the fecal samples. CONCLUSIONS The metabolites of Zhideke granules in rats mainly include baicalin， irisflorentin，chlorogenic acid， and the main metabolic pathways included methylation， hydroxylation， glucuronidation.

Optimization of the Extraction Technology of Zhideke Granules / 中国药房

OBJECTIVE：To optimize the extraction technology of Zhideke granules. ME THODS：The extraction technology （water extraction ，alcohol extraction ，water extraction and ethanol precipitation ）of Zhideke granules was initially screened by ammonia-induced cough experiment and xylene-induced ear swelling experiment in mice. Based on its preparation route ，the immersion time of medicinal materials containing volatile oil was investigated with water absorption as index firstly. The single factor test was adopted to investigate the amount of water added and the extraction time taking the volatile oil yield as index to optimize the extraction technology of medicinal materials containing volatile oil. Taking the contents of irisflorentin and total flavonoids as indicators ，on the basis of single factor investigation ，orthogonal test was adopted to examine the influence of three factors including the amount of water added ，extraction time and extraction frequency ，so as to optimize the water extraction technology of Zhideke granules and the validation tests were conducted. RESULTS ：The results of pharmacodynamics experiment showed that the cough latency of mice in water extract low-dose and high-dose groups （6.34，12.68 g/kg，by crude drug ）and water-extraction alcohol-precipitation extract high-dose group （12.68 g/kg，by crude drug ）were significantly longer than those inmodel group ，and the number of cough within 2 minutes was significantly reduced （P＜0.05 or P＜0.01）. Compared with model group ， the ear swelling of mice in water extract low-dose and high-dose groups （6.34，12.68 g/kg，by crude drug），ethanol extract high-dose group （12.68 g/kg，by crude drug） and water-extraction alcohol-precipitation extract hig dose group （12.68 g/kg，by crude drug ） were decreased significantly （P＜0.05 or P＜0.01）. The swelling inhibition rates were 42.26％，55.08％，33.49％，51.56％，39.57％ and 44.36％ in low-dose and high-dose groups of water extract ，alcohol extract ， water-extraction and alcohol-precipitation extract respectively ，indicating that the water extract had better antitussive and anti-inflammatory effects. The optimal extraction technology of volatile oil was adding 5-fold water ，soaking for 30 minutes，and extracting for 3 hours. The optimal water extraction technology was adding 12-fold water ，extracting for 3 times after soaked for 50 min，lasting for 1 h each time. Results of 3 times of validation tests showed that average content of irisflorentin in the extract obtained by optimal technology was 76.47 μg/g（RSD＝ 2.15％，n＝3）and the average content of total flavonoids was 92.45 mg/g（RSD＝0.48％，n＝3）. CONCLUSIONS ：The optimal extraction technology of Zhideke granules is stable and feasible.

Removal of Shield Needles from Graphene Oxide Thrombus and Preparation / 中国医疗器械杂志

Atherosclerosis is a chronic inflammatory disease commonly seen in clinical practice. It can lead to thickening of vascular intima, occlusion of lumen stenosis and thrombosis, leading to angina pectoris, hypertension, myocardial infarction and other diseases, posing a serious threat to human life and health. This study provides a method for removing shield needles from graphene oxide thrombus and its preparation. The graphene oxide shield needle mainly includes flexible rotating shaft, radial flexible rod, rotating needle, adsorption main pipe and dosing main pipe, laser measuring device, high definition camera and other structures, which has the following advantages:firstly, it achieves multi-angle rotation grinding thrombosis, precise rotation grinding, avoids vascular damage and infection; secondly, thrombolytic drugs can be applied in the process of rotary grinding and small thrombus can be adsorbed to effectively avoid secondary embolization of blood vessels; thirdly, it a coating of graphene oxide on a rotating needle, which protects against bacteria and infection. This study has practical reference value for the development of thrombotherapy and the application of graphene in the medical field.

Study on Chemical Constituents of Ethanol Extract from Yao Medicine Cissampelopsis spelaeicola / 中国药房

OBJECTIVE： To study the chemical constituents of ethanol extract from Yao medicine Cissampelopsis spelaeicola. METHODS： The petroleum ether， ethyl acetate and n-butanol fraction from 75％ ethanol extract of C. spelaeicola were isolated and purified by silica gel， SephadexLH-20 gel column and AB-8 macroporous resin column， etc. The structures of the compounds were analyzed and identified by physicochemical properties and spectral data （mass spectrometry， hydrogen spectrum， carbon spectrum）. RESULTS & CONCLUSIONS： Twelve compounds were isolated and identified from 75％ ethanol extract of C. spelaeicola. β-sitosterol（Ⅰ） and Stigmasterol（Ⅱ） were isolated from petroleum ether fraction； p-hydroxybenzoic acid（Ⅲ）， β-daucossterol（Ⅳ）， protocatechuic acid（Ⅴ）， 6β-hydroxyeremophi-7（11）-en-12，8β-olide（Ⅵ）， 10β-hydroxyeremophil-7（11）-en-8，12-olide（Ⅶ）， 10β-hydroxyeremophi-7（11），8（9）-dien-8，12-olide（Ⅷ）， Quercetin（Ⅸ）， Hyperin（Ⅹ） and 4α-hydroxy- eudesman-11-ene（Ⅺ） were isolated from ethyl acetate fraction； quercetin-3-O-robinobioside（Ⅻ） was isolated from n-butanol fraction. Compounds Ⅰ-Ⅻ are isolated from C. spelaeicola for the first time. The study can lay material foundation for activity stady of C. spelaeicola.

Prokaryotic expression of ATP5B and preparation and identification of its monoclonal antibody / 吉林大学学报(医学版)

Objective:To construct and identify the monoclonal antibody of ATP synthase beta subunit (ATP5B) with high purity, and to lay foundation for further study.Methods:The ATP5Bgene was amplified by PCR and cloned into the pET28avector and transformed into E.coli BL21 (DE3) .The protein expression was induced by IPTG and then the fusion protein was purified by nickel affinity chromatography column.The protein purity was detected by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) .Three female Balb/C mice were immunized with purified fusion protein and the tail vein blood was taken to detect the titer of ATP5Bantibody by indirect enzyme-linked immunosorbent assay (ELISA) .The spleen cells from the immunized mice with the highest serum titer were mixed with the SP2/0cells to establish the hybridoma cells and the fused cells were screened by indirect ELISA and monoclonally cultured.Karyotype analysis were performed in the positive cells.The hybridoma cells were intraperitoneally injected into 12weeks old BALB/C mice to estabilish the ascites models.The titer of ascites was detected by indirect ELISA.The purity of the antibody was detected by SDS-PAGE.The antibody subtype was detected by ELISA.Results:After PCR amplification, a specific band of 1 455bp was obtained, and the pET28aempty vector was ligated to obtain a recombinant pET28a/ATP5Bvector.The target protein was expressed in the IPTG-induced bacteria solution;the SDS-PAGE results showed that the protein band was found at51 000.The indirect ELISA results showed that the serum titer of the venous blood of immunized mice was up to1:64 000.In karyotype analysis, the total number of chromosomes in hybridoma cells was about the sum of myeloma cells and normal mouse spleen cells.The mouse ascites was prepared with the hybridoma cell line, and the highest titer of the antibody was 1:240 000.The subtype of the monoclonal antibody produced by the hybridoma cells was IgG1.Conclusion:The monoclonal antibody against ATP5Bprotein is successfully prepared by cloning, expressing and purifying the recombinant protein.

Expression of vascular endothelial growth factor gene in bone marrow mesenchymal stem cells / 吉林大学学报(医学版)

Objective To study the vascular endothelial growth factor(VEGF) expression and secretion of mesenchymal stem cells(MSCs) of rabbit transfected with pcDNA3-VEGF165 expression plasmid in order to construct a kind of tissue engineering artificial skull with more blood supply.Methods By gene reconstruction method,the VEGF165 gene was cloned into eukaryotic expression plasmid pcDNA3 and recombined eukaryotic expression plasmid pcDNA3-VEGF165 was constructed;By lipofectamine transfection method,pcDNA3-VEGF165 expression plasmid was transfected into MSCs of rabbit;By RT-PCR and Western blotting methods,the VEGF mRNA expression and VEGF protein secretion in the MSCs were detected.Results Recombined eukaryotic expression plasmid pcDNA3-VEGF165 was confirmed to be true by double enzyme digestion,two strips came to appear in 5400 bp and 600 bp of gelose electrophoresis and their sizes accorded with pcDNA3 plasmid and VEGF165 accordingly;Primarily cultured and subcultured MSCs of rabbit were successfully performed and the MSCs storehouse of rabbit was established,primary MSCs presented lymphoid form at first and then the morphology of them became circulara,polygonal or irregular forms,they were more and more like fibroblastic cells after subcultured cultivation.The expressions of VEGF mRNA and VEGF protein in the MSCs were found after transiently transfection by RT-PCR and Western blotting methods.Conclusion Recombined eukaryotic expression plasmid pcDNA3VEGF165 can be transfected into MSCs of rabbit effectively by lipofectamine and the VEGF expression can be detected in the MSCs after transfection.

Influence of predator stress on levels of IL-1?, IL-6 in BALB/C mice with brain asymmetry / 中国病理生理杂志

AIM: To investigate the influence of predator stress on hypothalamic-pituitary-adrenal (HPA) axis and IL-1?, IL-6 in mice with brain asymmetry. METHODS: By using paw preference test, right-pawed, left-pawed, and ambidextrous-pawed mice model were established. Mice with brain lateralization were exposed to their predator (cat). After acute and chronic predator stress by cats, EIA and ELISA were applied to detect plasma levels of corticosterone (CS), IL-1? and IL-6. RESULTS: (1) The level of plasma CS: in both acute and chronic predator stress group, right-pawed and ambidextrous mice had a higher level than that in their corresponding normal group (P

IL-1?,IL-6 levels and SOCS-3 gene expression in hypothalamus of BALB/c mice with brain asymmetry / 中国免疫学杂志

Objective:To investigate the effects of brain asymmetry on levels of IL-1?,IL-6 and suppressor of cytokine signaling-3(SOCS-3) in hypothalamus,to get better understanding of relationship between brain asymmetry and neuro-immuno-endocrine network at the molecular level.Methods:By using paw preference test,right-pawed,left-pawed,and ambidextrous mice model was established.After decapitation of the mice,hypothalamus was separated rapidly.Some samples were homogenized and used for detection of levels of interleukin-1?(IL-1?),interleukin-6(IL-6) by Enzyme-Linked-Immunosorbent Assay(ELISA).The other samples were applied to detect SOCS-3 expression level by RT-PCR.Results:(1)The level of IL-6 in hypothalamus:left-pawed were higher than right-pawed mice,there was significant difference in statistics( P 0.05).(3)The expression level of SOCS-3:the SOCS-3 expression of right-pawed showed higher than left-pawed mice in hypothalamus,revealing a significant difference( P

PREPARATION AND APPLICATION OF A RAT ANTI-HRP MONOCLONAL ANTIBODY / 解剖学报

The rat anti-HRP McAbs were prepared with rat-rat hybridoma and the rat McPAP complex was made by conjugating this McAb with HRP. The results of the present work indicated that the rat MePAP can be used for detection of antibodyantigen reaction by immunohistochemical techniques and ELISA. In both assay it gave strong staining reaction for positive antigen and with background staining appeared very pale. The sensitivity of rat McPAP and HRP labelling rabbit anti-rat IgG has been compared.