Preparation and characterization of a recombinant poly-epitopic vaccine EgG1Y162-2 (4) against cystic echinococcosis based on the linker GSGGSG / 中国血吸虫病防治杂志

Objective To perform prokaryotic expression and preliminary characterization of the recombinant poly-epitope vaccine EgG1Y162-2 (4) against cystic echinococcosis. Methods The recombinant poly-epitope vaccine EgG1Y162-2 (4) against Echinococcus granulosus based on the linker GSGGSG was subjected to structural three-dimensional (3D) modeling using immunoinformatics to analyze the structural changes and evaluate the antigenicity of the vaccine. The pET30a-EgG1Y162-2 (4) recombinant plasmid was generated using double digestion with EcoR I and Sal I, and then transformed into competent cells. Following protein induction with isopropyl-β-D-thiogalactoside (IPTG), the prokaryotic expression proteins were characterized using Western blotting, and the antigenicity of the recombinant protein was analyzed using sera from cystic echinococcosis patients and health volunteers. Results The four EgG1Y162-2 proteins coupled by the 3D structure of the recombinant vaccine EgG1Y162-2 (4) presented independent and effective expression and good antigenicity. The highest protein expression was detected in the supernatant following induction of the recombinant plasmid pET30a-EgG1Y162-2 (4) by 0.2 mmol/L IPTG at 37 °C for 4 h, and a pure protein component was seen following elution with 60 mmol/L imidazole. Western blotting analysis of the recombinant multiepitope protein HIS-EgG1Y162-2 (4) showed a band at approximately 39 kDa, and this band was recognized by sera from cystic echinococcosis patients. Conclusion A recombinant poly-epitope vaccine EgG1Y162-2 (4) against cystic echinococcosis has been successfully constructed, which provides a preliminary basis for researches on recombinant multi-epitope vaccine against cystic echinococcosis.

Separation and structural elucidation of C25 epimers of furostanol saponin from aerial parts of Paris polyphylla var. chinensis / 中国中药杂志

Paris polyphylla var. chinensis(PPC) is used as one of the origin plants of Paridis Rhizoma described in the Chinese Pharmacopoeia(2020 edition). Its resources shortage makes the planting scale gradually expand, and plenty of aerial parts are abandoned because of not being effectively used. On the basis of previous research, this study separated steroidal saponins to further clarify the chemical composition of the aerial parts of PPC. As a result, three pairs of 25R or 25S epimers of furostanol saponins were obtained by various column chromatography techniques. Their structures were identified as neosolanigroside Y6(1), solanigroside Y6(2), neoprotogracillin(3), protogracillin(4), neoprotodioscin(5) and protodioscin(6) by spectral data combining with chemical transformation. Compound 1 is a new compound, and compounds 2, 3 and 5 are isolated from Paris plants for the first time. Compounds 4 and 6 are isolated from this plant for the first time. Previously, only several spirostanol glycosides with 25S configuration were isolated from Paris plants. Guided by mass spectrometry, the present study isolated the furostanol saponins with 25S configuration from this genus for the first time, which further enriches the chemical information of Paris genus and provides a reference for the isolation of similar compounds.

Qualitative and quantitative analysis of Paris polyphylla var. chinensis by UPLC-Q-TOF-MS/MS and HPLC / 中国中药杂志

Paridis Rhizoma(PR) is prepared from the dried rhizome of Paris polyphylla var. yunnanensis(PPY) or P. polyphylla var. chinensis(PPC) in Liliaceae family. The rapid development of PPY or PPC planting industry resulted from resource shortage has caused the waste of a large number of non-medicinal resources. To clarify the chemical compositions in rhizomes, fibrous roots, stems, leaves, seeds and pericarps of PPC, and explore the comprehensive application value and development prospect of these parts, the qualitative and quantitative analyses on the different parts of PPC were carried out by ultra-high performance liquid chromatography tandem quadrupole time-of-flight mass spectrometry(UPLC-Q-TOF-MS/MS) and high performance liquid chromatography(HPLC). A total of 136 compounds were identified, including 112 steroidal saponins, 6 flavonoids, 11 nitrogen-containing compounds and 7 phytosterols. Rhizomes, fibrous roots, and seeds mainly contained protopennogenyl glycosides and pennogenyl glycosides; leaves and stems mainly contained protodiosgenyl glycosides and diosgenyl glycosides; pericarps mainly contained pennogenyl glycosides, followed by diosgenyl glycosides. The total level of four saponins was the highest in fibrous roots and rhizomes, followed by those in the pericarps and arillate seeds, and the lowest in the stems and exarillate seeds. This study can provide data support for the comprehensive development and rational application of non-medicinal parts of PPC.

Construction of Rho GDIa-sgRNAs plasmids by clustered regularly interspaced short palindromic repeats/associated protein 9 and the effect on migration of Hepa 1-6 cell line in mouse / 解剖学报

Objective To construct the recombinant plasmids of knocking down Rho guanine dissociation inhibitor α (GDIα ) gene by using clustered regularly interspaced short palindromic repeats/associated protein 9 (CRISPR/Cas9) technique, and investigate the effect of Rho GDIα interference on the migration of Hepa 1-6 cells of mouse in order to provide the method of prevention and treatment of liver cancer. Methods To construct and identify the PX458-Rho GDIα-single guide (sg) RNAs by using CRISPR/Cas9 technique. And the Hepa 1-6 cells were transfected by liposomes with PX458-Rho GDIα-sgRNAs for 48 hours respectively, and cells treated with PX458 plasmids were used as control. The migration ability of Hepa 1-6 was checked by wound healing assay and Transwell assay, respectively. Results The expression of Rho GDIα was depressed in group of PX458-Rho GDIα-sgRNAl transfection which was detected by using RT-PCR. The migration distance of Hepa 1-6 in PX458-Rho GDIα-sgRNAl transfection group was significantly promoted comparing with the control group which was transfected with PX458 only, and the cell number of PX458-Rho GDIα-sgRNAl group was more than that in control group by using transwell assay, indicating concluded that knocking down of Rho GDIα promoted the migration ability of Hepal-6 cells. Conclusion The result is explicit that in vivo, Rho GDIα may inhibit the migration of Hepal-6 partially. Overexpression of Rho GDIα might be used as an important method to prevent the metastasize of carcinoma.

Construction of forkhead box J2 gene knockout plasmids by CRISPR / Cas9 and the effects on expression of transforming growth factor￣β/ Smads and proliferation in hepatocellular carcinoma / 解剖学报

Objective To construct the clustered regularly interspaced short palindromic repeats / associated protein 9 (CRISPR/ Cas9) plasmid targeting forkhead box J2 (FOXJ2) gene and investigate the effects of FOXJ2 interference on the expression of transforming growth factor-β(TGF-β) / Smads and proliferation in hepatocellular carcinoma cells of mouse. Methods Small guide RNA(sgRNA) sequence of FOXJ2 was designed, linked with PX458 vector and transfected into competent E. coli for proliferation. The recombinant plasmids were sent for sequencing to confirm the accuracy of the sgRNA sequence. The PX458-FOXJ2-sgRNAs plasmids were transfected into Hepa1-6 cells by liposome transfection, respectively. The empty vectors of PX458 were transfected as control group. After 48 hours, the expression of FOXJ2, TGF-β and Smads were obtained by RT-PCR and agarose gel electrophoresis, respectively. The cell proliferation was detected by methylthio tetrazole (MTT) method . Results The CRISPR/ Cas9 plasmids of PX458-FOXJ2-sgRNAs were successfully constructed. The recombinant plasmid of PX458-FOXJ2-sgRNA2 could effectively inhibit FOXJ2 gene expression which induced increasing expression of TGF-β, Smad2 and Smad4 in Hepa1-6 cells comparing to the control group transfected with PX458 only. And the proliferation of Hepa1-6 was promoted in PX458-FOXJ2-sgRNA2 interference group. Conclusion In hepatocellular carcinoma cells of mouse, FOXJ2 gene inhibits the expression of TGF-β, Smad2, Smad4 and cell proliferation partially, which indicates the relationship between FOXJ2 and TGF-β signal pathway. The result provides the target molecule of FOXJ2 for the prevention and treatment of hepatocellular carcinoma.

Effect of Ru′ai Shuhou Prescription on Proliferation and Metastasis of Breast Cancer MDA-MB-453 Cells Through SDF-1/CXCR4 Biological Axis / 中国实验方剂学杂志

Objective:To investigate the effect of Ru′ai Shuhou prescription （RSR） drug-containing serum on the proliferation and invasion ability of breast cancer cells MDA-MB-453 based on the biological axis of stromal cell-derived factor-1（SDF-1）/chemokine receptor 4 （CXCR4）. Method:A model of MDA-MB-453 cells with SDF-1-induced high expression of CXCR4 was established， and the rat drug-serum containing RSR and blank rat serum were prepared respectively. The cells were divided into fetal bovine serum control group （Blank）， blank rat serum group， SDF-1+blank rat serum group， SDF-1+RSR group， AMD3100+ SDF-1+blank rat serum group， and AMD3100+ SDF-1+RSR group. After intervention for 48 h， cell proliferation was detected by cell counting kit-8 （CCK-8） assay， cell invasion ability was detected by transwell assay， and mRNA and protein expressions of CXCR4， matrix metalloproteinase-2 （MMP-2） and MMP-9 were detected by Real-time fluorescence quantitative polymerase chain reaction （Real-time PCR） and Western blot， respectively. Result:As compared with the blank serum group， the proliferation of MDA-MB-453 cells was promoted and expression of CXCR4 mRNA was increased significantly when SDF-1 was 100 μg·L-1 （P<0.05）. As compared with SDF-1+blank rat serum group， RSR inhibited the proliferation and invasion of MDA-MB-453 cells induced by SDF-1， and at the same time， down-regulated the mRNA and protein expressions of CXCR4， MMP-2 and MMP-9 （P<0.05）. After pre-treatment with AMD3100 for 24 h， the inhibitory effect of RSR to cell proliferation was significantly increased （P<0.05）， and meanwhile， the decreases in mRNA and protein expression of CXCR4， MMP-2 and MMP-9 were more obvious， with statistically significant differences （P<0.05）. Conclusion:Through SDF-1/CXCR4 biological axis， RSR could down-regulate the expression of MMP-2 and MMP-9， reduce the degradation of extracellular matrix （ECM）， and then inhibit the metastasis of MDA-MB-453 cells. In addition， it has a synergistic effect with CXCR4 inhibitor AMD3100.

Improving Quality Standard of Processed Slices of Paridis Rhizoma of Chinese Pharmacopoeia / 中国实验方剂学杂志

Objective:To study the appearance description,TLC examination and content determination was carried out, in order to improve the quality standard of processed slices of Paridis Rhizoma in the 2020 edition of Chinese Pharmacopoeia. Method:Based on the literature review and observation on the samples,the appearance description was described. TLC examination was used for the qualitative analysis. HPLC was used for the determination of polyphyllin Ⅰ,Ⅱ,Ⅵ and Ⅶ in the commercial and processed samples. UPLC was employed for the determination of 10 steroidal saponins,namely pseudoprotodioscin,polyphyllin Ⅶ,17-hydroxygracillin,polyphyllin H,polyphyllin Ⅵ,polyphyllin Ⅱ,dioscin,gracilin,polyphyllin Ⅰ and polyphyllin Ⅴ. Result:For the appearance description,color and luster,texture,odor and taste as well as the diameter of 1.0-4.5 cm were recorded. polyphyllin Ⅵ was not detected in the thin layer chromatograms of most of the tested samples derived from high-quality species but obviously detected in those of Trillium Rhizoma. Five of 13 commercial samples met the requirements that the total amounts of polyphyllin Ⅶ,Ⅵ,Ⅱ,and Ⅰ should be no less than 0.6%according to the current Chinese Pharmacopoeia. Because softening and drying had the obvious influence on the contents of steroidal saponins in the samples,soaking and sun-drying were preferred. Conclusion:Appearance description should be supplemented. Polyphyllin Ⅵ is not considered as one of quality markers for the TLC identification and HPLC determination of Paridis Rhizoma. Polyphyllin H was considered as a new marker for the quality control. It is recommended that the total amounts of polyphyllin Ⅶ,H,Ⅱ,and Ⅰ should be no less than 1.0%.

Efficacy and safety of vitamin D in the treatment of idiopathic oligoasthenozoospermia / 中华男科学杂志

<p><b>OBJECTIVE</b>To explore the efficacy and safety of vitamin D (VD) in the treatment of idiopathic oligoasthenozoospermia.</p><p><b>METHODS</b>This study included 86 infertile men with idiopathic oligoasthenozoospermia, who were randomized to a VD and a control group of equal number, the former given oral VD 200 IU/d and calcium 600 mg/d,qd, while the latter administered oral vitamin E 100 mg and vitamin C 100 mg, tid. After 3 months of medication, we compared the semen parameters, adverse reactions, and pregnancy rate between the two groups.</p><p><b>RESULTS</b>After medication, the count of progressively motile sperm per ejaculate was increased from (9.82 ± 3.72) x 10(6) to (21.47 ± 6.52) x 10(6) ( P < 0.05) and the proportion of progressively motile sperm from (18.41 ± 9.82)% to (28.27 ± 4.47)% (P < 0.05) in the VD group. In comparison, the count of progressively motile sperm per ejaculate was elevated from (9.51 ± 6.31) x 10(6) to (12.36 ± 4.43) x 10(6) (P > 0.05) and the proportion of progressively motile sperm from (17.79 ± 5.25)% to (21.35 ± 2.41)% (P > 0.05) in the control group. Pregnancy was achieved in 7 cases (16.3%) in the VD group, but only lease (2.3%) in the control (P < 0.05). No adverse reactions were observed in either of the groups.</p><p><b>CONCLUSION</b>Vitamin D, as a safe option for the treatment of idiopathic oligoasthenozoospermia, can effectively improve the semen quality, especially the progressive sperm motility of the patient.</p>

Effect of Xianlong granules on immunological function in rats of adjuvant arthritis / 中国中药杂志

<p><b>OBJECTIVE</b>To study the effect of Xianlong granules (XLG) on immunological function in the rat of adjuvant arthritis (AA).</p><p><b>METHOD</b>Rats were randomly divided into normal group, AA model group, prednisone group and low, middle and high dose XLG groups, 10 rats in each group. All rats were treated by intragastric administration from the 18 days after arthritis was induced by the complete Freud's adjuvant and the effect of XLG on toes swelling was observed. On the 30th days after modeling, proliferation of the splenic and thymic lymphocytes, and IgG secreted by splenocytes were detected respectively by MTT assay and ELISA.</p><p><b>RESULT</b>Compared with the model group, both the high and middle dose XLG groups had significant therapeutic effects on toes dwelling in the rat of AA (P < 0.05 or P < 0.01); The low, middle and high dose XLG groups strengthened the PHAM-inhibited proliferation of splenic lymphocytes (P < 0.05), and inhibited the PHAM-augmented proliferation of thymic lymphocytes (P < 0.05); XLG did not significantly effect on IgG level secreted by splenocytes in rats of AA.</p><p><b>CONCLUSION</b>XLG can cure toes swelling in rats of AA, which is related with regulation of the abnormal immunlological function.</p>

Study on Measuring Cord Blood Leptin Level and the Ralatioship between Leptin and Insulin in Premature Infants / 实用儿科临床杂志

0.05).2.Neonates umbilical serum leptin concentration was positively correlated with body mass index(r=0.520 P