Network pharmacological analysis and experimental validation of Anemarrhenae Rhizoma in treating Alzheimer 's disease / 浙江大学学报·医学版

OBJECTIVES@#To explore the mechanism of action of Anemarrhenae Rhizoma in treatment of Alzheimer's Disease (AD) through network pharmacology analysis and experimental validation.@*METHODS@#The active ingredients and targets of Anemarrhenae Rhizoma for treatment of AD were screened with network pharmacological methods, the protein-protein interaction (PPI) network was constructed and the core targets were analyzed, enriching Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways analysis was performed. The peripheral blood lymphocytes were extracted and lymphoblast like cell lines (LCL) were constructed and an in vitro cell model of LCL-SKNMC was established. MTT/CCK-8 method was used to detect the activity of SKNMC/LCL cells, 2 ´, 7 ´-dichlorodihydrofluorescein diacetate (DCFH-DA) probe was used to detect reactive oxygen species (ROS) generated, and immunofluorescence staining was used to detect the generation of Aβ1-42 in the co-cultured model; Western blotting was used to detect protein expression in the co-culture model. The lifespan of N2 nematodes was observed under oxidative stress, normal state, and heat stress; ROS generated by N2 nematodes was detected by DCFH-DA probes. The paralysis time of CL4176 N2 nematodes was evaluated by paralysis assay, and Aβ deposition in the pharynx was detected by Thioflavin S (Th S) staining.@*RESULTS@#Through network pharmacology, 15 potential active ingredients and 103 drug-disease targets were screened out. PPI analysis showed that the Anemarrhenae Rhizoma might play anti-AD roles through albumin, Akt1, tumor necrosis factor, epidermal growth factor receptor (EGFR), vascular endothelial growth factor (VEGFA), mammalian target of rapamycin (mTOR), amyloid precursor protein (APP), glycogen synthase kinas (GSK) 3β and other related targets. KEGG analysis showed that the pharmacological effects of Anemarrhenae Rhizoma might involve the biological processes of Alzheimer's disease, endocrine resistance, insulin resistance; and neuroactive ligand-receptor interaction, phosphatidylinositol 3-kinase (PI3K)-Akt signaling pathway, calcium signaling pathway, AGE-RAGE signaling pathway in diabetes complications, neurotrophic factor signaling pathway and others. The in vitro cell experiments showed that Anemarrhenae Rhizoma was able to reduce the production of ROS and Aβ1-42 (all P<0.01), inhibit the expression of β-secretase 1 (BACE1), APP and Aβ1-42 proteins (all P<0.05), up-regulate the expression of p-PI3K/PI3K, p-AKT/AKT, p-GSK3β/GSK3β in SKNMC cells (all P<0.05). Studies further confirmed that Anemarrhenae Rhizoma prolonged the lifespan of C. elegans under stress and normal conditions, reduced the accumulation of ROS and the toxicity of Aβ deposition.@*CONCLUSIONS@#Anemarrhenae Rhizoma may reduce the production of Aβ in AD and inhibit its induced oxidative stress, which may be achieved by regulating the PI3K/AKT/GSK-3β pathway.

The stability of the chromosomes during culture of lymphoblastoid cell lines from patients with herpes zoster / 国际检验医学杂志

Objective To evaluate the stability of the chromosomes during culture of lymphoblastoid cell lines (LCLs)from pa-tients with herpes zoster.Methods Established continuous cultures of LCLs for 6 months and analyzed the cells using conventional karyotyping.Results There were four LCLs with normal karyotyping had no change of chromosome structure during culturing, while one LCL with abnormal karyotyping showed chromosome aberrations and one LCL died.Conclusion It is feasible to main-tain LCLs with a normal genomic structure during the LCL establishment processes.

Establishment & characterization of lymphoblastoid cell lines from patients with multiple primary neoplasms in the upper aero-digestive tract & healthy individuals.

Background & objectives: A major drawback for genetic studies as well as long-term genotype-phenotype correlation studies in cancer is lack of representative human cell lines providing a continuous source of basic biomolecules and a system to carry out various experimental investigations. This can be overcome to some extent by establishing lymphoblastoid cell lines (LCLs) by infecting peripheral blood lymphocytes with Epstein Barr virus (EBV) which is known to immortalize human resting B cells in vitro giving rise to actively proliferating B-lymphoblastoid cell lines. The present study involves preparation and characterization of LCLs generated from patients with multiple primary neoplasms (MPN) of upper aero-digestive tract (UADT). Methods: Thirty seven LCLs were established from UADT MPN patients and healthy age, sex and habit matched controls using EBV crude stock. Characterization was done with respect to expression of CD-19 (Pan B-cell marker), CD3 (T cell specific marker), CD56 (NK-cell specific marker), cell morphology, ploidy analysis, genotype and gene expression comparison with the parent lymphocytes. Results: LCLs showed rosette morphology with doubling time of approximately 24 h. Ploidy analysis showed diploid DNA content which was maintained for at least 30 population doublings. When compared with parent lymphocytes there appeared no change at genetic and gene expression level. Interpretation & conclusions: Our results show that lymphoblastoid cell lines are a good surrogate of isolated lymphocytes bearing their close resemblance at genetic and phenotypic level to parent lymphocytes and are a valuable resource for understanding genotype-phenotype interactions.

Establishment of Immortalized B Lymphoblast Cell Line from Patients with Hepatitis B Virus Infection / 医学研究杂志

Objective To establish immortalized B lympho-blastcell line(LCL) from patients with hepatitis B virus(HBV) infection in vitro.Methods Immortalized B-lymphoblastoid cell lines were established by EB virus transformation of the peripheral blood B lymphocytes and Cyclosporin A(Cys A) restraining T cells.HLA-A gene were measured in blood mononuclear cells by PCR-SSP.Results Altogether 16 immortalized lymphoblastoid cell lines were established successfully in vitro.HLA-A alleles were detected,including 1101,0201,0101,2403,et al.Conclusion The LCLs by EB virus transformation provides a resource of target cell for further research on the cellular immunity of patients with HBV infection.

ESTABLISHMENT AND CHARACTERISTICS OF Epstein-Barr VIRUS TRANSFORMED B CELL LINES FROM HEPATITIS B PATIENTS / 解放军医学杂志

To study the characteristics of EBV transformed human peripheral blood B cell lines from hepatitis B patients and to provide a basis for further studies on the cellular immune function of hepatitis B patients.High titer of EBV was produced by B95 8 cell by induction with sodium n butyrate,and peripheral mononuclear cells from two different groups of hepatitis B patients were harvested and infected with EBV. The results showed that 4～8 weeks after infection 19 EBV transformed B cell lines from hepatitis B patients were established. It was found that colony formation of cells from A group appeared 2～3 weeks later than B group. Cells grew healthily and had good activity after 4 weeks of freezing.CD19 and CD20 were detected in 88 34% and 37 48% of the immortalized B cell membrane respectively. HBV DNA could not be detected in a11 19 immortalized B cell lines.It suggested that the time needed for the establishment of B cell lines was related with the immune state of the patients.HBV DNA could not exist persistently, and it would disappear finally in the immortalized B cell lines.

The Use of Canine Bone-Marrow Stromal Cell Line, DO64, as Feeder Cells for The Efficient Cloning of Human B Lymphoblastoid Cell Lines ( B-LCLs ) / 대한면역학회지

Epstein-Barr virus (EBV) is a potent inducer of polyclonal B lymphocyte proliferation and a tool for the establishment of human B lymphoblastoid cell lines (B-LCLs), which have proven useful for several human immunologic applications. B-LCLs serve as efficient antibody-producing cells and antigen-presenting cells. In spite of these advantages, the cloning efficiency of B-LCLs is less than 1%. In order to generate clones of B-LCLs, we cultured B-LCLs with and without canine stromal cell line, DO64, as feeder cell which was immortalized by transduction of retrovirus encoding E6 and E7 of the human papilloma virus type 16 (HPV-16), which was defined to produce various cytokines including stem cell factor (SCF) and interleukin- 6 (IL-6). After 3 weeks of B-LCLs cultured with DO64, 8.3% and 37.5% in 1 cell and 3 cells per well were efficiently cloned, respectively. There was no significant effect on growing of 8-LCLs without DO64 cells and on high concentration of FBS. The cloning efficiency of B-LCLs transduced by retrovirus cultured with and without DO64 cells was 4.2% and 0% in 3 cells per well, respectively, while that of stable transfectant 33.3% and 8.3% in 1 cell per well, respectively. Our results suggest that the use of DO64 cells as feeder cells might permit the cloning of B-LCLs. This efficient cloning of B-LCLs could be used for the convenient source of autologous antigen-presenting cells expressing foreign antigen for the study of human immune responses in vitro, and for a variety of additional purposes, such as the production of human monoclonal antibodies.