Alzheimer's disease (AD) is the most common neurodegenerative disorder causing dementia worldwide, and is mainly characterized by aggregated β-amyloid (Aβ). Increasing evidence has shown that plant extracts have the potential to delay AD development. The plant sterol β-Sitosterol has a potential role in inhibiting the production of platelet Aβ, suggesting that it may be useful for AD prevention. In the present study, we aimed to investigate the effect and mechanism of β-Sitosterol on deficits in learning and memory in amyloid protein precursor/presenilin 1 (APP/PS1) double transgenic mice. APP/PS1 mice were treated with β-Sitosterol for four weeks, from the age of seven months. Brain Aβ metabolism was evaluated using ELISA and Western blotting. We found that β-Sitosterol treatment can improve spatial learning and recognition memory ability, and reduce plaque load in APP/PS1 mice. β-Sitosterol treatment helped reverse dendritic spine loss in APP/PS1 mice and reversed the decreased hippocampal neuron miniature excitatory postsynaptic current frequency. Our research helps to explain and support the neuroprotective effect of β-Sitosterol, which may offer a novel pharmaceutical agent for the treatment of AD. Taken together, these findings suggest that β-Sitosterol ameliorates memory and learning impairment in APP/PS1 mice and possibly decreases Aβ deposition.
Alzheimer Disease ; Amyloid ; Animals ; Blood Platelets ; Blotting, Western ; Brain ; Cognition Disorders ; Dementia ; Dendritic Spines ; Enzyme-Linked Immunosorbent Assay ; Excitatory Postsynaptic Potentials ; Learning ; Memory ; Metabolism ; Mice ; Mice, Transgenic ; Neurodegenerative Diseases ; Neurons ; Neuroprotective Agents ; Plant Extracts ; Plants ; Plaque, Amyloid ; Spatial Learning

PURPOSE: This study aimed to investigate the effect of adipose-derived stem cell (ADSC) transplantation on atherosclerosis (AS) and its underlying mechanisms. MATERIALS AND METHODS: In our study, rat AS model was established, and ADSCs were isolated and cultured. Atherosclerotic plaque and pathological symptoms of thoracic aorta were measured by Oil Red O staining and Hematoxylin-Eosin staining, respectively. Total cholesterol (TC), triglyceride (TG), high-density lipoprotein cholesterol (HDL-C), and low-density lipoprotein cholesterol (LDL-C) levels were measured by an automatic biochemical analyzer. Expressions of vascular endothelial growth factor (VEGF), vascular cell adhesion molecule-1 (VCAM-1), intercellular adhesion molecule-1 (ICAM-1), aortic endothelin-1 (ET-1), interleukin-6 (IL-6), c-reactive protein (CRP), and tumor necrosis factor α (TNF-α) were measured by enzyme linked immunosorbent assay, VEGF, VCAM-1, ICAM-1, ET-1, respectively, and NF-κB p65 mRNA expressions were detected by quantitative real-time polymerase chain reaction. Protein expressions of VEGF, VCAM-1, ICAM-1, ET-1, NF-κB p65, p-NF-κB p65, and IκBα were measured by western blot. Moreover, NF-κB p65 expression was measured by immunofluorescence staining. RESULTS: ADSC transplantation alleviated the pathological symptoms of aortic AS. ADSC transplantation decreased the levels of TC, TG, and LDL-C and increased serum HDL-C level. Meanwhile, ADSC transplantation decreased the levels of IL-6, CRP, and TNF-α in AS rats. Moreover, the expressions of VEGF, ET-1, VCAM-1, and ICAM-1 were decreased by ADSC transplantation. ADSC transplantation inhibited phosphorylation of NF-κB p65 and promoted IκBα expression in AS rats. CONCLUSION: Our study demonstrated that ADSC transplantation could inhibit vascular inflammatory responses and endothelial dysfunction by suppressing NF-κB pathway in AS rats.
Animals ; Aorta, Thoracic ; Atherosclerosis ; Blotting, Western ; C-Reactive Protein ; Cholesterol ; Endothelin-1 ; Enzyme-Linked Immunosorbent Assay ; Fluorescent Antibody Technique ; Intercellular Adhesion Molecule-1 ; Interleukin-6 ; Lipoproteins ; Phosphorylation ; Plaque, Atherosclerotic ; Rats ; Real-Time Polymerase Chain Reaction ; RNA, Messenger ; Stem Cell Transplantation ; Stem Cells ; Triglycerides ; Tumor Necrosis Factor-alpha ; Vascular Cell Adhesion Molecule-1 ; Vascular Endothelial Growth Factor A

OBJECTIVE: Conventional methods for organotypic hippocampal tissue slice culture (OHSC) have shown several disadvantages or limitations regarding age of animals used, duration of culture and difficulty using neurodegenerative models. Therefore, we tried to establish OHSC from old 3xTg-Alzheimer’s disease (AD) mice for longer period (over 4 weeks) and to validate utility of this system as a valid platform for translational neuroscience of AD. METHODS: OHSC was performed with old 3xTg-AD mice (12–14 months), old wild type mice (12–14 months) and young 3xTg-AD mice (2–4 months) using serum-free medium for 4 weeks. Hippocampal structure was evaluated by 4’, 6-diamidino-2-phenylindole (DAPI) intensity and neuronal metabolism was measured by Alamarblue assay. Pathologic characteristics of AD were also investigated; β-amyloid levels by ELISA, amyloid plaque deposition by Thioflavin-S staining, and glial activation by immunohistochemistry. RESULTS: Following 4-week culture in serum-free media, hippocampal cells and layers were well preserved in cultured slices from old AD mice as was in those from young AD and old wild type mice. On the contrary, excessive regression of total visible cells was observed in conventional serum-containing medium regardless of genotype of mice. In parallel with this well preserved structure, major pathologic characteristics of AD were also well manifested in hippocampal slices from old AD mice. CONCLUSION: Our findings suggest that long-term OHSC from old 3xTg-AD mouse can serve as a promising ex vivo system for studies on pathophysiology of AD, especially with the minimum number of sacrifice of experimental animals.
Alzheimer Disease ; Animals ; Culture Media, Serum-Free ; Enzyme-Linked Immunosorbent Assay ; Genotype ; Hippocampus ; Immunohistochemistry ; Metabolism ; Mice ; Neurons ; Neurosciences ; Plaque, Amyloid

OBJECTIVE: Cytokines produced by various cells are strong local mediators of inflammation. Interleukin-1beta (IL-1β) and C-reactive protein (CRP) play essential roles in the development and progression of diabetes mellitus (DM). Thus periodontal diseases could be related to DM via the same mediators of inflammation. To evaluate plasma and gingival crevicular fluid (GCF) levels of IL-1β and CRP in adolescents with DM to further investigate whether DM has an impact on the levels of inflammation factors at an early stage, and to analyze the risk of developing periodontal diseases in adolescents with DM. METHODS: A total of 121 adolescents aged from ten to sixteen years were enrolled, 41 adolescents diagnosed with diabetes mellitus were collected in the DM group, and 80 nondiabetic adolescents as the control group. The periodontal indices of each individual were recorded, including plaque index (PLI), modified bleeding index (mBI), probing depth (PD) and attachment loss (AL). GCF and intravenous blood samples were collected, and CRP and IL-1β levels were detected by enzyme-linked immunosorbent assay (ELISA). RESULTS: (1) PLI of DM group and control group were 1.23±0.05 and 0.95±0.04 separately, with significant difference (P=0.001). DM group and control group had mBI of 0.80±0.08 and 0.51±0.06 separately, with significant difference (P=0.003). Attachment loss was found in none of the subjects. PDs of DM group and control group were (2.37±0.51) mm and (2.31±0.05) mm separately, and there was no significant difference. (2) CRP in GCF was only detectable in partial of the individuals, with a detectable rate of 22.9% (11/48) in total. The detectable rate of CRP in GCF was significantly higher in DM group (38.5%) than that in control group (4.5%, P=0.006). The plasma level of CRP in DM group [0.23 (0.15, 1.89) mg/L] was higher than that in control group [0.19 (0.12, 4.18) mg/L], but without significance (P=0.776). (3) The plasma levels of IL-1β in DM group and control group were (14.11±0.57) ng/L and (14.71±0.50) ng/L separately, but there was no significance (P=0.456). GCF levels of IL-1β in DM group and control group were (12.91±1.95) μg/L and (17.68±3.07) μg/L, without significant difference (P=0.185). CONCLUSION Periodontitis was not observed in adolescents with DM at an early stage. However, the rising levels of periodontal indices and CRP in GCF, might indicate that adolescents with DM have a higher risk of developing periodontal diseases in the future.
Adolescent ; C-Reactive Protein/analysis* ; Dental Plaque Index ; Diabetes Mellitus, Type 2 ; Disease Progression ; Enzyme-Linked Immunosorbent Assay ; Female ; Gingival Crevicular Fluid/chemistry* ; Humans ; Interleukin-1beta/analysis* ; Male ; Periodontal Diseases ; Periodontal Index ; Periodontitis ; Plasma

Bacterial infection and smoking are an important risk factors involved in the development and progression of periodontitis. However, the signaling mechanism underlying the host immune response is not fully understood in periodontal lesions. In this study, we determined the expression of janus kinase (JAK)/signal transducer and activator of transcription (STAT) on Porphyromonas gingivalis lipopolysaccharide (LPS)- and nicotine-induced cytotoxicity and the production of inflammatory mediators, using osteoblasts. The cells were cultured with 5 mM nicotine in the presence of 1 µg/ml LPS. Cell viability was determined using MTT assay. The role of JAK on inflammatory mediator expression and production, and the regulatory mechanisms involved were assessed via enzyme-linked immunosorbent assay, reverse transcription-polymerase chain reaction, and Western blot analysis. LPS- and nicotine synergistically induced the production of cyclooxgenase-2 (COX-2) and prostaglandin E₂ (PGE₂) and increased the protein expression of JAK/STAT. Treatment with an JAK inhibitor blocked the production of COX-2 and PGE₂ as well as the expression of pro-inflammatory cytokines, such as tumor necrosis factor-α, interleukin-1β (IL-1β), and IL-6 in LPS- and nicotine-stimulated osteoblasts. These results suggest that JAK/STAT is closely related to the LPS- and nicotine-induced inflammatory effects and is likely to regulate the immune response in periodontal disease associated with dental plaque and smoking.
Bacterial Infections ; Blotting, Western ; Cell Survival ; Cytokines ; Dental Plaque ; Enzyme-Linked Immunosorbent Assay ; Inflammation* ; Interleukin-6 ; Necrosis ; Nicotine ; Osteoblasts* ; Periodontal Diseases ; Periodontitis ; Phosphotransferases ; Porphyromonas gingivalis* ; Porphyromonas* ; Risk Factors ; Smoke ; Smoking ; Transducers

OBJECTIVETo distinguish the difference among the Clinacanthus nutans (Burm. f.) Lindau (C. nutans) and Clinacanthus siamensis Bremek (C. siamensis) by assessing pharmacognosy characteristics, molecular aspect and also to evaluate their anti-herpes simplex virus (HSV) type 1 and type 2 activities.

METHODSMacroscopic and microscopic evaluation were performed according to WHO Geneva guideline. Stomatal number, stomatal index and palisade ratio of leaves were evaluated. Genomic DNA was extracted by modified CTAB method and ITS region was amplified using PCR and then sequenced. Dry leaves were subsequently extracted with n-hexane, dichloromethane and methanol and antiviral activity was performed using plaque reduction assay and the cytotoxicity of the extracts on Vero cells was determined by MTT assay.

RESULTSCross section of midrib and stem showed similar major components. Leaf measurement index of stomatal number, stomatal index and palisade ratio of C. nutans were 168.32±29.49, 13.83±0.86 and 6.84±0.66, respectively, while C. siamensis were 161.60±18.04, 11.93±0.81 and 3.37±0.31, respectively. The PCR amplification of ITS region generated the PCR product approximately 700 bp in size. There were 34 polymorphisms within the ITS region which consisted of 11 Indels and 23 nucleotide substitutions. The IC50 values of C. nutans extracted with n-hexane, dichloromethane and methanol against HSV-1 were (32.05±3.63) µg/mL, (44.50±2.66) µg/mL, (64.93±7.00) µg/mL, respectively where as those of C. siamensis were (60.00±11.61) µg/mL, (55.69±4.41) µg/mL, (37.39±5.85) µg/mL, respectively. Anti HSV-2 activity of n-hexane, dichloromethane and methanol C. nutans leaves extracts were (72.62±12.60) µg/mL, (65.19±21.45) µg/mL, (65.13±2.22) µg/mL, respectively where as those of C. siamensis were (46.52±4.08) µg/mL, (49.63±2.59) µg/mL, (72.64±6.52) µg/mL, respectively.

CONCLUSIONSThe combination of macroscopic, microscopic and biomolecular method are able to authenticate these closely related plants and both of them have a potency to be an anti-HSV agent.

Acanthaceae ; chemistry ; genetics ; Antiviral Agents ; chemistry ; pharmacology ; Flowers ; chemistry ; cytology ; genetics ; Herpesvirus 1, Human ; drug effects ; Herpesvirus 2, Human ; drug effects ; Humans ; Phenotype ; Plant Extracts ; chemistry ; pharmacology ; Plant Leaves ; chemistry ; cytology ; genetics ; Simplexvirus ; drug effects ; Viral Plaque Assay ; Virus Replication ; drug effects

BACKGROUNDVulnerable plaques play an important role in the onset of sudden cardiac events and strokes. How to stabilize vulnerable plaques is still a challenge to medical science. Alprostadil is a biologically active substance with strong activity on vessel. Our study assessed the stabilizing effects of an alprostadil liposome microsphere preparation (ALMP) on vulnerable plaques in the brachiocephalic artery of apolipoprotein E (Apo E) knockout mice.

METHODSSeventy-two male Apo E-knockout mice were fed a high-fat diet beginning at eight weeks of age. At week 17, they were divided randomly into groups for treatment with a high dose (3.6 µg×kg(-1)×d(-1)) or low dose (1.8 µg×kg(-1)×d(-1)) of an ALMP, or 0.2 ml/d normal saline (control group). The drug was administered using a micro-capsule pump. Twenty weeks after drug administration, pathological changes in the vulnerable plaques within the brachiocephalic artery were assessed, and levels of anti-mouse monocyte/macrophage monoclonal antibody (MOMA-2) and superoxide anions in the plaques were detected using immunofluorescence. The soluble intercellular adhesion molecule-1 (ICAM-1) expression was measured by ELISA, and the expression of matrix metalloproteinase-9 (MMP-9) and CD40 mRNA was measured using RT-PCR. Thrombospindin-1 (TSP-1) expression was detected using Western blotting.

RESULTSCompared with the control group, ALMP treatment significantly reduced the plaque area in the brachiocephalic artery (P < 0.01), significantly lowered the contents of the lipid core (P < 0.01), significantly reduced the number of ruptured fibrous caps (P < 0.05), and increased the thickness of the fibrous cap and significantly reduced the incidence of intra-plaque hemorrhage (P < 0.05). ALMP treatment significantly reduced the expression of MOMA-2, superoxide anion, MMP-9, ICAM-1 and CD40 in the plaques (P < 0.01), decreased plasma ICAM-1 expression (P < 0.01), and increased the expression of TSP-1.

CONCLUSIONSTreatment with ALMP can stabilize vulnerable plaques by inhibiting inflammation.

Alprostadil ; chemistry ; therapeutic use ; Animals ; Enzyme-Linked Immunosorbent Assay ; Inflammation ; drug therapy ; metabolism ; pathology ; Intercellular Adhesion Molecule-1 ; metabolism ; Liposomes ; chemistry ; Male ; Mice ; Mice, Knockout ; Microscopy, Fluorescence ; Microspheres ; Plaque, Atherosclerotic ; drug therapy ; metabolism ; pathology ; Polymerase Chain Reaction

To establish the mouse-lethal model for pandemic H1N1 influenza virus, provide an animal model for studying the pathogenicity and host adaptation of 2009 pandemic H1N1 influenza virus, and find out the key amino acid mutations which may affect viral virulence and replication. A pandemic H1N1 influenza virus strain, A/Sichuan/SWL1/2009 (H1N1, SC/1) was passaged in mouse lung by 15 cycles with intranasal infection. The passaged viruses were all propagated in MDCK cells and sequenced. Based on the sequencing results, four mice in each group were inoculated with 6 selected viruses and their weight and survival rate were monitored during the following 14 days after infection. Additionally, SC/1-MA P14 and P15 viruses were sequenced after purification by Plague Assay. Viral virulence was increased after serial passages and the mortality of 100% was detected after 7 passages. Several amino acid residue mutations of passaged viruses which may contribute to the enhanced virulence were observed. The increased virulence of passaged viruses and mammalian host adaptation maybe associated with amino acid mutations in viral functional proteins. Finally, we established a mouse-lethal model.
Amino Acid Substitution ; Animals ; Base Sequence ; Cell Line ; China ; epidemiology ; Disease Models, Animal ; Dogs ; Female ; Humans ; Influenza A Virus, H1N1 Subtype ; genetics ; growth & development ; pathogenicity ; physiology ; Influenza, Human ; epidemiology ; virology ; Mice ; Mice, Inbred BALB C ; Pandemics ; Survival Analysis ; Viral Plaque Assay ; Virulence ; Virus Replication

OBJECTIVETo compare different methods commonly used for titering adenovirus and analyze the advantages and limitations of each method.

METHODSFour recombined adenoviruses (Ad-G-AT2R-EGFP, Ad-CMV-EGFP, Ad-mif-shRNA-EGFP and Ad-CBA-GFP) were amplified and purified, and each was titered by optical absorbance, real-time PCR, green fluorescent protein (GFP)-labeled method, immunoassay, and cytopathic effect (CPE). The results were then comparatively analyzed.

RESULTSNo significant difference was found in the titer amounts derived from GFP-labeled method, immunoassay, and cytopathic effect method (P>0.1). A positive correlation was noted in the titer amounts determined by real-time PCR and immunoassay (r=0.965), even though the value (vg/ml) obtained by real-time PCR was 10 times higher than that by immunoassay (ifu/ml).

CONCLUSIONGFP-labeled method and immunoassay allow rapid determination of the adenoviral titer. Real-time PCR can not directly determine the real infectious titer of the adenovirus, but the result is well correlated to that of immunoassay and reflects, though indirectly, the actual infectious titer of adenovirus. Considering the procedural convenience and shorter time consumption, real-time PCR is still a practical method for adenoviral titration.

Adenoviridae ; isolation & purification ; metabolism ; physiology ; Capsid Proteins ; metabolism ; DNA, Viral ; isolation & purification ; Green Fluorescent Proteins ; metabolism ; Real-Time Polymerase Chain Reaction ; methods ; Viral Plaque Assay ; methods ; Virus Replication

The plaque-forming characteristics of Newcastle disease viruses of chickens and geese source were compared on various cells. The result showed that there were obvious differences of plaque formation between F48E9 and NA-1 on Vero cells, chicken embryo fibroblast cells (CEF) and goose embryo fibroblast cells (GEF). The plaque-forming ability of NA-1 was higher than F48E9 on GEF, but lower than F48E9 on CEF. On Vero cells, the plaque-forming ability of NA-1 was slightly stronger than F48E9. It demonstrated that the plaque-forming characteristics were consistent with host tropism of virus. The morphogenesis of F48E9 and NA-1 on Vero cells was observed with transmission electron microscope. There were different replication processes between F48E9 and NA-1 on cells at different stages. NA-1 had stronger adaptability to host than F48E9 according to budding processes and envelope integrity.
Animals ; Cercopithecus aethiops ; Chick Embryo ; Chickens ; Geese ; Host-Pathogen Interactions ; Newcastle Disease ; virology ; Newcastle disease virus ; growth & development ; isolation & purification ; physiology ; ultrastructure ; Poultry Diseases ; virology ; Vero Cells ; Viral Plaque Assay