Physalin B (PB), one of the major active steroidal constituents of Solanaceae Physalis plants, has a wide variety of biological activities. We found that PB significantly down-regulated β-amyloid (Aβ) secretion in N2a/APPsw cells. However, the underlying mechanisms are not well understood. In the current study, we investigated the changes in key enzymes involved in β-amyloid precursor protein (APP) metabolism and other APP metabolites by treating N2a/APPsw cells with PB at different concentrations. The results indicated that PB reduced Aβ secretion, which was caused by down-regulation of β-secretase (BACE1) expression, as indicated at both the protein and mRNA levels. Further research revealed that PB regulated BACE1 expression by inducing the activation of forkhead box O1 (FoxO1) and inhibiting the phosphorylation of signal transducer and activator of transcription 3 (STAT3). In addition, the effect of PB on BACE1 expression and Aβ secretion was reversed by treatment with FoxO1 siRNA and STAT3 antagonist S3I-201. In conclusion, these data demonstrated that PB can effectively down-regulate the expression of BACE1 to reduce Aβsecretion by activating the expression of FoxO1 and inhibiting the phosphorylation of STAT3.
Alzheimer Disease ; Amyloid Precursor Protein Secretases/metabolism* ; Amyloid beta-Peptides/metabolism* ; Aspartic Acid Endopeptidases/metabolism* ; Down-Regulation ; Forkhead Box Protein O1/genetics* ; Humans ; Phosphorylation ; STAT3 Transcription Factor/metabolism* ; Secosteroids

BACKGROUNDAmyloid β (Aβ) has been established as a key factor for the pathological changes in the brains of patients with Alzheimer's disease (AD), and cellular senescence is closely associated with aging and cognitive impairment. However, it remains blurred whether, in the AD brains, Aβ accelerates the neuronal senescence and whether this senescence, in turn, impairs the cognitive function. This study aimed to explore the expression of senescence-associated genes in the hippocampal tissue from young to aged 5XFAD mice and their age-matched wild type (WT) mice to determine whether senescent neurons are present in the transgenic AD mouse model.

METHODSThe 5XFAD mice and age-matched wild type mice, both raised from 1 to 18 months, were enrolled in the study. The senescence-associated genes in the hippocampus were analyzed and differentially expressed genes (DEGs) were screened by quantitative real-time polymerase chain reaction. Cognitive performance of the mice was evaluated by Y-maze and Morris water maze tests. Oligomeric Aβ (oAβ) (1-42) was applied to culture primary neurons to simulate the in vivo manifestation. Aging-related proteins were detected by Western blotting analysis and immunofluorescence.

RESULTSIn 5XFAD mice, of all the DEGs, the senescence-associated marker p16 was most significantly increased, even at the early age. It was mainly localized in neurons, with a marginal expression in astrocytes (labeled as glutamine synthetase), nil expression in activated microglia (labeled as Iba1), and negatively correlated with the spatial cognitive impairments of 5XFAD mice. oAβ (1-42) induced the production of senescence-related protein p16, but not p53 in vitro, which was in line with the in vivo manifestation.

CONCLUSIONSoAβ-accelerated neuronal senescence may be associated with the cognitive impairment in 5XFAD mice. Senescence-associated marker p16 can serve as an indicator to estimate the cognitive prognosis for AD population.

Alzheimer Disease ; metabolism ; physiopathology ; Amyloid Precursor Protein Secretases ; genetics ; metabolism ; Amyloid beta-Peptides ; metabolism ; Amyloid beta-Protein Precursor ; metabolism ; Animals ; Aspartic Acid Endopeptidases ; genetics ; metabolism ; Brain ; metabolism ; physiopathology ; Cells, Cultured ; Cellular Senescence ; genetics ; physiology ; Cognition ; physiology ; Cognition Disorders ; metabolism ; physiopathology ; Disease Models, Animal ; Male ; Mice ; Mice, Inbred C57BL ; Mice, Transgenic ; Neurons ; metabolism ; pathology ; Real-Time Polymerase Chain Reaction

OBJECTIVETo study the diagnostic value of HNF-1β and Napsin A for ovarian clear cell carcinomas, serous carcinomas, endometrioid adenocarcinomas and metastatic Krukenberg tumors.

METHODSImmunohistochemical EnVision method was used to detect the expression of HNF-1β and Napsin A in 38 cases of ovarian clear cell carcinoma, 30 cases of high-grade serous carcinoma, 22 cases of endometrioid adenocarcinoma and 16 cases of metastatic Krukenberg tumor. Expression of HNF-1β and Napsin A were compared, and sensitivity and specificity of clear cell carcinoma of the ovary were analysed.

RESULTSThe positive rate of HNF-1β in the ovarian clear cell carcinoma was 100%(38/38), higher than those in high-grade serous carcinoma and endometrioid adenocarcinoma (P<0.05), although significant difference was not observed from that of metastatic Krukenberg tumor (P>0.05). Napsin A expressed in 97.4% (37/38) of ovarian clear cell carcinoma, 6.7% (2/30) of high-grade serous carcinoma, 22.7% (5/22) of endometrioid adenocarcinoma. Napsin A expression in clear cell carcinoma was higher than those in high-grade serous carcinoma and endometrioid adenocarcinoma (P<0.01), and no expression of Napsin A was seen in metastatic Krukenberg tumor (P>0.05). The sensitivity and specificity of HNF-1β in the diagnosis of ovarian clear cell carcinoma were 100% and 52.9%, those of Napsin A were 97.4% and 91.2%, those of both HNF-1β and Napsin A were 97.4% and 91.2%, respectively. The sensitivity and specificity of HNF-1β or Napsin A in the diagnosis of ovarian clear cell carcinoma were 100% and 52.9%, respectively.

CONCLUSIONSHNF-1β is a more sensitive marker for the diagnosis of ovarian clear cell carcinoma, whereas Napsin A is a more specific marker. The combined detection of HNF-1β and Napsin A may be helpful for the diagnosis of clear cell carcinoma of the ovary.

Adenocarcinoma, Clear Cell ; diagnosis ; Aspartic Acid Endopeptidases ; genetics ; Biomarkers, Tumor ; genetics ; Carcinoma, Endometrioid ; diagnosis ; Cystadenocarcinoma, Serous ; diagnosis ; Female ; Hepatocyte Nuclear Factor 1-alpha ; genetics ; Humans ; Immunohistochemistry ; Krukenberg Tumor ; diagnosis ; Ovarian Neoplasms ; diagnosis ; Sensitivity and Specificity

OBJECTIVETo explore the effect of Panax notoginseng saponin (PNS) on the activity and content of beta-secretase in the brain of senescence accelerated mouse-prone 8 (SAMP8) mice with Alzheimer's disease.

METHODSTotally 32 SAMP8 mice were randomly divided into the normal control group, the high dose PNS group (200 mg/kg), the low dose group (100 mg/kg), and the huperzine A group (0.3 mg/kg), 8 in each group. Equal volume of double distilled water was given to those in the normal control group. All medication was given by gastrogavage, once daily for two successive months. The activity of BACE1 was assayed by direct immunofluorescent method (DIF). The content of BACE1 protein was detected by Western blot.

RESULTSThe relative fluorescence units (RFU/microg) was 2.008 +/- 0.031 in the high dose PNS group, 2.221 +/- 0.029 in the low dose PNS group, and 2.267 +/- 0.076 in the huperzine A group, all lower than that in the normal control group (2.403 +/- 0.058; all P < 0.01). The content of BACE1 protein was 0.900 +/- 0.028 in the high dose PNS group, 1.000 +/- 0.032 in the low dose PNS group, and 0.837 +/- 0.080 in the huperzine A group, all lower than that in the normal control group (2.210 +/- 0.074, all P < 0.01).

CONCLUSIONPNS higher than 100 mg/kg could decrease the activity of BACE1 and down-regulate the content of BACE1 protein in the brain of SAMP8 mice.

Aging ; Alzheimer Disease ; metabolism ; Amyloid Precursor Protein Secretases ; metabolism ; Animals ; Aspartic Acid Endopeptidases ; metabolism ; Brain ; metabolism ; Disease Models, Animal ; Male ; Mice ; Panax notoginseng ; RNA, Messenger ; genetics ; Saponins ; pharmacology

OBJECTIVETo study the effects of Huannao Yicong Recipe (HNYCR)extract on the learning and memory ability, and the expressions of amyloid precursor protein (APP), beta-site APP-cleaving enzyme 1 (BACE1), presenilin-1 (PS-1), and beta amyloid protein (Abeta)in hippocampus CA1 area of APP transgenic mice, and to explore its mechanisms for treating Alzheimer's disease (AD).

METHODSTotally 3-month-old APP695V7171 transgenic mice were used to establish the AD model in this research. They were randomly divided into the model group, the Donepezil group, the large dose HNYCR extract group, the small dose HNYCR extract group, and the normal control group (C57BL/6J mice), 15 in each group. These animals were gavaged for 4 continuous months. Relevant indicators were detected: Morris water maze test was used to measure the spatial learning and memory ability. The immunohistochemical assay was used to detect the expressions of APP, BACE1, PS-1, and Abeta.

RESULTSThe times of crossing the original platform and the swimming time and distance in the fourth quadrant of the 7-month-old APP transgenic mice were significantly reduced in Morris water maze test, when compared with the normal control group (P < 0.01). The times of crossing original platform and the swimming time and distance in the fourth quadrant of all treatment groups significantly increased in Morris water maze test, when compared with the model group (P < 0.05). The expressions of APP, BACE1, PS-1, and Abeta in hippocampus CA1 area of 7-month-old model mice increased significantly (P < 0.01), when compared with the normal control group. The expressions of APP, BACE1, PS-1, and Abeta in each 7-month-old intervention groups were significantly reduced, when compared with the model group (P < 0.01).

CONCLUSIONEarly application of HNYCR extract can obviously improve the learning and memory ability of APP transgenic mice that has declined, reduce the expressions of APP, BACE1, PS-1, and Abeta in the hippocampal CA1 area, reduce the production of Abeta, and slow down the pathological process of brains in APP transgenic mice.

Alzheimer Disease ; metabolism ; Amyloid Precursor Protein Secretases ; genetics ; metabolism ; Amyloid beta-Peptides ; genetics ; metabolism ; Amyloid beta-Protein Precursor ; genetics ; metabolism ; Animals ; Aspartic Acid Endopeptidases ; genetics ; metabolism ; Brain ; drug effects ; metabolism ; CA1 Region, Hippocampal ; drug effects ; metabolism ; Disease Models, Animal ; Drugs, Chinese Herbal ; pharmacology ; Female ; Male ; Maze Learning ; drug effects ; Memory ; drug effects ; Mice ; Mice, Inbred C57BL ; Mice, Transgenic ; Presenilin-1 ; genetics ; metabolism

BACKGROUNDEpithelial-mesenchymal transition is a cellular process characterized by the loss of cell adhesion, inhibition of E-cadherin expression, and increased cell mobility. Cells without Napsin A are susceptible to transition. Further studies are required to investigate whether this transition can be reversed by restoration of Napsin A.

METHODSA Napsin A expression vector PLJM1-Napsin A plasmid was constructed and then transfected into the epithelial cell line A549 by lentivirus transfection to obtain A549-PLJM1-Napsin A cell line. Cell proliferation was assayed by 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyltetrazolium bromide and cell cycle was measured by flow cytometry. The E-cadherin, type I collagen, and focal adhesion kinase mRNA level was detected by reverse transcription-polymerase chain reaction. The Napsin A, E-cadherin, type I collagen, and focal adhesion kinase protein level in A549 cells was detected by Western blotting.

RESULTSTransforming growth factor-b1 induced epithelial-mesenchymal transition in A549 cells, as demonstrated by significant reduction of E-cadherin mRNA and protein levels (P < 0.01) as well as up-regulation of type I collagen (P < 0.01). Transfection of Napsin A in A549 cells can partially block the transforming growth factor-b1-regulated expression of E-cadherin and type I collagen (P < 0.01). In addition, transforming growth factor-b1-induced cell proliferation was inhibited by Napsin A (P < 0.01). Further study demonstrated that Napsin A caused G(0)/G(1) arrest and inhibited the expression of focal adhesion kinase (P < 0.01), a key protein in the integrin signaling pathway, in the in vitro epithelial-mesenchymal transition model.

CONCLUSIONSSustained Napsin A expression in A549 cells can inhibit the transforming growth factor-b1-induced epithelial-mesenchymal transition. This may be due to the Napsin A-mediated inhibition of focal adhesion kinase expression and integrin signaling pathway.

Aspartic Acid Endopeptidases ; genetics ; metabolism ; Cadherins ; genetics ; metabolism ; Cell Line ; Collagen Type I ; genetics ; metabolism ; Epithelial-Mesenchymal Transition ; drug effects ; genetics ; Focal Adhesion Protein-Tyrosine Kinases ; genetics ; metabolism ; Humans ; Transfection ; Transforming Growth Factor beta1 ; pharmacology

OBJECTIVETo investigate the influence of inhibited α7 neuronal nicotinic acetylcholine receptor (nAChR) by small interference RNA (siRNA) in SH-SY5Y cells and to explore the connection of these changes with the β-amyloid precursor protein (APP) metabolism and the pathogenesis of Alzheimer's disease (AD).

METHODSThe siRNA of α7 nAChR was transfected into SH-SY5Y cells, and the expression of α7 nAChR and two subtypes of β-secretases (BACE1 and BACE2) at mRNA and protein levels was studied by real-time PCR and Western blot, respectively. The variation of Aβ(1-42) content was detected by ELISA.

RESULTSAs compared with controls, the expression of α7 nAChR at mRNA and protein levels in the SH-SY5Y cells transfected with the α7 nAChR siRNA were decreased by 84% and 79% (P < 0.01), respectively. The expressions of BACE1 mRNA and protein levels was increased by 527% and 71% (P < 0.01), respectively, while the expression of BACE2 decreased by 58% and 75% (P < 0.01), respectively. The Aβ(1-42) content increased by 208% (P < 0.01).

CONCLUSIONSAn inhibited α7 nAChR mRNA induced by siRNA may markedly stimulate the production of Aβ through the mechanism of increased expression of BACE1 and inhibited expression of BACE2, which may be related to the pathogenesis of AD.

Amyloid Precursor Protein Secretases ; genetics ; metabolism ; Amyloid beta-Peptides ; metabolism ; Aspartic Acid Endopeptidases ; genetics ; metabolism ; Cell Line, Tumor ; Humans ; Neuroblastoma ; metabolism ; pathology ; Peptide Fragments ; metabolism ; RNA Interference ; RNA, Messenger ; metabolism ; RNA, Small Interfering ; genetics ; Receptors, Nicotinic ; genetics ; metabolism ; Transfection ; alpha7 Nicotinic Acetylcholine Receptor

The purpose of this study was to investigate the therapeutic effects of small hairpin RNA (shRNA) targeting endothelin-converting enzyme (ECE)-1 in monocrotaline (MCT)-induced pulmonary hypertensive rats. Ninty-four Sprague-Dawley rats were divided into three groups: control (n = 24), MCT (n = 35) and shRNA (n = 35). Four-week survival rate in the shRNA group was significantly increased compared to that in the MCT group. The shRNA group showed a significant improvement of right ventricular (RV) pressure compared with the MCT group. The MCT and shRNA groups also showed an increase in RV/(left ventricle + septum) ratio and lung/body weight. Plasma endothelin (ET)-1 concentrations in the shRNA group were lower than those in the MCT group. Medial wall thickness of pulmonary arterioles were increased after MCT injection and was significantly decreased in the shRNA group. The number of intra-acinar muscular pulmonary arteries was decreased in the shRNA group. The mRNA expressions of ET-1 and ET receptor A (ETA) were significantly decreased in the shRNA group in week 4. The protein levels of ETA were decreased in the shRNA group in week 2. The protein levels of tumor necrosis factor-alpha and vascular endothelial growth factor were decreased in the shRNA group in week 4. In conclusion, the gene silencing with lentiviral vector targeting ECE-1 could be effective against hemodynamic, histopathological and gene expression changes in pulmonary hypertension.
Animals ; Aspartic Acid Endopeptidases/*antagonists & inhibitors/blood/genetics ; Body Weight ; Heart Ventricles/physiopathology ; Hypertension, Pulmonary/chemically induced/*enzymology/mortality ; Lentivirus/genetics ; Lung/anatomy & histology/metabolism/pathology ; Male ; Metalloendopeptidases/*antagonists & inhibitors/blood/genetics ; Monocrotaline/toxicity ; Pulmonary Artery/drug effects/physiopathology ; RNA, Small Interfering/*metabolism ; Rats ; Rats, Sprague-Dawley ; Receptor, Endothelin A/genetics/metabolism ; Survival Rate ; Tumor Necrosis Factor-alpha/metabolism ; Vascular Endothelial Growth Factor A/metabolism

To generate active recombinant human beta-secreatase (BACE1) for studying its interaction with its inhibitors, we constructed two recombinant plasmids, pPIC9K-MetBACE22 (bearing pro-bace1 gene) and pPIC9K-MetBACE46 (bearing bace1 gene). These two plasmids were then transformed into Pichia pastoris GS115 by electroporation to obtain the recombinant strains 9k-B22 and 9k-B46. After induction in buffered methanol complex medium, we found the supernatant activity of 9k-B22 significantly higher than that of 9k-B46. The culture filtrate of 9k-B22 was concentrated, and then purified by HisTrap affinity column. The purified proteins, showing good BACE1 protease activity, were found to be a mixture of glycoproteins because they can be stained by periodic acid-Schiff reagent. After this mixture was treated with Endo H(f) (a recombinant protein of endoglycosidase H), we found two new adjacent bands around 50 kDa on SDS-PAGE. These two bands were cut and subjected to peptide mass fingerprint analysis, and identified as proBACE1 and BACE1 proteins. Enzyme assays revealed that the activities of both BACE1 proteins in glycosylated and deglycosylated form were lower than that of commercial BACE1 (expressed in HEK-293), inferring glycosylation and the type of glycosylation are crucial to the activity. However, we found no apparent difference in the inhibition of those all above three enzyme forms by one known BACE1 inhibitor. This observation demonstrated that the glycosylation of BACE1 by Pichia pastoris does not affect its interaction with this inhibitor. After optimization of culture conditions, the production of BACE1 in Pichia pastoris was enhanced to about 1 mg/L. This work enables us to further investigate the interaction of BACE1 and its inhibitors, and assists in discovering and optimizing BACE1 inhibitors as anti-Alzheimer's disease agents.
Amyloid Precursor Protein Secretases ; biosynthesis ; genetics ; Aspartic Acid Endopeptidases ; biosynthesis ; genetics ; Electroporation ; Humans ; Pichia ; genetics ; metabolism ; Plasmids ; genetics ; Recombinant Proteins ; biosynthesis ; genetics ; isolation & purification

Bla g 2 is a cockroach allergen of great importance. This study was conducted to identify IgE-binding epitope(s) of Bla g 2 using the recombinant protein technique. Approximately 50% of tested sera showed IgE reactivity to Pichiaexpressed Bla g 2 (PrBla g 2) and E. coli-expressed Bla g 2 (ErBla g 2). Only 5.3% of serum samples showed stronger reactivity to PrBla g 2 than ErBla g 2, indicating that serum was reactive to conformational or carbohydrate epitopes. The full-length and 5 peptide fragments of Bla g 2 were produced in E. coli. All fragments showed IgE-binding activity to the cockroach-allergy patients' sera. Specifically, peptide fragments of amino acid residue 1-75 and 146-225 appeared to be important for IgE-binding. The information about the IgE-binding epitope of Bla g 2 can aid in the diagnosis and treatment for cockroach allergies.
Adolescent ; Adult ; Aged ; Amino Acid Sequence ; Animals ; Antibodies ; Aspartic Endopeptidases/chemistry/genetics/*immunology ; Child ; Cockroaches/immunology ; Epitopes ; Gene Expression Regulation ; Humans ; Immunoglobulin E/*immunology ; Middle Aged ; Young Adult