Effect of Toll-like receptor 4 deficiency on clinical severity and expression of Th1/Th2/Th17-associated cytokines in a murine model of experimental autoimmune neuritis.

Introduction: The aim was to observe the effect of Toll-like receptor 4 (TLR4) deficiency on clinical severity and expression of Th1/Th2/Th17-associated cytokines in experimental autoimmune neuritis (EAN). Material and methods: We selected C57BL/10 wild type (WT) mice and TLR4 knockout (KO) mice with the C57BL/10 background for induction of the EAN model by immunizing mice twice (days 0 and 8) via subcutaneous injection of 180 µg P0 peptide 180-199 emulsion in 25 µl of PBS and 0.5 mg Mycobacterium tuberculosis (Difco, USA) in 25 µl of Freund's incomplete adjuvant into the back of mice. The concentrations of serum cytokines (IL-2, IL-4, IL-6, IL-10, IL-17A, IFN-γ and TNF) were determined using the Ms Th1/Th2/Th17 CBA kit. Results: We found that TLR4 deficiency could attenuate the clinical severity and delay the onset of EAN. Moreover, our data showed that the sera levels of IFN-γ, TNF, IL-6 and IL-17A were elevated in the WT mice with EAN when compared with the naive WT mice, but only the production of IL-17A was significantly lower in the TLR4 KO mice with EAN than in their WT counterparts. Conclusions: Based on these findings, TLR4 may contribute to the pathogenesis of EAN by regulating Th17 cells and the production of Th17-associated factors. However, the exact mechanism remains unclear and more evidence is needed to elucidate its role in EAN.

Levetiracetam alleviates cognitive decline in Alzheimer's disease animal model by ameliorating the dysfunction of the neuronal network.

Background: Patients with Alzheimer's disease (AD) have a significantly higher risk of seizures than other individuals in an age-matched population, suggesting a close association between epilepsy and AD. We aimed to examine the effects of levetiracetam (LEV)-a drug for treating seizures-on learning and memory and the neuropathological features of AD. Methods: We crossbred APP23 mice with microtubule-associated protein tau (MAPT) transgenic mice to generate APP23/MAPT mice. These mice were treated with different concentrations of LEV in the presence of kainic acid (KA) for 3 months. Results: Low doses of LEV alleviated the effects of KA on memory defects in APP23/MAPT mice. Mechanistic investigations showed that low concentrations of LEV decreased tau phosphorylation by reducing the activities of cyclin-dependent kinase 5 and glycogen synthase kinase 3α/ß, thus rescuing neurons from synaptic dystrophy and apoptosis. Low doses of LEV inhibited the effects of KA (i.e., inducing neuroinflammation and impairing the autophagy of amyloid ß-peptide), thus improving cognitive decline. High concentrations of LEV decreased the production and deposition of amyloid ß-peptide (Aß) by reducing the expression of ß-site APP-cleaving enzyme 1 and presenilin 1. However, high concentrations of LEV also induced neuronal apoptosis, decreased movement ability in mice, and did not alleviate cognitive decline in AD mice. Conclusion: Our results support the hypothesis that aberrant network activity contributes to the synaptic and cognitive deficits in APP23/MAPT mice. A low concentration of LEV may help ameliorate abnormalities of AD; however, a high LEV concentration did not induce similar results.

Sesquiterpene lactones from Artemisia vulgaris L. as potential NO inhibitors in LPS-induced RAW264.7 macrophage cells.

Twelve new guaianolide sesquiterpene lactones (1-12), along with ten known analogs (13-22) were isolated from an EtOH extract of the dried aerial parts of Artemisia vulgaris L. The new structures were elucidated via abundant spectroscopic data analyses (HRESIMS, IR, 1D, and 2D NMR), and the absolute configurations of these compounds were determined by X-ray crystallography and ECD calculations. The compounds (1-22) were identified as guaiane-type sesquiterpenes with characteristic α-methylene-γ-lactone and α,ß-unsaturated carbonyl moieties. All compounds were tested for their inhibitory activity against NO production in lipopolysaccharide-stimulated RAW264.7 macrophages. The isolated sesquiterpenoids dose-dependently exhibited an NO production inhibitory activity by inhibiting the expression of inducible NO oxidase (iNOS) and cyclooxygenase-2 (COX-2) with IC50 values ranging from 1.0 to 3.6 µM. The inhibitory effect on the NO production of the compounds (1-4 and 6-22) is better than that of the positive control (dexamethasone). The different substitutions of compounds on C-8 influence anti-inflammatory effects, as evidenced by the in silico analysis of related binding interactions of new compounds (1-12) with iNOS.

Association of rs610604 in TNFAIP3 and rs17728338 in TNIP1 gene polymorphisms with psoriasis susceptibility: a meta-analysis of case-control studies.

BACKGROUND: To date, the fundamental pathophysiology underlying the occurrence and progression of psoriasis are still unanswered questions. Genome-wide association surveys have revealed that TNFAIP3 and TNIP1 were key biomarkers for psoriasis. Here, we intended to conduct a survey on the association between TNFAIP3 and TNIP1 gene polymorphisms and psoriasis risk. METHODS: A comprehensive search of four online databases-China National Knowledge Infrastructure (CNKI), PubMed, Embase, and Cochrane Library was undertaken up to August 25, 2019. We chose allele genetic model to deal with the original data. Newcastle-Ottawa scale (NOS) was used to evaluate the risk bias of each study. The RevMan 5.3 software was used to calculate the combined odds ratio and 95% confidence interval. RESULTS: In total, we included 13 case-control studies consist of 13,908 psoriasis patients and 20,051 controls in this work. Our results demonstrated that rs610604 in TNFAIP3 polymorphism was significantly associated with psoriasis risk using random-effect model (G vs. T, OR = 1.19, 95% CI: 1.09-1.31, P = 0.0002), and a significant association between rs17728338 in TNIP1 polymorphism and psoriasis vulnerability using fixed-effect model (A vs. G, OR = 1.69, 95% CI:1.58-1.80, P < 0.00001). CONCLUSIONS: Our findings indicated that rs610604 in TNFAIP3 and rs17728338 in TNIP1 gene polymorphisms were associated with psoriasis susceptibility.

Association of Interleukin-23R Gene Polymorphisms with Behcet's Disease Susceptibility: A Meta-Analysis of Case-control Studies.

BACKGROUND: The pathological mechanisms associated with the occurrence and development of Behcet's disease (BD) are not yet known. Two large genome-wide association surveys revealed an association between interleukin (IL)-23R single nucleotide polymorphism and BD. This study aimed to investigate the association between IL-23R gene polymorphisms and BD susceptibility. METHODS: Comprehensive literature search was performed across four online databases - PubMed, Embase, Cochrane Library, and Web of science. The included studies had to be published before May 15, 2019. The Newcastle-Ottawa scale was used to assess the quality of every included study, and pooled odds ratios (ORs) and 95% confidence intervals (95% CIs) were calculated using the allele model of inheritance to evaluate the potential associations between IL-23R gene polymorphisms and BD risk. RESULTS: In all, 12 case-control studies comprising 6,926 BD patients and 10,211 controls were identified and included in this meta-analysis, in which 5 loci of IL-23R gene polymorphisms were investigated. Of these 5 loci, 2 were found to be significantly associated with BD susceptibility: rs17375018 (G vs. A, OR = 1.50, 95% CI: 1.34-1.68, P < .00001) and rs924080 (T vs. C, OR = 1.36, 95% CI: 1.29-1.43, P < .00001). Only a systematic review was conducted for rs12119179, rs11209032, and rs12141431, owing to the lack of sufficient data. CONCLUSION: This meta-analysis indicated that rs17375018 (G/A) and rs924080 (T/C) were associated with BD susceptibility. However, association of the other IL-23R polymorphisms could not be estimated owing to the lack of studies. ABBREVIATIONS: BD: Behcet's disease; SNP: single nucleotide polymorphism; HLA: human leukocyte antigen; IL: interleukin; OR: odds ratio; CI: confidence interval; HWE: Hardy-Weinberg equilibrium; UK: United Kingdom; NOS: Newcastle-Ottawa scale; GWAS: genome-wide association study; TNF-α: tumor necrosis factor-α.

Kainic acid hyperphosphorylates tau via inflammasome activation in MAPT transgenic mice.

The excitotoxicity induced by kainic acid (KA) is thought to contribute to the development of Alzheimer's disease (AD); however, the mechanisms underlying this excitotoxicity remain unknown. In the current study, we investigated the dynamic changes in tau phosphorylation and their associations with the excitotoxicity induced by intraperitoneal injection of KA in the mouse brain. We found that KA-induced excitotoxicity led to sustained hyperphosphorylation of tau in MAPT transgenic (Tg) mice. By using cultured microglia and mouse brains, we showed that KA treatment specifically induced endoplasmic reticulum (ER) stress, which was characterized by activation of the major biomarkers of ER, such as ATF6, GRP78, and IRE1, and resulted in stimulation of inflammasomes. KA receptors (KARs), such as Girk1, were determined to be involved in this KA-induced ER stress. ER stress was also shown to activate inflammasomes by stimulating the expression of the two major components of inflammasomes, nucleotide binding oligomerization domain (NOD)-like receptor (NLR) protein 3 (NLRP3) and nuclear factor (NF)-κB, and eventually causing the production of interleukin-1ß (IL-1ß). Inhibition of NLRP3 or NF-κB by Bay11-7082 resulted in reduction of KA-induced IL-1ß production. Our results also revealed the positive effects of IL-1ß on tau phosphorylation, which was blocked by Bay11-7082. Notably, the results indicate that Bay11-7082 acts against KA-induced neuronal degeneration, tau phosphorylation, and memory defects via inflammasomes, which further highlight the protective role of Bay11-7082 in KA-induced neuronal defects.

The Regulatory Effects of CDKN1A on Relevant Genes in Kaposi's Sarcoma Cells.

The cyclin-dependent kinase inhibitor 1A (CDKN1A) gene plays important roles in different types of cancer; however, its mechanism in Kaposi's Sarcoma (KS) is far less known. The aim of this study is to investigate the regulatory effects of CDKN1A on relevant genes in KS cells, and ultimately determine the role of CDKN1A in KS. In the study, the CDKN1A overexpression group, or siRNA group, was transfected into KS cells, respectively. Western-Blot (WB) and quantitative real-time polymerase chain reactions (q-PCR) were performed to detect the expression of cell cycle protein D1 (CyclinD1) and cell cycle protein E (CyclinE). Q-PCR was performed to determine the expression of kshv-mir-k12-1-5p. The results prove that CDKN1A negatively regulates the expression of CyclinD1, CyclinE, and kshv-mir-k1-2-1-5p. Therefore, CDKN1A may be involved in the formation and development of KS through the regulation of kshv-mir-k12-1-5p, CyclinD1, and CyclinE. CDKN1A may become a new choice for targeted therapy in KS.

Bioinformatics analysis of key biomarkers and pathways in KSHV infected endothelial cells.

Kaposi sarcoma (KS) is an endothelial tumor etiologically related to Kaposi sarcoma herpesvirus (KSHV) infection. The aim of our study was to screen out candidate genes of KSHV infected endothelial cells and to elucidate the underlying molecular mechanisms by bioinformatics methods. Microarray datasets GSE16354 and GSE22522 were downloaded from Gene Expression Omnibus (GEO) database. the differentially expressed genes (DEGs) between endothelial cells and KSHV infected endothelial cells were identified. And then, functional enrichment analyses of gene ontology (GO) and Kyoto encyclopedia of genes and genomes (KEGG) pathway analysis were performed. After that, Search Tool for the Retrieval of Interacting Genes (STRING) was used to investigate the potential protein-protein interaction (PPI) network between DEGs, Cytoscape software was used to visualize the interaction network of DEGs and to screen out the hub genes. A total of 113 DEGs and 11 hub genes were identified from the 2 datasets. GO enrichment analysis revealed that most of the DEGs were enrichen in regulation of cell proliferation, extracellular region part and sequence-specific DNA binding; KEGG pathway enrichments analysis displayed that DEGs were mostly enrichen in cell cycle, Jak-STAT signaling pathway, pathways in cancer, and Insulin signaling pathway. In conclusion, the present study identified a host of DEGs and hub genes in KSHV infected endothelial cells which may serve as potential key biomarkers and therapeutic targets, helping us to have a better understanding of the molecular mechanism of KS.

Kainic acid Induces production and aggregation of amyloid ß-protein and memory deficits by activating inflammasomes in NLRP3- and NF-κB-stimulated pathways.

Kainic acid (KA) treatment causes neuronal degeneration, which is a feature of Alzheimer's disease (AD) symptoms such as amyloid ß-protein production and memory deficits. Inflammasomes are known to be critical for the progression of AD. However, the underlying mechanism by which inflammasomes influence AD progression remains unknown. The present study investigated the damaging effect of KA on neurons by focusing on the inflammasome-mediated signaling pathways. Assessments using cultured microglia and mouse brains demonstrated that KA treatment specifically induced inflammasome activation. Mechanistic evaluations showed that KA activated two major components of inflammasomes, nucleotide binding oligomerization domain (NOD)-like receptor (NLR) protein 3 (NLRP3) and nuclear factor (NF)-κB, which resulted in the production of interleukin-1ß (IL-1ß) and brain-derived neurotrophic factor (BDNF). Inhibition of NLRP3 or NF-κB by Bay11-7082 caused a reduction in the KA-induced expression of interleukin (IL)-1ß and BDNF. Moreover, knockdown of the expression of KA receptors (KARs) such as Grik1 and Grik3 induced suppression of NLRP3 and NF-κB, suggesting that KARs function upstream of NLRP3 and NF-κB to mediate the effects of KA on regulation of IL-1ß and BDNF expression. Notably, IL-1ß was shown to exert positive effects on the expression of BACE1, which is blocked by Bay11-7082. Overall, our results revealed that Bay11-7082 acts against KA-induced neuronal degeneration, amyloid ß-protein (Aß) deposition, and memory defects via inflammasomes and further highlighted the protective role of Bay11-7082 in KA-induced neuronal defects.

Association of rs2910164 polymorphism in MiR-146a gene with psoriasis susceptibility: A meta-analysis.

The rs2910164 single nucleotide polymorphism (SNP) in miR-146a has been implicated in the etiology of psoriasis in different relevant studies with contradictory conclusions and limited sample size. Therefore, the aim of this study was to undertake a systematic review and meta-analysis to estimate the association between rs2910164 SNP and psoriasis. We searched the databases of PubMed, EMBASE, Web of Science, WanFang, and Chinese National Knowledge Infrastructure (CNKI) to identify relevant literatures published before July 15, 2018. Four case-control studies including 2212 cases and 2274 healthy controls from 4 different countries met the predetermined criteria. The effect size was pooled by odds ratios (ORs) and 95% confidence intervals (95%CIs). Recessive model (CC vs CG+GG) was confirmed to be the optimal model. The results indicated that rs2910164 SNP was significantly associated with psoriasis (OR = 0.74, 95%CI 0.60-0.91, P = .004), and individuals with CC-genotype were predisposed to have decreased risk of psoriasis.

Vitamin D receptor gene polymorphism, serum 25-hydroxyvitamin D levels, and risk of vitiligo: A meta-analysis.

OBJECTIVES: To explore the relationship among the vitamin D receptor (VDR) gene polymorphisms, serum 25-hydroxyvitamin D levels, and vitiligo. METHODS: Databases including PubMed, Cochrane Library, Ovid, Web of Science, CNKI, SinoMed, and Wanfang Data were systematically searched. The association was assessed using odds ratios (ORs), standard mean difference (SMD), and 95% confidence intervals (CIs). The statistical tests were performed using Review Manager 5.3.3. RESULTS: We identified a total of 17 studies. The relationship between VDR gene polymorphisms (BsmI, ApaI, TaqI, and FokI), serum 25 (OH)D levels, and incidence of vitiligo was investigated. The results of this meta-analysis showed that the dominant genetic model (CC+AC vs AA, P = .007, OR = 1.41, 95% CI = 1.10-1.80), recessive genetic model (CC vs AC+AA, P = .01, OR = 4.10, 95% CI = 1.36-12.35), and allelic contrast model (C vs A, P = .005, OR = 1.87, 95% CI = 1.21-2.90) of VDR Apal locus increased the risk of vitiligo, and BsmI, TaqI, and FokI loci and the risk of vitiligo have no obvious correlation. Serum 25 (OH)D deficiency was positively associated with the incidence of vitiligo (P < .0001, SMD = -0.94, 95% CI = -1.39, -0.48). CONCLUSION: This meta-analysis revealed that VDR Apal polymorphism increased the susceptibility risk of vitiligo, and there is a positive correlation between serum 25 (OH)D deficiency and the incidence of vitiligo.

Ankle-brachial index and brachial-ankle pulse wave velocity are risk factors for ischemic stroke in patients with Type 2 diabetes.

The incidence of ischemic stroke in patients with diabetes is increasing. While brachial-ankle pulse wave velocity (BaPWV) and ankle-brachial index (ABI) are known to be associated with ischemic cardiovascular and cerebrovascular diseases, whether these measures predict the risk of ischemic cerebrovascular disease in diabetic patients remains unclear. 117 patients with type 2 diabetes were enrolled in this study. According to the results of head magnetic resonance imaging, the patients were divided into a diabetes-only group (n = 55) and a diabetes and ischemic stroke group (n = 62). We then performed ABI and BaPWV examinations for all patients. Compared with the diabetes-only group, we found decreased ABI and increased BaPWV in the diabetes and ischemic stroke group. Multivariate logistic regression analyses revealed that BaPWV and ABI were risk factors for ischemic stroke in patients with type 2 diabetes. Our findings indicate that decreased ABI and increased BaPWV are objective indicators of increased risk of ischemic stroke in patients with type 2 diabetes.

miR-218 Involvement in Cardiomyocyte Hypertrophy Is Likely through Targeting REST.

MicroRNAs (miRNAs) have been identified as key players in cardiomyocyte hypertrophy, which is associated with significant risks of heart failure. However, many microRNAs are still not recognized for their functions in pathophysiological processes. In this study, we evaluated effects of miR-218 in cardiomyocyte hypertrophy using both in vitro and in vivo models. We found that miR-218 was evidently downregulated in a transverse aortic constriction (TAC) mouse model. Overexpression of miR-218 is sufficient to reduce hypertrophy, whereas the suppression of miR-218 aggravates hypertrophy in primary cardiomyocytes induced by isoprenaline (ISO). In addition, we identified RE1-silencing transcription factor (REST) as a novel target of miR-218; it negatively regulated the expression of REST in hypertrophic cardiomyocytes and the TAC model. These results showed that miR-218 plays a crucial role in cardiomyocyte hypertrophy, likely via targeting REST, suggesting a potential candidate target for interfering hypertrophy.

MicroRNA-126-3p suppresses cell proliferation by targeting PIK3R2 in Kaposi's sarcoma cells.

Kaposi's sarcoma is a highly vascular tumor of lymphatic endothelial origin. Many deregulated miRNAs, including miR-126-3p, have been identified in Kaposi's sarcoma tissues. miR-126-3p is the most highly endothelial-specific miRNA that regulates vascular integrity and angiogenesis. In this study, we aimed to determine the effect of miR-126-3p on Kaposi's sarcoma cells through transfection of a miRNA mimic and inhibitor. Moreover, we searched the target gene (PIK3R2) of miR-126-3p using bioinformatics software and further verified PIK3R2 using luciferase reporter assays, Real-time quantitative PCR (qRT-PCR) and western blot. The results demonstrated that miR-126-3p inhibited cell proliferation, arrested cell cycle progression, induced cell apoptosis, and inhibited cell invasion of SLK cells. The bioinformatics analysis and luciferase reporter assay revealed that PIK3R2 mRNA is a direct target of miR-126-3p. Moreover, the level of expression of the PIK3R2 gene was downregulated in SLK cells transfected with miR-126-3p siRNAs. Therefore, our data demonstrated that miR-126-3p is a tumor suppressor miRNA that acts by targeting PIK3R2 in Kaposi's sarcoma cells. These findings contribute to our understanding of the molecular mechanisms underlying Kaposi's sarcoma.

Role of the immunogenic and tolerogenic subsets of dendritic cells in multiple sclerosis.

Multiple sclerosis (MS) is an immune-mediated disorder in the central nervous system (CNS) characterized by inflammation and demyelination as well as axonal and neuronal degeneration. So far effective therapies to reverse the disease are still lacking; most therapeutic drugs can only ameliorate the symptoms or reduce the frequency of relapse. Dendritic cells (DCs) are professional antigen presenting cells (APCs) that are key players in both mediating immune responses and inducing immune tolerance. Increasing evidence indicates that DCs contribute to the pathogenesis of MS and might provide an avenue for therapeutic intervention. Here, we summarize the immunogenic and tolerogenic roles of DCs in MS and review medicinal drugs that may affect functions of DCs and have been applied in clinic for MS treatment. We also describe potential therapeutic molecules that can target DCs by inducing anti-inflammatory cytokines and inhibiting proinflammatory cytokines in MS.

The expression profiles of microRNAs in Kaposi's sarcoma.

Kaposi's sarcoma (KS) is a multicentric angioproliferative tumor of mesenchymal origin. The molecular and biologic aspects of KS are not fully understood. MicroRNAs are non-protein-coding small RNAs in the size range 19-25 nucleotides (nt) that play important roles in biological processes, including cellular differentiation, proliferation, and death. We performed a miRNA microarray analysis by detecting six paired KS and matched adjacent healthy tissues using the 7th generation of miRCURY(TM) LNA Array (v.18.0) (Exiqon) containing 3100 capture probes. We selected 10 significant differentially expressed miRNAs, which were confirmed by quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR) in 18 paired KS and matched adjacent healthy tissue specimens. We also investigated the associations between clinical features and miRNA expression. Among the 3100 human miRNA probes in the microarrays, we identified 170 differentially expressed miRNAs (69 upregulated and 101 downregulated miRNAs) in KS versus adjacent healthy tissues. Among the most significantly upregulated miRNAs were miR-126-3p, miR-199a-3p, miR-16-5p, and the 13 KSHV-related miRNAs. The most significantly downregulated miRNAs included miR-125b-1-3p and miR-1183. Eight upregulated miRNAs, miR-181b-5p, miR-199a-3p, miR-15a-5p, miR-126-3p, miR-1297, kshv-miR-k12-12-3p, kshv-miR-k12-1-5p, and miR-16-5p, and two downregulated miRNAs, miR-125b-1-3p and miR-1183, were confirmed by qRT-PCR in 18 paired KS samples. The qRT-PCR results for 10 miRNAs were consistent with our microarray results. The miR-125b-1-3p and miR-16-5p had statistically significant associations with HHV-8 and HIV infections in KS. The results of miRNA profiling showed that KS appears to have unique expression patterns when compared with paired adjacent healthy tissues, suggesting that deregulation of miRNAs plays an important role in the progression of KS. These differentially expressed miRNAs may provide novel diagnostic and prognostic tools.

[Identification of interaction between BPHL and PML-C].

OBJECTIVE: To explore the interaction between BPHL and PML-C by co-immunoprecipitation and yeast two-hybird system. METHODS: The recombination expression plasmids pGBKT7-PML-C and pACT2-BPHL were cotransformed into yeast AH109, to investigate their interaction in vivo. The expression vector of HA-tagged fusion protein (pCMV-HA-PML-C) and the expression vector of myc-tagged fusion protein (pCMV-myc-BPHL) were constructed and identified respectively, and cotransfected into human embryo kidney 293 (HEK293) cells. The interaction between PML-C and BPHL was investigated by co-immunoprecipitation in vitro. RESULTS: Blue clones were found in QDO/5-bromo-4-chloro-3-indolyl-alpha-D-galactoside (X-alpha-gal) plate, eukaryotic expression vectors named as pCMV-HA-PML-C and pCMV-myc-BPHL were constructed and confirmed with double restriction enzyme digestion and co-transfected into HEK 293 cells successfully. After immunoprecipitation of HA-PML-C with anti-HA polyclonal antibody, expressed myc-BPHL protein was identified by Western blot with anti-c-myc monoclonal antibody from immunoprecipitated complex. CONCLUSION: The eukaryotic expression vector of PCMV-HA-PML-C and PCMV-myc-BPHL were constructed successfully. The interaction between PML-C and BPHL was identified by co-immunoprecipitation and yeast two-hybird technique.

Clinical utilization of microembolus detection by transcranial Doppler sonography in intracranial stenosis-occlusive disease.

OBJECTIVE: To discuss the clinical ultiliazation and significance of microembolus detection by transcranial Doppler (TCD) sonography in intracranial stenosis-occlusive disease. DATA SOURCES: All related articles in this review were mainly searched from PubMed published in English from 1996 to 2012 using the terms of microembolic signal, transcranial Doppler, intracranial stenosis, stroke. STUDY SELECTION: Original articles and reviews were selected if they were related to the clinical utilization of microembolus detection in intracranial stenosis-occlusive disease. RESULTS: Intracranial stenosis is a significant cause of cerebral emboli, and microembolus detection by TCD sonography were widely used in exploring the mechanisms of ischemic stroke with intracranial stenosis (including the middle cerebral artery stenosis and the vertebral-basilar stenosis), evaluating the prognosis of acute stroke, evaluating the therapeutic effects, and predicting the recurrent events of stroke. CONCLUSION: Microembolus detection by TCD sonography plays an important role in the cerebral ischemic stroke patients with intracranial stenosis.

[Effect of recombinant adenovirus carrying NLS-RARalpha gene on the proliferation of HL-60 cell and the differentiation of HL-60 cells induced by ATRA and relevant mechanism].

OBJECTIVE: To explore the effect and mechanism of recombined adenovirus carrying NLS-RARalpha gene on proliferation of HL-60 cells and the differentiation of HL-60 cells induced by ATRA. METHODS: HL-60 cells was infected with Ad-NLS-RARalpha and control virus Ad-KZ. The efficiency of infection was detected by FCM. The mRNA and protein levels of NLS-RARalpha were assessed by Real-time PCR (RT-PCR) and Western blot, respectively. MTT assay were applied to determine proliferation of HL-60 cells. Cell surface differentiation antigen CD11b of infected HL-60 cell induced by ATRA was examined by FCM. The mRNA and protein levels of C-MYC of infected HL-60 cell induced by ATRA were determined by Real-time PCR (RT-PCR) and Western blot assay. RESULTS: The efficiency of infection of Ad-NLS-RARalpha and Ad-KZ on HL-60 cell was 70%-80%. The mRNA and protein levels of NLS-RARalpha gene of HL-60 cells which infected with Ad-NLS-RARalpha were both obviously higher than that of the cells which infected with Ad-KZ and non-infected (P < 0.05). The proliferation ability of HL-60 cell infected with Ad-NLS-RARalpha was significantly increased (P < 0.05). The level of CD11b of HL-60 cell infected with Ad-NLS-RARalpha and induced by ATRA was clearly decreased than control groups (P < 0.05). The mRNA and protein levels of C-MYC gene of HL-60 cells infected with Ad-NLS-RARalpha and induced by ATRA were both obviously higher than that of the cells which infected with Ad-KZ and non-infected (P < 0.05). CONCLUSION: The recombined adenovirus Ad-NLS-RARalpha can increase the proliferation ability of HL-60 cell, and inhibit the differentiation of HL-60 cell through reduce the expression level of C-MYC gene.

[Identification of differentially expressed genes in rats and preliminary analysis in regression of vascular calcification].

OBJECTIVE: To investigate the differentially expressed genes in rat in the process of regression of vascular calcification by using the suppression subtractive hybridization (SSH). METHODS: 24 SD male rats which aged 6 weeks and specific pathogen free grade were selected and randomly divided into 3 groups (n = 8): control group, calcification group and regression group respectively. Vascular calcification model (vitamin D3 plus nicotine, VDN) were made from rats in calcification group and regression group, and rats in control group were intragastric administered with normal saline and lavaged with peanut oil. Rats were bred for 8 weeks in calcification group and control group, while rats in regression group were fed for 16 weeks. All rats were killed to measure concentration of calcium in the arterial tissue and examine the pathological lesion changes. Subtractive hybridization among vascular cDNA sequences from calcification group and regression group were established. The cDNA fragments which expressed higher or lower in regression group than those in calcification group were isolated. Differentially expressed genes with cDNA fragment were inserted into PMD18-T plasmid vector and transformed competent DH-5alpha, cDNA libraries of differentially expressed gene between calcification group and regression group were then constructed. Recombinant vectors were analyzed by colony PCR, positive genes were randomly selected for sequencing and analyzed by BLAST. 4 genes were randomly selected for RT-PCR certification combined with semi-quantitative analysis of DNA bands. RESULTS: VDN model of rats were successfully constructed. Concentration of tissue calcium in calcification group (15.34 mg/g +/- 2.51 mg/g) was significantly increased compared to that in control group (5.20 mg/g +/- 0.75 mg/g, P < 0.001), while in comparison with calcification group (15.34 mg/g +/- 2.51 mg/g), calcium in regression group was relatively lower (12.73 mg/g +/- 1.89 mg/g, P < 0.05). 28 up-regulated genes and 22 down-regulated genes were gained through sequencing and BLAST analysis among positive clones. RT-PCR validation indicated that 4 genes such as prdx3 and Ank2 had increasedly expressed in regression group than those in calcification group, the average fold change was 1.7. CONCLUSION: Rat vascular calcification tissue had characteristic of active regression. Genes in relation to pyrophosphoric acid synthesis, glutamate signal peptides, anti-oxidant and ant-apoptosis were up-regulated, at the same time many genes related to ossification and oxidation activity were down-regulated in the process of calcification regression. Increased expression of calcification suppressor genes accompanying decreased expression of calcification promoting genes might be the intrinsic mechanisms which initiated the active regression of calcified tissues.