LncRNA SNHG12 regulated by WTAP aggravated the oxygen-glucose deprivation/reperfusion-induced injury in bEnd.3 cell.

OBJECTIVES: Previous studies have identified abnormal expression of lncRNA SNHG12 in ischemic stroke, but the underlying molecular mechanism remains unclear. MATERIALS AND METHODS: Through database predictions, m6A methylation sites were found on SNHG12, suggesting post-transcriptional modification. To further elucidate the role of SNHG12 and m6A methyltransferase WTAP in oxygen-glucose deprivation/reperfusion (OGD/R)-induced damage in cerebral microvascular endothelial cells, we conducted investigations. Additionally, we examined the impact of m6A methyltransferase WTAP on SNHG12 expression. RESULTS: Overexpressing SNHG12 in bEnd.3 cells was found to inhibit cell proliferation and promote apoptosis, as well as activate the production of reactive oxygen species and inflammatory cytokines (E-selectin, IL-6 and MCP-1), along with angiogenic proteins (VEGFA and FGFb). Conversely, SNHG12 knockdown alleviated OGD/R-induced damage to BEnd.3 cells, resulting in improved cell proliferation, reduced apoptosis, decreased ROS and LDH production, as well as diminished expression of inflammatory cytokines (E-selectin, IL-6 and MCP-1) and angiogenic proteins (VEGFA and FGFb). Furthermore, WTAP was found to positively regulate SNHG12 expression, and WTAP knockdown in bEnd.3 cells under the OGD/R conditions inhibited cell proliferation, promoted apoptosis, and increased ROS and LDH production. CONCLUSION: These findings suggest that WTAP may play a crucial role in SNHG12-mediated OGD/R-induced damage in bEnd.3 cells. More molecular experiments are needed to further analyze its mechanism. Overall, our study helps to enrich our understanding of the dysregulation of SNHG12 in ischemic stroke.

CYP4 subfamily V member 2 (CYP4V2) polymorphisms were associated with ischemic stroke in Chinese Han population.

BACKGROUND: CYP4 subfamily V member 2 (CYP4V2) polymorphisms are related to venous thromboembolism. However, the influence of CYP4V2 polymorphisms on the susceptibility to ischemic stroke (IS) remains undetermined. METHODS: We selected and genotyped five polymorphisms of CYP4V2 in 575 cases and 575 controls to test whether CYP4V2 variants were associated with the risk for IS in a Chinese Han population. Genotyping of CYP4V2 polymorphisms was performed using the Agena MassARRAY platform. Logistic regression analysis was used to assess the association between CYP4V2 polymorphisms and IS risk by calculating odds ratios (ORs) and 95% confidence interval (CI). False-positive report probability analysis was applied to assess the noteworthy relationship of the significant findings. RESULTS: CYP4V2 rs1398007 might be a risk factor for IS (OR = 1.34, 95% CI 1.05-1.71, p = 0.009). Specially, confounding factors (age, gender, smoking and drinking status) might affect the relationship between rs1398007 and IS susceptibility. Moreover, rs1053094 and rs56413992 were associated with IS risk in males. Multifactor dimensionality reduction analysis showed the combination of rs13146272 and rs3736455 had the strongest interaction effect (information gain value of 0.40%). Furthermore, genotypes of rs1398007 (p = 0.006) and rs1053094 (p = 0.044) were associated with the levels of high-density lipoprotein cholesterol (HDL-C) among healthy controls. CONCLUSION: Our results first provided evidence that CYP4V2 rs1398007 might be a risk factor for IS, which provides instructive clues for studying the mechanisms of CYP4V2 to the pathogenesis of IS.

LncRNA SNHG12 ameliorates brain microvascular endothelial cell injury by targeting miR-199a.

Long non-coding RNAs regulate brain microvascular endothelial cell death, the inflammatory response and angiogenesis during and after ischemia/reperfusion and oxygen-glucose deprivation/reoxygenation (OGD/R) insults. The long non-coding RNA, SNHG12, is upregulated after ischemia/reperfusion and OGD/R in microvascular endothelial cells of the mouse brain. However, its role in ischemic stroke has not been studied. We hypothesized that SNHG12 positively regulates ischemic stroke, and therefore we investigated its mechanism of action. We established an OGD/R mouse cell model to mimic ischemic stroke by exposing brain microvascular endothelial cells to OGD for 0, 2, 4, 8, 16 or 24 hours and reoxygenation for 4 hours. Quantitative real-time polymerase chain reaction showed that SNHG12 levels in brain microvascular endothelial cells increased with respect to OGD exposure time. Brain microvascular endothelial cells were transfected with pcDNA-control, pcDNA-SNHG12, si-control, or si-SNHG12. After exposure to OGD for 16 hours, these cells were then analyzed by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide, trypan blue exclusion, western blot, and capillary-like tube formation assays. Overexpression of SNHG12 inhibited brain microvascular endothelial cell death and the inflammatory response but promoted angiogenesis after OGD/R, while SNHG12 knockdown had the opposite effects. miR-199a was identified as a target of SNHG12, and SNHG12 overexpression reversed the effect of miR-199a on brain microvascular endothelial cell death, the inflammatory response, and angiogenesis. These findings suggest that SNHG12 suppresses endothelial cell injury induced by OGD/R by targeting miR-199a.

Associations of TF Gene Polymorphisms with the Risk of Ischemic Stroke.

Ischemic stroke (IS) is the main cause of mortality and disability in China; thus, this study aimed to examine the association between six variants and their haplotypes within the transferrin (TF) gene and the risk of IS in the Southern Chinese Han population. Genotyping was performed using the Sequenom MassARRAY platform for 249 IS patients and 249 age- and sex-matched controls. The association between polymorphisms and IS risk was tested by Chi squared test and haplotype and stratification analysis. Odds ratios (ORs) and confidence intervals (CIs) were estimated by unconditional logistic regression analysis. The results of genetic model analyses indicated that the two SNPs (rs1880669 and rs2692695) were associated with decreased IS risk under the co-dominant, dominant, and additive models. Additionally, rs4525863 was also associated with decreased IS risk both under the dominant and additive models in males. Moreover, the CG haplotype of TF (rs1880669 and rs2692695) was significantly associated with a decreased risk of IS in the total population and males. Our findings suggested that polymorphisms (rs4525863, rs1880669, and rs2692695) of the TF gene might be a protective factor for IS in Southern Chinese Han population. Further large prospective studies are required to confirm these findings.

Using Mobile Health Intervention to Improve Secondary Prevention of Coronary Heart Diseases in China: Mixed-Methods Feasibility Study.

BACKGROUND: Coronary heart disease (CHD) is the leading cause of cardiovascular mortality worldwide, yet implementation of evidence-based strategies for secondary prevention remains suboptimal. OBJECTIVE: This study aimed to evaluate the feasibility, specifically the usability and acceptability, and estimate the preliminary effectiveness of a mobile health (mHealth) intervention targeting both physicians and patients to improve adherence to evidence-based medications and lifestyle modifications. METHODS: We conducted a 12-week pre-post interventional pilot study at two sites in Shanghai and Hainan, China. Physicians used the app designed in this study to prescribe evidence-based medicines and record patient information. Eligible and consenting patients received automatic text messages or voice calls 4 to 5 times per week for 12 weeks on medication adherence and healthy behaviors. Interviews were conducted among 10 physicians and 24 patients at the two sites for their thoughts on medication adherence and feedback on the usability and acceptability. Questions on usability and acceptability were also asked in a patient follow-up survey. With regard to estimating effectiveness, the primary outcome was medication adherence (as estimated by the Morisky Green Levine Scale) at 12 weeks. Secondary outcomes included physical activity, smoking status, fruits and vegetables consumption, and facility visit frequency. RESULTS: Interview findings and patient survey showed the good usability and acceptability of the intervention. Among 190 patients who completed the intervention, there was a significant increase in medication adherence (odds ratio [OR] 1.80, 95% CI 1.14-2.85). The study also showed decrease of smokers' percentage (-5%, P=.05), increase of daily vegetables consumption frequency (+0.3/day, P=.01), and community health care center visit frequency (+3 in 3 months, P=.04). The following site-specific differences were noted: medication adherence appeared to increase in Hainan (OR 14.68, 95% CI 5.20-41.45) but not in Shanghai (OR 0.61, 95% CI 0.33-1.12). CONCLUSIONS: Our study demonstrated that the intervention was feasible in both a tertiary care center and an urban community health center in China. Preliminary results from pre-post comparison suggest the possibility that provider and patient-linked mHealth interventions may improve medication adherence and lifestyle modifications among CHD patients, especially in resource-scarce settings. Randomized controlled trials are needed to verify the findings.

Association of RTEL1 gene polymorphisms with stroke risk in a Chinese Han population.

We investigated the associations between single nucleotide polymorphisms (SNPs) in the regulator of telomere elongation helicase 1 (RTEL1) gene and stroke in the Chinese population. A total of 400 stroke patients and 395 healthy participants were included in this study. Five SNPs in RTEL1 were genotyped and the association with stroke risk was analyzed. Odds ratios (ORs) and 95% confidence intervals (95% CIs) were calculated using unconditional logistic regression analysis. Multivariate logistic regression analysis was used to identify SNPs that correlated with stroke. Rs2297441 was associated with an increased risk of stroke in an allele model (odds ratio [OR] = 1.24, 95% confidence interval [95% CI] = 1.01-1.52, p = 0.043). Rs6089953 was associated with an increased risk of stroke under the genotype model ([OR] = 1.862, [CI] = 1.123-3.085, p = 0.016). Rs2297441 was associated with an increased risk of stroke in an additive model (OR = 1.234, 95% CI = 1.005, p = 0.045, Rs6089953, Rs6010620 and Rs6010621 were associated with an increased risk of stroke in the recessive model (Rs6089953:OR = 1.825, 95% CI = 1.121-2.969, p =0.01546; Rs6010620: OR = 1.64, 95% CI = 1.008-2.669, p =0.04656;Rs6010621:OR = 1.661, 95% CI = 1.014-2.722, p =0.04389). Our findings reveal a possible association between SNPs in the RTEL1 gene and stroke risk in Chinese population.

Effects of a health promotion program on medication adherence to antiplatelet therapy among ischemic stroke patients in Hainan Province, China.

Survivors of ischemic stroke are still at a significant risk for recurrence. Antiplatelet agents are the treatment of first choice for long-term secondary prevention of vascular events. This study aims to assess a health promotion program on medication adherence to antiplatelet therapy among ischemic stroke patients in Hainan province, China. In five hospitals from the intervention group, four highly experienced physicians trained 62 neurologists, who in turn trained 613 stroke patients to improve their awareness and adherence to antiplatelet therapy. Physicians and patients of the control group received usual stroke management programs. After one-year follow-up, the proportion of patients who took the antiplatelet therapy increased significantly in the intervention group, reaching 73.2%, with a pre-post difference between two arms of 22.9% ( P < 0.01). There was also a significant net increase in the proportion of patients with awareness of antiplatelet therapy (24.4%, P < 0.01). Multivariate analysis illustrated health promotion program, higher education, annual household income, insurance, and medical status affected antiplatelet drug use in stroke patients. In conclusion, the health promotion program, based on a train-the-trainer approach, showed positive effects on awareness of and adherence to antiplatelet therapy, which has the potential to be scaled up to other resource-limited areas.

Endophilin-1 regulates blood-brain barrier permeability via EGFR-JNK signaling pathway.

Endophilin-1 (Endo1), a multifunctional protein, is essential for synaptic vesicle endocytosis. However, the role and mechanism of endophilin-1 in blood-brain barrier (BBB) function are still unclear. This study was performed to determine whether endophilin-1 regulated BBB permeability via the EGFR-JNK signaling pathway. In the present study, we found that endophilin-1 over-expression in human cerebral microvascular endothelial cell (hCMEC/D3) increased BBB permeability and meanwhile reduced the expression levels of epidermal growth factor receptor (EGFR), phosphorylated c-Jun N-terminal kinase (p-JNK). While endophilin-1 knockdown led to the contrary results. After JNK inhibitor SP600125 was administered to the endophilin-1 silenced hCMEC/D3 cells, the transendothelial electrical resistance (TEER) value was decreased and the permeability coefficient values to 4kDa and 40kDa FITC-dextran were increased. Results observed by Transmission electron microscopy (TEM) showed that tight junctions (TJs) were opened. Moreover, immunofluorescence and Western blot assays revealed the discontinuous distribution of TJ-associated proteins ZO-1, occludin on cell-cell boundaries and a significant decrease in protein expressing levels. Therefore, these results indicated that endophilin-1 positively regulated BBB permeability via the EGFR-JNK signaling pathway in hCMEC/D3 cells, which would provide an experimental basis for further research on endophilin-1 mediated the opening of BBB.

Evaluation on the compliance with secondary prevention and influence factors of ischemic stroke in Hainan province, China.

Survivors of ischemic stroke are still at a significant risk for recurrence. Numerous effective strategies for the secondary prevention of ischemic stroke have now been established; however, these guidelines are not widely known. In this retrospective, a multicenter study was conducted from January 2011 to February 2012 in 10 general hospitals, which included 1300 elderly patients who had previously been diagnosed with ischemic stroke and re-admitted to hospitals. Logistic regression models were fitted to determine the relationship between compliance with secondary prevention therapy and each variable of interest. The treatment rates of antihypertensive, antiplatelet and lipid-lowering therapy were only 56.3%, 48.9% and 19.6%, respectively. Multivariate analysis presented that cardiovascular risk factors would motivate patients with hypertension and hyperlipidemia to receive corresponding treatments. However, it is worth noting that they did not influence the use of antiplatelet therapy. In addition, high education, health education and insurance promote the use of secondary prevention in patients. In conclusion, the importance of antiplatelet therapy should not be ignored any more. Besides, health education will raise patients' attention to ischemic stroke.

[Cloning and expression of fox growth hormone gene in Pichia pastoris].

To prepare recombinant fox growth hormone (fGH), we amplified its cDNA from silver fox pituitary tissue by RT-PCR and cloned into yeast shuttle vector pPIC9K down stream of a-factor signal peptide sequence by SnaB I and Not I restriction sites. The recombinant secretion vector pPIC9K/fGH, linearized by Sal I, was transformed into histidine-deficient Pichia pastoris strain GS115 by electroporation. We selected His+ -transformed methylotropic (His+, Mut+) yeast using histidine-absent medium containing dextrose (MD) or methanol (MM) as the only carbon source, and then screened the recombinant GS115 with multi-copy fGH genes by G418. The secretive expression of fGH was performed under the induction of methanol in shaking flask culture. The results showed that the fGH cDNA sequence amplified in this paper was basically in consistence with the published in GenBank. We achieved the secretive expression of recombinant fGH identified by SDS-PAGE and Western blotting. The fGH expression level was 119 mg/L, accounted for 34% of total proteins in fermentation medium.

Involvement of ERK1/2 and p38 MAPK in up-regulation of 14-3-3 protein induced by hydrogen peroxide preconditioning in PC12 cells.

OBJECTIVE: To investigate the protective effects of hydrogen peroxide preconditioning (HPP) on the pheochromocytoma (PC12) cells treated with 1-methyl-4-phenylpyridinium (MPP(+)) and to explore the potential mechanisms. METHODS: The viability and apoptosis of PC12 cells were determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and 4',6'-diamidino-2-phenylindole (DAPI) staining, respectively. The expressions of 14-3-3 protein and phosphorylated p38 mitogen-activated protein kinase (MAPK) were determined by Western blot. Enzyme-linked immunosorbent assay (ELISA) was used to measure the activity of extracellular signal-regulated protein kinase 1/2 (ERK1/2). RESULTS: The cell viability decreased and the number of apoptotic cells increased dramatically in MPP(+) group compared with that in Control group. HPP induced a significant increase in cell viability and a marked decrease in population of apoptotic cells of the MPP(+)-treated PC12 cells, accompanied with up-regulation of 14-3-3 protein and increase of ERK1/2 and p38 MAPK activities. The 14-3-3 protein expression was positively correlated with the phosphorylation of ERK1/2. Furthermore, inhibition of the ERK1/2 with PD98059 abolished the 14-3-3 protein up-regulation in PC12 cells induced by HPP. CONCLUSION: HPP protects PC12 cells against MPP(+) toxicity by up-regulating 14-3-3 protein expression through the ERK1/2 and p38 MAPK signaling pathways.

Correlation between angiotensinogen gene and primary hypertension with cerebral infarction in the Li nationality of China.

OBJECTIVE: To investigate the relationship of four single nucleotide polymorphism (SNP) haplotypes in the angiotensinogen (AGT) gene to the primary hypertension with or without cerebral infarction in the Li nationality of Hainan, China. METHODS: Total 300 subjects were allocated into three different groups: Group 1, 100 patients who have primary hypertension; Group 2, 100 patients who have primary hypertension with cerebral infarction; and control group, 100 healthy individuals. The genotypes of all subjects were determined by PCR-sequencing to analyze the four polymorphisms at position -152 (G-A), -20 (A-C), -18 (C-T), and -6 (A-G) in the promoter region of AGT. RESULTS: The frequencies of CT genotype of AGT-18 and T allele in Group 1 (P = 0.003, P = 0.004) and Group 2 (P = 0.002, P = 0.002) were both significantly higher than in healthy controls. The frequency of G allele of AGT-6 was significantly higher in Group 2 than in the control group (P = 0.016), while there is no significant difference between Group 1 and the control. Haplotype analysis revealed that H6 haplotype frequency which included -20C and -6G was significantly increased in Group 2 (P = 0.003) compared with the control group, while H5 haplotype frequency which included -20C and -18T was significantly increased in Group 1 (P = 0.006) versus the control. CONCLUSION: The -20 (A-C) and -18 (C-T) of the AGT may play an important role in pathogenesis of primary hypertension; and -20 (A-C), -18 (C-T), and -6 (A-G) may be the genetic risk factors for the onset of primary hypertension with cerebral infarction in the Li nationality of Hainan, China.