Genetic and functional variants of the TBX20 gene promoter in dilated cardiomyopathy.

BACKGROUND: Dilated cardiomyopathy (DCM) is a major cause of heart failure and sudden cardiac death. As DCM is a genetically heterogeneous disease, genetic variants of cardiac transcription factor genes may play an important role. Transcription factor TBX20, an indispensable factor in normal heart development, is involved in the regulation of cardiac structure and function. Although the TBX20 gene is associated with the occurrence and development of DCM, the influence of genetic variants of the TBX20 gene promoter region on DCM has not been reported. METHODS: We conducted a case-control study consisting of 107 DCM patients and 210 healthy controls. Genetic variants within TBX20 gene promoter region were identified using sequencing techniques and were functionally analyzed by dual-luciferase reporting assay. Electrophoretic mobility shift assay (EMSA) was used to investigate DNA-protein interactions. RESULTS: In this study cohort (n = 317), we identified eight variants within TBX20 gene promoter. One novel DNA sequence variants (DSV) (g.4275G>T) and four single-nucleotide polymorphisms (SNPs) [g.4169G>A (rs1263874255), g.4949C>T (rs1191745927), g.5114G>A (rs112076877), g.5252C>T (rs1356932911)] were identified in DCM patients, but in none of controls. Among them, the DSV (g.4275G>T) and three SNPs [g.4949C>T (rs1191745927), g.5114G>A (rs112076877) and g.5252C>T (rs1356932911)] significantly altered the transcription activity of TBX20 gene promoter by dual-luciferase reporting assay (p < 0.05). Further, EMSA assay indicated that the DSV (g.4275G>T) and three SNPs [g.4949C>T (rs1191745927), g.5114G>A (rs112076877) and g.5252C>T (rs1356932911)] affected the binding of transcription factors. CONCLUSIONS: These data indicate that the DSV (g.4275G>T) and three SNPs [g.4949C>T (rs1191745927), g.5114G>A (rs112076877) and g.5252C>T (rs1356932911)] increase transcription activity of TBX20 gene promoter in both HEK-293 and neonatal rat cardiomyocytes (NRCMs) cell lines by affecting the binding of transcription factors. But the mechanism remains to be verified in vivo.

Correlation Analysis of CTSB Promoter Polymorphism and Function in Patients with Dilated Cardiomyopathy.

Dilated cardiomyopathy (DCM) is caused by a combination of genetic susceptibility and environmental factors. Cathepsin B affects the pathogenesis of DCM; however, its molecular mechanism is still unclear. In this study, we examined the association of rare CTSB variants with the occurrence of DCM. This case-control study involved 394 participants: 142 patients with DCM and 252 healthy controls. DNA was extracted from the peripheral leukocytes of all participants, and CTSB variants were analyzed and identified using polymerase chain reaction amplification. Functional analysis was performed using the dual-luciferase reporter assay, and the ability of genetic CTSB variants to bind to transcription factors (TFs) was analyzed and validated using the electrophoretic mobility shift assay (EMSA). Two single-nucleotide polymorphisms (SNPs) were identified in the study population. One SNP, g.4803 T > C (rs1293312), was more common in patients with DCM. A second SNP, g.4954 T > A (rs942670850), was identified in two patients with DCM. Both SNPs significantly enhanced the transcriptional activity of CTSB promoters. An analysis using the TRANSFAC database revealed that these SNPs affect TF binding, which was confirmed using the EMSA. Our results demonstrate that within the CTSB promoter, the genetic variants g.4803T>C (rs1293312) and g.4954 T > A (rs942670850) are rare risk factors for DCM development.

Association Lp-PLA2 Gene Polymorphisms with Coronary Heart Disease.

Objectives: The study evaluated the association between lipoprotein-associated phospholipase A2 (Lp-PLA2) gene polymorphisms and coronary heart disease (CHD), in order to explore the molecular genetics of CHD. Methods: Groups of CHD patients (n = 283) and healthy controls (n = 261) were involved in this study. R92H, V279F, and A379V polymorphisms of LP-PLA2 gene were confirmed using polymerase chain reaction (PCR) and direct DNA sequencing. These polymorphisms and their interaction were also analyzed as potential risk factors of CHD. Results: In this study population, the genotypes of R92H (GG, GA, and AA), V279F (CC, AC, and AA) and A379V (GG, GA, and AA) were studied. There was a significantly difference in frequencies of R92H between CHD patients and controls (P < 0.05). In contrast, no significant difference in frequencies of V279F and A379V existed between CHD patients and controls. Furthermore, R92H and A379V were in strong linkage disequilibrium. Conclusions: These results suggested that R92H polymorphism might contribute to increased risk of CHD.

Identification and functional study of GATA4 gene regulatory variants in type 2 diabetes mellitus.

BACKGROUND: Type 2 diabetes mellitus (T2D) is a common and complex disease. Dysfunction of pancreatic ß cells, which cannot release sufficient insulin, plays a central role in T2D. Genetics plays a critical role in T2D etiology. Transcription factor GATA4 is required for the pancreatic development, and GATA4 gene mutations are implicated in neonatal or childhood-onset diabetes. In this study, we aimed to investigate whether regulatory variants in GATA4 gene may change GATA4 levels, conferring susceptibility to T2D development. METHODS: The promoter region of GATA4 gene was analyzed by targeted sequencing in T2D patients (n = 255) and ethnic-matched controls (n = 371). Dual luciferase activity assay was used for functional study, and EMSA (electrophoretic mobility shift assay) was performed for detecting transcription factor binding. RESULTS: Thirteen regulatory variants including 5 SNPs were identified. A novel heterozygous variant (32124C > T) and one SNP [31487C > G (rs1053351749)] were only identified in T2D. Both regulatory variants significantly affected GATA4 gene promoter activity in cultured HEK-293 and INS-1 cells. Furthermore, the variant (32124C > T) evidently enhanced the binding of unknown transcriptional activator. CONCLUSIONS: Our data suggested that GATA4 gene regulatory variants may contribute to T2D development as a rare risk factor.

Heterodimerization of the kappa opioid receptor and neurotensin receptor 1 contributes to a novel ß-arrestin-2-biased pathway.

Together with its endogenous ligands (dynorphin), the kappa opioid receptor (KOR) plays an important role in modulating various physiological and pharmacological responses, with a classical G protein-coupled pathway mediating analgesia and non-G protein-dependent pathway, especially the ß-arrestin-dependent pathway, eliciting side effects of dysphoria, aversion, drug-seeking in addicts, or even relapse to addiction. Although mounting evidence has verified a functional overlap between dynorphin/KOR and neurotensin/neurotensin receptor 1 (NTSR1) systems, little is known about direct interaction between the two receptors. Here, we showed that KOR and NTSR1 form a heterodimer that functions as a novel pharmacological entity, and this heterodimer, in turn, brings about a switch in KOR-mediated signal transduction, from G protein-dependent to ß-arrestin-2-dependent. This was simultaneously verified by analyzing a KOR mutant (196th residue) that lost the ability to dimerize with NTSR1. We also found that dual occupancy of the heterodimer forced the ß-arrestin-2-dependent pathway back into Gi protein-dependent signaling, according to KOR activation. These data provide new insights into the interaction between KOR and NTSR1, and the newly discovered role of NTSR1 acting as a switch between G protein- and ß-arrestin-dependent pathways of KOR also suggests a new approach for utilizing pathologically elevated dynorphin/KOR system into full play for its analgesic effect with limited side effects.

Lateral intracerebroventricular injection of Apelin-13 inhibits apoptosis after cerebral ischemia/reperfusion injury.

Apelin-13 inhibits neuronal apoptosis caused by hydrogen peroxide, yet apoptosis following cerebral ischemia-reperfusion injury has rarely been studied. In this study, Apelin-13 (0.1 µg/g) was injected into the lateral ventricle of middle cerebral artery occlusion model rats. TTC, TUNEL, and immunohistochemical staining showed that compared with the cerebral ischemia/reperfusion group, infarct volume and apoptotic cell number at the ischemic penumbra region were decreased in the Apelin-13 treatment group. Additionally, Apelin-13 treatment increased Bcl-2 immunoreactivity and decreased caspase-3 immunoreactivity. Our findings suggest that Apelin-13 is neuroprotective against cerebral ischemia/reperfusion injury through inhibition of neuronal apoptosis.

3-methyladenine, an autophagic inhibitor, attenuates therapeutic effects of sirolimus on scopolamine-induced cognitive dysfunction in a rat model.

Previous studies have demonstrated that sirolimus has therapeutic effects for Alzheimer's disease which characterized by cognitive dysfunction. However, its underlying mechanisms have not been fully elucidated. In the present study, we aimed to investigate the mechanisms of therapeutic effects of sirolimus for cognitive dysfunction rat model which induced by chronic administration of scopolamine. Forty Wistar rats were randomly divided into 4 groups (n=10 each): saline group and scopolamine group, sirolimus plus scopolamine group and 3-methyladenine pretreatment group. Morris water maze test was applied to measure the cognitive function of rat. After behavioral test, rats were sacrificed and prefrontal cortex and hippocampus were harvested for measuring amyloid-ß (Aß), Beclin-1 and mammalian target of rapamycin (mTOR). Compared with saline group, scopolamine administered significantly decreased the cognitive performance of rats during the Morris water maze test and changed Aß, Beclin-1 and mTOR levels in rat prefrontal cortex and hippocampus (P<0.05); In addition, rats in sirolimus plus scopolamine group significantly reversed scopolamine-induced effects (P<0.05). Most importantly, 3-methyladenine abrogated the effects of sirolimus on scopolamine-induced cognitive dysfunction (P<0.05). In conclusion, the mechanism of sirolimus exerting therapeutic effects for scopolamine-induced cognitive dysfunction is likely related to the activation of autophagy.

Chronic lipopolysaccharide exposure induces cognitive dysfunction without affecting BDNF expression in the rat hippocampus.

Previous studies have shown that lipopolysaccharide (LPS) has the potential to cause cognitive dysfunction. However, the underlying pathogenesis has yet to be fully elucidated. Increasing attention is being focused on infection in the central nervous system. Therefore, the present study aimed to investigate the behavioral performance of rats receiving intraperitoneal injections of LPS and to determine the expression levels of amyloid-ß (Aß), brain-derived neurotrophic factor (BDNF) and pro-inflammatory cytokines in the hippocampus. In total, 30 male Wistar rats were randomly divided into 3 groups (each n=10): Control and 3 and 7 day LPS administration groups. The rats were intraperitoneally injected with saline or LPS for 3 or 7 days. Following this, rats performed the Morris water maze test, in which the latency to the platform and proportion of time spent in the target quadrant were recorded. Rats were then sacrificed and the hippocampi were harvested for determination of interleukin (IL)-1ß, IL-6, tumor necrosis factor-α (TNF-α), Aß and BDNF expression levels. LPS administration for 3 and 7 days significantly increased the latency to the platform and decreased the proportion of time spent in the target quadrant compared with those in the control group, (P<0.05). Administration of LPS for 3 and 7 days induced statistically significant increases in the expression levels of IL-1ß, IL-6 and TNF-α in the hippocampus, compared with those in the control group (P<0.05). Additionally, the administration of LPS for 7 days induced a statistically significant increase in the expression level of Aß in the hippocampus, compared with that in the control group (P<0.05). However, the administration of LPS did not elicit a statistically significant change in the expression level of BDNF in the hippocampus, compared with that in the control group (P>0.05). The results indicate that LPS induces cognitive dysfunction, which is associated with increased expression levels of pro-inflammatory cytokines and Aß, but does not affect the expression of BDNF in the hippocampus.

Effects of BD1047, a σ1 receptor antagonist, on the expression of mTOR, Camk2γ and GSK-3ß in fluvoxamine-treated N2a cells.

Fluvoxamine, a common antidepressant agent, is designed to exert its pharmacological effect by inhibiting synaptic serotonin reuptake. However, increasing evidence has demonstrated that σ1 receptors are likely to be involved in the mechanism of action of fluvoxamine. The present study aimed to observe the effects of fluvoxamine on the expression levels of mammalian target of rapamycin (mTOR), Ca2+/calmodulin-dependent protein kinase 2γ (Camk2γ) and glycogen synthase kinase-3ß (GSK-3ß) in fluvoxamine-treated N2a cells and attempted to elucidate whether σ1 receptors mediate the pharmacological effects of fluvoxamine. The N2a cells were randomly divided into three groups (each n=6): DMEM group (D group), 0.5 µmol/l fluvoxamine group (F group) and 0.2 µmol/l BD1047 (a σ1 receptor antagonist) + 0.5 µmol/l fluvoxamine group (BF group). Western blotting was used to determine the expression levels of mTOR, Camk2γ and GSK-3ß in the cultured N2a cells after two days of incubation. The F group exhibited significant increases in the expression levels of mTOR and Camk2γ and a significant reduction in the expression levels of GSK-3ß compared with those in the D group (P<0.01). By contrast, the BF group demonstrated significant reductions in the expression levels of mTOR and Camk2γ and a significant increase in the expression levels of GSK-3ß, compared with those in the F group (P<0.01). These results suggest that σ1 receptors mediate fluvoxamine-elicited changes in the expression levels of mTOR, Camk2γ and GSK-3ß in N2a cells, which indicates that σ1 receptors are likely to be involved in the pharmacological effects of fluvoxamine.

Ketamine attenuates the lipopolysaccharide-induced inflammatory response in cultured N2a cells.

The use of ketamine is recommended in patients with sepsis undergoing surgery due to its anti-inflammatory effects. However, a paucity of data exists with regard to the anti-inflammatory effects of ketamine in the central nervous system. Therefore, the present study aimed to investigate the effect of ketamine on lipopolysaccharide (LPS)­induced inflammatory responses in cultured Neuro2a (N2a) cells and to elucidate its potential mechanism of action. N2a cells were randomly divided into the following 3 groups (n=6): The DMEM culture solution administration alone group, the 0.5 µmol/l LPS administration alone group and the 1 µmol/l ketamine plus 0.5 µmol/l LPS administration group. The expression levels of interleukin (IL)-1ß, IL-6, tumor necrosis factor (TNF)-α, nuclear factor (NF)-κB and inducible nitric oxide synthase (iNOS) were determined. LPS-treated N2a cells exhibited a significant increase in the expression levels of IL-1ß, IL-6, TNF-α, NF-κB and iNOS, while the administration of ketamine eliminated the LPS-induced production of IL-1ß, IL-6, TNF-α, NF-κB and iNOS. Based on our data, we hypothesized that the anti-inflammatory effect exerted by ketamine on N2a cells was potentially due to the inhibition of NF-κB and iNOS.

Prehospital therapeutic hypothermia after cardiac arrest: a systematic review and meta-analysis of randomized controlled trials.

BACKGROUND: Therapeutic hypothermia has been recommended for the treatment of cardiac arrest patients who remain comatose after the return of spontaneous circulation. However, the optimal time to initiate therapeutic hypothermia remains unclear. The objective of the present study is to assess the effectiveness and safety of prehospital therapeutic hypothermia after cardiac arrest. METHODS: Databases such as MEDLINE, Embase, and Cochrane Library were searched from their establishment date to May of 2012 to retrieve randomized control trials on prehospital therapeutic hypothermia after cardiac arrest. Thereafter, the studies retrieved were screened based on predefined inclusion and exclusion criteria. Data were extracted and the quality of the included studies was evaluated. A meta-analysis was performed by using the Cochrane Collaboration Review Manager 5.1.6 software. RESULTS: Five studies involving 633 cases were included, among which 314 cases were assigned to the treatment group and the other 319 cases to the control group. The meta-analysis indicated that prehospital therapeutic hypothermia after cardiac arrest produced significant differences in temperature on hospital admission compared with in-hospital therapeutic hypothermia or normothermia (patient data; mean difference=-0.95; 95% confidence interval -1.15 to -0.75; I(2)=0%). However, no significant differences were observed in the survival to the hospital discharge, favorable neurological outcome at hospital discharge, and rearrest. The risk of bias was low; however, the quality of the evidence was very low. CONCLUSION: This review demonstrates that prehospital therapeutic hypothermia after cardiac arrest can decrease temperature on hospital admission. On the other hand, regarding the survival to hospital discharge, favorable neurological outcome at hospital discharge, and rearrest, our meta-analysis and review produces non-significant results. Using the Grading of Recommendations, Assessment, Development and Evaluation methodology, we conclude that the quality of evidence is very low.

[The nonlinear relationship between the resuscitation therapy and intensive insulin therapy in severe sepsis and septic shock patients].

OBJECTIVE: To study the relationship between the resuscitation therapy and intensive insulin therapy on stress-induced hyperglycemia in severe sepsis and septic shock patients, and to evaluate the value on nonlinear viewpoint in the treatment of patients with sepsis. METHODS: The data of 129 hospitalized patients with severe sepsis and septic shock were analyzed and they were divided into eight groups every 6 hours in ascending order for full recovery. The resuscitation therapy time of each group was compared with insulin dosage in each unit time with nonlinear least square method. RESULTS: The relationship of the exponential function fit very well between the resuscitation therapy time of each group and the insulin dosage in each unit time. The exponential curve equation was y=e0.739 3-0.015 2x2 (a=0.739 3, b=0.015 2) and the curve fit very well (R2=0.976 943 6). CONCLUSION: It conforms to the nonlinear viewpoint that the resuscitation therapy time is closely correlated with recovery of dysfunction of endocrine system during the treatment for patients with severe sepsis and septic shock. Therefore, the essence of successful treatment is to concentrate on helping the body rebuild the disorganized network and the recovery of physiological harmony rather than to support and repair the damaged organs.