Dual-modular immunosensor for bongkrekic acid detection using specific monoclonal antibody.

Bongkrekic acid (BA) is a mitochondrial toxin that causes high mortality but is often mistakenly categorized as other food poisonings. The immunoassay of BA is still challenging since the specific antibody is unavailable. In this work, a monoclonal antibody specific to BA was first generated and a dual-modular immunosensor for on-site and laboratory detection was established. The antibody showed good affinity (Kd=0.33 µM) and sensitivity (IC50 =17.9 ng/mL in ELISA) with negligible cross-reactivity with common mycotoxins. In dual-modular conditions, fluorescence assay (FA) was conducted based on the inner filter effect of carbon dots (CDs) and oxidized 3,3',5,5'-tetramethylbenzidine (TMB), while the colorimetric assay (CA) was conducted using TMB2+-mediated rapid surface etching of gold nanostars (Au NSs). The proposed immunosensor showed good sensitivity and reproducibility to BA in food samples, with a limit of detection lower than 10 ng/mL and recovery ranging from 80.0% to 103.6%, which was in good consistence with that of standard LC-MS/MS. Overall, the proposed immunosensor is an ideal tool for screening BA contaminants in food with good sensitivity and high effectivity.

Berberine Protects Against Simulated Ischemia/Reperfusion Injury-Induced H9C2 Cardiomyocytes Apoptosis In Vitro and Myocardial Ischemia/Reperfusion-Induced Apoptosis In Vivo by Regulating the Mitophagy-Mediated HIF-1α/BNIP3 Pathway.

Berberine (BBR) has a variety of pharmacological activities and is widely used in Asian countries. However, the clinical application of BBR still lacks scientific basis, what protective mechanism of BBR against myocardial ischemia-reperfusion injury (MIRI). In vitro experiments, BBR pretreatment regulated autophagy-related protein expression, induced cell proliferation and autophagosome formation, and reduced the mitochondrial membrane potential (ΔΨm) increase in H9C2 cells. In vivo experiments, BBR reduced the myocardial infarct size, decreased cardiomyocyte apoptosis, and markedly decreased myocardial enzyme (CK-MB, LDH, and AST) activity-induced I/R. In addition, upon BNIP3 knockdown, the regulatory effects of BBR on the above indicators were weakened both in H9C2 cells and in vivo. Luciferase reporter and ChIP assays indicated that BBR mediated BNIP3 expression by enhancing the binding of HIF-1α to the BNIP3 promoter. BBR protects against myocardial I/R injury by inducing cardiomyocytes proliferation, inhibiting cardiomyocytes apoptosis, and inducing the mitophagy-mediated HIF-1α/BNIP3 pathway. Thus, BBR may serve as a novel therapeutic drug for myocardial I/R injury.

Berberine attenuates mitochondrial dysfunction by inducing autophagic flux in myocardial hypoxia/reoxygenation injury.

Berberine (BBR) is routinely prescribed in many Asian countries to treat diarrhea. Evidence from both animal and clinical investigations suggests that BBR exerts diverse pharmacological activities, including antidiabetic, antineoplastic, antihypertensive, and antiatherosclerotic effects. This study aimed to explore the cardioprotective mechanisms of BBR and to elucidate the modulations between autophagy and mitochondrial function during hypoxia/reoxygenation (H/R) in H9c2 cells. The degree of autophagic flux was assessed by pretreating H9c2 cells with BBR prior to H/R exposure and measuring the expression levels of Beclin-1 and green fluorescent protein (GFP)-labeled LC3B fusion proteins as well as the LC3II/LC3I ratio. The mitochondrial membrane potential (△Ψm) in H9c2 cells was evaluated by detecting rhodamine-123 fluorescence using flow cytometry. The results revealed that pretreatment with BBR upregulated autophagic flux and protected against the loss of the △Ψm in H9c2 cells subjected to H/R. We conclude that BBR attenuates mitochondrial dysfunction by inducing autophagic flux.

Y-box protein 1 promotes hypoxia/reoxygenation- or ischemia/reperfusion-induced cardiomyocyte apoptosis via SHP-1-dependent STAT3 inactivation.

Cardiomyocyte apoptosis induced by hypoxia and ischemia plays important roles in heart dysfunction after acute myocardial infarction (AMI). However, the mechanism of apoptosis induction remains unclear. A previous study reported that Y-box protein 1 (YB1) is upregulated after myocardial hypoxia/reoxygenation or ischemia/reperfusion (H/R or I/R, respectively) injury; however, whether YB1 is associated with H/R-induced cardiomyocyte apoptosis is completely unknown. In the present study, we investigated the roles of YB1 in H/R-induced cardiomyocyte apoptosis and the possible underlying molecular mechanisms. In vitro, H/R treatment upregulated the YB1 expression in H9C2 cells, whereas YB1 knockdown inhibited H/R-induced cardiomyocyte apoptosis and induced H9C2 cell proliferation via Src homology region 2 domain-containing phosphatase 1 (SHP-1)-mediated activation of signal transducer and activator of transcription 3 (STAT3). In vivo, YB1 knockdown ameliorated AMI, reducing infarct size, cardiomyocyte apoptosis, and oxidative stress, via SHP-1-mediated inactivation of STAT3. Additionally, YB1 knockdown inhibited H/R- or I/R-induced oxidative stress in vitro and in vivo. H/R and I/R increase YB1 expression, and YB1 knockdown ameliorates AMI injury via SHP-1-dependent STAT3 inactivation.

[Protective effect and mechanism of curcumin on aorta in rats with metabolic syndrome].

To investigate the protective effect and mechanism of curcumin on aorta in rats with metabolic syndrome,72 SD rats were randomly divided into blank control group,model control group,positive control group,curcumin low,middle and high dose groups.The rat model of metabolic syndrome was established in all groups except the blank control group. After the intervention by curcumin,the blood pressure,blood lipid,blood glucose,serum insulin and insulin sensitivity index were measured. The contents of serum leptin(LP),adiponectin(ADP) and tumor necrosis factor-α(TNF-α) in rat aorta were detected by enzyme-linked immunosorbent assay(ELISA),and the pathological changes of rat thoracic aorta were observed by HE staining and electron microscope scanning. Western blot assay was used to detect the expression of inducible nitric oxide synthase(i NOS) and endothelial nitric oxide synthase(e NOS) in rats. The results showed that the blood lipid level,fasting blood glucose,fasting insulin,insulin sensitivity index,systolic blood pressure,LP,TNF-α and intima/media thickness ratio in the model control group were significantly higher than those in the blank control group. As compared with the model control group,the levels of blood lipids,fasting blood glucose,fasting insulin,insulin sensitivity index,systolic blood pressure,LP,TNF-α and intima/media thickness ratio were significantly decreased in positive control group,low,middle and high dose curcumin groups. The difference was statistically significant. The results of HE staining showed that the intima of the thoracic aorta in the model group was significantly thickened; the endothelial cell membrane was wrinkled and the organelle was ruptured. The intima of the thoracic aorta in the positive control group was slightly thickened and the structure of endothelial cells was intact,with no foam cells and no abnormality in the adventitia. There was no significant thickening of the thoracic aorta in the low,middle and high dose curcumin groups,and the endothelial cells were still intact. The results of Western blot assay showed that the expression levels of i NOS and e NOS were decreased significantly in the model group,while the expression levels of i NOS and e NOS were increased significantly in the positive control group and curcumin groups. The results indicated that curcumin had a certain protective effect on the aorta of rats with metabolic syndrome and improves the aortic endothelial dysfunction,and its mechanism may be related to the fact that curcumin could reduce the production of oxygen free radicals and up-regulate the expression of i NOS and e NOS in aorta.

Y-box binding protein 1 regulates ox-LDL mediated inflammatory responses and lipid uptake in macrophages.

AIMS: Y-box protein 1 (YB1) is a key regulator of inflammatory mediators. However, the roles of YB1 in oxidized low-density lipoprotein (ox-LDL)-induced macrophage inflammation and lipid uptake remain less understood. Thus, we explored the roles of YB1 in ox-LDL-induced macrophage inflammation and lipid uptake and its underlying molecular mechanisms. METHODS: An ox-LDL-induced atherosclerosis (AS) model was used in this study. Western blotting, RT-PCR, immunofluorescence, ELISA, dil-ox-LDL staining, a dual-luciferase reporter assay, RNA-binding protein immunoprecipitation (RIP) and in vivo experiments were used to detect each target. RESULTS: ox-LDL downregulates YB1 expression in THP-1-derived macrophages and human monocyte-derived macrophages (hMDMs) via the NF-κB pathway. Downregulation of YB1 is facilitated by lipid uptake in macrophages, and CD36 is involved in this process. Furthermore, YB1 suppresses CD36 protein levels by directly binding to the coding sequence of the CD36 gene to promote CD36 mRNA decay but does not affect its mRNA transcription. Additionally, YB1 knockdown enhances the inflammatory response and lipid deposition via the NF-κB pathway in vivo. CONCLUSION: ox-LDL decreases YB1 expression in macrophages, resulting in enhanced inflammatory responses by affecting NF-κB and facilitating lipid uptake by promoting scavenger receptor CD36 mRNA decay.

Retracted Article: Exosomal miR-25-3p derived from hypoxia tumor mediates IL-6 secretion and stimulates cell viability and migration in breast cancer.

Hypoxia is a major hallmark of solid tumors and is associated with malignant phenotypes. Exosomal miRNAs derived from hypoxia tumor cells are implicated in the modulation of cancer progression, whereas, the mechanisms underlying the association between hypoxia and exosomal miR-25-3p during breast cancer progression remain to be further clarified. The present study aimed to investigate the role of exosomal miR-25-3p in regulating breast cancer progression. Herein, we found that miR-25-3p expression was increased in hypoxia tumor-derived exosomes a HIF-1α-dependent manner. Hypoxia exosomes markedly stimulated the viability and migration of normoxia breast cancer cells, which was reversed by miR-25-3p depletion. Inhibition of exosomes miR-25-3p lowered hypoxic-induced the expression of IL-6 and NF-κB from THP-1 and RAW264.7 cells in a TLR7/8-dependent way. Treatment of macrophage supernatant (MS) initially incubated with hypoxic-responsed exosomes accelerated the viability and migration of breast cancer cells, and miR-25-3p depletion relieved the stimulatory effects of hypoxic on cell viability and migration. Moreover, miR-25-3p knockdown dramatically suppressed HIF-1α-induced tumor growth in vivo via inactivation of IL-6/STAT3 signaling pathway, reflected by the abated abundances of IL-6 and p-STAT3. These data suggested that absence of exosomal miR-25-3p rescued breast cancer aggressiveness through inhibiting cell viability and migration by regulation of IL-6 secretion from macrophages, providing a potential biomarker for breast cancer treatment.

Effects of miR-181a targeting XIAP gene on apoptosis of cardiomyocytes induced by hypoxia/reoxygenation and its mechanism.

To investigate the effect of miR-181a targeting XIAP gene on the apoptosis of cardiomyocytes induced by hypoxia/reoxygenation (H/R) and its mechanism. The primary cultured cardiomyocytes were treated with hypoxia for 3 hours and reoxygenation for 4 hours to construct H/R cell model. The expression of miR-181a and XIAP messenger RNA in cardiomyocytes was detected by reverse-transcription polymerase chain reaction, and the expression of XIAP protein in cardiomyocytes was detected by Western blot analysis. H/R cardiomyocytes with low expression of miR-181a and overexpression of XIAP were constructed, and the effects of low expression of miR-181a and upregulation of XIAP on cardiomyocyte apoptosis were detected by flow cytometry. A dual luciferase reporter assay was used to detect the target relationship between miR-181a and XIAP. Further, H/R myocardial cells with low XIAP expression were constructed to observe the effect of downregulation of XIAP expression on apoptosis of myocardial cells with low expression of microarray-181a. The expression of apoptosis-related proteins Bax and Bcl-2 in myocardial cells was detected by Western blot analysis. After H/R treatment, the expression of microRNAs-181a was high but that of XIAP was low. The apoptosis of cardiomyocytes could be inhibited by both the low expression of miR-181a and the upregulation of XIAP. The results of dual luciferase reporter gene showed that XIAP was a potential target gene for miR-181a. The inhibitory effect of low expression of miR-181a on myocardial apoptosis could be reversed and the inhibitory effect of low expression of miR-181a on Bax protein expression and the promotion of Bcl-2 protein expression could be reversed by the downregulation of XIAP. MiR-181a can inhibit the apoptosis of hypoxic-reoxygenated cardiomyocytes by targeting XIAP to downregulate Bax and upregulate Bcl expression.

Direct matching methods for coils and preamplifiers in MRI.

In this paper, direct matching methods for coils and preamplifiers in receiver arrays are presented. Instead of compensating the reactance of the input impedance of preamplifiers, in our method, the reactance was used to resonate with the coil matching networks and thus to decouple the coils. Furthermore, coil matching networks and preamplifier input matching networks were combined, meaning the coil loop can be matched to the transistor in the preamplifier directly. These matching methods and, for comparison, the conventional matching method were implemented with custom-made preamplifiers and coils. Decoupling and noise-matching performance were compared between these three configurations. Phase shifting networks between coils and preamplifiers are not necessary in our matching methods. With fewer components, these matching networks showed lower noise factors, while similar preamplifier-decoupling performance was found for all three methods.

The noise factor of receiver coil matching networks in MRI.

In typical MRI applications the dominant noise sources in the received signal are the sample, the coil loop and the preamplifier. We hypothesize that in some cases (e.g. for very small receiver coils) the matching network noise has to be considered explicitly. Considering the difficulties of direct experimental determinations of the noise factor of matching networks with sufficient accuracy, it is helpful to estimate the noise factor by calculation. A useful formula of the coil matching network is obtained by separating commonly used coil matching network into different stages and calculating their noise factor analytically by a combination of the noise from these stages. A useful formula of the coil matching network is obtained. ADS simulations are performed to verify the theoretical predictions. Thereafter carefully-designed proof-of-concept phantom experiments are carried out to qualitatively confirm the predicted SNR behavior. The matching network noise behavior is further theoretically investigated for a variety of scenarios. It is found that in practice the coil matching network noise can be improved by adjusting the coil open port resonant frequency.

Design of a 3T preamplifier which stability is insensitive to coil loading.

In MRI (magnetic resonance imaging), preamplifiers are needed to amplify signals obtained from MRI receiver coils. Under various loading conditions of the corresponding receiver coils, preamplifiers see different source impedance at their input and may become unstable. Therefore preamplifiers which stability is not sensitive to coil loading are desirable. In this article, a coil-loading-insensitive preamplifier for MRI is presented, derived from an unstable preamplifier. Different approaches to improve stability were used during this derivation. Since a very low noise factor is essential for MRI preamplifiers, noise contributions from passive components in the MRI preamplifier have to be considered during the stabilization process. As a result, the initially unstable preamplifier became stable with regard to coil loading, while other MRI requirements, as the extremely low noise factor, were still fulfilled. The newly designed preamplifier was manufactured, characterized and tested in the MRI spectrometer. Compared to a commercially available preamplifier, the newly designed preamplifier has similar imaging performance but other advantages like smaller size and better stability. Furthermore, presented stabilization approaches can be generalized to stabilize other unstable low-noise amplifiers.

Sulforaphane prevents rat cardiomyocytes from hypoxia/reoxygenation injury in vitro via activating SIRT1 and subsequently inhibiting ER stress.

AIM: Sulforaphane (SFN), a natural dietary isothiocyanate, is found to exert beneficial effects for cardiovascular diseases. This study aimed to investigate the mechanisms underlying the protective effects of SFN in a model of myocardial hypoxia/reoxygenation (H/R) injury in vitro. METHODS: Cultured neonatal rat cardiomyocytes pretreated with SFN were subjected to 3-h hypoxia followed by 3-h reoxygenation. Cell viability and apoptosis were detected. Caspase-3 activity and mitochondrial membrane potential (ΔΨm) was measured. The expression of ER stress-related apoptotic proteins were analyzed with Western blot analyses. Silent information regulator 1 (SIRT1) activity was determined with SIRT1 deacetylase fluorometric assay kit. RESULTS: SFN (0.1-5 µmol/L) dose-dependently improved the viability of cardiomyocytes, diminished apoptotic cells and suppressed caspase-3 activity. Meanwhile, SFN significantly alleviated the damage of ΔΨm and decreased the expression of ER stress-related apoptosis proteins (GRP78, CHOP and caspase-12), elevating the expression of SIRT1 and Bcl-2/Bax ratio in the cardiomyocytes. Co-treatment of the cardiomyocytes with the SIRT1-specific inhibitor Ex-527 (1 µmol/L) blocked the SFN-induced cardioprotective effects. CONCLUSION: SFN prevents cardiomyocytes from H/R injury in vitro most likely via activating SIRT1 pathway and subsequently inhibiting the ER stress-dependent apoptosis.

Comparison of the degree of autophagy in neonatal rat cardiomyocytes and H9c2 cells exposed to hypoxia/reoxygenation.

BACKGROUND: Hypoxia/reoxygenation (H/R) is an important in vitro model for exploring the molecular mechanisms and functions of autophagy during myocardial ischemia/reperfusion (I/R). Neonatal rat cardiomyocytes (NRCM) and H9c2 cells are widely used to study H/R. METHODS: The degree of autophagy in NRCM and H9c2 cells exposed to H/R was assessed by detecting the markers of autophagy, Beclin-1 and LC3II. Autophagosomes were confirmed in both NRCM and H9c2 cells exposed to H/R using MDC staining and TEM. RESULTS: The expression levels of Beclin-1 and LC3II were significantly increased in NRCM under H/R conditions (p < 0.05 vs. control). In contrast, the expression levels of Beclin-1 and LC3II were significantly reduced in H9c2 cells exposed to H/R (p < 0.05 vs. control). The fluorescence intensity and the number of MDC-labeled particles were greater in NRCM exposed to H/R, compared to H9c2 cells (p < 0.05 vs. control). The number of autophagosomes exposed to H/R by TEM was greater in NRCM, compared to H9c2 cells, which was similar to the levels of autophagy markers observed in NRCM and H9c2 cells (p < 0.05 vs. control). CONCLUSIONS: NRCM may be more suitable to study autophagy during H/R than H9c2 cells.

Alpha-lipoic acid protects cardiomyocytes against hypoxia/reoxygenation injury by inhibiting autophagy.

Hypoxia/reoxygenation (H/R) is an important in vitro model for exploring the molecular mechanisms and functions of autophagy during myocardial ischemia/reperfusion (I/R). Alpha-lipoic acid (LA) plays an important role in the etiology of cardiovascular disease. Autophagy is widely implicated in myocardial I/R injury. We assessed the degree of autophagy by pretreatment with LA exposed to H/R in H9c2 cell based on the expression levels of Beclin-1, LC3II/LC3I, and green fluorescent protein-labeled LC3 fusion proteins. Autophagic vacuoles were confirmed in H9c2 cells exposed to H/R using transmission electron microscopy. Our findings indicated that pretreatment with LA inhibited the degree of autophagy in parallel to the enhanced cell survival and decreased total cell death in H9c2 cells exposed to H/R. We conclude that LA protects cardiomyocytes against H/R injury by inhibiting autophagy.

[Experimental study of electroacupuncture improving the obstruction of vestibular microcirculation in vertebrobasilar insufficiency and the effect on vestibulo-ocular reflex].

AIM: To investigate the mechanism of EA improving the obstruction of inner ear microcirculation and the effect on the vestibulo-ocular reflex (VOR) by comparing the effects of electroacupuncture (EA) with sibelium (flunarizine hydrochloride) on vertebrobasilar insufficiency(VBI). METHODS: Injected with sclerosant-775 injection into the solt tissue on the left side of cervical vertebral transverse processes of rabbits to set up the vertebral artery type of cervical spondylosis (VCS) models. Electronystagmography (ENG) induced by linear acceleration (LA) and horizontal rotation (HR), the transcranial Doppler (TCD), laser Doppler flowmetry (LDF) and hemorheology were used to measure changes of the frequencies of nystagmus, the hemodynamics in the basilar artery (BA), inner ear blood flow(IEBF) and blood viscosity in VBI rabbits. RESULTS: The frequencies of ENG, the velocity of blood flow in BA and IEBF decreased obviously, and whole blood middle where viscosity, whole blood lower where viscosity and erythrocyte distortion index ( EDI) increased significantly in the model group. Sibelium could reduce whole blood viscosity and EDI, and increased the systolic phase velocity (Vs) of blood flow in BA, but had no effect on diastolic phase velocity (Vd) and mean velocity (Vm). EA could not reduce the viscosity of blood and EDI, but had more significant effects on improving IEBF and ENG induced by LA than those of sibelium,and had the tendency of increasing Vs, Vd and Vm. EA and sibelium had no effect on improving ENG induced by HR. CONCLUSION: Inner ear microcirculation obstruction caused by VBI can induce dysfunctions of vestibule cyst macula and horizontal semicircular canals. EA may depend upon the neurohumoral regulation to improve VBI, and ameliorate inner ear blood supply obstruction by enhancing mechanism of local adjusting for microcirculation in the inner ear to recover vestibular cyst macula irritability for LA chiefly. There exist complicated mechanism that EA adjusts blood flow distribution and vestibular signal transduction in vestibular organ in VBI model likely, and remain to be researched deeply.