Brain-specific loss of Abcg1 disturbs cholesterol metabolism and aggravates pyroptosis and neurological deficits after traumatic brain injury.

Based on accumulating evidence, cholesterol metabolism dysfunction has been suggested to contribute to the pathophysiological process of traumatic brain injury (TBI) and lead to neurological deficits. As a key transporter of cholesterol that efflux from cells, the ATP-binding cassette (ABC) transporter family exerts many beneficial effects on central nervous system (CNS) diseases. However, there is no study regarding the effects and mechanisms of ABCG1 on TBI. As expected, TBI resulted in the different time-course changes of cholesterol metabolism-related molecules in the injured cortex. Considering ABCG1 is expressed in neuron and glia post-TBI, we generated nestin-specific Abcg1 knockout (Abcg1-KO) mice using the Cre/loxP recombination system. These Abcg1-KO mice showed reduced plasma high-density lipoprotein cholesterol levels and increased plasma lower-density lipoprotein cholesterol levels under the base condition. After TBI, these Abcg1-KO mice were susceptible to cholesterol metabolism turbulence. Moreover, Abcg1-KO exacerbated TBI-induced pyroptosis, apoptosis, neuronal cell insult, brain edema, neurological deficits, and brain lesion volume. Importantly, we found that treating with retinoid X receptor (RXR, the upstream molecule of ABCG1) agonist, bexarotene, in Abcg1-KO mice partly rescued TBI-induced neuronal damages mentioned above and improved functional deficits versus vehicle-treated group. These data show that, in addition to regulating brain cholesterol metabolism, Abcg1 improves neurological deficits through inhibiting pyroptosis, apoptosis, neuronal cell insult, and brain edema. Moreover, our findings demonstrate that the cerebroprotection of Abcg1 on TBI partly relies on the activation of the RXRalpha/PPARgamma pathway, which provides a potential therapeutic target for treating TBI.

Chronic restraint stress exacerbates neurological deficits and disrupts the remodeling of the neurovascular unit in a mouse intracerebral hemorrhage model.

Growing evidences have shown that patients recovering from stroke experience high and unremitting stress. Chronic restraint stress (CRS) has been found to exacerbate neurological impairments in an experimental focal cortical ischemia model. However, there have been no studies reporting the effect and mechanism of CRS on intracerebral hemorrhage (ICH). This study aimed to evaluate the effect of CRS on a mouse ICH model. Adult male C57BL mice were subjected to infusion of collagenase IV (to induce ICH) or saline (for sham) into the left striatum. After ICH, animals were stressed with application of CRS protocol for 21 days. Our results showed that CRS significantly exacerbated neurological deficits (Garcia test, corner turn test, and wire grip test) and the ipsilateral brain atrophy and reduced body weight gain after ICH. Immunofluorescence staining indicated that CRS exerted significant suppressive effects on neuron, astrocyte, vascular endothelial cell and pericyte and excessively activated microglia post ICH. All of the key cellular components mentioned above are involved in the neurovascular unit (NVU) remodeling in the peri-hemorrhagic region after ICH. Western blot results showed that matrix metalloproteinase (MMP)-9 and tight junction (TJ) proteins including zonula occludens-1, occludin and claudin-5 were increased after ICH, but MMP-9 protein was further up-regulated and TJ-related proteins were down-regulated by CRS. In addition, ICH-induced activation of endoplasmic reticulum stress and apoptosis were further strengthened by CRS. Collectively, CRS exacerbates neurological deficits and disrupts the remodeling of the peri-hemorrhagic NVU after ICH, which may be associated with TJ proteins degradation and excessive activation of MMP-9 and endoplasmic reticulum stress-apoptosis.LAY SUMMARYCRS exacerbates neurological deficits and disrupts the remodeling of the NVU in the recovery stage after ICH, which suggest that monitoring chronic stress levels in patients recovering from ICH may merit consideration in the future.

Salubrinal offers neuroprotection through suppressing endoplasmic reticulum stress, autophagy and apoptosis in a mouse traumatic brain injury model.

Traumatic brain injury (TBI) is a complex injury that can cause severe disabilities and even death. TBI can induce secondary injury cascades, including but not limited to endoplasmic reticulum (ER) stress, apoptosis and autophagy. Although the investigators has previously shown that salubrinal, the selective phosphatase inhibitor of p-eIF2α, ameliorated neurologic deficits in murine TBI model, the neuroprotective mechanisms of salubrinal need further research to warrant the preclinical value. This study was undertaken to characterize the effects of salubrinal on cell death and neurological outcomes following TBI in mice and the potential mechanisms. In the current study, ER stress-related proteins including p-eIF2α, GRP78 and CHOP showed peak expressions both in the cortex and hippocampus from day 2 to day 3 after TBI, indicating ER stress was activated in our TBI model. Immunofluorescence staining showed that CHOP co-located NeuN-positive neuron, GFAP-positive astrocyte, Iba-1-positive microglia, CD31-positive vascular endothelial cell and PDGFR-ß-positive pericyte in the cortex on day 2 after TBI, and these cells mentioned above constitute the neurovascular unit (NVU). We also found TBI-induced plasmalemma permeability, motor dysfunction, spatial learning and memory deficits and brain lesion volume were alleviated by continuous intraperitoneal administration of salubrinal post TBI. To investigate the underlying mechanisms further, we determined that salubrinal suppressed the expression of ER stress, autophagy and apoptosis related proteins on day 2 after TBI. In addition, salubrinal administration decreased the number of CHOP+/TUNEL+ and CHOP+/LC3+ cells on day 2 after TBI, detected by immunofluorescence. In conclusion, these data imply that salubrinal treatment improves morphological and functional outcomes caused by TBI in mice and these neuroprotective effects may be associated with inhibiting apoptosis, at least in part by suppressing ER stress-autophagy pathway.

IL-33/ST2L Signaling Provides Neuroprotection Through Inhibiting Autophagy, Endoplasmic Reticulum Stress, and Apoptosis in a Mouse Model of Traumatic Brain Injury.

Interleukin-33 (IL-33) is a member of the interleukin-1 (IL-1) cytokine family and an extracellular ligand for the orphan IL-1 receptor ST2. Accumulated evidence shows that the IL-33/ST2 axis plays a crucial role in the pathogenesis of central nervous system (CNS) diseases and injury, including traumatic brain injury (TBI). However, the roles and molecular mechanisms of the IL-33/ST2 axis after TBI remain poorly understood. In this study, we investigated the role of IL-33/ST2 signaling in mouse TBI-induced brain edema and neurobehavioral deficits, and further exploited underlying mechanisms, using salubrinal (SAL), the endoplasmic reticulum (ER) stress inhibitor and anti-ST2L. The increase in IL-33 level and the decrease in ST2L level at injured cortex were first observed at 24 h post-TBI. By immunofluorescent double-labeled staining, IL-33 co-localized in GFAP-positive astrocytes, and Olig-2-positive oligodendrocytes, and predominantly presented in their nucleus. Additionally, TBI-induced brain water content, motor function outcome, and spatial learning and memory deficits were alleviated by IL-33 treatment. Moreover, IL-33 and SAL alone, or their combination prevented TBI-induced the increase of IL-1ß and TNF-α levels, suppressed the up-regulation of ER stress, apoptosis and autophagy after TBI. However, anti-ST2L treatment could significantly invert the above effects of IL-33. Together, these data demonstrate that IL-33/ST2 signaling mitigates TBI-induced brain edema, motor function outcome, spatial learning and memory deficits, at least in part, by a mechanism involving suppressing autophagy, ER stress, apoptosis and neuroinflammation.

IL-33 Exerts Neuroprotective Effect in Mice Intracerebral Hemorrhage Model Through Suppressing Inflammation/Apoptotic/Autophagic Pathway.

Interleukin-33 (IL-33) is a recently identified member of the IL-1 family that exerts biologic functions by binding to a heterodimer composed of IL-1 receptor-related protein ST2L and IL-1RAcP. However, the role of IL-33 and whether IL-33 accounts for inflammation, apoptotic, and autophagic neuropathology after intracerebral hemorrhage (ICH) are not clear. Here, we established a mouse ICH model in this study, to determine the role of IL-33 and explore the underlying mechanism. Male mice were subjected to an infusion of type IV collagenase/saline into the left striatum to induce ICH/sham model. IL-33, soluble ST2 (sST2), or saline were also administered by a single intracerebroventricular (i.c.v.) injection, respectively. The results showed that the expression level of IL-33 markedly decreased within 6 h and reached the valleys at 6 and 72 h after ICH vs. sham group. In parallel, ST2L (a transmembrane form receptor of IL-33) significantly increased within 6 h and reached the peaks at 6 h and 24 h after ICH vs. sham group. In addition, administration of IL-33 alleviated cerebral water contents, reduced the number of PI- and TUNEL-positive cells, and improved neurological function after ICH. Moreover, IL-33 treatment apparently suppressed the expression of pro-inflammation cytokines IL-1ß and TNF-α, evidently increased Bcl-2 but decreased cleaved-caspase-3, and obviously decreased the levels of autophagy-associated proteins LC3-II and Beclin-1 but maintained P62 at high level after ICH. On the contrary, treatment with sST2, a decoy receptor of IL-33, exacerbated ICH-induced brain damage and neurological dysfunction by promoting apoptosis, and enhancing autophagic activity. In conclusion, IL-33 provides neuroprotection through suppressing inflammation, apoptotic, and autophagic activation in collagenase-induced ICH model.

IL-33 Provides Neuroprotection through Suppressing Apoptotic, Autophagic and NF-κB-Mediated Inflammatory Pathways in a Rat Model of Recurrent Neonatal Seizure.

Interleukin-33 (IL-33) is a novel identified chromatin-associated cytokine of IL-1 family cytokines. It signals through a heterodimer comprised of ST2L and IL-1RAcp, and plays a crucial role in many diseases. However, very little is known about the role and underlying intricate mechanisms of IL-33 in recurrent neonatal seizure (RNS). To determine whether IL-33 plays an important regulatory role, we established a neonatal seizure model in this study. Rats were subjected to recurrent seizures induced by inhaling volatile flurothyl. Recombinant IL-33 or PBS were also administered by intraperitoneally (IP) before surgery, respectively. Here, our current results indicated that RNS contributed to a significant reduction in IL-33 and its specific receptor (ST2L) expressions in cortex. While, in hippocampus, RNS induced an increase in IL-33 and ST2L evidently, compared with Sham group. After injection with IL-33, however, a remarkable increase in total IL-33 was detected both in brain cortex and hippocampus. In addition, IL-33 was mainly co-localized in the nuclear of GFAP+ astrocytes and the cytoplasm of the Iba-1+ microglia and IL-33+/NeuN+ merged cells. In parallel, ST2L was expressed mainly in the membrane of GFAP+ astrocytes, Iba-1+ microglia and NeuN+ neurons, respectively. Furthermore, administration of IL-33 improved RNS-induced behavioral deficits, promoted bodyweight gain, and ameliorated spatial learning and memory ability. Moreover, IL-33 pretreatment blocked the activation of NF-κB, resisted inflammatory cytokines IL-1ß and TNF-α increase, as well as suppressed apoptosis and autophagy activation after RNS. Collectively, IL-33 provides potential neuroprotection through suppressing apoptosis, autophagy and at least in part by NF-κB-mediated inflammatory pathways after RNS.

Dynamin-related protein 1 (Drp1) mediating mitophagy contributes to the pathophysiology of nervous system diseases and brain injury.

As the main source of energy (celluar ATP) in eukaryotic cells, mitochondria are involved in cellular physiology and pathology. The balance of mitochondrial dynamic, fission and fusion regulated by quality control mechanisms, provides a guarantee for maintaining mitochondrial function, even celluar function. Worn out mitochondria would be removed through mitophagy which is regulated by autophagy related proteins and mitochondrial membrane proteins. Drp1, dynamic-related protein 1, is regarded as one of the most important proteins to evaluate mitochondrial fission mediating mitophagy in neurodegenerative diseases (eg. Alzheimer's, Parkinson's, Huntington's, amyotrophic lateral sclerosis) and heart failure. Recent studies have focused on the roles of Drp1 in ischemia-induced mitophagy in the hippocampal CA3 region, and traumatic brain injury (TBI)-induced cell death together with functional deficits. However, the exact mechanisms have not been well characterized. In this review, we will discuss and clarify the role of Drp1 and mitophagy in nervous system diseases and brain injury therein, with a special emphasis on their molecular mechanisms mediating mitochondrial dynamics and mitophagy.

Mitochondrial division inhibitor 1 (Mdivi-1) offers neuroprotection through diminishing cell death and improving functional outcome in a mouse model of traumatic brain injury.

Mitochondria dysfunction, an enormous potential crisis, has attracted increasing attention. Disturbed regulation of mitochondrial dynamics, the balance of mitochondrial fusion and fission, has been implicated in neurodegenerative diseases, such as Parkinson׳s disease and cerebral ischemia/reperfusion. However the role of mitochondrial dynamics in traumatic brain injury (TBI) has not been illuminated. The aim of the present study was to investigate the role of Mdivi-1, a small molecule inhibitor of a key mitochondrial fission protein dynamin-related protein 1 (Drp1), in TBI-induced cell death and functional outcome deficits. Protein expression of Drp1 was first investigated. Outcome parameters consist of motor test, Morris water maze, brain edema and lesion volume. Cell death was detected by propidium iodide (PI) labeling, and mitochondrial morphology was assessed using transmission electron microscopy. In addition, the expression of apoptosis-related proteins cytochrome c (cyt-c) and caspase-3 was investigated. Our findings showed that up-regulation of Drp1 expression started at 1h post-TBI and peaked at 24 h, but inhibition of Drp1 by Mdivi-1 significantly alleviated TBI-induced behavioral deficits and brain edema, reduced morphological change of mitochondria, and decreased TBI-induced cell death together with lesion volume. Moreover, treatment with Mdivi-1 remarkably inhibited TBI-induced the release of cyt-c from mitochondria to cytoplasm, and activation of caspase-3 at 24 h after TBI. Taken together, these data imply that inhibition of Drp1 may help attenuate TBI-induced functional outcome and cell death through maintaining normal mitochondrial morphology and inhibiting activation of apoptosis.

JMJD2A contributes to breast cancer progression through transcriptional repression of the tumor suppressor ARHI.

INTRODUCTION: Breast cancer is a worldwide health problem and the leading cause of cancer death among females. We previously identified Jumonji domain containing 2A (JMJD2A) as a critical mediator of breast cancer proliferation, migration and invasion. We now report that JMJD2A could promote breast cancer progression through transcriptional repression of the tumor suppressor aplasia Ras homolog member I (ARHI). METHODS: Immunohistochemistry was performed to examine protein expressions in 155 cases of breast cancer and 30 non-neoplastic tissues. Spearman correlation analysis was used to analyze the correlation between JMJD2A expression and clinical parameters as well as several tumor regulators in 155 cases of breast cancer. Gene and protein expressions were monitored by quantitative polymerase chain reaction (qPCR) and Western blot. Results from knockdown of JMJD2A, overexpression of JMJD2A, Co-immunoprecipitation (Co-IP) assay, dual luciferase reporter gene assay and chromatin immunoprecipitation (ChIP) elucidated molecular mechanisms of JMJD2A action in breast cancer progression. Furthermore, the effects of ARHI overexpression on JMJD2A-mediated tumor progression were investigated in vitro and in vivo. For in vitro experiments, cell proliferation, wound-healing, migration and invasion were monitored by cell counting, scratch and Boyden Chamber assays. For in vivo experiments, control cells and cells stably expressing JMJD2A alone or together with ARHI were inoculated into mammary fat pads of mice. Tumor volume, tumor weight and metastatic nodules were measured by caliper, electronic balance and nodule counting, respectively. RESULTS: JMJD2A was highly expressed in human breast cancers and positively correlated with tumor progression. Knockdown of JMJD2A increased ARHI expression whereas overexpression of JMJD2A decreased ARHI expression at both protein and mRNA levels. Furthermore, E2Fs and histone deacetylases were involved in the transcriptional repression of ARHI expression by JMJD2A. And the aggressive behavior of JMJD2A in breast cancers could be reversed by re-expression of ARHI in vitro and in vivo. CONCLUSION: We demonstrated a cancer-promoting effect of JMJD2A and defined a novel molecular pathway contributing to JMJD2A-mediated breast cancer progression.

MicroRNA­21 regulation of the progression of viral myocarditis to dilated cardiomyopathy.

MicroRNAs (miRNAs) comprise a broad class of small non­coding RNAs that control the expression of complementary target messenger RNAs. The dysregulation of miRNAs by several mechanisms has been described in various disease states, including cardiac disease. Although an etiological link between viral myocarditis (VMC) and idiopathic dilated cardiomyopathy (DCM) has long been recognized, the true extent of this association is uncertain. Previous studies of the two diseases have focused on protein degradation systems. In the present study, miR­21 expression and its potential role in VMC and DCM was investigated. The expression levels of miR­21, its target gene sprouty homolog 1 (SPRY1) and mitogen­activated protein kinase (MAPK) were measured by quantitative polymerase chain reaction. The protein levels of SPRY1 and MAPK were also determined by western blotting. miR­21 levels were significantly increased in cardiac myocytes from VMC and DCM in comparison with control samples. The levels of SPRY1 were decreased and MAPK activity was increased. Using a bioinformatics­based approach, an identical potential binding site was identified in mouse miR­21 and the SPRY 3' untranslated region (3' UTR), suggesting a regulatory role for miR­21. In cultured, miRNA­transfected myocardial cells, the overexpression of miR­21 was associated with a decrease in SPRY1 protein expression and an increased expression of the MAPK protein. These findings revealed that changes in the expression of miRNAs may contribute to the pathogenesis of VMC to DCM and establish the therapeutic efficacy of miRNA targeted intervention in a cardiovascular disease setting.

Expression of JMJD2A in infiltrating duct carcinoma was markedly higher than fibroadenoma, and associated with expression of ARHI, p53 and ER in infiltrating duct carcinoma.

Jumonji Domain Containing 2A (JMJD2A) may be a cancer-associated gene involved in human breast cancer. With a view to investigating expression of JMJD2A in human breast cancer and benign lesion tissues as well as relationship between JMJD2A and tumor related proteins, histological and immunohistochemical analysis, Western blot and quantitative real-time PCR in infiltrating duct carcinoma and fibroadenoma for JMJD2A and immunohistochemical analysis and quantitative real-time PCR in infiltrating duct carcinoma for tumor related proteins (ARHI, p53, ER, PR and CerbB-2) were performed. Histological examination validated the clinical diagnosis. The JMJD2A positive rate of infiltrating duct carcinoma was significantly higher than fibroadenoma by immunohistochemical analysis. The mean optical density of JMJD2A in infiltrating duct carcinoma was higher than fibroadenoma by western blot. JMJD2A mRNA level in infiltrating duct carcinoma was higher than fibroadenoma by quantitative real-time PCR. Spearman correlation analysis revealed that the expression of JMJD2A was associated with ARHI, p53 and ER from immunohistochemical results respectively. Pearson correlation analysis revealed that the expression of JMJD2A was associated with ARHI, p53 and ER from quantitative real-time PCR results respectively. Expression of JMJD2A in infiltrating duct carcinoma was higher, and associated with ARHI, p53 and ER. The results may take JMJD2A as a potential diagnostic and therapeutic target in human breast cancer.

The selection of endogenous genes in human postmortem tissues.

Precisely determining the postmortem interval (PMI), which is crucial to criminal and forensic cases, is a research in which quantitative RT-PCR (also known as qRT-PCR or real-time RT-PCR) has been used to analyse gene expression levels and data normalisation should be required to eliminate the differences among the samples. Therefore, it is quite necessary to find stable molecular biological markers in PMI determination research. In this study, we compared nine commonly used endogenous markers (containing ACTB, GAPDH, B2M, U6, 18S rRNA, hsa-mir-1, hsa-mir-9, hsa-mir-194-1 and hsa-mir-203) in the 109 human tissue samples obtained from autopsy at the aim of finding stable markers in human tissues with consideration of the impact of parameters (PMI and cause of death). After RNA was extracted from four tissues (heart, brain, kidney, skin), the Ct values of nine endogenous markers were obtained by qRT-PCR and assessed by geNorm software. The results showed that U6, GAPDH and 18S rRNA were the suitable markers in our set of samples in various corpse conditions, that B2M and ACTB were reliable internal controls in heart tissue only, and that microRNAs had such high M values that they should not be chosen for endogenous control genes.

Lipoxin A4 attenuates brain damage and downregulates the production of pro-inflammatory cytokines and phosphorylated mitogen-activated protein kinases in a mouse model of traumatic brain injury.

The present study was designed to investigate the effects of lipoxin A4 (LXA4) on traumatic brain injury (TBI) and to analyze the possible mechanism. Outcome parameters consist of blood-brain barrier (BBB) breakdown, brain edema and lesion volume. Using western blot and quantitative real-time PCR, we examined the levels of pro-inflammatory cytokines (TNF-α, IL-1ß, IL-6) and activation of mitogen-activated protein kinases (MAPKs) (including ERK, JNK, p38) following TBI. To investigate the cell types in which the LXA4 receptor (ALXR) staining was localized, brain sections pulsed with ALXR were subjected to immunofluorescence staining with antibodies against cell type-specific antigens. Our findings show that LXA4 decreases BBB permeability, attenuates brain edema, and reduces TBI-induced lesion volume. In addition, LXA4 inhibits TBI-induced elevation of mRNA and protein levels of pro-inflammatory cytokines (TNF-α, IL-1ß, IL-6). In the injured cortex at 24h post-TBI, the phosphorylated-ERK (p-ERK) and -JNK (p-JNK), but not -p38 (p-p38) levels were increased. The p-ERK and p-JNK production were attenuated by their respective inhibitors (PD98059 and SP600125), as well as LXA4. Moreover, ALXR was found to label more GFAP positive cells, whereas few CD11b-positive cells were labeled by ALXR within the layers of the injured cortex at 24h post-TBI. The activation of ALXR in astrocytes was partially enhanced by LXA4 treatment. Taken together, these data indicate that TBI activates pro-inflammatory cytokines, the MAPK pathways together with ALXR in astrocytes, and these mechanisms may be exploited by pharmacological interventions.

Therapeutic effect of SN50, an inhibitor of nuclear factor-κB, in treatment of TBI in mice.

NF-κB upregulation has been demonstrated in neurons and glial cells in response to experimental injury and neuropathological disorders, where it has been related to both neurodegenerative and neuroprotective activities. It has been generally recognized that NF-κB plays important roles in the regulation of apoptosis and inflammation as well as innate and adaptive immunity. However, the regulatory mechanism of NF-κB in apoptosis remained to be determined. The present study sought to first investigate the effect of a NF-κB inhibitor SN50, which inhibits NF-κB nuclear translocation, on cell death and behavioral deficits in our mice traumatic brain injury (TBI) models. Additionally, we tried to elucidate the possible mechanisms of the therapeutic effect of SN50 through NF-κB regulating apoptotic and inflammatory pathway in vivo. Encouragingly, the results showed that pretreatment with SN50 remarkably attenuated TBI-induced cell death (detected by PI labeling), cumulative loss of cells (detected by lesion volume), and motor and cognitive dysfunction (detected by motor test and Morris water maze). To analyze the mechanism of SN50 on cell apoptotic and inflammatory signaling pathway, we thus assessed expression levels of TNF-α, cathepsin B and caspase-3, Bid cleavage and cytochrome c release in SN50-pretreated groups compared with those in saline vehicle groups. The results imply that through NF-κB/TNF-α/cathepsin networks SN50 may contribute to TBI-induced extrinsic and intrinsic apoptosis, and inflammatory pathways, which partly determined the fate of injured cells in our TBI model.

Poloxamer 188 attenuates in vitro traumatic brain injury-induced mitochondrial and lysosomal membrane permeabilization damage in cultured primary neurons.

Acute membrane damage due to traumatic brain injury (TBI) is a critical precipitating event. However, the subsequent effects of the mechanical trauma, including mitochondrial and lysosomal membrane permeability (MOMP and LMP) remain elusive. The main objective of the current study was to assess the role of a putative membrane-resealing agent poloxamer 188 (P188) in MOMP and LMP in response to a well-defined mechanical insult. Using an in vitro cell shearing device (VCSD), mechanical injury resulted in immediate disruption of membrane integrity in cultured primary neurons, and neurons were treated with P188 or a cathepsin B inhibitor (CBI) after VCSD 10 min. The protective effect of P188 on cultured primary neurons was first detected visually with a light microscope, and measured by MTT assay and LDH assay. The validity of monitoring changes in mitochondrial membrane potential (ΔΨm) was measured by JC-1 staining, and Western blot for cytochrome c and truncated Bid (tBid) in purified mitochondria was also performed. In addition, lysosomal integrity was detected by blotting for cathepsin B and tBid in purified lysosomes. Our results showed post-injury P188 treatment moderated the dissipation of ΔΨm in mitochondria, and inhibited VCSD-induced cytochrome c release from mitochondria as well as cathepsin B from lysosomes. Cathepsin B inhibition (CBI) could also increase cell viability, maintain mitochondrial membrane potential, and repress VCSD-induced release of cytochrome c from mitochondria to cytosol. Both P188 and CBI treatment decreased the cytosolic accumulation of tBid in supernatant of purified lysosomes, and the amount of mitochondrial localized tBid. These data indicate injured neurons have undergone mitochondrial and lysosomal membrane permeability damage, and the mechanism can be exploited with pharmacological interventions. P188's neuroprotection appears to involve a relationship between cathepsin B and tBid-mediated mitochondrial initiation of cell death.

Effects of siRNA-mediated knockdown of jumonji domain containing 2A on proliferation, migration and invasion of the human breast cancer cell line MCF-7.

Jumonji domain containing 2A (JMJD2A) is a potential cancer-associated gene that may be involved in human breast cancer. The present study aimed to investigate suppressive effects on the MCF-7 human breast cancer cell line by transfection with JMJD2A-specific siRNA. Quantitative real-time PCR and western blot analysis were used to detect the expression levels of JMJD2A. Flow cytometric (FCM) analysis and WST-8 assay were used to evaluate cell proliferation. Boyden chambers were used in cell migration and invasion assays to evaluate the cell exercise capacity. Expression levels of JMJD2A mRNA and protein in the siRNA group were both downregulated successfully by transfection. FCM results showed that the percentage of cells in the G0/G1 phase in the siRNA group was significantly greater than that in the blank (P<0.05) and negative control groups (P<0.05). Additionally, the mean absorbance in the siRNA group was significantly lower (P<0.05), as observed by WST-8 assay. Moreover, a decreased number of migrated cells in the siRNA group was observed (P<0.05) using a cell migration and invasion assay. These data indicated that knockdown of JMJD2A may cause inhibition of proliferation, migration and invasion of MCF-7 cells. This study provides a new perspective in understanding the molecular mechanisms underlying the progression of breast cancer and offers a potential therapeutic target for breast cancer.

MicroRNA- 1 represses Cx43 expression in viral myocarditis.

MicroRNAs (miRNAs) are increasingly reported to have important roles in diverse biological and pathological processes. Changes in abundance of muscle-specific microRNA, miR-1, have been implicated in cardiac disease, including arrhythmia and heart failure. However, the specific molecular targets and cellular mechanisms involved in the miR-1 function in the heart are only beginning to emerge. In this study, we investigated miR-1 expression and its potential role in the mouse model of viral myocarditis (VMC). The expression levels of miR-1 and its target gene Connexin 43 (Cx43) were measured by real-time PCR and western blotting, respectively. The miR-1 expression levels were significantly increased in cardiac myocytes from VMC mice in comparison with control samples (relative expression: 10 ± 2.5 vs. 31 ± 7.6, P < 0.05). Among the target genes of miR-1, the expression Cx43 protein was significantly reduced in such mice while there was no significant difference in the its mRNA levels. Our results revealed an inverse correlation between miR-1 levels and Cx43 protein expression in VMC samples. Using a bioinformatics-based approach, we found two identical potential binding sites were found in mouse miR-1 and Cx43 3'- untranslated region, this confirms a possible regulatory role of miR-1. In cultured, miRNA transfected myocardial cells, we show overexpression of miR-1 accompanied by a decrease in Cx43 protein's expression. There was only a slight (not statistically significant) drop in Cx43 mRNA levels. Our results indicate that miR-1 is involved in VMC via post-transcriptional repression of Cx43, and might constitute potentially valuable data for the development of a new approach in the treatment of this disease.

Effects of RNA interference-mediated gene silencing of JMJD2A on human breast cancer cell line MDA-MB-231 in vitro.

Previous data demonstrate that JMJD2A is a cancer-associated gene and may be involved in human breast cancer by demethylation of H3K9me3. The aim of this study was to investigate depressive effects on JMJD2A by transfection with JMJD2A-sepcific siRNA in human breast cancer cell line MDA-MB-231 and effects on cell proliferation, invasion and migration. JMJD2A-specific siRNA was chemically synthesised and transfected into human breast cancer cell line MDA-MB-231. Expression levels of JMJD2A were detected by quantitative real-time PCR and Western blot analysis. Cells proliferation was evaluated by using flow cytometric anlysis and MTT assay. The abilities of invasion and migration were evaluated by cell migration and invasion assay with Boyden chambers. The results showed that the transfection was successful and expression levels of JMJD2A mRNA and protein in siRNA group were both down-regulated. By MTT assay, the mean actual absorbance in siRNA group was significantly lower than that in blank control group (P < 0.05) and negative control group (P < 0.05). In addition, the percentage of cells in G0/G1 phase in siRNA group was significantly more than that in blank control group (P < 0.05) and negative control group (P < 0.05). Furthermore, by cell invasion and migration assay, the decreased number of migrated cells in siRNA group was observed (P < 0.05). These data imply that silencing JMJD2A gene could result in cell cycle change and proliferation inhibition, and lead to suppress tumor cell invasion and migration. It provides a new perspective in understanding the pleiotropic functions of JMJD2A and its contribution to human breast cancer.

Cathepsin B contributes to traumatic brain injury-induced cell death through a mitochondria-mediated apoptotic pathway.

It has been reported that lysosomal proteases play important roles in ischemic and excitotoxic neuronal cell death. We have previously reported that cathepsin B expression increased remarkably after traumatic brain injury (TBI). The present study sought to investigate the effects of a selective cathepsin B inhibitor (CBI) [N-L-3-trans-prolcarbamoyloxirane-2-carbonyl)-L-isoleucyl-L-proline] on cell death and behavioral deficits in our model. We examined the levels of cathepsin B enzymatic activity and its expression by double labelling damaged cells in the brain slice with propidium iodide (PI) and anticathepsin B. The results showed an elevated enzymatic activity associated with TBI-induced increase in a mature form of cathepsin B, suggesting that cathepsin B may play a role in TBI-induced cell injury. PI was found to label cells positive for the neuronal-specific nuclear marker NeuN, whereas fewer GFAP-positive cells were labelled by PI, suggesting that neurons are more sensitive to cell death induced by TBI. Additionally, we found that pretreatment with CBI remarkably attenuated TBI-induced cell death, lesion volume, and motor and cognitive dysfunction. To analyze the mechanism of action of cathepsin B in the cell death signaling pathway, we assessed DNA fragmentation by electrophoresis, Bcl-2/Bax protein expression levels, Bid cleavage, cytochrome c release, and caspase-3 activation. The results imply that cathepsin B contributes to TBI-induced cell death through the present programmed cell necrosis and mitochondria-mediated apoptotic pathways.