MiR-137 mediated high expression of TIGD1 promotes migration, invasion, and suppresses apoptosis of lung adenocarcinoma.

OBJECTIVES: Tigger transposable element-derived 1 (TIGD1) expression and its underlying functions and regulatory mechanisms in lung adenocarcinoma (LUAD) remain unknown. Therefore, we intended to explore the expression, potential functions, and regulatory mechanisms of TIGD1 in LUAD. MATERIALS AND METHODS: TIGD1 expression in LUAD tissues was determined by immunohistochemistry analysis of a tissue microarray. Functional experiments were conducted to determine how TIGD1 affects LUAD tumorigenesis and metastasis. The molecular mechanisms by which TIGD1 induces LUAD progression were determined. RESULTS: TIGD1 was upregulated in LUAD tissues and was related to lymph node metastases. TIGD1 knockdown suppressed LUAD cell proliferation, migration, and invasion, while promoted cell apoptosis. Furthermore, decreased metastatic nodules were observed in the TIGD1 knockdown mouse metastasis model. Moreover, microarray analysis was performed to determine the potential downstream genes of TIGD1 in LUAD. Hallmark pathway analysis revealed that the downstream genes of TIGD1 were involved in epithelial-mesenchymal transition (EMT). Western blotting confirmed that vimentin and TWIST was downregulated in TIGD1 knockdown cells, while E-cadherin was upregulated. Ingenuity pathway and hallmark pathway analyses revealed that TIGD1 regulated the interleukin-6 signaling pathway and related gene members. Western blotting, quantitative real-time polymerase chain reaction, enzyme-linked immunosorbent assay indicated that downregulation of TIGD1 decreased interleukin-6 and CXCL1 expression. TIGD1 expression was negatively correlated with immune infiltration in LUAD. The upstream microRNA of TIGD1 was predicted, and subsequent luciferase reporter gene experiments confirmed the interactions between miR-137 and TIGD1. The expression of miR-137 was significantly downregulated in LUAD tissues and miR-137 suppressed the proliferation, migration, and invasion of LUAD cells, partially through negatively regulating the expression of TIGD1. CONCLUSION: Our findings suggest that TIGD1, which was regulated by miR-137, contributed to LUAD progression by promoting cell proliferation, migration, invasion, and EMT and suppressing cell apoptosis.

Bi-directional neuro-immune dysfunction after chronic experimental brain injury.

BACKGROUND: It is well established that traumatic brain injury (TBI) causes acute and chronic alterations in systemic immune function and that systemic immune changes contribute to posttraumatic neuroinflammation and neurodegeneration. However, how TBI affects bone marrow (BM) hematopoietic stem/progenitor cells chronically and to what extent such changes may negatively impact innate immunity and neurological function has not been examined. METHODS: To further understand the role of BM cell derivatives on TBI outcome, we generated BM chimeric mice by transplanting BM from chronically injured or sham (i.e., 90 days post-surgery) congenic donor mice into otherwise healthy, age-matched, irradiated CD45.2 C57BL/6 (WT) hosts. Immune changes were evaluated by flow cytometry, multiplex ELISA, and NanoString technology. Moderate-to-severe TBI was induced by controlled cortical impact injury and neurological function was measured using a battery of behavioral tests. RESULTS: TBI induced chronic alterations in the transcriptome of BM lineage-c-Kit+Sca1+ (LSK+) cells in C57BL/6 mice, including modified epigenetic and senescence pathways. After 8 weeks of reconstitution, peripheral myeloid cells from TBI→WT mice showed significantly higher oxidative stress levels and reduced phagocytic activity. At eight months after reconstitution, TBI→WT chimeric mice were leukopenic, with continued alterations in phagocytosis and oxidative stress responses, as well as persistent neurological deficits. Gene expression analysis revealed BM-driven changes in neuroinflammation and neuropathology after 8 weeks and 8 months of reconstitution, respectively. Chimeric mice subjected to TBI at 8 weeks and 8 months post-reconstitution showed that longer reconstitution periods (i.e., time post-injury) were associated with increased microgliosis and leukocyte infiltration. Pre-treatment with a senolytic agent, ABT-263, significantly improved behavioral performance of aged C57BL/6 mice at baseline, although it did not attenuate neuroinflammation in the acutely injured brain. CONCLUSIONS: TBI causes chronic activation and progressive dysfunction of the BM stem/progenitor cell pool, which drives long-term deficits in hematopoiesis, innate immunity, and neurological function, as well as altered sensitivity to subsequent brain injury.

Knockdown of LRRK2 inhibits the progression of lung cancer by regulating TLR4/NF-κB pathways and NLRP3 inflammasome.

Leucine-rich repeat kinase 2 (LRRK2) plays an important role in a variety of inflammatory diseases, as well as peripheral and central immune responses. At present, there are few reports about the role of LRRK2 in lung cancer, and need to be further explored. The main purpose of this study is to explore the role and mechanism of LRRK2 in lung cancer. The results revealed that the expression of LRRK2 was increased in the tissues of lung cancer patient and lung cancer cells. Further studies found that interference with LRRK2 expression significantly induced the apoptosis, and promoted the expression of caspase-3, caspase-9, and Bax. More importantly, si-LRRK2 inhibited the expression of VEGF and P-gp, indicating inhibition of cell proliferation and drug resistance. What's more, LRRK2 regulated TLR4/NF-κB signaling pathways and NLRP3 inflammasome, and TLR4/NF-κB pathways was involved in the molecular mechanism of LRRK2 on lung cancer cells. In conclusion, this study suggested that the mechanism of si-LRRK2 inhibiting the progression of lung cancer is to regulate the TLR4/NF-κB signaling pathways and NLRP3 inflammasome.

The brain-bone marrow axis and its implications for chronic traumatic brain injury.

Traumatic brain injury (TBI) causes acute and chronic alterations in systemic immune function which contribute to posttraumatic neuroinflammation and neurodegeneration. However, how TBI affects bone marrow (BM) hematopoietic stem/progenitor cells chronically and to what extent such changes may negatively impact innate immunity and neurological function has not been examined. To further understand the role of BM cell derivatives on TBI outcome, we generated BM chimeric mice by transplanting BM from chronically injured or sham congenic donor mice into otherwise healthy, age-matched, irradiated hosts. After 8 weeks of reconstitution, peripheral myeloid cells from TBI→WT mice showed significantly higher oxidative stress levels and reduced phagocytic activity. At eight months after reconstitution, TBI→WT chimeric mice were leukopenic, with continued alterations in phagocytosis and oxidative stress responses, as well as persistent neurological deficits. Gene expression analysis revealed BM-driven changes in neuroinflammation and neuropathology after 8 weeks and 8 months of reconstitution, respectively. Chimeric mice subjected to TBI showed that longer reconstitution periods were associated with increased microgliosis and leukocyte infiltration. Thus, TBI causes chronic activation and progressive dysfunction of the BM stem/progenitor cell pool, which drives long-term deficits in innate immunity and neurological function, as well as altered sensitivity to subsequent brain injury.

Inhibition of autophagy in microglia and macrophages exacerbates innate immune responses and worsens brain injury outcomes.

Excessive and prolonged neuroinflammation following traumatic brain injury (TBI) contributes to long-term tissue damage and poor functional outcomes. However, the mechanisms contributing to exacerbated inflammatory responses after brain injury remain poorly understood. Our previous work showed that macroautophagy/autophagy flux is inhibited in neurons following TBI in mice and contributes to neuronal cell death. In the present study, we demonstrate that autophagy is also inhibited in activated microglia and infiltrating macrophages, and that this potentiates injury-induced neuroinflammatory responses. Macrophage/microglia-specific knockout of the essential autophagy gene Becn1 led to overall increase in neuroinflammation after TBI. In particular, we observed excessive activation of the innate immune responses, including both the type-I interferon and inflammasome pathways. Defects in microglial and macrophage autophagy following injury were associated with decreased phagocytic clearance of danger/damage-associated molecular patterns (DAMP) responsible for activation of the cellular innate immune responses. Our data also demonstrated a role for precision autophagy in targeting and degradation of innate immune pathways components, such as the NLRP3 inflammasome. Finally, inhibition of microglial/macrophage autophagy led to increased neurodegeneration and worse long-term cognitive outcomes after TBI. Conversely, increasing autophagy by treatment with rapamycin decreased inflammation and improved outcomes in wild-type mice after TBI. Overall, our work demonstrates that inhibition of autophagy in microglia and infiltrating macrophages contributes to excessive neuroinflammation following brain injury and in the long term may prevent resolution of inflammation and tissue regeneration.Abbreviations: Becn1/BECN1, beclin 1, autophagy related; CCI, controlled cortical impact; Cybb/CYBB/NOX2: cytochrome b-245, beta polypeptide; DAMP, danger/damage-associated molecular patterns; Il1b/IL1B/Il-1ß, interleukin 1 beta; LAP, LC3-associated phagocytosis; Map1lc3b/MAP1LC3/LC3, microtubule-associated protein 1 light chain 3 beta; Mefv/MEFV/TRIM20: Mediterranean fever; Nos2/NOS2/iNOS: nitric oxide synthase 2, inducible; Nlrp3/NLRP3, NLR family, pyrin domain containing 3; Sqstm1/SQSTM1/p62, sequestosome 1; TBI, traumatic brain injury; Tnf/TNF/TNF-α, tumor necrosis factor; Ulk1/ULK1, unc-51 like kinase 1.

Sesquiterpenoids from Seriphidium transiliense and Their Melanogenic Activity.

A sesquiterpenoid with an unprecedented 5/5/4 tricyclic skeleton (1), a nor-sesquiterpenoid with a rare 6/7 bicyclic skeleton (2), 10 new sesquiterpenoids (3-12), and six known analogues (13-18) were isolated from the whole plants of Seriphidium transiliense. The structures of compounds 1-12 were elucidated by spectroscopic data analysis. Compound 7 showed melanogenic promotion activity in murine melanoma (B16) cells more potent than the positive control used, 8-methoxypsoralen (8-MOP). Further mechanistic studies indicated that compound 7 promotes melanogenesis through activating the transcription of microphthalmia-associated transcription factor (MITF) and tyrosinase family genes in B16 cells. Moreover, compound 7 also inhibited the expression of IFN-γ-chemokine through the JAK/STAT signaling pathway in immortalized human keratinocyte (HaCaT) cells. These results suggest that the sesquiterpenoid 7 shows potential activity for treating vitiligo.

Circ_0040929 Serves as Promising Biomarker and Potential Target for Chronic Obstructive Pulmonary Disease.

Background: Circular RNAs (circRNAs) can act as essential regulators in many diseases, including chronic obstructive pulmonary disease (COPD). We aimed to explore the role and underlying mechanism of circ_0040929 in COPD. Methods: A cellular model of COPD was constructed by treating human bronchial epithelial cells (16HBE) with cigarette smoke extract (CSE). The levels of circ_0040929, microRNA-515-5p (miR-515-5p) and insulin-like growth factor-binding protein 3 (IGFBP3) were measured by quantitative real-time PCR. Cell proliferation was assessed by Cell Counting Kit-8 and 5-ethynyl-2'-deoxyuridine assays. Cell apoptosis was evaluated by flow cytometry. Protein expression was measured using Western blot assay. The levels of inflammatory factors and airway remodeling were assayed via enzyme-linked immunosorbent assay. The interaction between miR-515-5p and circ_0040929/IGFBP3 was confirmed by dual-luciferase reporter, RNA pull-down and RNA immunoprecipitation assays. Exosomes were detected using transmission electron microscopy. Results: Circ_0040929 expression and IGFBP3 expression were upregulated in the serum of smokers (n = 22) compared to non-smokers (n = 22) and more significantly upregulated in the serum of COPD patients (n = 22). However, miR-515-5p expression was decreased in the serum of smokers compared to non-smokers and further reduced in the serum of COPD. Circ_0040929 knockdown attenuated CSE-induced cell injury by increasing proliferation and reducing apoptosis, inflammation, and airway remodeling in 16HBE cells. MiR-515-5p was a direct target of circ_0040929, and miR-515-5p inhibition reversed the effect of circ_0040929 knockdown in CSE-treated 16HBE cells. IGFBP3 was a direct target of miR-515-5p, and miR-515-5p overexpression alleviated CSE-induced cell injury via targeting IGFBP3. Moreover, circ_0040929 regulated IGFBP3 expression by targeting miR-515-5p. Importantly, circ_0040929 was upregulated in serum exosomes from COPD patients. Conclusion: Circ_0040929 played a promoting role in CSE-induced COPD by regulating miR-515-5p/IGFBP3 axis, suggesting that it might be a novel potential target for COPD treatment.

Targeted Multiple Reaction Monitoring Analysis of CSF Identifies UCHL1 and GPNMB as Candidate Biomarkers for ALS.

The neurodegenerative diseases amyotrophic lateral sclerosis (ALS) and Parkinson's disease (PD) share some common molecular deficits including disruption of protein homeostasis leading to disease-specific protein aggregation. While insoluble protein aggregates are the defining pathological confirmation of diagnosis, patient stratification based on early molecular etiologies may identify distinct subgroups within a clinical diagnosis that would respond differently in therapeutic development programs. We are developing targeted multiple reaction monitoring (MRM) mass spectrometry methods to rigorously quantify CSF proteins from known disease genes involved in lysosomal, ubiquitin-proteasomal, and autophagy pathways. Analysis of CSF from 21 PD, 21 ALS, and 25 control patients, rigorously matched for gender, age, and age of sample, revealed significant changes in peptide levels between PD, ALS, and control. In patients with PD, levels of two peptides for chromogranin B (CHGB, secretogranin 1) were significantly reduced. In CSF of patients with ALS, levels of two peptides from ubiquitin carboxy-terminal hydrolase like protein 1 (UCHL1) and one peptide each for glycoprotein non-metastatic melanoma protein B (GPNMB) and cathepsin D (CTSD) were all increased. Analysis of patients with ALS separated into two groups based on length of survival after CSF sampling revealed that the increases in GPNMB and UCHL1 were specific for short-lived ALS patients. While analysis of additional cohorts is required to validate these candidate biomarkers, this study suggests methods for stratification of ALS patients for clinical trials and identifies targets for drug efficacy measurements during therapeutic development.

Inhibition of microRNA-711 limits angiopoietin-1 and Akt changes, tissue damage, and motor dysfunction after contusive spinal cord injury in mice.

Spinal cord injury (SCI) causes neuronal cell death and vascular damage, which contribute to neurological dysfunction. Given that many biochemical changes contribute to such secondary injury, treatment approaches have increasingly focused on combined therapies or use of multi-functional drugs. MicroRNAs (miRs) are small (20-23 nucleotide), non-protein-coding RNAs and can negatively regulate target gene expression at the post-transcriptional level. As individual miRs can potentially modulate expression of multiple relevant proteins after injury, they are attractive candidates as upstream regulators of the secondary SCI progression. In the present study we examined the role of miR-711 modulation after SCI. Levels of miR-711 were increased in injured spinal cord early after SCI, accompanied by rapid downregulation of its target angiopoietin-1 (Ang-1), an endothelial growth factor. Changes of miR-711 were also associated with downregulation of the pro-survival protein Akt (protein kinase B), another target of miR-711, with sequential activation of glycogen synthase kinase 3 and the pro-apoptotic BH3-only molecule PUMA. Central administration of a miR-711 hairpin inhibitor after SCI limited decreases of Ang-1/Akt expression and attenuated apoptotic pathways. Such treatment also reduced neuronal/axonal damage, protected microvasculature and improved motor dysfunction following SCI. In vitro, miR-711 levels were rapidly elevated by neuronal insults, but not by activated microglia and astrocytes. Together, our data suggest that post-traumatic miR-711 elevation contributes to neuronal cell death after SCI, in part by inhibiting Ang-1 and Akt pathways, and may serve as a novel therapeutic target.

Inhibition of NOX2 signaling limits pain-related behavior and improves motor function in male mice after spinal cord injury: Participation of IL-10/miR-155 pathways.

NADPH oxidase (NOX2) is an enzyme that induces reactive oxygen species (ROS) and serves as a switch between the pro-inflammatory and neurorestorative microglial/macrophage phenotypes; such changes play an important role in neuropathic pain and motor dysfunction. Increased NOX2 expression after spinal cord injury (SCI) has been reported, and inhibition of NOX2 improves motor function. However, the underlying mechanisms of NOX2 in post-traumatic pain and motor deficit remain unexplored. In the present study, we report that depletion of NOX2 (NOX2-/-) or inhibition of NOX2 using NOX2ds-tat significantly reduced mechanical/thermal cutaneous hypersensitivity and motor dysfunction after moderate contusion SCI at T10 in male mice. Western blot (WB, 3 mm lesion area) and immunohistochemistry (IHC) showed that SCI elevates NOX2 expression predominantly in microglia/macrophages up to 8 weeks post-injury. Deletion of NOX2 significantly reduced CD11b+/CD45hiF4/80+ macrophage infiltration at 24 h post-injury detected by flow cytometry and 8-OHG+ ROS production at 8 weeks post-injury by IHC in both lesion area and lumbar enlargement. NOX2 deficiency also altered microglial/macrophage pro-inflammatory and anti-inflammatory balance towards the neurorestorative response. WB analysis showed robust increase of Arginase-1 and YM1 proteins in NOX2-/- mice. Furthermore, qPCR analysis showed significant up-regulation of anti-inflammatory cytokine IL-10 levels in NOX2-/- mice, associated with reduced microRNA-155 expression. These findings were confirmed in CD11b+ microglia/macrophages isolated from spinal cord at 3 days post-injury. Taken together, our data suggest an important role for IL-10/miR-155 pathway in regulating NOX2-mediated SCI-dysfunction. Thus, specific targeting of NOX2 may provide an effective strategy for treating neurological dysfunction in SCI patients.

ECG conduction disturbances and ryanodine receptor expression levels in occupational lead exposure workers.

OBJECTIVES: A significant number of researches have evidenced that occupational lead (Pb) exposure increased risks of cardiovascular disease. However, evidences about the potential effects of Pb on the cardiac conduction system are sparse and inconclusive. Besides, ryanodine receptors (RyRs) induced dysfunction of cardiac excitation contraction coupling which is considered to be one of the mechanisms in cardiovascular diseases. Therefore, we examined the association between occupational Pb exposure and ECG conduction abnormalities, as well as RyRs in Pb-induced ECG abnormalities. METHODS: We investigated 529 Pb smelter workers, and measured blood lead (BPb), zinc protoporphyrin (ZPP), ECG outcomes and RyR expression levels. Based on BPb levels, the workers were divided into three groups: the BPb not elevated group, the BPb elevated group and the Pb poisoning group. Descriptive and multivariable analyses were performed. RESULTS: Compared with the BPb not elevated group, the Pb poisoning group had a higher incidence of high QRS voltage, and a lower level of RyR1 gene expression (p<0.05). Further unconditional multivariable logistic regression analyses showed that high QRS voltage was positively related to BPb (OR=1.045, 95% CI 1.014 to 1.078) and inversely associated with RyR1 expression (OR=0.042, 95% CI 0.002 to 0.980) after adjusting for potential confounders. In addition, multiple linear regression analyses showed that the QTc interval was positively associated with ZPP (ß=0.299, 95% CI 0.130 to 0.468) after adjusting for potential confounders. CONCLUSIONS: Our study provided evidences that occupational exposure to Pb may be associated with worse ECG outcomes (high QRS voltage), which might be related to decreased levels of RyR1.

Lysosomal damage after spinal cord injury causes accumulation of RIPK1 and RIPK3 proteins and potentiation of necroptosis.

Necroptosis, a regulated necrosis pathway mediated by the receptor-interacting protein kinases 1 and 3 (RIPK1 and RIPK3), is induced following spinal cord injury (SCI) and thought to contribute to neuronal and glial cell death. However, mechanisms leading to activation of necroptosis after SCI remain unclear. We have previously shown that autophagy, a catabolic pathway facilitating degradation of cytoplasmic proteins and organelles in a lysosome-dependent manner, is inhibited following SCI in rats. Our current data confirm that inhibition of autophagy also occurs after thoracic contusive SCI in the mouse model, as indicated by accumulation of both the autophagosome marker, LC3-II and autophagy cargo protein, p62/SQSTM1. This was most pronounced in the ventral horn neurons and was caused by rapid inhibition of lysosomal function after SCI. Interestingly, RIPK1, RIPK3, and the necroptosis effector protein MLKL also rapidly accumulated after SCI and localized to neurons with disrupted autophagy, suggesting that these events may be related. To determine if lysosomal dysfunction could contribute to induction of necroptosis, we treated PC12 cells and primary rat cortical neurons with lysosomal inhibitors. This led to rapid accumulation of RIPK1 and RIPK3, confirming that they are normally degraded by the lysosomal pathway. In PC12 cells lysosomal inhibition also sensitized cells to necroptosis induced by tumor necrosis factor α (TNFα) and caspase inhibitor. Imaging studies confirmed that RIPK1 partially localized to lysosomes in both untreated and lysosomal inhibitor treated cells. Similarly, we detected presence of RIPK1, RIPK3 and MLKL in both cytosol and at lysosomes after SCI in vivo. Furthermore, stimulation of autophagy and lysosomal function with rapamycin treatment led to decreased accumulation of RIPK1 and attenuated cell death after SCI. These data suggest that lysosomal dysfunction after SCI may contribute to both inhibition of autophagy and sensitize cells to necroptosis by promoting RIPK1 and RIPK3 accumulation.

Endoplasmic Reticulum Stress and Disrupted Neurogenesis in the Brain Are Associated with Cognitive Impairment and Depressive-Like Behavior after Spinal Cord Injury.

Clinical and experimental studies show that spinal cord injury (SCI) can cause cognitive impairment and depression that can significantly impact outcomes. Thus, identifying mechanisms responsible for these less well-examined, important SCI consequences may provide targets for more effective therapeutic intervention. To determine whether cognitive and depressive-like changes correlate with injury severity, we exposed mice to sham, mild, moderate, or severe SCI using the Infinite Horizon Spinal Cord Impactor and evaluated performance on a variety of neurobehavioral tests that are less dependent on locomotion. Cognitive impairment in Y-maze, novel objective recognition, and step-down fear conditioning tasks were increased in moderate- and severe-injury mice that also displayed depressive-like behavior as quantified in the sucrose preference, tail suspension, and forced swim tests. Bromo-deoxyuridine incorporation with immunohistochemistry revealed that SCI led to a long-term reduction in the number of newly-generated immature neurons in the hippocampal dentate gyrus, accompanied by evidence of greater neuronal endoplasmic reticulum (ER) stress. Stereological analysis demonstrated that moderate/severe SCI reduced neuronal survival and increased the number of activated microglia chronically in the cerebral cortex and hippocampus. The potent microglial activator cysteine-cysteine chemokine ligand 21 (CCL21) was elevated in the brain sites after SCI in association with increased microglial activation. These findings indicate that SCI causes chronic neuroinflammation that contributes to neuronal loss, impaired hippocampal neurogenesis and increased neuronal ER stress in important brain regions associated with cognitive decline and physiological depression. Accumulation of CCL21 in brain may subserve a pathophysiological role in cognitive changes and depression after SCI.

TGF-ß1 induces apoptosis of bone marrow-derived mesenchymal stem cells via regulation of mitochondrial reactive oxygen species production.

Bone marrow-derived mesenchymal stem cells (BMSCs) are the most promising seed cells in regenerative medicine. Our previous study demonstrated that transforming growth factor (TGF)-ß1 induced BMSC senescence in vitro. Whether TGF-ß1 affects the apoptosis of BMSCs has not been examined; therefore the aim of the present study was to investigate this effect. BMSCs were isolated from mouse bone marrow, and the third-passage cells were exposed to 0, 10 and 20 ng/ml TGF-ß1 for 24 h. Cell proliferation was measured by MTT assay; apoptosis was assessed using DAPI staining; and the apoptotic signals Annexin V, B-cell lymphoma (Bcl)-2 and Bcl-2-associated X protein (Bax) were measured using western blotting. Mitochondrial reactive oxygen species (ROS) were measured by flow cytometry following staining with MitoSOX™ Red mitochondrial superoxide indicator. The MTT assay showed that 10 and 20 ng/ml TGF-ß1 inhibited BMSC proliferation. DAPI staining demonstrated that 10 and 20 ng/ml TGF-ß1 promoted BMSC apoptosis, which was further confirmed by a western blotting assay showing a significant increase in the pro-apoptotic signals Annexin V and Bax but a decrease in the anti-apoptotic signal Bcl-2. It was also found that TGF-ß1 markedly increased the mitochondrial ROS levels in BMSCs. It is well known that mitochondrial ROS are strong stimulators of cell apoptosis. These findings indicate that TGF-ß1 can induce BMSC apoptosis, and the mechanism may involve mitochondrial ROS generation.

Ablation of the transcription factors E2F1-2 limits neuroinflammation and associated neurological deficits after contusive spinal cord injury.

Traumatic spinal cord injury (SCI) induces cell cycle activation (CCA) that contributes to secondary injury and related functional impairments such as motor deficits and hyperpathia. E2F1 and E2F2 are members of the activator sub-family of E2F transcription factors that play an important role in proliferating cells and in cell cycle-related neuronal death, but no comprehensive study have been performed in SCI to determine the relative importance of these factors. Here we examined the temporal distribution and cell-type specificity of E2F1 and E2F2 expression following mouse SCI, as well as the effects of genetic deletion of E2F1-2 on neuronal cell death, neuroinflammation and associated neurological dysfunction. SCI significantly increased E2F1 and E2F2 expression in active caspase-3(+) neurons/oligodendrocytes as well as in activated microglia/astrocytes. Injury-induced up-regulation of cell cycle-related genes and protein was significantly reduced by intrathecal injection of high specificity E2F decoy oligodeoxynucleotides against the E2F-binding site or in E2F1-2 null mice. Combined E2F1+2 siRNA treatment show greater neuroprotection in vivo than E2F1 or E2F2 single siRNA treatment. Knockout of both E2F1 and E2F2 genes (E2Fdko) significantly reduced neuronal death, neuroinflammation, and tissue damage, as well as limiting motor dysfunction and hyperpathia after SCI. Both CCA reduction and functional improvement in E2Fdko mice were greater than those in E2F2ko model. These studies demonstrate that SCI-induced activation of E2F1-2 mediates CCA, contributing to gliopathy and neuronal/tissue loss associated with motor impairments and post-traumatic hyperesthesia. Thus, E2F1-2 provide a therapeutic target for decreasing secondary tissue damage and promoting recovery of function after SCI.

Small concentrations of TGF-ß1 promote proliferation of bone marrow-derived mesenchymal stem cells via activation of Wnt/ß-catenin pathway.

Bone marrow-derived mesenchymal stem cells (BMSCs) are the most promising seed cells for cell transplant. The proliferation of BMSCs is one of the most important determinants of the efficiency of MSC-based transplant therapy. It has been reported that transforming growth factor-ß1 (TGF-ß1) activates Wnt/ß-catenin signaling and regulates cell proliferation. In this study, we investigated the effect of low concentrations of TGF-ß1 on proliferation of BMSCs and the related mechanisms. BMSCs were treated with 0, 1, 5 and 10 ng/L recombinant mouse TGF-ß1 for 12 h. Cell proliferation was measured by cell counting and MTT assay, and the proliferation-related signals p53, Mdm2, Aktl, Wnt3, phospho-Akt and ß-catenin were measured by quantitative polymerase chain reaction (qPCR) and/or Western blot. Our results showed that TGF-ß1 at low concentrations induced BMSC proliferation and expression of Mdm2, Aktl, phospho-Akt, Wnt3 and ß-catenin, and inhibited p53 expression in dose dependent manner. Importantly, ß-catenin siRNA significantly inhibited TGF-ß1-induced BMSC proliferation. These findings suggest that low concentrations of TGF-ß1 can stimulate proliferation of BMSCs, which is at least partially dependent on the activation of Wnt/ß-catenin pathway.

Association between endothelin-1/endothelin receptor A and inflammation in mouse kidneys following acute ischemia/reperfusion.

Renal ischemia/reperfusion (I/R) is a common risk factor for renal failure. Expression of endothelin­1 (ET­1) and its receptor ETA were also reported to be involved in the development of acute and chronic renal disease. The present study was designed to investigate the association between inflammation and ET­1/ETA expression in mouse kidneys following acute I/R. The results demonstrated that acute renal I/R caused a significant increase in ET­1 and ETA gene and transcriptional levels compared with those of the sham group (P<0.01). Ischemia alone also resulted in a marked increase of ET­1 and ETA expression compared with that of the sham group (P<0.05). In addition, ET­1 and ETA expression was significantly increased in the I/R group compared with that of the ischemia group (P<0.05 or P<0.01). Of note, the altered expression levels of inflammatory cytokines tumor necrosis factor (TNF)­α and interleukin (IL)­6 in kidneys following I/R and ischemia alone were correlated with the expression of ET­1 and ETA. Hypoxia is the most important stimulus of I/R for tissue injury. In kidneys, ET­1 is primarily produced by renal glomerular endothelial cells (RGECs). In the present study, treatment with hypoxia alone or hypoxia/reoxygenation were found to increase ET­1 and ETA expression in human RGECs (P<0.05 or P<0.01). In order to elucidate the role of inflammation in the ischemia­ and hypoxia­induced upregulation of ET­1 and ETA, human RGECs were exposed to different concentrations of TNF­α. As expected, TNF­α increased ET­1 and ETA expression in a dose­dependent manner; furthermore, application of the TNF­α inhibitor CAY10500 partially inhibited hypoxia­induced ET­1 and ETA expression. In conclusion, these results indicated that I/R induced upregulation of ET­1 and ETA in the kidneys, which was, at least in part, dependent on the production of inflammatory cytokines.

Voluntary Exercise Preconditioning Activates Multiple Antiapoptotic Mechanisms and Improves Neurological Recovery after Experimental Traumatic Brain Injury.

Physical activity can attenuate neuronal loss, reduce neuroinflammation, and facilitate recovery after brain injury. However, little is known about the mechanisms of exercise-induced neuroprotection after traumatic brain injury (TBI) or its modulation of post-traumatic neuronal cell death. Voluntary exercise, using a running wheel, was conducted for 4 weeks immediately preceding (preconditioning) moderate-level controlled cortical impact (CCI), a well-established experimental TBI model in mice. Compared to nonexercised controls, exercise preconditioning (pre-exercise) improved recovery of sensorimotor performance in the beam walk task, as well as cognitive/affective functions in the Morris water maze, novel object recognition, and tail-suspension tests. Further, pre-exercise reduced lesion size, attenuated neuronal loss in the hippocampus, cortex, and thalamus, and decreased microglial activation in the cortex. In addition, exercise preconditioning activated the brain-derived neurotrophic factor pathway before trauma and amplified the injury-dependent increase in heat shock protein 70 expression, thus attenuating key apoptotic pathways. The latter include reduction in CCI-induced up-regulation of proapoptotic B-cell lymphoma 2 (Bcl-2)-homology 3-only Bcl-2 family molecules (Bid, Puma), decreased mitochondria permeabilization with attenuated release of cytochrome c and apoptosis-inducing factor (AIF), reduced AIF translocation to the nucleus, and attenuated caspase activation. Given these neuroprotective actions, voluntary physical exercise may serve to limit the consequences of TBI.

Downregulation of miR-23a and miR-27a following experimental traumatic brain injury induces neuronal cell death through activation of proapoptotic Bcl-2 proteins.

MicroRNAs (miRs) are small noncoding RNAs that negatively regulate gene expression at the post-transcriptional level. To identify miRs that may regulate neuronal cell death after experimental traumatic brain injury (TBI), we profiled miR expression changes during the first several days after controlled cortical impact (CCI) in mice. miR-23a and miR-27a were rapidly downregulated in the injured cortex in the first hour after TBI. These changes coincided with increased expression of the proapoptotic Bcl-2 family members Noxa, Puma, and Bax. In an etoposide-induced in vitro model of apoptosis in primary cortical neurons, miR-23a and miR-27a were markedly downregulated as early as 1 h after exposure, before the upregulation of proapoptotic Bcl-2 family molecules. Administration of miR-23a and miR-27a mimics attenuated etoposide-induced changes in Noxa, Puma, and Bax, reduced downstream markers of caspase-dependent (cytochrome c release and caspase activation) and caspase-independent (apoptosis-inducing factor release) pathways, and limited neuronal cell death. In contrast, miRs hairpin inhibitors enhanced etoposide-induced neuronal apoptosis and caspase activation. Importantly, administration of miR-23a and miR-27a mimics significantly reduced activation of Puma, Noxa, and Bax as well as attenuated markers of caspase-dependent and -independent apoptosis after TBI. Furthermore, miR-23a and miR-27a mimics significantly attenuated cortical lesion volume and neuronal cell loss in the hippocampus after TBI. These findings indicate that post-traumatic decreases in miR-23a and miR-27a contribute to neuronal cell death after TBI by upregulating proapoptotic Bcl-2 family members, thus providing a novel therapeutic target.

TGF-ß1 induces senescence of bone marrow mesenchymal stem cells via increase of mitochondrial ROS production.

BACKGROUND: Bone marrow derived mesenchymal stem cells (bmMSCs) are multipotent cells that can differentiate into diverse cell types, including cardiomyocytes. BmMSC-based transplantation is capable of repairing acute and chronic myocardial infarction. Prior to the transplantation, MSCs are usually induced in vitro by biological reagents and chemicals for directional differentiation. Transforming growth factor beta (TGF-ß) is one of the most commonly used biological reagents for induction of cardiomyocyte differentiation of bmMSCs. Previous studies have shown that TGF-ß induces senescence in several cell types. However, whether TGF-ß affects senescence of bmMSCs has not been elucidated. The goal of this study was to investigate the effect of TGF-ß1 on senescence of bmMSCs and the underlying mechanisms. RESULTS: We found that TGF-ß1 increased activity of senescence-associated-galactosidase (SA-Gal) and production of mitochondrial reactive oxygen species (mtROS) in bmMSCs in a dose-dependent manner. TGF-ß1 also significantly decreased expression of superoxide dismutase 2 (SOD2) and Id1, and increased expression of 4-Hydroxynonenal (4-HNE) subunits and p16 in bmMSCs in a dose-dependent manner. Pre-treatment with mtROS inhibitor acetyl-L-carnitine (ALCAR, 0.1 mM) significantly inhibited TGF-ß1-induced mtROS production and SA-Gal activity. CONCLUSION: TGF-ß1 can induce senescence of bmMSCs, which at least partially depends on mtROS production.