Melatonin reverses H2O2‐induced premature senescence in mesenchymal stem cells via the SIRT1‐dependent pathway

Mesenchymal stem cells (MSCs) represent an attractive source for stem cell‐based regenerative therapy, but they are vulnerable to oxidative stress‐induced premature senescence in pathological conditions. We previously reported antioxidant and antiarthritic effects of melatonin on MSCs against proinflammatory cytokines. In this study, we hypothesized that melatonin could protect MSCs from premature senescence induced by hydrogen peroxide (H₂O₂) via the silent information regulator type 1 (SIRT1)‐dependent pathway. In response to H₂O₂ at a sublethal concentration of 200 μm , human bone marrow‐derived MSCs (BM‐MSCs) underwent growth arrest and cellular senescence. Treatment with melatonin before H₂O₂ exposure cannot significantly prevent premature senescence; however, treatment with melatonin subsequent to H₂O₂ exposure successfully reversed the senescent phenotypes of BM‐MSCs in a dose‐dependent manner. This result was made evident by improved cell proliferation, decreased senescence‐associated β‐galactosidase activity, and the improved entry of proliferating cells into the S phase. In addition, treatment with 100 μm melatonin restored the osteogenic differentiation potential of BM‐MSCs that was inhibited by H₂O₂‐induced premature senescence. We also found that melatonin attenuated the H₂O₂‐stimulated phosphorylation of p38 mitogen‐activated protein kinase, decreased expression of the senescence‐associated protein p16^INK4α, and increased SIRT1. Further molecular experiments revealed that luzindole, a nonselective antagonist of melatonin receptors, blocked melatonin‐mediated antisenescence effects. Inhibition of SIRT1 by sirtinol counteracted the protective effects of melatonin, suggesting that melatonin reversed the senescence in cells through the SIRT1‐dependent pathway. Together, these findings lay new ground for understanding oxidative stress‐induced premature senescence and open perspectives for therapeutic applications of melatonin in stem cell‐based regenerative medicine.     

p53 and ATF-2 partly mediate the overexpression of COX-2 in H2O2-induced premature senescence of human fibroblasts

Cyclooxygenase-2 and release of prostaglandin E2 are up-regulated in replicative senescence of dermal and prostate fibroblasts and in H₂O₂-induced premature senescence of IMR-90 lung fibroblasts expressing the catalytic subunit of telomerase. Inhibition of cyclooxygenase-2 activity by specific chemical inhibitor or siRNA attenuates the H₂O₂-induced increase of senescence associated β-galactosidase positive cells and attenuates growth arrest. In this work, p38^MAPK activation and increased DNA binding activities of ATF-2 and p53 are shown to mediate cyclooxygenase-2 overexpression in premature senescence.     

Proteomic and metabolomic analysis of H2O2-induced premature senescent human mesenchymal stem cells

Stress induced premature senescence (SIPS) occurs after exposure to many different sublethal stresses including H₂O₂, hyperoxia, or tert-butylhydroperoxide. Human mesenchymal stem cells (hMSCs) exhibit limited proliferative potential in vitro, the so-called Hayflick limit. According to the free-radical theory, reactive oxygen species (ROS) might be the candidates responsible for senescence and age-related diseases. H₂O₂ may be responsible for the production of high levels of ROS, in which the redox balance is disturbed and the cells shift into a state of oxidative stress, which subsequently leads to premature senescence with shortening telomeres. H₂O₂ has been the most commonly used inducer of SIPS, which shares features of replicative senescence (RS) including a similar morphology, senescence-associated β-galactosidase activity, cell cycle regulation, etc. Therefore, in this study, the senescence of hMSC during SIPS was confirmed using a range of different analytical methods. In addition, we determined five differentially expressed spots in the 2-DE map, which were identified as Annexin A2 (ANXA2), myosin light chain 2 (MLC2), peroxisomal enoyl-CoA hydratase 1 (ECH1), prosomal protein P30-33K (PSMA1) and mutant β-actin by ESI-Q-TOF MS/MS. Also, proton (¹H) nuclear magnetic resonance spectroscopy (NMR) was used to elucidate the difference between metabolites in the control and hMSCs treated with H₂O₂. Among these metabolites, choline and leucine were identified by ¹H-NMR as up-regulated metabolites and glycine and proline were identified as down-regulated metabolites.     

Bcl‐2 overexpression in hepatic stellate cell line CFSC‐2G,induces a pro‐fibrotic state

Background and Aim: Development of hepatic fibrosis is a complex process that involves oxidative stress (OS) and an altered balance between pro‐ and anti‐apoptotic molecules. Since Bcl‐2 overexpression preserves viability against OS, our objective was to address the effect of Bcl‐2 overexpression in the hepatic stellate cells (HSC) cell‐line CFSC‐2G under acetaldehyde and H₂O₂ challenge, and explore if it protects these cells against OS, induces replicative senescence and/or modify extracellular matrix (ECM) remodeling potential. Methods: To induce Bcl‐2 overexpression, HSC cell line CFSC‐2G was transfected by lipofection technique. Green fluorescent protein‐only CFSC‐2G cells were used as a control. Cell survival after H₂O₂ treatment and total protein oxidation were assessed. To determine cell cycle arrest, proliferation‐rate, DNA synthesis and senescence were assessed. Matrix metalloproteinases (MMP), tissue‐inhibitor of MMP (TIMP), transglutaminases (TG) and smooth muscle a‐actin (α‐SMA) were evaluated by western blot in response to acetaldehyde treatment as markers of ECM remodeling capacity in addition to transforming growth factor‐β (TGF‐β) mRNA. Results: Cells overexpressing Bcl‐2 survived ≈ 20% more than control cells when exposed to H₂O₂ and ≈ 35% proteins were protected from oxidation, but Bcl‐2 did not slow proliferation or induced senescence. Bcl‐2 overexpression did not change α‐SMA levels, but it increased TIMP‐1 (55%), tissue transglutaminases (tTG) (25%) and TGF‐β mRNA (49%), when exposed to acetaldehyde, while MMP‐13 content decreased (47%). Conclusions: Bcl‐2 overexpression protected HSC against oxidative stress but it did not induce replicative senescence. It increased TIMP‐1, tTG and TGF‐β mRNA levels and decreased MMP‐13 content, suggesting that Bcl‐2 overexpression may play a key role in the progression of fibrosis in chronic liver diseases.     

Resveratrol attenuates cigarette smoke extract induced cellular senescence in human airway epithelial cells by regulating the miR-34a/SIRT1/NF-κB pathway

Chronic obstructive pulmonary disease (COPD) is characterized by accelerated lung aging. Smoking is the critical risk factor for COPD. Cellular senescence of airway epithelial cells is the cytological basis of accelerated lung aging in COPD, and the regulation of microRNAs (miRNAs) is the central epigenetic mechanism of cellular senescence. Resveratrol (Res) is a polyphenol with anti-aging properties. This study investigated whether Res attenuates cigarette smoke extract (CSE)-induced cellular senescence in human airway epithelial cells (BEAS-2B) through the miR-34a/SIRT1/nuclear factor-kappaB (NF-κB) pathway. BEAS-2B cells were treated with Res, CSE and transfected with miR-34a-5p mimics. Cellular senescence was evaluated by senescence -related β-galactosidase (SA-β-gal) staining and expression of senescence-related genes (p16, p21, and p53). The expressions of miR-34a-5p, SIRT1, and NF-κB p65 were examined using quantitative real time polymerase chain reaction and western blotting. The senescence-associated secretory phenotype (SASP) cytokines (IL-1β, IL-6, IL-8, TNF-α) were assessed by enzyme-linked immunosorbent assay. The binding between miR-34a-5p and SIRT1 was confirmed by dual-luciferase reporter assay. The results showed that CSE dose-dependently decreased cell viability and elevated cellular senescence, characterized by increased SA-β-gal staining and senescence-related gene expressions (p16, p21, and p53). Further, CSE dose-dependently increased the expression of miR-34a-5p and SASP cytokines (IL-1β, IL-6, IL-8, TNF-α) in BEAS-2B cells. Pretreatment with Res inhibited CSE-induced cellular senescence and secretion of SASP cytokines (IL-1β, IL-6, IL-8, TNF-α) in a dose-dependent manner. Moreover, Res reversed the CSE-induced down-regulation of SIRT1 and up-regulation of miR-34a-5p and NF-κB p65. SIRT1 is a target of miR-34a-5p. Overexpression of miR-34a-5p via transfection with miR-34a-5p mimic in BEAS-2B cells attenuated the inhibitory effect of Res on cellular senescence, accompanied by reversing the expression of SIRT1 and NF-κB p65. In conclusion, Res attenuated CSE-induced cellular senescence in BEAS-2B cells by regulating the miR-34a/SIRT1/NF-κB pathway, which may provide a new approach for COPD treatment.     

Retarding the senescence of human vascular endothelial cells induced by hydrogen peroxide: effects of 17beta-estradiol (E2) mediated mitochondria protection

This study investigates the influence of 17beta-estradiol (E₂) on hydrogen peroxide (H₂O₂)-induced human vascular endothelial cell (HUVEC) senescence. HUVECs were divided into four groups, namely control group, H₂O₂ stimulation group, E2 intervention group and ICI182780 (ICI) intervention group. The aging-related β-galactosidase activities, cytochrome C oxidase activities, intracellular ATP levels, intracellular reactive oxygen species (ROS) levels and phosphorylated Rb protein expressions were mainly observed. Of which, senescence-associated β-galactosidase activities were detected using immunohistochemical staining, cytochrome C oxidase activities and intracellular ATP levels were detected using commercial kits, ROS levels were detected by fluorescence microscopy and fluorescence microplate reader, immunoblotting was used to quantitatively detect the expressions of phosphorylated Rb proteins. After continuous treatment of H₂O₂, the senescent phenotypes appeared in the HUVECs. The percentage of positive SA-βgal staining cells and the phosphorylated Rb expressions were significantly increased; intracellular ROS levels, cytochrome C oxidase activities and intracellular ATP levels were elevated. Compared with the H₂O₂ stimulation group, E2 intervention significantly decreased the positive rate of SA-β-gal staining, the phosphorylated Rb protein levels, the intracellular ROS levels, cytochrome C oxidase activities and intracellular ATP levels. Pretreatment of estrogen receptor blocker ICI182780 weakened the role of E2. These results indicated that H₂O₂ could induce HUVEC senescence; 17beta-E2 might relieve H₂O₂-induced mitochondrial damage through estrogen receptor and delay the vascular endothelial cell senescence.     

Rb1 protects endothelial cells from hydrogen peroxide-induced cell senescence by modulating redox status

Objectives Endothelial senescence has been proposed to be involved in endothelial dysfunction and atherogenesis. This study investigates the effects of ginsenoside Rbl, a major constituent of ginseng,on H₂O₂-induced endothelial senescence.Methods Primary human umbilical vein endothelial cells（HUVECs） senescence was induced by H₂O₂ as judged by senescence-associated P-galactosidase assay （SA-P-gal）.Fntracellur superoxide dismutase（S0D1） activity and malondialdehyde（MDA） level were determined by commercial kit.S0D1 mRNA and protein expression were analyzed by real time PCR and Western blot.Reactive oxygen species（ROS） were determined by flow cytometry.Results Rb1 was found to reverse endothelial senescence,as witnessed by a significant decrease of senescent cell numbers. Rbl could markedly increase intracellular SOD activity, decrease the MDA level,and suppress the generation of intracellular ROS in H₂O₂-treated HUVECs.Consistent with these findings,Rbl can effectively restore SOD1 mRNA and protein expression which decreased in H₂O₂ treated cells. Conclusions Our report demonstrates thatRbl can exert reversal effects on H₂O₂-induced cellular senescence through modulating cellular redox status.     

MicroRNA-21 protects against the H2O2-induced injury on cardiac myocytes via its target gene PDCD4

Reactive oxygen species (ROS)-induced cardiac cell injury via expression changes of multiple genes plays a critical role in the pathogenesis of numerous heart diseases. MicroRNAs (miRNAs) comprise a novel class of endogenous, small, noncoding RNAs that negatively regulate about 30% of the genes in a cell via degradation or translational inhibition of their target mRNAs. Currently, the effects of ROS on miRNA expression and the roles of miRNAs in ROS-mediated injury on cardiac myocytes are uncertain. Using quantitative real-time RT-PCR (qRT-PCR), we demonstrated that microRNA-21 (miR-21) was upregulated in cardiac myocytes after treatment with hydrogen peroxide (H₂O₂). To determine the potential roles of miRNAs in H₂O₂-mediated gene regulation and cellular injury, miR-21 expression was downregulated by miR-21 inhibitor and upregulated by pre-miR-21. H₂O₂-induced cardiac cell death and apoptosis were increased by miR-21 inhibitor and was decreased by pre-miR-21. Programmed cell death 4 (PDCD4) that was regulated by miR-21 and was a direct target of miR-21 in cardiac myocytes. Pre-miR-21-mediated protective effect on cardiac myocyte injury was inhibited in H₂O₂-treated cardiac cells via adenovirus-mediated overexpression of PDCD4 without miR-21 binding site. Moreover, Activator protein 1 (AP-1) was a downstream signaling molecule of PDCD4 that was involved in miR-21-mediated effect on cardiac myocytes. The results suggest that miR-21 is sensitive to H₂O₂ stimulation. miR-21 participates in H₂O₂-mediated gene regulation and functional modulation in cardiac myocytes. miR-21 might play an essential role in heart diseases related to ROS such as cardiac hypertrophy, heart failure, myocardial infarction, and myocardial ischemia/reperfusion injury.     

Therapeutic Effects of SRT2104 on Lung Injury in Rats with Emphysema via Reduction of Type II Alveolar Epithelial Cell Senescence

Abstract

Chronic obstructive pulmonary disease (COPD) is one of the most prevalent and severe diseases worldwide with high societal and health care costs. The pathogenesis of COPD is very complicated, and no curative treatment is available. Cellular senescence promotes the development of COPD. Type II alveolar epithelial cells (AECII) play a momentous role in lung tissue repair and maintenance of alveolar homeostasis. Sirtuin 1 (SIRT1), an antiaging molecule involved in the response to chronic inflammation and oxidative stress, regulates many pathophysiological changes including stress resistance, apoptosis, inflammation, and cellular senescence. This study aimed to investigate whether the pharmacological SIRT1 activator SRT2104 protects against AECII senescence in rats with emphysema. Our findings confirmed that SRT2104 administration reduced the pathological characteristics of emphysema and improved lung function parameters, including pulmonary resistance, pulmonary dynamic compliance, and peak expiratory flow. Moreover, SRT2104 treatment upregulated the expression of surfactant proteins A and C, SIRT1, and forkhead box O 3a (FoxO3a), decreased senescence-associated-β-galactosidase (SA-β-gal) activity, increased SIRT1 deacetylase activity, and downregulated the levels of p53 and p21. Therefore, SRT2104 administration protected against AECII senescence in rats with emphysema via SIRT1/FoxO3a and SIRT1/p53 signaling pathways and may provide a novel potential therapeutic strategy for COPD.     

Catabolic stress induces features of chondrocyte senescence through overexpression of caveolin 1: Possible involvement of caveolin 1–induced down‐regulation of articular chondrocytes in the pathogenesis of osteoarthritis

Objective

Articular chondrocyte senescence is responsible, at least in part, for the increased incidence of osteoarthritis (OA) with increased age. Recently, it was suggested that caveolin 1, a 21–24‐kd membrane protein, participates in premature cellular senescence. Caveolin 1 is the principal structural component of caveolae, vesicular invaginations of the plasma membrane. This study was undertaken to investigate whether the catabolic factors oxidative stress and interleukin‐1β (IL‐1β) induce features of premature senescence of articular chondrocytes through up‐regulation of caveolin 1 expression.

Methods

Caveolin 1 expression was investigated in human OA cartilage by real‐time polymerase chain reaction and in rat OA cartilage by immunohistologic analysis. We studied whether IL‐1β and H₂O₂ induce caveolin 1 expression in OA chondrocytes and analyzed the relationship between cellular senescent phenotypes and caveolin 1 expression in human chondrocytes.

Results

In human and rat OA articular cartilage, caveolin 1 positivity was associated with cartilage degeneration. Both IL‐1β and H₂O₂ up‐regulated caveolin 1 messenger RNA and protein levels, and both treatments induced marked expression of senescent phenotypes: altered cellular morphology, cell growth arrest, telomere erosion, and specific senescence‐associated β‐galactosidase activity. Caveolin 1 overexpression induced p38 MAPK activation and impaired the ability of chondrocytes to produce type II collagen and aggrecan. In contrast, down‐regulation of caveolin 1 with antisense oligonucleotide significantly inhibited the features of chondrocyte senescence induced by catabolic factors. Caveolin 1 induction and stresses with both IL‐1β and H₂O₂ up‐regulated p53 and p21 and down‐regulated phosphorylated retinoblastoma (Rb), suggesting that the p53/p21/Rb phosphorylation pathway, as well as prolonged p38 MAPK activation, may mediate the features of chondrocyte senescence induced by stress.

Conclusion

Our findings suggest that IL‐1β and oxidative stress induce features of premature senescence in OA chondrocytes, mediated, at least in part, by stress‐induced caveolin 1 expression. This indicates that caveolin 1 plays a role in the pathogenesis of OA via promotion of chondrocyte down‐regulation.

     

Dendritic cell SIRT1–HIF1α axis programs the differentiation of CD4+ T cells through IL-12 and TGF-β1

The differentiation of naive CD4⁺ T cells into distinct lineages plays critical roles in mediating adaptive immunity or maintaining immune tolerance. In addition to being a first line of defense, the innate immune system also actively instructs adaptive immunity through antigen presentation and immunoregulatory cytokine production. Here we found that sirtuin 1 (SIRT1), a type III histone deacetylase, plays an essential role in mediating proinflammatory signaling in dendritic cells (DCs), consequentially modulating the balance of proinflammatory T helper type 1 (T_H1) cells and antiinflammatory Foxp3⁺ regulatory T cells (T_reg cells). Genetic deletion of SIRT1 in DCs restrained the generation of T_reg cells while driving T_H1 development, resulting in an enhanced T-cell–mediated inflammation against microbial responses. Beyond this finding, SIRT1 signaled through a hypoxia-inducible factor-1 alpha (HIF1α)-dependent pathway, orchestrating the reciprocal T_H1 and T_reg lineage commitment through DC-derived IL-12 and TGF-β1. Our studies implicates a DC-based SIRT1–HIF1α metabolic checkpoint in controlling T-cell lineage specification.CD4⁺ T cells are essential components of the adaptive immune system that regulate immune responses against foreign antigen. Upon antigen recognition, naive CD4⁺ T cells undergo activation and expansion, and, depending on inflammatory contexts and cytokine milieus, differentiate into functional and phenotypic T helper (T_H) subsets characterized by distinct cytokine production profile and function (1–3). T_H1 cells produce IFN-γ and elicit cellular immunity in responding to intracellular pathogens; T_H2 cells produce IL-4 and IL-5 and promote humoral immunity in responding to extracellular bacteria and helminthes; and T_H17 cells produce IL-17 and mediate antifungal defense and inflammation (4, 5). Additionally, regulatory T cells, often known as “induced regulatory T cells” (iT_reg cells), which act in synergy with naturally occurring T_reg cells (nT_reg cells), produce IL-10 and TGF-β1 and dampen immune responses elicited from T_H1, T_H2, and T_H17 (6–9).Dendritic cells (DCs), an essential component in the innate immune system, play a critical role in initiating front-line primary immune responses and directing subsequent pathogen-specific adaptive immune responses (2). In addition to presenting antigens and modulating cell surface costimulatory molecules, DC-derived cytokines and chemokines can result in either a proinflammatory or antiinflammatory environment, engaging distinct T-cell differentiation programs on naive CD4⁺ T cells (1, 10–16). For example, DC-producing IL-12 can support T_H1 development, whereas DC-producing IL-10 or TGF-β1 can support T_reg development. Recent studies from us and others have shown that innate signaling in DCs mediated by G protein-coupled receptor S1P1, the mitogen-activated kinases (MAPKs), and Wnt–β-catenin plays important roles in stimulating adaptive immune responses through directing native CD4⁺ T-cell differentiation (17–20). However, other critical signaling components in DCs that may play a role in shaping T-cell lineage engagement remain to be identified.SIRT1 is a mammalian homolog of the yeast NAD⁺-dependent protein deacetylase Sirt2, and plays a role in a variety of essential biological processes, including cell cycle progression, apoptosis, cell survival, gene silencing, heterochromatin formation, tumorigenesis, metabolism, and development (21, 22). SIRT1 has also been implicated in regulating immune responses. In T cells, SIRT1 is required to maintain T-cell tolerance (23, 24) and also play a role in inhibiting the function of T_reg cells in allograft survival (25). In myeloid cells, SIRT1 limits the inflammatory process by inhibiting the expression of proinflammatory cytokines (26, 27), while promoting DC maturation and T_H2 response in airway allergy (28). However, whether SIRT1 is involved in bridging the innate immune signal to adaptive immune response remains unexplored.Here, we found that SIRT1 plays a critical role in determining the T-cell lineage fate by directing DC-derived cytokine production, which links innate and adaptive immune modulation. Largely through a HIF1α–dependent signaling pathway, SIRT1 is required for the reciprocal production of IL-12 and TGF-β1 production in DCs as well as the expression of IL-12Rβ2 and TGF-βR2 in responding T cells, resulting in a differential lineage engagement of T_H1 and iT_reg in the microbial-induced inflammation.     

Drosophila TRPA1 isoforms detect UV light via photochemical production of H2O2

The transient receptor potential A1 (TRPA1) channel is an evolutionarily conserved detector of temperature and irritant chemicals. Here, we show that two specific isoforms of TRPA1 in Drosophila are H₂O₂ sensitive and that they can detect strong UV light via sensing light-induced production of H₂O₂. We found that ectopic expression of these H₂O₂-sensitive Drosophila TRPA1 (dTRPA1) isoforms conferred UV sensitivity to light-insensitive HEK293 cells and Drosophila neurons, whereas expressing the H₂O₂-insensitive isoform did not. Curiously, when expressed in one specific group of motor neurons in adult flies, the H₂O₂-sensitive dTRPA1 isoforms were as competent as the blue light-gated channelrhodopsin-2 in triggering motor output in response to light. We found that the corpus cardiacum (CC) cells, a group of neuroendocrine cells that produce the adipokinetic hormone (AKH) in the larval ring gland endogenously express these H₂O₂-sensitive dTRPA1 isoforms and that they are UV sensitive. Sensitivity of CC cells required dTRPA1 and H₂O₂ production but not conventional phototransduction molecules. Our results suggest that specific isoforms of dTRPA1 can sense UV light via photochemical production of H₂O₂. We speculate that UV sensitivity conferred by these isoforms in CC cells may allow young larvae to activate stress response—a function of CC cells—when they encounter strong UV, an aversive stimulus for young larvae.Light is an important sensory cue that has a wide-ranging influence on animal physiology and behavior. In addition to its role in vision, light detection also contributes to circadian rhythm regulation, sleep, phototaxis, and even mood control (1–5). Animals of different species have evolved diverse light sensors and light-detection cells to regulate various light-dependent physiological processes. For example, whereas rods and cones in mammals are critical for detecting and relaying image-forming visual information to the visual cortex, the intrinsically light-sensitive melanopsin-expressing retinal ganglion cells relay nonimage-forming visual information primarily to the suprachiasmatic nucleus to regulate circadian rhythm (6, 7).Similar to the diversity of cell types that sense light, the molecular mechanisms for light detection also vary. For example, in mammalian rods and cones, light activates a Rhodopsin-dependent phototransduction pathway that hyperpolarizes the cells via closing a cyclic nucleotide-gated channel (5). In the intrinsically light-sensitive retinal ganglion cells, however, light activates a melanopsin-dependent pathway that depolarizes the cells via opening TRP channels (6, 8). Further, in Drosophila PDF neurons, light has been shown to activate cryptochrome signaling to depolarize the cells (9). And in the ASJ sensory neuron in Caenorhabditis elegans, UV and blue light have been shown to activate the cell via signaling a specific GPCR known as LITE-1 (high energy light unresponsive protein 1) (10, 11).It has been shown that short wavelength UV and blue light can trigger reactive oxygen species (ROS) and H₂O₂ production in cultured cells (12), and H₂O₂ can modulate ion channel activities directly or indirectly (13–15). We therefore reasoned that sensing H₂O₂ might constitute another light-sensing mechanism. Indeed, a recent finding has suggested that two specific GPCRs—GUR-3 and LITE-1—both of which play important roles in light sensing in C. elegans, may also be H₂O₂ sensitive (16). Moreover, TRPA1, an evolutionarily conserved TRP channel known for its role in sensing many chemical irritants, has been shown to sense H₂O₂ (17–24), thus TRPA1 may be activated by light via sensing light-induced H₂O₂ production. Some recent evidence has demonstrated a role for TRPA1 in light sensing. First, in human melanocyte, TRPA1 has been shown to act to increase melanin production in response to UV (25). Moreover, in Drosophila, TRPA1 has been shown to increase neuronal activities of a specific group of larval somatosensory neurons (also known as the C4da neurons) in response to UV (26). However, in both cases, GPCRs—rhodopsin in the case of human melanocyte and Gr28b in the case of C4da neurons—are thought to mediate light-dependent activation of TRPA1 (25, 26); thus, it is not clear whether TRPA1 can be activated by light through H₂O₂ production.Here, we show that two specific isoforms of Drosophila dTRPA1 are H₂O₂ sensitive and that their H₂O₂ sensitivity allows them to detect UV without relying on conventional phototransduction molecules. We found that ectopic expression of these H₂O₂-sensitive dTRPA1 isoforms conferred UV sensitivity to light-insensitive cultured HEK293 cells and a few types of light-insensitive Drosophila neurons. The light and H₂O₂ sensitivities are specific to certain dTRPA1 isoforms, consistent with previous findings that different dTRPA1 isoforms exhibit distinct thermal sensitivities (20, 21). Strikingly, the H₂O₂-sensitive dTRPA1 was as effective as channelrhodopsin-2 (ChR2) in triggering light-produced motor responses when expressed in a specific group of motor neurons that control proboscis extension in adult Drosophila. We further discovered that the corpus cardiacum (CC) cells, a group of adipokinetic hormone (AKH)-producing cells that reside in the Drosophila larval ring gland, expressed the H₂O₂-sensitive dTRPA1 isoforms endogenously, and that these cells were UV and H₂O₂ sensitive. Their sensitivity required dTRPA1 and H₂O₂ production: Reducing dual oxidase (DUOX) or increasing catalase (cat) expression in them reduced their UV sensitivity significantly. In contrast, reducing PLC or GR28b in CC cells had little impact. Our results suggest specific H₂O₂-sensitive TRPA1 isoforms can be activated by UV via a photochemical transduction cascade and that these isoforms may be exploited as a UV-dependent optogenetic tool for controlling neuronal activities.     

氯通道ClC-3反义寡核苷酸对过氧化氢诱导的大鼠主动脉平滑肌细胞凋亡的影响

目的研究ClC3反义寡核苷酸对H2O2诱导的大鼠主动脉平滑肌细胞凋亡的影响。方法蛋白免疫印迹法检测ClC3蛋白表达;形态学方法、DNA琼脂糖电泳、MTT法和流式细胞仪观察和分析H2O2诱导的大鼠主动脉平滑肌细胞形态学改变、DNA断裂、细胞存活率和凋亡率及ClC3反义寡核苷酸转染对其影响。结果ClC3反义寡核苷酸转染抑制内源性ClC3蛋白表达后,可加重H2O2诱导大鼠主动脉平滑肌细胞形态学改变及DNA断裂,细胞凋亡率由52.8%±13.6%增至75.7%±5.8%(n=6,P<0.01),而细胞存活率由48.9%±4.3%进一步降低为31.3%±4.3%(n=6,P<0.01)。结论ClC3反义寡核苷酸转染促进H2O2诱导的大鼠主动脉平滑肌细胞凋亡。     

Long noncoding RNA H19 mediates melatonin inhibition of premature senescence of c‐kit+ cardiac progenitor cells by promoting miR‐675

Melatonin, a hormone secreted by the pineal gland, possesses multiple biological activities such as antitumor, antioxidant, and anti‐ischemia. C‐kit⁺ cardiac progenitor cells (CPCs) have emerged as a promising tool for the treatment of heart diseases. However, the senescence of CPCs due to pathological stimuli leads to the decline of CPCs' functions and regenerative potential. This study was conducted to demonstrate whether melatonin antagonizes the senescence of CPCs in response to oxidative stress. Here, we found that the melatonin treatment markedly inhibited the senescent characteristics of CPCs after exposed to sublethal concentration of H₂O₂, including the increase in senescence‐associated β‐galactosidase (SA‐β‐gal)‐positive CPCs, senescence‐associated heterochromatin loci (SAHF), secretory IL‐6 level, and the upregulation of p53 and p21 proteins. Senescence‐associated proliferation reduction was also attenuated by melatonin in CPCs. Luzindole, the melatonin membrane receptor blocker, may block the melatonin‐mediated suppression of premature senescence in CPCs. Interestingly, we found that long noncoding RNA H19 and its derived miR‐675 were downregulated by H₂O₂ in CPCs, but melatonin treatment could counter this alteration. Furthermore, knockdown of H19 or miR‐675 blocked antisenescence actions of melatonin on H₂O₂‐treated CPCs. It was further verified that H19‐derived miR‐675 targeted at the 3′UTR of USP10, which resulted in the downregulation of p53 and p21 proteins. In summary, melatonin antagonized premature senescence of CPCs via H19/miR‐675/USP10 pathway, which provides new insights into pharmacological actions and potential applications of melatonin on the senescence of CPCs.     

Initial Processes of Atomic Layer Deposition of Al2O3 on InGaAs: Interface Formation Mechanisms and Impact on Metal-Insulator-Semiconductor Device Performance

Interface-formation processes in atomic layer deposition (ALD) of Al₂O₃ on InGaAs surfaces were investigated using on-line Auger electron spectroscopy. Al₂O₃ ALD was carried out by repeating a cycle of Al(CH₃)₃ (trimethylaluminum, TMA) adsorption and oxidation by H₂O. The first two ALD cycles increased the Al KLL signal, whereas they did not increase the O KLL signal. Al₂O₃ bulk-film growth started from the third cycle. These observations indicated that the Al₂O₃/InGaAs interface was formed by reduction of the surface oxides with TMA. In order to investigate the effect of surface-oxide reduction on metal-insulator-semiconductor (MIS) properties, capacitors and field-effect transistors (FETs) were fabricated by changing the TMA dosage during the interface formation stage. The frequency dispersion of the capacitance-voltage characteristics was reduced by employing a high TMA dosage. The high TMA dosage, however, induced fixed negative charges at the MIS interface and degraded channel mobility.