Role of circular RNAs in visceral organ fibrosis.

Circular RNAs (circRNAs) are a novel class of noncoding RNAs produced during pre-mRNA splicing and are emerging as new members of the gene regulatory network. Unlike linear RNAs, circRNAs have a unique structure with a covalently closed loop formed from the ligation of exons, introns, or both. CircRNAs are widely expressed in various organisms in a species-, tissue-, developmental stage- and disease-specific manner; circRNAs have been demonstrated to play a vital role in the pathogenesis and progression of human diseases. Fibrosis is characterized by an abnormal excessive deposition of extracellular matrix (ECM) in the extracellular space and plays important roles in many different pathologies of various organs. CircRNAs function as master regulators of gene expression to "sponge" or sequester other genes and target gene expression, transcription, splicing, etc. Increasing evidence has revealed that circRNAs are tightly associated with fibrotic diseases in various organs, including the lungs, liver, heart and kidneys. Herein, we provide the current understanding of the molecular characteristics of circRNAs and summarize the findings from circRNA studies in which the functions and mechanisms of action of circRNAs in organ fibrosis were proposed.

circDLPAG4/HECTD1 mediates ischaemia/reperfusion injury in endothelial cells via ER stress.

Background: Vascular endothelial cell dysfunction, characterized by cell apoptosis and migration, plays a crucial role in ischaemia/reperfusion (I/R) injury, a common aspect of cardiovascular diseases. Recent studies have suggested that non-coding RNAs, such as circular RNAs (circRNA), play a role in cell dysfunction in I/R injury, although the detailed mechanism is unclear.Methods: Human umbilical vein endothelial cells (HUVECs) were used for in vitro I/R model. Protein expression was detected by western blotting (WB) and immunocytochemistry. The CRISPR/Cas9 system, WB, cell viability assays, Hoechst staining and a 3D migration model were used to explore functional changes. RNA expression was evaluated using quantitative real-time PCR and a FISH assay combined with lentivirus transfection regulating circRNAs and miRNAs. A mouse myocardial I/R model using C57 mice was established to confirm the in vitro findings.Results: In HUVECs, I/R induced a significant time-dependent decrease in HECTD1 associated with an approximately 45% decrease in cell viability and increases in cell apoptosis and migration, which were attenuated by HECTD1 overexpression. I/R-induced upregulation of endoplasmic reticulum stress was also attenuated HECTD1 overexpression. Moreover, miR-143 mimics inhibited HECTD1 expression, which was restored by circDLGAP4 overexpression, providing insight as to the molecular mechanism of I/R-induced HECTD1 in endothelial cell dysfunction.Conclusion: Our results suggest a critical role for circDLGAP4 and HECTD1 in endothelial cell dysfunction induced by I/R, providing novel insight into potential therapeutic targets for the treatment of myocardial ischaemia.

CircHECTD1 mediates pulmonary fibroblast activation via HECTD1.

BACKGROUND: Circular RNA (circRNA), a new class of noncoding RNA, has been shown to be important in silicosis due to its unique role as a transcription regulator or as a sponge of small RNA regulators. Here, the mechanisms underlying circHECTD1/HECTD1 in fibroblast activation and subsequent fibrosis induced by SiO2 were investigated. METHODS: Primary human pulmonary fibroblasts (HPF-a) were utilized, combined with quantitative real-time PCR (qRT-PCR) and fluorescence in situ hybridization (FISH) assays. LC3B-LV-RFP lentivirus was used to evaluate the role of autophagy. The CRISPR/Cas9 system was applied to specifically knock down HECTD1, combined with MTT, BrdU, and migration assays, to explore the functional changes induced by SiO2. RESULTS: After exposure to SiO2, the circHECTD1 level was decreased, which was associated with an increase in HECTD1 in HPF-a cells. SiO2-induced autophagy was reversed by either circHECTD1 overexpression or HECTD1 knockdown in HPF-a cells, with restored SiO2-induced fibroblast activation, proliferation, and migration via downstream autophagy. The lungs of mice exposed to SiO2 confirmed the upregulation of HECTD1 in pulmonary fibroblasts. CONCLUSIONS: Our data suggested a link between circHECTD1/HECTD1 and fibroblast activation with subsequent fibrosis induced by SiO2, providing novel insight into the potential of circHECTD1/HECTD1 to be a therapeutic target for silicosis.

SPIO nanoparticle-labeled bone marrow mesenchymal stem cells inhibit pulmonary EndoMT induced by SiO2.

Endothelial-mesenchymal transition (EndoMT) is a key step during lung fibrosis. Studies have shown that bone marrow mesenchymal stem cells (BMSCs) may act as therapeutic candidates for lung fibrosis. However, the effects of BMSCs on EndoMT induced by SiO2 have not been elucidated, and means to label and track grafted cells have been lacking. The current study explored whether BMSCs prevented pulmonary fibrosis by targeting EndoMT, as well as analyzed the distribution of BMSCs labeled with superparamagnetic iron oxide (SPIO) nanoparticles during treatment. TIE2-GFP mice, human umbilical vein endothelial cells (HUVECs), and BMSCs labeled with SPIO nanoparticles were used to explore the distributions and therapeutic effects of BMSCs in vivo and in vitro. We found that BMSCs reversed lung fibrosis by targeting EndoMT in vivo. Furthermore, we show that BMSCs labeled with SPIO nanoparticles could be used to track stem cells reliably in the lungs for 14 days. Conditioned medium from BMSCs attenuated the increased functional changes and reversed the SiO2-induced upregulation of ER stress and autophagy markers irrespective of whether they were nanoparticle labeled or not. Our findings identify novel methods to track labeled BMSCs with therapeutic potential.

CircRNA-012091/PPP1R13B-mediated Lung Fibrotic Response in Silicosis via Endoplasmic Reticulum Stress and Autophagy.

Silicosis is a progressive fibrotic disease of lung tissue caused by long-term inhalation of SiO2. However, relatively few studies of the direct effects of SiO2 on lung fibroblasts have been performed. PPP1R13B is a major member of the apoptosis-stimulating proteins of the p53 family, but its role in pulmonary fibrosis is unclear. To elucidate the role of PPP1R13B in the pathological process of silicosis, we explored the molecular mechanisms related to PPP1R13B and the functional effects of proliferation and migration of fibroblasts. Through lentivirus transfection, Western blotting, and fluorescent in situ hybridization experiments, we found that SiO2 downregulated circRNA-012091 (circ-012091) expression in lung fibroblasts and induced upregulation of downstream PPP1R13B. Transfection of L929 cells with PPP1R13B CRISPR NIC plasmid inhibited the upregulation of endoplasmic reticulum stress (ERS) and autophagy-related protein expression in lung fibroblasts treated with SiO2, and induced decreases in cell proliferation, migration, and viability. Transfection of L929 cells with the PPP1R13B CRISPR ACT plasmid induced increases in cell proliferation, migration, and viability. In addition, the ERS inhibitor salubrinal and the autophagy inhibitor 3-methyladenine inhibited the increased migration of L929 cells transfected with the PPP1R13B CRISPR ACT plasmid. These results suggest that PPP1R13B regulated by circ-012091 promotes the proliferation and migration of lung fibroblasts through ERS and autophagy, and plays a crucial role in the development of pulmonary fibrosis in silicosis.

circHIPK2-mediated σ-1R promotes endoplasmic reticulum stress in human pulmonary fibroblasts exposed to silica.

Silicosis is characterized by fibroblast accumulation and excessive deposition of extracellular matrix. Although the roles of SiO2-induced chemokines and cytokines released from alveolar macrophages have received significant attention, the direct effects of SiO2 on protein production and functional changes in pulmonary fibroblasts have been less extensively studied. Sigma-1 receptor, which has been associated with cell proliferation and migration in the central nervous system, is expressed in the lung, but its role in silicosis remains unknown. To elucidate the role of sigma-1 receptor in fibrosis induced by silica, both the upstream molecular mechanisms and the functional effects on cell proliferation and migration were investigated. Both molecular biological assays and pharmacological techniques, combined with functional experiments, such as migration and proliferation, were applied in human pulmonary fibroblasts from adults to analyze the molecular and functional changes induced by SiO2. SiO2 induced endoplasmic reticulum stress in association with enhanced expression of sigma-1 receptor. Endoplasmic reticulum stress promoted migration and proliferation of human pulmonary fibroblasts-adult exposed to SiO2, inducing the development of silicosis. Inhibition of sigma-1 receptor ameliorated endoplasmic reticulum stress and fibroblast functional changes induced by SiO2. circHIPK2 is involved in the regulation of sigma-1 receptor in human pulmonary fibroblasts-adult exposed to SiO2. Our study elucidated a link between SiO2-induced fibrosis and sigma-1 receptor signaling, thereby providing novel insight into the potential use of sigma-1 receptor/endoplasmic reticulum stress in the development of novel therapeutic strategies for silicosis treatment.

Protective Effects of Cerium Oxide Nanoparticles on MC3T3-E1 Osteoblastic Cells Exposed to X-Ray Irradiation.

BACKGROUND/AIMS: Exposure to ionizing radiation can result in bone damage, including decreased osteocyte number and suppressed osteoblastic activity. However, molecular mechanisms remain to be elucidated, and effective prevention strategies are still limited. This study was to investigate whether cerium oxide nanoparticles (CeO2 NP) can protect MC3T3-E1 osteoblast-like cells from damaging effects of X-ray irradiation, and to study the underpinning mechanism(s). METHODS: MC3T3-E1, a osteoblast-like cell line, was exposed to X-ray irradiation and treated with different concentration of CeO2 nanoparticles. The micronucleus frequency was counted under a fluorescence microscope. Cell viability was evaluated using MTT assay. The effects of irradiation and CeO2 nanoparticles on alkaline phosphatase activity and MC3T3-E1 mineralization were further assayed. RESULTS: We found that the ratio of micronuclei to binuclei was dose-dependently increased with X-ray irradiation (from 2 to 6 Gy), but diminished with the increased concentration of CeO2 NP treatment (from 50 to 100 nM). Exposure to X-rays (6 Gy) decreased cell viability, differentiation and the mineralization, but CeO2 NP treatment (100 nM) attenuated the deteriorative effects of irradiation. Both intracellular reactive oxygen species (ROS) production and extracellular H2O2 concentration were increased after X-ray irradiation, but reduced following CeO2 NP treatment. Similar to irradiation, exposure to H2O2 (10 µM) elevated the frequency of micronuclei and diminished cell viability and mineralization, while these changes were ameliorated following CeO2 NP treatment. CONCLUSIONS: Taken together, our findings suggest that CeO2 nanoparticles exhibit astonishing protective effects on irradiation-induced osteoradionecrosis in MC3T3-E1 cells, and the protective effects appear to be mediated, at least partially, by reducing oxidative stress.

MCPIP1 Regulates Alveolar Macrophage Apoptosis and Pulmonary Fibroblast Activation After in vitro Exposure to Silica.

BACKGROUND: Silicosis is a fatal and fibrotic pulmonary disease caused by the inhalation of silica. After arriving at the alveoli, silica is ingested by alveolar macrophages (AMOs), in which monocyte chemotactic protein-induced protein 1 (MCPIP1) plays an essential role in controlling macrophage-mediated inflammatory responses. However, the mechanism of action of MCPIP1 in silicosis is poorly understood. METHODS: Primary rat AMOs were isolated and treated with SiO2 (50 µg/cm(2)). MCPIP1 and AMO activation/apoptosis markers were detected by immunoblotting. MCPIP1 was down-regulated using siRNA in AMOs. The effects of AMOs on fibroblast activation and migration were evaluated using a gel contraction assay, a scratch assay, and a nested collagen matrix migration model. RESULTS: After exposure to SiO2, MCPIP1 was significantly increased in rat AMOs. Activation and apoptosis markers in AMOs were up-regulated after exposure to SiO2 Following siRNA-mediated silencing of MCPIP1 mRNA, the markers of AMO activation and apoptosis were significantly decreased. Rat pulmonary fibroblasts (PFBs) cultured in conditional medium from AMOs treated with MCPIP1 siRNA and SiO2 showed significantly less activation and migration compared with those cultured in conditional medium from AMOs treated with control siRNA and SiO2 CONCLUSION: Our data suggest a vital role for MCPIP1 in AMO apoptosis and PFB activation/migration induced by SiO2.

MCPIP1 mediates silica-induced cell migration in human pulmonary fibroblasts.

Silicosis is a systemic disease caused by inhaling silicon dioxide (SiO2). Phagocytosis of SiO2 in the lungs initiates an inflammatory cascade that results in fibroblast proliferation and migration followed by fibrosis. According to previous data from our laboratory, monocyte chemotactic protein-1 (MCP-1) plays a critical role in fibroblast proliferation and migration in conventional two-dimensional (2D) monolayer cultures. The present study aimed to explore the downstream cascade of MCP-1 in both 2D and three-dimensional (3D) cell culture models of silicosis. Experiments using primary cultured adult human pulmonary fibroblasts (HPF-a) demonstrated the following: 1) SiO2 treatment induces expression of MCP-1-induced protein (MCPIP1) in a time- and dose-dependent manner in both 2D and 3D cultures; 2) the MAPK and phosphatidylinositol-3-kinase (PI3K)/Akt pathways are involved in SiO2-induced MCPIP1 expression; and 3) MCPIP1 induction mediates the SiO2-induced increase in cell migration in both 2D and 3D cultures. The effect of MCP-1 in silicosis occurs mainly through MCPIP1, which, in turn, mediates the observed SiO2-induced increase in pulmonary fibroblast migration. However, the time frame for MCPIP1 induction differed between 2D and 3D cultures, indicating that, compared with conventional 2D cell culture systems, 3D culture may be useful for analyses of fibroblast physiology under conditions that more closely resemble in vivo environments. Our study determined the link between fibroblast-derived MCPIP1 and SiO2-induced cell migration, and this finding provides novel evidence of the potential of MCPIP1 in the development of novel therapeutic strategies for silicosis.

Exposure to soil, house dust and decaying plants increases gut microbial diversity and decreases serum immunoglobulin E levels in BALB/c mice.

To assess the impact of sanitation of a living environment on gut microbiota and development of the immune system, we raised BALB/c mice under three distinct environmental conditions: a specific pathogen-free animal room (SPF), a general animal room (XZ) and a farmhouse (JD). All other variables like diet, age, genetic background, physiological status and original gut microbiota were controlled for in the three groups. Using high-throughput sequencing of the 16S rRNA gene, we found that each mouse group had a specific structure of the gut microbial community. Groups JD and XZ harboured a significantly more diverse and richer gut microbiota than did group SPF. Bacteroidetes were significantly more abundant in groups XZ and JD than in group SPF, whereas Firmicutes showed the inverse pattern. Total serum immunoglobulin E (IgE) levels were significantly lower in groups XZ and JD than in group SPF. There were no significant differences in gut microbiota diversity and serum IgE concentration between groups JD and XZ, but we found higher abundance of dominant genera in the gut microflora of group JD. We conclude that exposure to soil, house dust and decaying plant material enhances gut microbial diversity and innate immunity. Our results seem to provide new evidence supporting the hygiene hypothesis.

p53/PUMA expression in human pulmonary fibroblasts mediates cell activation and migration in silicosis.

Phagocytosis of SiO2 into the lung causes an inflammatory cascade that results in fibroblast proliferation and migration, followed by fibrosis. Clinical evidence has indicated that the activation of alveolar macrophages by SiO2 produces rapid and sustained inflammation characterized by the generation of monocyte chemotactic protein 1, which, in turn, induces fibrosis. However, the details of events downstream of monocyte chemotactic protein 1 activity in pulmonary fibroblasts remain unclear. Here, to elucidate the role of p53 in fibrosis induced by silica, both the upstream molecular mechanisms and the functional effects on cell proliferation and migration were investigated. Experiments using primary cultured adult human pulmonary fibroblasts led to the following results: 1) SiO2 treatment resulted in a rapid and sustained increase in p53 and PUMA protein levels; 2) the MAPK and PI3K pathways were involved in the SiO2-induced alteration of p53 and PUMA expression; and 3) RNA interference targeting p53 and PUMA prevented the SiO2-induced increases in fibroblast activation and migration. Our study elucidated a link between SiO2-induced p53/PUMA expression in fibroblasts and cell migration, thereby providing novel insight into the potential use of p53/PUMA in the development of novel therapeutic strategies for silicosis treatment.

MCPIP1 Regulates Fibroblast Migration in 3-D Collagen Matrices Downstream of MAP Kinases and NF-κB.

The fibroblast-populated three-dimensional (3-D) collagen matrix has been used to model matrix contraction, cell motility, and general fibroblast biology. MCPIP1 (monocyte chemotactic protein-induced protein 1) has been shown to regulate inflammation, angiogenesis, and cellular motility. In the present study, we demonstrated induction of MCPIP1 in human fibroblasts embedded in the stress-released 3-D collagen matrix, which occurred through activation of mitogen-activated protein kinases, phosphoinositide 3-kinase, and NF-κB. Furthermore, MCPIP1 induction was associated with inhibition of fibroblast migration out of the nested collagen matrix. MCPIP1 induction or ectopic expression also upregulated p53. RNA interference of p53 prevented the inhibition of migration produced by induction or ectopic expression of MCPIP1. Our findings suggest a new role for MCPIP1 as a molecular switch that regulates fibroblast migration in the nested collagen matrix model.

Role of human pulmonary fibroblast-derived MCP-1 in cell activation and migration in experimental silicosis.

BACKGROUND: Silicosis is a systemic disease caused by inhaling silicon dioxide (SiO2). Phagocytosis of SiO2 in the lung initiates an inflammatory cascade that results in fibroblast proliferation and migration and subsequent fibrosis. Clinical evidence indicates that the activation of alveolar macrophages by SiO2 produces rapid and sustained inflammation that is characterized by the generation of monocyte chemotactic protein 1 (MCP-1), which induces fibrosis. Pulmonary fibroblast-derived MCP-1 may play a critical role in fibroblast proliferation and migration. METHODS AND RESULTS: Experiments using primary cultured adult human pulmonary fibroblasts (HPF-a) demonstrated the following results: 1) SiO2 treatment resulted in the rapid and sustained induction of MCP-1 as well as the elevation of the CC chemokine receptor type 2 (CCR2) protein levels; 2) pretreatment of HPF-a with RS-102895, a specific CCR2 inhibitor, abolished the SiO2-induced increase in cell activation and migration in both 2D and 3D culture systems; and 3) RNA interference targeting CCR2 prevented the SiO2-induced increase in cell migration. CONCLUSION: These data demonstrated that the up-regulation of pulmonary fibroblast-derived MCP-1 is involved in pulmonary fibroblast migration induced by SiO2. CCR2 was also up-regulated in response to SiO2, and this up-regulation facilitated the effect of MCP-1 on fibroblasts. Our study deciphered the link between fibroblast-derived MCP-1 and SiO2-induced cell migration. This finding provides novel insight into the potential of MCP-1 in the development of novel therapeutic strategies for silicosis.

Acetaminophen attenuates glomerulosclerosis in obese Zucker rats via reactive oxygen species/p38MAPK signaling pathways.

Focal segmental glomerulosclerosis is a critical pathological lesion in metabolic syndrome-associated kidney disease that, if allowed to proceed unchecked, can lead to renal failure. However, the exact mechanisms underlying glomerulosclerosis remain unclear, and effective prevention strategies against glomerulosclerosis are currently limited. Herein, we demonstrate that chronic low-dose ingestion of acetaminophen (30 mg/kg/day for 6 months) attenuates proteinuria, glomerulosclerosis, podocyte injury, and inflammation in the obese Zucker rat model of metabolic syndrome. Moreover, acetaminophen treatment attenuated renal fibrosis and the expression of profibrotic factors (fibronectin, connective tissue growth factor, transforming growth factor ß), reduced inflammatory cell infiltration into the glomeruli, and decreased the expression of monocyte chemoattractant protein, glutathione (GSH) reductase, and nuclear factor erythroid 2-related factor 2, but increased the level of GSH synthetase in obese animals. Further in vivo and in vitro studies using human renal mesangial cells exposed to high glucose or hydrogen peroxide suggested that the renoprotective effects of acetaminophen are characterized by diminished renal oxidative stress and p38MAPK hyperphosphorylation.

Involvement of PPARγ in emodin-induced HK-2 cell apoptosis.

Emodin, a major compound in total rhubarb anthraquinones (TRAs), has exhibited nephrotoxicity in Sprague Dawley rats and cytotoxicity to HK-2 cells, a human proximal tubular epithelial cell line, in our previous study. However, the exact molecular mechanisms underlying emodin-induced cytotoxicity remain undefined. In this study, the exposure of HK-2 cells to emodin led to decreased cell viability, caspase 3 cleavage and activation, loss of mitochondrial membrane potential (DWm), and cytochrome c release from mitochondria to cytosol. Meanwhile, the levels of peroxisome proliferator-activated receptor gamma (PPARγ) mRNA and protein expression were elevated. GW9662, an antagonist of PPARγ, dramatically ameliorated the release of cytochrome c, the activation of caspase 3, and the reduction of cell viability induced by emodin. Importantly, emodin at the concentration causing apoptosis enhanced the stability of PPARγ mRNA. Taken together, these findings suggest that PPARγ might mediate, at least in part, emodin-induced HK-2 cell apoptosis via mitochondrial pathway.

Acetaminophen attenuates obesity-related renal injury through ER-mediated stress mechanisms.

BACKGROUND/AIMS: Obesity is an independent risk factor for the development of kidney disease. The purpose of this study was to determine how obesity may contribute to renal damage and whether acetaminophen ingestion can diminish obesity-associated renal cell injury in the obese Zucker rat model. METHODS: Male obese Zucker rats (4 weeks old, n=6) were treated with acetaminophen (30 mg / kg body weight / day) for 26 weeks. Age matched obese control (OC), obese vehicle (OV, 0.073 mL DMSO/kg/d), and lean Zucker rats (LC) were used to determine the effects of treatment and obesity. RESULTS: Compared to lean control rats, renal lipid deposition, expression of the endoplasmic reticulum (ER) stress protein GRP78 and activation of the ER stress-related eIF2α-ATF4-CHOP, caspase 12, and JNK-MAPK signaling pathways were increased in the obese control and obese vehicle rats. These alterations were associated with the elevated renal cell apoptosis and urinary albumin excretion. Acetaminophen treatment decreased renal lipid deposition, ER-stress related signaling, apoptosis and albuminuria. CONCLUSION: These data suggest that the protective effects of low dose acetaminophen on renal injury are mediated, at least in part, through attenuation of ER stress.

Possible roles of astrocytes in estrogen neuroprotection during cerebral ischemia.

17ß-Estradiol (E2), one of female sex hormones, has well-documented neuroprotective effects in a variety of clinical and experimental disorders of the central cerebral ischemia, including stroke and neurodegenerative diseases. The cellular mechanisms that underlie these protective effects of E2 are uncertain because a number of different cell types express estrogen receptors in the central nervous system. Astrocytes are the most abundant cells in the central nervous system and provide structural and nutritive support of neurons. They interact with neurons by cross-talk, both physiologically and pathologically. Proper astrocyte function is particularly important for neuronal survival under ischemic conditions. Dysfunction of astrocytes resulting from ischemia significantly influences the responses of other brain cells to injury. Recent studies demonstrate that estrogen receptors are expressed in astrocytes, indicating that E2 may exert multiple regulatory actions on astrocytes. Cerebral ischemia induced changes in the expression of estrogen receptors in astrocytes. In the present review, we summarize the data in support of possible roles for astrocytes in the mediation of neuroprotection by E2 against cerebral ischemia.

Metabolic syndrome-induced tubulointerstitial injury: role of oxidative stress and preventive effects of acetaminophen.

The prevalence of metabolic syndrome persistently increases and affects over 30% of U.S. adults. To study how metabolic syndrome may induce tubulointerstitial injury and whether acetaminophen has renal-protective properties, 4-week-old obese Zucker rats were randomly assigned into three groups, control (OC), vehicle dimethyl sulfoxide (OV), and acetaminophen treatment (30 mg/kg/day for 26 weeks), and lean Zucker rats served as healthy controls. Significant tubulointerstitial injuries were observed in both OC and OV animals, evidenced by increased tubular cell death, tubular atrophy/dilation, inflammatory cell infiltration, and fibrosis. These tubulointerstitial alterations were significantly reduced by treatment with a chronic but low dose of acetaminophen, which acted to diminish NADPH oxidase isoforms Nox2 and Nox4 and decrease tubulointerstitial oxidative stress (reduced tissue superoxide and macromolecular oxidation). Decreased oxidative stress by acetaminophen was paralleled by the reduction of tubular proapoptotic signaling (diminished Bax/Bcl-2 ratio and caspase 3 activation) and the alleviation of tubular epithelial-to-mesenchymal transition (decreased transforming growth factor ß, connective tissue growth factor, α-smooth muscle actin, and laminin). These data suggest that increased oxidative stress plays a critical role in mediating metabolic syndrome-induced tubulointerstitial injury and provide the first evidence suggesting that acetaminophen may be of therapeutic benefit for the prevention of tubulointerstitial injury.

Effects of estrogen on neuronal KCNQ2/3 channels expressed in PC-12 cells.

We previously reported that 17ß-estradiol (E2) improves long term potentiation (LTP) in hippocampal neurons after global ischemia in rat. In the present study, we investigated if E2 can directly modulate the activity of neuronal KCNQ2/3 channels, the molecular entity of neuronal M-current in hippocampus, expressed in the PC-12 cells. We found that exogenous E2 inhibits the KCNQ2/3 channels in a dose-dependent fashion. The minimal inhibitory concentration of E2 is 10 µM. At testing membrane potential of +90 mV, the whole cell current density was reduced to 56.5, 49.3 and 31.9% of the control by 50, 20 and 10 µM of E2, respectively. The voltage-dependency of the KCNQ2/3 currents was also affected. E2 at 10, 20 and 50 µM shifted the half maximal activation voltage (V1/2) from 13.8 ± 2.3 mV (n=12) to 20.6 ± 1.9 mV (n=8, p<0.05), 26.0 ± 1.9 mV (n=8, p<0.001) and 27.6 ± 3.5 mV (n=8, p<0.001), respectively. Our data indicate that exogenous E2 can directly affect the activity of KCNQ2/3 channels at pharmacological levels via a non-genomic pathway.

Neurosteroid estradiol rescues ischemia-induced deficit in the long-term potentiation of rat hippocampal CA1 neurons.

Increasing evidence indicates that neurosteroid 17beta-Estradiol (E2), a type of female sex hormone, has a neuroprotective effect against cerebral injury. However, it remains unknown whether E2 can also protect the hippocampal CA1 neurons from functional deficits in synaptic transmission and plasticity caused by ischemia. To address this issue, adult male Wistar rats were subjected to mild global cerebral ischemia created by four-vessel occlusion (4VO) for 10min, and the effects of E2 administration against the ischemic injury were investigated. The electrophysiological properties of Schaffer collateral-CA1 synapses were examined 7days after ischemia by applying a real-time optical recording technique to the hippocampal slices stained with a voltage-sensitive dye (RH482). The ischemic brain showed a decreased basal synaptic transmission and an impairment of LTP induction, but no alteration in paired-pulse facilitation. The administration of E2 (1mg/kg) 3h before ischemia was able to protect CA1 neurons from these ischemia-induced synaptic dysfunctions. The estrogen receptor-alpha (ERalpha) selective agonist, propyl pyrazole triol (PPT, 2mg/kg), exerted a similar protective effect, but the estrogen receptor-beta (ERbeta) agonist, diarylpropiolnitrile (DPN, 8mg/kg), failed to do so. A histological examination revealed that the transient global cerebral ischemia markedly reduced the density of pyramidal neurons in the CA1 region. The cell loss was significantly attenuated by E2 and PPT but not by DPN, as observed in synaptic functions. These findings suggest that E2 can protect neurons not only from cell death but also from functional damages due to a relatively mild degree of transient cerebral ischemia, and this effect is mediated by ERalpha, but not by ERbeta.