The inflammation and reactive oxygen species regulated by Nrf2 and NF-κB signaling pathways in 630-nm light-emitting diode irradiation treated THP-1 monocytes/macrophages.

Because of a large number of macrophages and its secreted pro-inflammatory factors in the synovial fluid of patients with rheumatoid arthritis, the present study aimed to investigate the effect and mechanism of 630-nm LED exposure on monocytes/macrophages and its anti-inflammatory effect. The THP-1 monocytes and PMA-induced THP-1 macrophages (THP-1 macrophages) were employed and irradiated by 630-nm LED for different time and times, and then measure the pro-inflammatory cytokines production by RT-qPCR and Milliplex MAP Multiplex assay, the proteins involved in inflammation pathway and reactive oxygen species (ROS) levels in the cells were detected by Western blot and DCFH-DA method. The exposure dose of red LED (15.3 J/cm2, 30.6 J/cm2) were determined as no-influence on the cell proliferation, the pro-inflammatory factors TNF-α and IL-1ß mRNAs, and secretions in supernatant of THP-1 macrophages were significantly decreased after LED exposure. The ROS production was blocked in THP-1 monocytes and THP-1 macrophages after treatment of LED. Finally, the phosphorylated NF-κB proteins which involved in inflammation pathway significantly decreased, and its inhibitors Nrf2 were slightly upregulated. The effects of LED anti-inflammation response are dependent on the mechanism of inhibiting ROS level and regulating NF-κB signaling pathways by increasing Nrf2 expression in the cells. It is suggested that 630-nm LED could decrease pro-inflammation in immune cells, and it may be a beneficial adjunct therapy in relieving inflammation of patients with rheumatoid arthritis.

Expression of HMGB1 and RAGE in rat and human brains after traumatic brain injury.

BACKGROUND: Increasing evidence suggests that an inflammatory reaction contributes to the secondary brain injury that plays a critical role in the clinical outcome of patients with traumatic brain injury (TBI). Recently, high-mobility group box 1 (HMGB1) has been identified as a key cytokine in the inflammatory reaction and may represent a new target for the treatment of TBI. However, the expression of HMGB1 during this injury process has not yet been studied. METHODS: In this study, the levels of both HMGB1 and receptor for advanced glycation end products (RAGE) in the rat brain were analyzed by Western blot at different time points after TBI. Immunohistochemistry was also performed to examine the expression pattern of HMGB1 and RAGE in both the rat and the human brain after TBI. RESULTS: In the rat brain, HMGB1 levels significantly declined below the basal level at 6 hours after TBI and then gradually returned to the basal level 2 days later. RAGE expression increased 6 hours after TBI and reached its peak after 1 day; this level then slowly decreased but remained higher than the sham-injury group until 6 days after TBI. In both rat and human brains, HMGB1 either disappeared or was translocated from the nucleus to the cytoplasm at early stages after TBI and then was localized to the cytoplasm of phagocytic microglia at later stages. RAGE expression increased in the region surrounding the contused area after TBI in both rat and human brains. At later stages, RAGE was mainly expressed in microglia. CONCLUSION: HMGB1 is involved in both early and later stages after TBI. Targeting HMGB1 signaling may be a promising therapeutic approach for the treatment of TBI.

[Expression of FAPalpha and TGF-beta1 in sudden deaths due to acute myocardial ischemia].

OBJECTIVE: To explore the expression of fibroblast activation protein alpha (FAPalpha) and transforming growth factor-beta1 (TGF-beta1) in myocardial cytoplasm for the cases of sudden death due to acute myocardial ischemia. METHODS: The heart tissues of 47 cases were collected. All cases were divided into three groups: control group, acute myocardial infarction group and recurrent myocardial infarction group. FAPalpha and TGF-beta1 expressions were explored in myocardial cytoplasm by immunohistochemistry technology. The staining results were collected by image analysis system and then the positive area ratio and average optical density were detected. The positive signal differences were compared among the groups. RESULTS: Strong FAPalpha and TGF-beta1 expressions were detected in myocardial cytoplasm in both acute and recurrent myocardial infarction groups. The expression of FAPalpha was not detected in myocardial cytoplasm in control group and TGF-beta1 expression showed a weak positive result. FAPalpha and TGF-beta1 expressions showed the statistical difference (P < 0.05) in myocardial infarction (acute and recurrent) groups and control group. CONCLUSION: FAPalpha and TGF-beta1 can be the diagnostic markers for determing acute myocardial infarction.

Post-conditioning protecting rat cardiomyocytes from apoptosis via attenuating calcium-sensing receptor-induced endo(sarco)plasmic reticulum stress.

Our previous studies demonstrated that caclium-sensing receptor (CaR) stimulation elicited phospholipase C (PLC)-mediated inositol triphosphate (IP(3)) formation, leading to an elevation in [Ca(2+)](i) released from the endo(sarco)plasmic reticulum (ER) to induce ER stress and perturbations of ER function, which cause cardiomyocyte apoptosis during ischemia/reperfusion (I/R). The aim of this study was to determine whether the protection of post-conditioning (PC) from I/R heart injury involved relieving calcium-sensing receptor (CaR)-induced ER stress. Male Wistar rats were subjected to 30 min of ischemia followed by 2 h of reperfusion. The rats were post-conditioned after the 30 min of ischemia by three cycles of 10 s of reperfusion followed by 10 s of ischemia at the onset of reperfusion. Meanwhile, GdCl(3), an activator of CaR, and NPS-2390, a specific inhibitor, were administered. We found that the PC and PC with NPS-2390 groups improved the recovery of cardiac function during reperfusion compared to the IR and PC groups with GdCl(3), respectively. [Ca(2+)](i) and [Ca(2+)](ER) were determined using Fluo-4 AM and Fluo-5N AM, respectively, using laser confocal microscopy. [Ca(2+)](i) was significantly increased, whereas [Ca(2+)](ER) was significantly decreased in the I/R and PC groups with GdCl(3). The rate of apoptotic cells was significantly decreased as shown by TUNEL (Terminal deoxy-nucleotidyl transferase-mediated dUTP nick end labeling) assay in PC and PC with NPS-2390 groups compared to the I/R and PC groups with GdCl(3). In the I/R and PC groups with GdCl(3), the activated fragments of caspase-12, the cleavage products of activating transcription factor 6 (ATF6) and phospho-JNK (c-Jun NH(2)-terminal kinase) were increased compared to the PC and PC with GdCl(3) groups. These results demonstrated that PC could protect the myocardium from I/R injury by inhibiting CaR-induced sarcoplasmic reticulum stress.

Small interfering RNA against the 2C genomic region of coxsackievirus B3 exerts potential antiviral effects in permissive HeLa cells.

Coxsackievirus B3 (CVB3) is the most important causal agent of viral heart muscle disease, but no specific antiviral drug is currently available. Small interfering RNA (siRNA) has been used as an antiviral therapeutic strategy via posttranscriptional gene silencing. In this study, eleven siRNAs were designed to target seven distinct regions of the CVB3 genome including VP1, VP2, VP3, 2A, 2C, 3C, and 3D. All of the siRNAs were individually transfected into HeLa cells, which were subsequently infected with CVB3. The impacts of RNA interference (RNAi) on viral replication were evaluated using five measures: cytopathic effect (CPE), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, 50% tissue culture infectious dose (TCID(50)), real-time RT-PCR, and Western blot. Five of the eleven siRNAs were highly efficient at inhibiting viral replication. This was especially true for siRNA-5, which targeted the ATPase 2C. However, antiviral activity varied significantly among siRNA-9, -10, and -11 even though that they all targeted the 3D region. Our results revealed several effective targets for CVB3 silencing, and provided evidence that sequences except CRE within the 2C region may also be potential targets for CVB3-specific siRNAs design. These data supported a potential role of RNA interference in future antiviral intervention therapies.

Calcium-sensing receptor activating phosphorylation of PKCδ translocation on mitochondria to induce cardiomyocyte apoptosis during ischemia/reperfusion.

The calcium-sensing receptor (CaSR) is a G protein-coupled receptor (GPCR) that activates intracellular effectors; for example, it causes inositol phosphate (IP) and 1,2 diacylglycerol (DAG) accumulation, stimulating the release of intracellular calcium and the activation of the protein kinase Cs (PKCs). The activation of CaSR by ischemia/reperfusion (I/R) induces cardiomyocyte apoptosis through the mitochondrial apoptotic pathway; however, the underlying mechanisms remain unclear. In this study, rat hearts were subjected to 30 min of ischemia followed by 2 h of reperfusion in the presence of a CaSR activator, GdCl(3). Our results revealed that, under these conditions, the expression of CaSR was increased, the number of apoptotic cardiomyocytes was significantly increased (as shown by terminal deoxy-nucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay) and the cells with a typical apoptotic morphology were observed using transmission electron microscopy. Our data further showed that mitochondria isolated from hearts that had undergone I/R combined with GdCl(3) exhibited a significant increase in the translocation of phosphorylated PKCδ to the mitochondria, an increase in cytochrome c (cyt c) release from the mitochondria and a marked decrease in mitochondrial potential. The administration of rottlerin, an inhibitor of PKCδ, significantly reduced reperfusion-induced apoptosis, phospho-PKCδ translocation to the mitochondria and the release of cyt c from the mitochondria. Thus, the involvement of CaSR in cardiac apoptosis through the mitochondrial pathway during I/R with GdCl(3) is related to phospho-PKCδ translocation to the mitochondria.

Parathyroid hormone(1-34)-induced apoptosis in neuronal rat PC12 cells: implications for neurotoxicity.

Based on accumulated evidence, we speculate that a high concentration of parathyroid hormone (PTH) may cause neurotoxicity in patients with uremia through apoptosis-induced neuropathy. In this study, we demonstrated that in vitro stimulation with PTH(1-34) induced a significant decrease in PC12 cell numbers in both dosage- and time-dependent fashions when these cells were treated with PTH(1-34) at concentrations of 0.01, 0.1 or 1.0 µM for 24, 48, 72, and 96 h, respectively, as assessed by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) method. Decreased numbers of PC12 cells were caused by PTH(1-34)-induced apoptotic and cytotoxic processes, as determined by DNA fragmentation, flow cytometry, and lactate dehydrogenase (LDH)-leakage assays. Upregulation of the extracellular signal-regulated kinase (ERK) and p38 signaling pathway was the underlying mechanism responsible for 1.0 µM PTH(1-34)-induced apoptosis in PC12 cells, as elucidated by Western blotting analysis and confirmed with ERK and p38 inhibitors. Furthermore, 1.0 µM PTH(1-34)-induced apoptosis was accompanied by a release of cytochrome c and subsequent caspase-3 activation. These data suggest that a high concentration of PTH(1-34)-induced cytotoxicity and apoptosis in PC12 cells was associated with upregulation of ERK and p38 through a mitochondria-mediated apoptotic pathway.

[Immunohistochemical expression of HO-1 in traumatic human brain tissue].

OBJECTIVE: To investigate the expression of heme oxygenase-1 (HO-1) at different intervals and to provide evidence for estimation on injury intervals after brain contusion in human. METHODS: Twenty-four patients died of serious brain injury were assigned as injury group and 4 patients died of non-brain injury were served as control group. HO-1 expression was analyzed in brain tissue at different time intervals (3 h, 6-9 h, 12-24 h, 36 h-3d, 5-8d, 17-20d) by immunohistochemistry and auto-image analysis system. RESULTS: The level of HO-1 expression started to increase in 3 h after brain contusion compared to the control group (P < 0.05). The level of HO-1 expression highest level in 12-24 h group, and maintained high level in 36 h-3 d, then decreased gradually. CONCLUSION: The expression of HO-1 might be a strong evidence for human brain contusion time estimation.

Alterations of bone marrow sinusoidal endothelium in rat and patients with liver cirrhosis.

Whether bone marrow changes occur and potentially contribute to the hematological abnormalities in liver cirrhosis remain unclear. In this study, we established a rat model of liver cirrhosis induced by carbon tetrachloride. Electron microscopy examination showed focal lesions in bone marrow sinusoidal endothelium and hematopoietic cells in animals with cirrhosis. With the persistence of liver cirrhosis, injuries of bone marrow sinusoidal endothelium progressed from mild mitochondrial changes to nuclear pycnosis and cell disruption, and the trilineage hematopoietic cells showed apoptosis and necrosis. Immunohistochemistry revealed increased expression of E-selectin, P-selectin and vWF in bone marrow sinusoidal endothelium of the cirrhotic rats, which was consistent with the data from semiquantitative reverse transcriptase-polymerase chain reaction analysis. Autopsy specimens from patients with liver cirrhosis (in the absence of other disease) showed the same findings as detected by immunohistochemistry in animal models. The results provide evidence of the association between liver cirrhosis and bone marrow alterations by demonstrating the bone marrow sinusoidal endothelium lesions in both a rat model and patients. It also indicates that activation or injury of bone marrow sinusoidal endothelium mediated by E-selectin, P-selectin, and vWF might have a role in pathogenesis of bone marrow changes during liver cirrhosis. The lesions of bone marrow sinusoidal endothelium might contribute to the hematological abnormalities in the end stage of liver disease.

Fas/FasL-dependent apoptosis of hepatocytes induced in rat and patients with Clonorchis sinensis infection.

To investigate the injury and pathogenesis of Clonorchis sinensis (C. sinensis) to hepatocytes, the liver samples from Wistar rats and patients with C. sinensis infection were examined. The typical histopathological findings of clonorchiasis were observed in rats 4 to 12 weeks postinfection, and majority hepatocytes exhibited hydropic degeneration, even some hepatocytes showed densely condensed nuclei suggesting apoptosis in liver tissue. Apoptosis was found around the central vein or portal areas of liver tissue in rat infected with C. sinensis by transferase uridyl nick end labeling (TUNEL) assay. Compared with normal control, TUNEL-positive cells in liver tissue increase from 4 to 12 weeks postinfection with the peak at 8 weeks. Furthermore, the expression of mRNA and protein of Fas, FasL, and caspase-3 was stronger in infected group than normal control using semiquantitative reverse transcriptase polymerase chain reaction (RT-PCR) and immunohistochemistry. Autopsy specimens from four patients infected with C. sinensis have the same findings detected by histopathology, TUNEL, and immunohistochemistry. These findings suggest that the C. sinensis can stimulate both hepatocytic apoptosis and hydropic degeneration that may be responsible for relationship between clinical manifestations and liver lesions in patients with clonorchiasis. These data also indicate a role for Fas/FasL-mediated pathway in the apoptosis that occurs in response to C. sinensis infection.

Anti-apoptotic PTD-FNK protein suppresses lipopolysaccharide-induced acute lung injury in rats.

The present study was aimed at clarifying the effects of an anti-apoptotic protein for modulating symptoms in acute lung injury (ALI). From Bcl-x(L), a Bcl-2 family member, we constructed an artificial protein (FNK) and fused it with the protein transduction domain (PTD) of the HIV/Tat protein (PTD-FNK) to facilitate its permeation into cells. ALI was induced by intratracheal infusion of lipopolysaccharide (LPS) into Sprague-Dawley male rats. PTD-FNK was injected into the peritoneal cavity of the animals either 2 h before, or 3 h or 6 h after LPS challenge. All rats were sacrificed 24 h after the last treatment. Cell differential ratios and albumin concentration were estimated in bronchoalveolar lavage fluid. We examined histological change, myeloperoxidase activity, TUNEL assay, caspase-3/caspase-3-like activity and immunohistochemical reaction for caspase 3 (active form). In animals with PTD-FNK treatment, the albumin leakage was significantly attenuated with protection of tissue damage. Also, the apoptosis of alveolar wall cells was reduced by PTD-FNK treatment, while a total cell number and the neutrophil ratio were not changed. Human umbilical vein endothelial cells (HUVEC) and cells of an alveolar epithelial cell line (A549) were exposed to LPS or TNF-alpha with or without PTD-FNK treatment in vitro. Cell survival rates examined by trypan-blue exclusion assay were increased by PTD-FNK treatment in a concentration-dependent manner. Thus, PTD-FNK could play a protective role in ALI by suppressing apoptosis of alveolar epithelial cells and capillary endothelial cells despite of some effect on neutrophil activity.