Bone marrow-derived mesenchymal stem cells accelerate angiogenesis in pregnant experimentally induced deep venous thrombosis rat model via up-regulation of pro-angiogenic secretogranin II.

The present study investigated whether bone marrow-derived mesenchymal stem cells (BMMSCs) facilitate angiogenesis and improve outcomes of pregnancy with obstetric deep venous thrombosis (DVT) and explored the underlying mechanism. A pregnant DVT rat model was established using a "stenosis" method on the lower segment of the inferior vena cava (IVC). The extent of vascularization in thrombosed IVC was examined by immunohistochemistry. In addition, the effect of BMMSCs on DVT pregnancy outcomes was evaluated. We also characterized the effect of BMMSC-derived conditioned medium (BM-CM) on the impaired human umbilical vein endothelial cells (HUVECs). Thereafter, transcriptome sequencing was employed to identify the differentially expressed genes in thrombosed IVC tissues of DVT and DVT plus BMMSCs (thrice) groups. Lastly, the candidate gene's role in the promotion of angiogenesis was demonstrated in vitro and in vivo. The DVT model was successfully established using IVC stenosis. The injection of three consecutive BMMSC doses into pregnant SD rats with DVT was demonstrated to be the most effective treatment, which significantly reduced the length and weight of the thrombus, induced the highest level of angiogenesis, and ameliorated the embryo absorption rate. In vitro, BM-CM efficiently increased the abilities of impaired endothelial cells to proliferate, migrate, invade, and form vessel-like tubes, while inhibiting their apoptosis. Transcriptome sequencing revealed that BMMSCs induced a prominent upregulation of a variety of pro-angiogenic genes, including secretogranin II (SCG2). When SCG2 expression was knocked down by lentivirus, the BMMSCs' and BM-CM-induced pro-angiogenic effects on pregnant DVT rats and HUVECs were markedly attenuated. In conclusion, the study results suggest that BMMSCs enhance angiogenesis via up-regulation of SCG2, providing an effective alternative regenerative agent and novel target for the therapy of obstetric DVT.

Salidroside ameliorates severe acute pancreatitis-induced cell injury and pyroptosis by inactivating Akt/NF-κB and caspase-3/GSDME pathways.

Our previous studies showed that Salidroside (Sal), a glucoside of the phenylpropanoid tyrosol isolated from Rhodiola rosea L, alleviated severe acute pancreatitis (SAP) by inhibiting inflammation. However, the detailed mechanism remains unclear. Recent evidence has indicated a critical role of Sal in ameliorating inflammatory disorders by regulating pyroptosis. The present study aimed to explore the involvement of Sal and pyroptosis in the pathogenesis of SAP and investigate the potential mechanism. The effects of Sal on pyroptosis were first evaluated using SAP rat and cell model. Our results revealed that Sal treatment significantly decreased SAP-induced pancreatic cell damage and pyroptosis in vivo and in vitro, as well as reduced the release of lactate dehydrogenase (LDH), IL-1ß and IL-18. Search Tool for Interacting Chemicals (STITCH) online tool identified 4 genes (CASP3, AKT1, HIF1A and IL10) as candidate targets of Sal in both rattus norvegicus and homo sapiens. Western blot and immunohistochemistry staining validated that Sal treatment decreased the phosphorylation levels of Akt and NF-κB p65, as well as cleaved caspase-3 and N-terminal fragments of GSDME (GSDME-N), suggesting that Sal might suppress pyroptosis through inactivating Akt/NF-κB and Caspase-3/GSDME pathways. Furthermore, overexpression of AKT1 or CASP3 could partially reverse the inhibitory effects of Sal on cell injury and pyroptosis, while downregulation of AKT1 or CASP3 promoted the inhibitory effects of Sal. Taken together, our data indicate that Sal suppresses SAP-induced pyroptosis through inactivating Akt/NF-κB and Caspase-3/GSDME pathways.

Single-cell RNA sequencing for traumatic spinal cord injury.

Traumatic spinal cord injury (tSCI) is a severe injury of the central nervous system (CNS) with complicated pathological microenvironment that results in hemorrhage, inflammation, and scar formation. The microenvironment of the injured spinal cord comprises heterogeneous neurons, glial cells, inflammatory cells, and stroma-related cells. Increasing evidence has indicated that the altered cellular and molecular microenvironment following tSCI is a key factor impeding functional recovery. Single-cell RNA sequencing (scRNA-seq) has provided deep insights into the dynamic cellular and molecular changes in the microenvironment by comprehensively characterizing the diversity of spinal cord cell types. Specifically, scRNA-seq enables the exploration of the molecular mechanisms underlying tSCI by elucidating intercellular communication in spinal cord samples between normal and injury conditions at a single-cell resolution. Here, we first described the pathological and physiological processes after tSCI and gave a brief introduction of the scRNA-seq technology. We then focused on the recent scRNA-seq researches in tSCI, which characterized diverse cell-type populations and specific cell-cell interactions in tSCI. In addition, we also highlighted some potential directions for the research of scRNA-seq in tSCI in the future.

Salidroside alleviates severe acute pancreatitis-triggered pancreatic injury and inflammation by regulating miR-217-5p/YAF2 axis.

BACKGROUND: Our previous studies have shown that salidroside (Sal) exerted a protective effect in severe acute pancreatitis (SAP) via inhibiting the inflammatory response. However, the molecular mechanism has not been fully elucidated. METHODS: Using SAP rat model and miRNA microarray, the effect of Sal on miRNA expression profiling was determined and then validated their changes by quantitative Real-time PCR (qRT-PCR). Then, SAP cell model, enzyme-linked immunosorbent assay (ELISA) and Cell Counting Kit-8 (CCK-8) assay were used to explore the biological function of miR-217-5p in vitro. Bioinformatics analysis, luciferase reporter assay and miRNA pulldown assay were performed to investigate the underlying mechanism of miR-217-5p in the protection of Sal against SAP. RESULTS: Compared with SAP group, 21 differentially expressed miRNAs were identified in SAP + Sal group. The target genes of these miRNAs were strongly associated with regulation of transcription, Axon guidance, Pathways in cancer and MAPK signaling pathway. Among these miRNAs, miR-217-5p was the most downregulated miRNA. Sal treatment alleviated cell injury and reduced the production of pro-inflammatory cytokines. Whereas overexpression of miR-217-5p reversed the effects of Sal. We identified YY1 associated factor 2 (YAF2) as a direct target gene of miR-217-5p and Sal treatment could upregulate YAF2 expression via targeting miR-217-5p. Furthermore, knockdown of YAF2 counteracted Sal-induced alleviation of cell injury and inflammation. Moreover, Sal could suppress the activation of p38 MAPK pathway by regulating miR-217-5p/YAF2 axis. CONCLUSIONS: Our findings for the first time highlighted that Sal alleviated pancreatic injury and inhibited inflammation by regulating miR-217-5p/YAF2 axis, which might provide new therapeutic strategies for SAP treatment.

In Situ Hybridization Combined with Immunohistochemistry in Cryosectioned Zebrafish Embryos.

As a vertebrate, the zebrafish has been widely used in biological studies. Zebrafish and humans share high genetic homology, which allows its use as a model for human diseases. Gene function study is based on the detection of gene expression patterns. Although immunohistochemistry offers a powerful way to assay protein expression, the limited number of commercially available antibodies in zebrafish restricts the application of costaining. In situ hybridization is widely used in zebrafish embryos to detect mRNA expression. This protocol describes how to obtain images by combining in situ hybridization and immunohistochemistry for zebrafish embryo sections. In situ hybridization was performed prior to cryosectioning, followed by antibody staining. Immunohistochemistry and the imaging of a single cryosection were performed after in situ hybridization. The protocol is helpful to unravel the expression pattern of two genes, first by in situ transcript detection and then by immunohistochemistry against a protein in the same section.

Therapeutic effects of salidroside vs pyrrolidine dithiocarbamate against severe acute pancreatitis in rat.

OBJECTIVE: To evaluate the therapeutic effectiveness of salidroside (Sal) and pyrrolidine dithiocarbamate (PDTC) against severe acute pancreatitis (SAP) in a rat model. METHODS: Rat models of SAP were established by retrograde infusion of sodium taurocholate solution. SAP rats were randomly divided into 6 groups: SAP 3 h group, SAP 24 h group, low-dose Sal treatment group (Sal L+S), middle-dose Sal treatment group (Sal M+S), high-dose Sal treatment group (Sal H+S) and PDTC treatment group (PDTC+S). The serum amylase, tumor necrosis factor-α (TNF-α), interleukin-1ß (IL-1ß) and interleukin-10 (IL-10) levels were determined by optical turbidimetry and enzyme-linked immunosorbent assay. The expression of Beclin-1, microtubule-associated protein light chain 3II (LC3 II ), lysosome associated membrane protein 2 (LAMP2), interleukin-1 receptor associated kinase 1 (IRAK1) inhibitor α of nuclear transcription factor-kB (IkBα), nuclear transcription factor-kB 65 (p65) in the pancreas tissues were detected by quantitative real-time polymerase chain reaction and Western blot, while the pIkBα and p-p65 levels were detected by Western blot. Pathological changes of the pancreas and all the other indexes were observed at 3 and 24 h after operation. RESULTS: The serum IL-10 level, IkBα and LAMP2 levels in Sal M+S, Sal H+S and PDTC+S groups were higher than those in SAP 24 h group, while all the other indexes in these three groups were all lower significantly than those in SAP 24 h group. There was no significant difference in all indexes between Sal H+S and PDTC+S groups. CONCLUSION: High-dose Sal has an effectively therapeutic effect on SAP in rats, which was similar to PDTC.

Heterogeneity and Molecular Markers for CNS Glial Cells Revealed by Single-Cell Transcriptomics.

Glial cells, including astrocytes, oligodendrocytes, and microglia, are the major components in the central nervous system (CNS). Studies have revealed the heterogeneity of each glial cell type and that they each may play distinct roles in physiological processes and/or neurological diseases. Single-cell sequencing (scRNA-seq) technology developed in recent years has extended our understanding of glial cell heterogeneity from the perspective of transcriptome profiling. This review summarizes the marker genes of major glial cells in the CNS and reveals their heterogeneity in different species, CNS regions, developmental stages, and pathological states (Alzheimer's disease and spinal cord injury), expanding our knowledge of glial cell heterogeneity on both molecular and functional levels.

Profile of the RNA in exosomes from astrocytes and microglia using deep sequencing: implications for neurodegeneration mechanisms.

Glial cells play an important role in signal transduction, energy metabolism, extracellular ion homeostasis and neuroprotection of the central nervous system. However, few studies have explained the potential effects of exosomes from glial cells on central nervous system health and disease. In this study, the genes expressed in exosomes from astrocytes and microglia were identified by deep RNA sequencing. Kyoto Encyclopedia of Genes and Genomes analysis indicated that several pathways in these exosomes are responsible for promoting neurodegenerative diseases, including Alzheimer's disease, Parkinson's disease and Huntington's disease. Gene ontology analysis showed that extracellular exosome, mitochondrion and growth factor activity were enriched in exosomes from the unique astrocyte group, while extracellular exosome and mitochondrion were enriched in exosomes from the unique microglia group. Next, combined with the screening of hub genes, the protein-protein interaction network analysis showed that exosomes from astrocytes influence neurodegenerative diseases through metabolic balance and ubiquitin-dependent protein balance, whereas exosomes from microglia influence neurodegenerative diseases through immune inflammation and oxidative stress. Although there were differences in RNA expression between exosomes from astrocytes and microglia, the groups were related by the hub genes, ubiquitin B and heat shock protein family A (Hsp70) member 8. Ubiquitin B appeared to be involved in pleiotropic regulatory functions, including immune regulation, inflammation inhibition, protein catabolism, intracellular protein transport, exosomes and oxidative stress. The results revealed the clinical significance of exosomes from glia in neurodegenerative diseases. This study was approved by the Animal Ethics Committee of Nantong University, China (approval No. S20180102-152) on January 2, 2018.

Inhibit inflammation and apoptosis of pyrroloquinoline on spinal cord injury in rat.

BACKGROUND: Pyrroloquinoline quinone (PQQ) is a redox cofactor that can participate in a variety of physiological and biochemical processes, such as anti-inflammatory, cytoprotection, anti-aging, and anti-apoptosis. PQQ plays an important protective role in the central nervous system (CNS). However, the effects of PQQ on astrocytes of the CNS and spinal cord injury (SCI) of rats is still unclear. The present study investigates the role of PQQ in inflammation, apoptosis, and autophagy after SCI in rats. And the effect of PQQ on lipopolysaccharide (LPS)-induced apoptosis and inflammation of astrocytes in vitro, to explore the neuroprotective mechanism of PQQ. METHODS: Sixty specific pathogen free (SPF) SD male rats (200-250 g) were randomly divided into Normal group, Sham group, SCI group, and SCI + PQQ group, with 15 rats in each group. BBB score, HE staining, Nissl staining, Western blot, immunofluorescence, and other methods were used for detection. RESULTS: Our results showed that PQQ could upregulate BBB score in SCI rats. In the second place, PQQ can increase the number and improve the morphology of neurons after SCI. The expression of IL-1ß, TNF-α, IL-6 was significantly decreased after PQQ treatment. And then, the ratio of B-cell lymphoma-2 (Bcl-2)/Bcl-2 associated X protein (Bax) increased significantly, and the positive signal of NeuN increased obviously after PQQ treatment. There are a large number of co-localizations between Bcl-2 and NeuN. Meanwhile, PQQ could down-regulate the expression of Active-Caspase3, and PQQ treatment could reverse the transfer of Active-Caspase3/Caspase3 from the cytoplasm to the nucleus in neurons and astrocytes after SCI. At the same time, PQQ had no significant effect on the LC3b/a ratio. PQQ could decrease the LAMP2 expression in spinal cord after injury. The expression level of phospho-Akt (p-AKT) increased after SCI and decreased after PQQ treatment. In primary astrocytes, LPS could induce the expression levels of IL-1ß, TNF-α, and IL-6, and which were inhibited by PQQ treatment at 12 hours. After treatment with LPS, the expression level of Active-Caspase3 increased, which could be reversed by PQQ treatment for 24 h. CONCLUSIONS: These results suggest that PQQ can ameliorate the motor function of hind limbs and the pathological changes of neurons and injured spinal cord after SCI, down-regulate the expressions of IL-1ß, TNF-α, and IL-6, inhibit apoptosis after SCI, and inhibit LPS-induced apoptosis and inflammation of astrocytes.

Salidroside alleviates taurolithocholic acid 3-sulfate-induced AR42J cell injury.

OBJECTIVES: To investigate the protective effects of Salidroside (Sal) on AP cell model induced by taurolithocholic acid 3-sulfate (TLC-S) as well as its underlying mechanism. METHODS: AR42J cells were divided into normal group (N group), AP cell model group (Mod group), Sal treated alone group (S+N group) and Sal treated AP cell model group (S+Mod group). The cell viability was examined by CCK-8 assay. Secretion of lipase and trypsin by AR42J cells, quantified using commercial assay kits, was used as the markers of TLC-S-induced pancreatitis. The levels of TNF-α, IL-1ß, IL-8, IL-6 and IL-10 in the cell supernatant were measured by ELISA. The effect of Sal on molecules in the NF-κB signaling pathway and autophagy was investigated by qRT-PCR and western blot. RESULTS: The decreased cell viability in Mod group was increased by Sal (P < 0.01). The upheaved activities of lipase and trypsin in AP cell model were declined by Sal (P < 0.01). The levels of TNF-α, IL-1ß, IL-8 and IL-6 in the cell supernatant, Beclin-1 and LC3-Ⅱ mRNA and protein, p-p65/p65 protein, which were increased in AP cell model, were decreased by Sal; and IL-10 in the cell supernatant, LAMP2 mRNA and protein, p-IκBα/IκBα protein which was declined in AP cell model, was increased by Sal (P < 0.05 or 0.01). There were no significant differences in all indexes between the N and S+N groups (P > 0.05). CONCLUSIONS: Sal alleviated AR42J cells injury induced by TLC-S, inhibited the inflammatory responses and modulated the autophagy, mainly through inhibiting the NF-κB signaling pathway.

Pirfenidone ameliorates the formation of choroidal neovascularization in mice.

The formation and development of choroidal neovascularization (CNV) is accompanied by inflammation and fibrosis. Existing treatments are expensive and can cause irreversible complications. Pirfenidone (PFD) exerts anti­inflammatory and anti­fibrotic effects; however, its applications in the eye remain unclear. Male C57BL/6J mice (aged 6­8 weeks) were used to explore whether PFD can inhibit the formation of laser­induced CNV. The localization of transforming growth factor ß2 (TGFß2) was determined through immunofluorescent staining. After laser photocoagulation, the vehicle and PFD groups were intravitreally injected with 1 µl PBS and 1 µl 0.5% PFD, respectively. At day 7 after intravitreal injection, the expression of TGFß2 and vascular endothelial growth factor (VEGF) was assessed. Fundus fluorescein angiography was performed to investigate the extent of fluorescence leakage, and the CNV areas were analyzed using a choroidal flat mount. The results demonstrated that, on day 7 after photocoagulation, the expression of TGFß2 and VEGF was reduced in the experimental group. In addition, fluorescein angiography showed that the leakage area of CNV was significantly smaller in the PFD injection group than those observed in the control and vehicle groups. Moreover, the areas of CNV in the PFD injection group were smaller compared with those reported in the other two injection groups. Histopathological and TUNEL analyses performed on day 28 revealed that there were no notable abnormalities on the layers of the neural retina of PFD­treated mice. In conclusion, intravitreal injection of PFD inhibited the formation of CNV in mice, likely via the downregulation of VEGF and TGFß2, which did not cause damage to the mouse retina after 28 days of treatment.

Receptor for activated C kinase 1 mediates the chronic constriction injury-induced neuropathic pain in the rats' peripheral and central nervous system.

Peripheral and central sensitization has been reported as significant features in the course of the occurrence and development of neuropathic pain (NP). Receptor for activated C kinase 1 (RACK1), a scaffold protein, participated in fundamental cellular activities and various neuronal functions. Peripheral and central sensitization are a state that the morphology of neuronal cell bodies as well as the corresponding function change, whether this process can be regulated by RACK1 is still unknown. In this study, the biological effects and mechanisms of RACK1 contributes to the pathogenesis of chronic constriction injury (CCI)-induced neuropathic pain were investigated. By western blot and staining, we found that RACK1 protein changed in dorsal root ganglion (DRG) neurons and spinal cord (SC) neurons except glial cells after CCI. Especially, RACK1 was co-located with IB4-, CGRP-positive neurons, suggesting it was related to integrate nociceptive information from the primary aff ;erents in DRG. The successful establishment of CCI models also directly led to mechanical allodynia and heat hyperalgesia, which could be reversed by intrathecal injection of RACK1 siRNA. Furthermore, intrathecal injection of RACK1 siRNA reduced the expression of RACK1 and accompanying spinal c-fos, which is the transcription factor and marker of neuronal activation. These results suggested that targeting RACK1 be a sensible approach for treating NP.

Interleukin-1 Receptor Associated Kinase 1 Mediates the Maintenance of Neuropathic Pain after Chronic Constriction Injury in Rats.

Neuropathic pain (NP) has complicated pathogenesis as it mainly involves a lesion or dysfunction of the somatosensory nervous system and its clinical treatment remains challenging. Chronic constriction injury (CCI) model is a widely used neuropathic pain model and involved in mechanisms including both nerve inflammatory and injury. Cytokines and their receptors play essential roles in the occurrence and persistence of neuropathic pain, but the underlying mechanisms have not well been understood. Therefore, Interleukin-1 receptor-associated kinase 1 (IRAK1) is chosen to explore the possible mechanisms of NP. In the present study, IRAK1 was found to persistently increase in the dorsal root ganglion (DRG) and spinal cord (SC) during CCI detected by western blot. The staining further confirmed that IRAK1 was mainly co-located in the DRG astrocytes or SC neurons, but less in the DRG microglia or SC astrocytes. Moreover, the region of increased IRAK1 expression was observed in superficial laminae of the spinal dorsal horn, which was the nociceptive neuronal expression domain, suggesting that IRAK1 may mediated CCI-induced pain by nociceptive primary afferent. In addition, intrathecal injection of Toll-like receptor 4 (TLR4) inhibitor or IRAK1 siRNA decreased the expression of IRAK1 accompanied with the alleviation of CCI-induced neuropathic pain. The upregulation of p-NF-κB expression was reversed by IRAK1 siRNA in SC, and intrathecal injection of p-NF-κB inhibitor relieved neuropathic pain. Taking together, targeting IRAK1 may be a potential treatment for chronic neuropathic pain.

Chemokine CXCL10/CXCR3 signaling contributes to neuropathic pain in spinal cord and dorsal root ganglia after chronic constriction injury in rats.

Inflammatory cytokines and chemokines play essential roles in the occurrence and persistence of neuropathic pain (NP). Chronic constriction injury (CCI) enhances the activation of p-ERK, which is involved in neuropathic pain. Although the chemokine CXCL10 and its receptor CXCR3 are implicated in the pathophysiology of itch, it is largely unexplored for neuropathic pain. In this study, we determined the role of the CXCL10-CXCR3 axis in NP using a well-established CCI model. CCI significantly induced mechanical allodynia and thermal hyperalgesia. Following the pain course, a significant increase of CXCL10 and CXCR3 in both dorsal root ganglion (DRG) neurons and spinal cord (SC) neurons was detected in rats. Furthermore, intrathecal injection of CXCR3 inhibitor AMG487 was found to attenuate pain hypersensitivity in a dose-dependent manner in CCI. The expression of p-ERK was also depressed after intrathecal injection of AMG487 associated with a significant laxation of hyperalgesia, which demonstrated that the interaction of CXCL10/CXCR3 possibly took part in neuropathic pain by regulating p-ERK signaling in the SC. Overall, these findings demonstrate that the CXCL10/CXCR3 signaling pathway is critical in CCI.

Translocation Associated Membrane Protein 1 Contributes to Chronic Constriction Injury-Induced Neuropathic Pain in the Dorsal Root Ganglion and Spinal Cord in Rats.

Neuropathic pain is a severe debilitating state caused by injury or dysfunction of somatosensory nervous system, and the clinical treatment is still challenging. Translocation associated membrane protein 1 (TRAM1), an adapter protein, participates in a variety of transduction pathways and mediates the biological functions such as cell proliferation, activation, and differentiation. However, whether TRAM1 is involved in the pathogenesis of neuropathic pain is still unclear. In our study, we reported the role of TRAM1 in the maintenance of neuropathic pain induced by chronic constriction injury (CCI) on rats. By western blot and staining, we found that TRAM1 increased in the dorsal root ganglion (DRG) neurons and spinal cord (SC) neurons after CCI. Being similar to IB4-, CGRP-positive expressed area, TRAM1 also expressed in the superficial laminae of the spinal cord dorsal horn (SCDH), suggesting it was related to the innervations of the primary afferents. Moreover, intrathecal injection of TRAM1 siRNA or Toll-like receptor 4 (TLR4) inhibitor induced low expression of TRAM1 in SC, which alleviated the pain response induced by CCI. The upregulation of p-NF-κB expression was reversed by TRAM1 siRNA in SC and DRG, and intrathecal injection of p-NF-κB inhibitor relieved neuropathic pain. All the data indicated that TRAM1 could take part in CCI-induced pain and might be a potential treatment for chronic neuropathic pain.

The Role of Smurf1 in Neuronal Necroptosis after Lipopolysaccharide-Induced Neuroinflammation.

The role of inflammation in neurological disorders such as Alzheimer's disease and Parkinson's disease is gradually recognized and leads to an urgent challenge. Smad ubiquitination regulatory factor 1 (Smurf1), one member of the HECT family, is up-regulated by proinflammatory cytokines and associated with apoptosis in acute spinal cord injury. However, the function of Smurf1 through promoting neuronal necroptosis is still limited in the central nervous system (CNS). Hence, we developed a neuroinflammatory model in adult rats following lipopolysaccharide (LPS) lateral ventral injection to elaborate whether Smurf1 is involved in necroptosis in CNS injury. The up-regulation of Smurf1 detected in the rat brain cortex was similar to the necroptotic marker RIP1 expression in a time-dependent manner after LPS-induced neuroinflammation. Meanwhile, Smurf1 knockdown with siRNA inhibited neuronal necroptosis following LPS-stimulated rat pheochromocytomal PC12 cells. Thus, it was indicated that LPS-induced necroptosis could be promoted by Smurf1. In short, these studies suggest that Smurf1 might promote neuronal necroptosis after LPS-induced neuroinflammation, which might act as a novel and potential molecular target for the treatment of neuroinflammation associated diseases.

Mitochondrial DNA damage and oxidative damage in HL-60 cells exposed to 900MHz radiofrequency fields.

HL-60 cells, derived from human promyelocytic leukemia, were exposed to continuous wave 900MHz radiofrequency fields (RF) at 120µW/cm2 power intensity for 4h/day for 5 consecutive days to examine whether such exposure is capable damaging the mitochondrial DNA (mtDNA) mediated through the production of reactive oxygen species (ROS). In addition, the effect of RF exposure was examined on 8-hydroxy-2'-dexoyguanosine (8-OHdG) which is a biomarker for oxidative damage and on the mitochondrial synthesis of adenosine triphosphate (ATP) which is the energy required for cellular functions. The results indicated a significant increase in ROS and significant decreases in mitochondrial transcription factor A, mtDNA polymerase gamma, mtDNA transcripts and mtDNA copy number in RF-exposed cells compared with those in sham-exposed control cells. In addition, there was a significant increase in 8-OHdG and a significant decrease in ATP in RF-exposed cells. The response in positive control cells exposed to gamma radiation (GR, which is also known to induce ROS) was similar to those in RF-exposed cells. Thus, the overall data indicated that RF exposure was capable of inducing mtDNA damage mediated through ROS pathway which also induced oxidative damage. Prior-treatment of RF- and GR-exposed the cells with melatonin, a well-known free radical scavenger, reversed the effects observed in RF-exposed cells.

Potential Vaccine Targets against Rabbit Coccidiosis by Immunoproteomic Analysis.

The aim of this study was to identify antigens for a vaccine or drug target to control rabbit coccidiosis. A combination of 2-dimensional electrophoresis, immunoblotting, and mass spectrometric analysis were used to identify novel antigens from the sporozoites of Eimeria stiedae. Protein spots were recognized by the sera of New Zealand rabbits infected artificially with E. stiedae. The proteins were characterized by matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF/TOF-MS) analysis in combination with bioinformatics. Approximately 868 protein spots were detected by silver-staining, and a total of 41 immunoreactive protein spots were recognized by anti-E. stiedae sera. Finally, 23 protein spots were successfully identified. The proteins such as heat shock protein 70 and aspartyl protease may have potential as immunodiagnostic or vaccine antigens. The immunoreactive proteins were found to possess a wide range of biological functions. This study is the first to report the proteins recognized by sera of infected rabbits with E. stiedae, which might be helpful in identifying potential targets for vaccine development to control rabbit coccidiosis.

Up-regulation of MCM3 Relates to Neuronal Apoptosis After Traumatic Brain Injury in Adult Rats.

Minichromosome maintenance complex component 3, one of the minichromosome maintenance proteins, functions as a part of pre-replication complex to initiate DNA replication in eukaryotes. Minichromosome maintenance complex component 3 (MCM3) was mainly implied in cell proliferation and tumorigenesis. In addition, MCM3 might play an important role in neuronal apoptosis. However, the functions of MCM3 in central nervous system are still with limited acquaintance. In this study, we performed a traumatic brain injury (TBI) model in adult rats. Western blot and immunohistochemistry staining showed up-regulation of MCM3 in the peritrauma brain cortex. The expression patterns of active caspase-3 and Bax, Bcl-2 were parallel with that of MCM3. Immunofluorescent staining and terminal deoxynucleotidyl transferase-mediated biotinylated-dUTP nick-end labeling suggested that MCM3 was involved in neuronal apoptosis. In conclusion, our data indicated that MCM3 might play an important role in neuronal apoptosis following TBI. Further understanding of these insights could serve as the basis for broadening the therapeutic scope against TBI.

Increased Expression of Ubiquitin-Specific Protease 4 Participates in Neuronal Apoptosis After Intracerebral Hemorrhage in Adult Rats.

Ubiquitinating enzymes catalyze protein ubiquitination, a reversible process countered by deubiquitinating enzyme (DUB) action. Ubiquitin-specific protease 4 (USP4) is a member of the ubiquitin-specific protease (USP) family of DUBs that has a role in spliceosome regulation. In the present study, we demonstrated that USP4 may be involved in neuronal apoptosis in the processes of intracerebral hemorrhage (ICH). We obtained a significant up-regulation of USP4 in neurons adjacent to the hematoma following ICH by the results of Western blot, immunohistochemistry, and immunofluorescence. Increasing USP4 level was found to be accompanied by the up-regulation of active caspase-3, γH2AX, Bax, and decreased expression of Bcl-2. In addition, USP4 co-localized well with γH2AX in the nucleus in the ICH model and hemin-induced apoptosis model. Moreover, in vitro study, knocking down USP4 by USP4-specific siRNA in PC12 cells reduced active caspase-3 expression. All these results above suggested that USP4 may be involved in neuronal apoptosis after ICH.