Hydrogen gas inhalation ameliorates LPS-induced BPD by inhibiting inflammation via regulating the TLR4-NFκB-IL6/NLRP3 signaling pathway in the placenta.

INTRODUCTION: Hydrogen (H2) is regarded as a novel therapeutic agent against several diseases owing to its inherent biosafety. Bronchopulmonary dysplasia (BPD) has been widely considered among adverse pregnancy outcomes, without effective treatment. Placenta plays a role in defense, synthesis, and immunity, which provides a new perspective for the treatment of BPD. This study aimed to investigate if H2 reduced the placental inflammation to protect the neonatal rat against BPD damage and potential mechanisms. METHODS: We induced neonatal BPD model by injecting lipopolysaccharide (LPS, 1 µg) into the amniotic fluid at embryonic day 16.5 as LPS group. LPS + H2 group inhaled 42% H2 gas (4 h/day) until the samples were collected. We primarily analyzed the neonatal outcomes and then compared inflammatory levels from the control group (CON), LPS group and LPS + H2 group. HE staining was performed to evaluate inflammatory levels. RNA sequencing revealed dominant differentially expressed genes. Bioinformatics analysis (GO and KEGG) of RNA-seq was applied to mine the signaling pathways involved in protective effect of H2 on the development of LPS-induced BPD. We further used qRT-PCR, Western blot and ELISA methods to verify differential expression of mRNA and proteins. Moreover, we verified the correlation between the upstream signaling pathways and the downstream targets in LPS-induced BPD model. RESULTS: Upon administration of H2, the inflammatory infiltration degree of the LPS-induced placenta was reduced, and infiltration significantly narrowed. Hydrogen normalized LPS-induced perturbed lung development and reduced the death ratio of the fetus and neonate. RNA-seq results revealed the importance of inflammatory response biological processes and Toll-like receptor signaling pathway in protective effect of hydrogen on BPD. The over-activated upstream signals [Toll-like receptor 4 (TLR4), nuclear factor kappa-B p65 (NF-κB p65), Caspase1 (Casp1) and NLR family pyrin domain containing 3 (NLRP3) inflammasome] in LPS placenta were attenuated by H2 inhalation. The downstream targets, inflammatory cytokines/chemokines [interleukin (IL)-6, IL-18, IL-1ß, C-C motif chemokine ligand 2 (CCL2) and C-X-C motif chemokine ligand 1 (CXCL1)], were decreased both in mRNA and protein levels by H2 inhalation in LPS-induced placentas to rescue them from BPD. Correlation analysis displayed a positive association of TLR4-mediated signaling pathway both proinflammatory cytokines and chemokines in placenta. CONCLUSION: H2 inhalation ameliorates LPS-induced BPD by inhibiting excessive inflammatory cytokines and chemokines via the TLR4-NFκB-IL6/NLRP3 signaling pathway in placenta and may be a potential therapeutic strategy for BPD.

The role of hydrogen in the prevention and treatment of coronary atherosclerotic heart disease.

Coronary atherosclerotic heart disease (CHD) is a primary cardiovascular disease caused by atherosclerosis (AS), which is characterized by chronic inflammation and lipid oxidative deposition. Molecular hydrogen (H2) is an effective anti-inflammatory agent and has potential to ameliorate glycolipid metabolism disorders, which is believed to exert beneficial effects on the prevention and treatment of CHD. It is suggested that H2 reduces inflammation in CHD by regulating multiple pathways, including NF-κB inflammatory pathway, pyroptosis, mitophagy, endoplasmic reticulum (ER) stress, and Nrf2 antioxidant pathway. Additionally, H2 may improve glycolipid metabolism by mediation of PI3K and AMPK signalling pathways, contributing to inhibition of the occurrence and development of CHD. This review elaborates pathogenesis of CHD and evaluates the role of H2 in CHD. Moreover, possible molecular mechanisms have been discussed and speculated, aiming to provide more strategies and directions for subsequent studies of H2 in CHD.

A fucoidan plant drink reduces Helicobacter pylori load in the stomach: a real-world study.

Background: Helicobacter pylori (Hp) infection is highly prevalent globally and is predominantly managed by antibiotics. Recently, the anti-adhesive, antioxidant, antitoxin, immunomodulatory, anti-coagulant, and anti-infective activities of fucoidan, a polysaccharide extracted from brown seaweeds, have been widely studied, and the results showed promise. Fucoidan has the potential to be utilized in Hp eradication therapy. Our present clinical study was designed to evaluate the efficiency of Lewuyou®, a fucoidan plant drink (FPD) in eradicating Hp in humans. Methods: This multi-center, clinical study was conducted between October 2020 and July 2021. Hp infection was confirmed by urea breath test (UBT). A total of 122 patients with confirmed Hp infection were enrolled; after exclusion of incomplete data, 85 eligible patients (37 males and 48 females aged 20-81 years) were included in the final analysis. FPD (50 mL per vial) was orally administered twice daily for a 4-week cycle, and 41 patients completed an 8-week cycle. Results: No adverse event (AE) was reported in all 122 participants who had consumed FPD. The Hp eradication rate and clearance rate were 77.6% (66/85) and 20.0% (17/85), respectively, after 4 weeks of FPD consumption and 80.5% (33/41) and 26.8 (11/41) , respectively, after 8 weeks of consumption. Conclusions: The 4- and 8-week protocols of FPD consumption were safe and effective at reducing Hp load on the gastric mucosa, with Hp eradicated in the majority of participants.

Potent anti-inflammatory responses: Role of hydrogen in IL-1α dominated early phase systemic inflammation.

Introduction: It has been proven that hydrogen has obvious anti-inflammatory effects in animal experiments and clinical practice. However, the early dynamic process of the inflammatory response caused by lipopolysaccharide (LPS) and the anti-inflammatory effect of hydrogen has not been definitively reported. Methods: Inflammation in male C57/BL6J mice or RAW264.7 cells was induced with LPS, for which hydrogen was immediately administered until samples were taken. Pathological changes in lung tissue were assessed using hematoxylin and eosin (HE) staining. Levels of inflammatory factors in serum were determined using liquid protein chip. The mRNA levels of chemotactic factors in lung tissues, leukocytes, and peritoneal macrophages were measured by qRT-PCR. The expression levels of IL-1α and HIF-1α were measured by immunocytochemistry. Results: Hydrogen alleviated LPS-induced inflammatory infiltration in the lung tissues of mice. Among the 23 inflammatory factors screened, LPS-induced upregulation of IL-1α etc. was significantly inhibited by hydrogen within 1 hour. The mRNA expression of MCP-1, MIP-1α, G-CSF, and RANTES was inhibited obviously by hydrogen at 0.5 and 1 h in mouse peritoneal macrophages. In addition, hydrogen significantly blocked LPS or H2O2-induced upregulation of HIF-1α, and IL-1α in 0.5 h in RAW264.7 cells. Discussion: The results suggested that hydrogen is potentially inhibitive against inflammation by inhibiting HIF-1α and IL-1α release at early occurrence. The target of the inhibitive LPS-induced-inflammatory action of hydrogen is chemokines in macrophages in the peritoneal cavity. This study provides direct experimental evidence for quickly controlling inflammation with the translational application of a hydrogen-assisted protocol.

Hydrogen gas alleviates lipopolysaccharide-induced acute lung injury and inflammatory response in mice.

BACKGROUND: Chronic inflammation and oxidant/antioxidant imbalance are two main pathological features associated with lipopolysaccharide (LPS)-induced acute lung injury (ALI). The following study investigated the protective role of hydrogen (H2), a gaseous molecule without known toxicity, in LPS-induced lung injury in mice and explored its potential molecular mechanisms. METHODS: Mice were randomly divided into three groups: H2 control group, LPS group, and LPS + H2 group. The mice were euthanized at the indicated time points, and the specimens were collected. The 72 h survival rates, cytokines contents, pathological changes, expression of Toll-like receptor 4 (TLR4), and oxidative stress indicators were analyzed. Moreover, under different culture conditions, RAW 264.7 mouse macrophages were used to investigate the potential molecular mechanisms of H2 in vitro. Cells were divided into the following groups: PBS group, LPS group, and LPS + H2 group. The cell viability, intracellular ROS, cytokines, and expression of TLR4 and nuclear factor kappa-B (NF-κB) were observed. RESULTS: Hydrogen inhalation increased the survival rate to 80%, reduced LPS-induced lung damage, and decreased inflammatory cytokine release in LPS mice. Besides, H2 showed remarked anti-oxidative activity to reduce the MDA and NO contents in the lung. In vitro data further indicated that H2 down-regulates the levels of ROS, NO, TNF-α, IL-6, and IL-1ß in LPS-stimulated macrophages and inhibits the expression of TLR4 and the activation of nuclear factor kappa-B (NF-κB). CONCLUSION: Hydrogen gas alleviates lipopolysaccharide-induced acute lung injury and inflammatory response most probably through the TLR4-NF-κB pathway.

Hydrogen Repairs LPS-Induced Endothelial Progenitor Cells Injury via PI3K/AKT/eNOS Pathway.

Endotoxins and other harmful substances may cause an increase in permeability in endothelial cells (ECs) monolayers, as well as ECs shrinkage and death to induce lung damage. Lipopolysaccharide (LPS) can impair endothelial progenitor cells (EPCs) functions, including proliferation, migration, and tube formation. EPCs can migrate to the damaged area, differentiate into ECs, and participate in vascular repair, which improves pulmonary capillary endothelial dysfunction and maintains the integrity of the endothelial barrier. Hydrogen (H2) contributes to the repairment of lung injury and the damage of ECs. We therefore speculate that H2 protects the EPCs against LPS-induced damage, and it's mechanism will be explored. The bone marrow-derived EPCs from ICR Mice were treated with LPS to establish a damaged model. Then EPCs were incubated with H2, and treated with PI3K inhibitor LY294002 and endothelial nitric oxide synthase (eNOS) inhibitor L-NAME. MTT assay, transwell assay and tube formation assay were used to detect the proliferation, migration and angiogenesis of EPCs. The expression levels of target proteins were detected by Western blot. Results found that H2 repaired EPCs proliferation, migration and tube formation functions damaged by LPS. LY294002 and L-NAME significantly inhibited the repaired effect of H2 on LPS-induced dysfunctions of EPCs. H2 also restored levels of phosphor-AKT (p-AKT), eNOS and phosphor-eNOS (p-eNOS) suppressed by LPS. LY294002 significantly inhibited the increase of p-AKT and eNOS and p-eNOS expression exposed by H2. L-NAME significantly inhibited the increase of eNOS and p-eNOS expression induced by H2. H2 repairs the dysfunctions of EPCs induced by LPS, which is mediated by PI3K/AKT/eNOS signaling pathway.

Hydrogen inhalation promotes recovery of a patient in persistent vegetative state from intracerebral hemorrhage: A case report and literature review.

BACKGROUND: Persistent vegetative state (PVS) is a devastating and long-lasting clinical condition with high morbidity and mortality; currently, there are no available effective interventions. CASE SUMMARY: We report the case of an 11-year-old boy with PVS caused by severe intracerebral bleeding in the left hemisphere following anticoagulation treatment. The patient's PVS severity showed no notable improvement after 2-mo neuroprotective treatment and rehabilitation, including nerve growth factor and baclofen, hyperbaric oxygen, and comprehensive bedside rehabilitation therapies. Daily inhalation treatment (4-6 h) of high-concentration hydrogen (H2) gas (66.6% H2 + 33.3% O2) was provided. Surprisingly, the patient's orientation, consciousness, ability to speak, facial expressions, and locomotor function were significantly restored, along with improvements in essential general health status, after H2 gas inhalation treatment, which was consistent with stabilized neuropathology in the left hemisphere and increased Hounsfield unit values of computed tomography in the right hemisphere. The patient finally recovered to a near normal conscious state with a Coma Recovery Scale-Revised Score of 22 from his previous score of 3. CONCLUSION: Phase 1 clinical trials are needed to explore the safety and efficacy of H2 gas inhalation in patients with PVS.

Pre-inhalation of hydrogen-rich gases protect against caerulein-induced mouse acute pancreatitis while enhance the pancreatic Hsp60 protein expression.

BACKGROUND: Acute pancreatitis (AP) lacks targeted prevention and treatment measures. Some key points in the pathogenesis of AP remain unclear, such as early activation of pancreatic enzymes. Several recent reports have shown the protective effect of hydrogen on several AP animal models, and the mechanism is related to antioxidant activity. Heat shock protein 60 (Hsp60) is known to accompany pancreatic enzymes synthesis and secretion pathway of in pancreatic acinar cells, while role of hsp60 in AP remains a topic. Aim of this study was to investigate effect of hydrogen pretreatment on AP and the mechanisms, focusing on pancreatic oxidative stress and Hsp60 expression. METHODS: 80 mice were randomly assigned into four groups: HAP group, AP group, HNS group, and NS group and each group were set 3 observation time point as 1 h, 3 h and 5 h (n = 6-8). Mouse AP model was induced by intraperitoneal injection of 50 µg/kg caerulein per hour for 6 injections both in AP and HAP groups, and mice in NS group and HNS group given normal saline (NS) injections at the same way as control respectively. Mice in HAP group and HNS group were treated with hydrogen-rich gases inhalation for 3 days before the first injection of caerulein or saline, while mice in AP group and NS group in normal air condition. Histopathology of pancreatic tissue, plasma amylase and lipase, plasma IL-1 and IL-6, pancreatic glutathione (GSH) and malondialdehyde (MDA), and Hsp60 mRNA and protein expression were investigated. Comparisons were made by one-way analysis of variance. RESULTS: The pancreatic pathological changes, plasma amylase and lipase activity, and the increase of plasma IL-1 and IL-6 levels in AP mice were significantly improved by the hydrogen-rich gases pretreatment, Meanwhile, the pancreatic GSH content increased and the pancreatic MDA content decreased. And, the hydrogen-rich gases pretreatment improved the Hsp60 protein expression in pancreatic tissues of AP mice at 1 h and 5 h. CONCLUSIONS: Pre-inhalation of hydrogen-rich gases have a good protective effect on AP mice, and the possible mechanisms of reduced oxidative stress and the early increased pancreatic Hsp60 protein deserve attention.

Hydrogen, a Novel Therapeutic Molecule, Regulates Oxidative Stress, Inflammation, and Apoptosis.

Molecular hydrogen (H2) is a colorless and odorless gas. Studies have shown that H2 inhalation has the therapeutic effects in many animal studies and clinical trials, and its application is recommended in the novel coronavirus pneumonia treatment guidelines in China recently. H2 has a relatively small molecular mass, which helps it quickly spread and penetrate cell membranes to exert a wide range of biological effects. It may play a role in the treatment and prevention of a variety of acute and chronic inflammatory diseases, such as acute pancreatitis, sepsis, respiratory disease, ischemia reperfusion injury diseases, autoimmunity diseases, etc.. H2 is primarily administered via inhalation, drinking H2-rich water, or injection of H2 saline. It may participate in the anti-inflammatory and antioxidant activity (mitochondrial energy metabolism), immune system regulation, and cell death (apoptosis, autophagy, and pyroptosis) through annihilating excess reactive oxygen species production and modulating nuclear transcription factor. However, the underlying mechanism of H2 has not yet been fully revealed. Owing to its safety and potential efficacy, H2 has a promising potential for clinical use against many diseases. This review will demonstrate the role of H2 in antioxidative, anti-inflammatory, and antiapoptotic effects and its underlying mechanism, particularly in coronavirus disease-2019 (COVID-19), providing strategies for the medical application of H2 for various diseases.

Transient sublethal hypoxia in neonatal rats causes reduced dendritic spines, aberrant synaptic plasticity, and impairments in memory.

Hypoxic/ischemic insult, a leading cause of functional brain defects, has been extensively studied in both clinical and experimental animal research, including its etiology, neuropathogenesis, and pharmacological interventions. Transient sublethal hypoxia (TSH) is a common clinical occurrence in the perinatal period. However, its effect on early developing brains remains poorly understood. The present study was designed to investigate the effect of TSH on the dendrite and dendritic spine formation, neuronal and synaptic activity, and cognitive behavior of early postnatal Day 1 rat pups. While TSH showed no obvious effect on gross brain morphology, neuron cell density, or glial activation in the hippocampus, we found transient hypoxia did cause significant changes in neuronal structure and function. In brains exposed to TSH, hippocampal neurons developed shorter and thinner dendrites, with decreased dendritic spine density, and reduced strength in excitatory synaptic transmission. Moreover, TSH-treated rats showed impaired cognitive performance in spatial learning and memory. Our findings demonstrate that TSH in newborn rats can cause significant impairments in synaptic formation and function, and long-lasting brain functional deficits. Therefore, this study provides a useful animal model for the study of TSH on early developing brains and to explore potential pharmaceutical interventions for patients subjected to TSH insult.

LINC01133 inhibits breast cancer invasion and metastasis by negatively regulating SOX4 expression through EZH2.

Mounting evidence highlights long non-coding RNAs (lncRNAs) as crucial regulators in multiple types of biological processes and contributing to tumourigenesis. LINC01133, located in chromosome 1q23.2, was a recently identified novel lncRNA with a length of 1154nt. It was involved in the development of colorectal cancer and non-small cell lung cancer. However, its clinical relevance, biological functions and potential molecular mechanism in breast cancer are still unclear. In this study, we found that the LINC01133 expression was significantly down-regulated in breast cancer samples and was associated with progression and poor prognosis of breast cancer. Further experiments demonstrated that overexpression of LINC01133 inhibited invasion and metastasis in breast cancer both in vitro and in vivo. Mechanistic investigations revealed that LINC01133 repressed SOX4 expression by recruiting EZH2 to SOX4 promoter. Moreover, rescue experiments further confirmed that LINC01133 functional acted as an anti-oncogene, at least partly, via repressing SOX4 in breast cancer. Taken together, these findings imply that LINC01133 could serve as a novel prognostic biomarker and potential therapeutic target for breast cancer.

A case of pulmonary benign metastasizing leiomyoma: clinicopathologic, immunohistochemical, and PET/CT analyses.

The pulmonary benign metastasizing leiomyoma (BML) is an extremely rare disease case, of which the pathogenesis was not elucidated. The accumulation of additional case reports and analyses is needed. A case of pulmonary BML is presented. A 48-year-old asymptomatic female patient that underwent a hysteromyomectomy 11 years ago was found to have abnormal shadows on a chest X-ray during a regular health check-up in 2012. Multiple nodular was observed in both lungs and uterine under computed tomography (CT) examination. Transbronchial lung biopsy surgery with a resection of the lesion was performed. The results revealed the pulmonary nodule was formed by benign smooth muscle cells which displayed very low mitotic activity. The samples showed highly positive signals for α-smooth muscle actin (αSMA), Desmin and Vimentin. Estrogen receptor (ER) and progesterone receptor (PR) were also immunoreactive positive. HMB-45, CD117 (C-kit) and S-100 were found to be negative with low Ki-67 index in the tumor cells. The pathological diagnosis was pulmonary BML. A total hysterectomy with adnexectomy were performed for the multiple uterine leiomyomas and pulmonary BML one week after diagnosis was made. From then on, the pulmonary nodules in both lungs were stable in 7 years postoperation, based on chest CT scan. When a solitary lung nodule is detected in a patient with a history of uterine leiomyoma, as an option, pulmonary BML, accurately diagnosing and proper treatment to has important implications for clinical outcome.

Beneficial effects of hydrogen gas inhalation on a murine model of allergic rhinitis.

Allergic rhinitis (AR) is a common chronic inflammatory condition. It has been previously indicated that oxidative stress may contribute to allergic inflammation, including AR. Although molecular hydrogen (H2), an antioxidative agent, has been effective in treatment of numerous oxidative stress-associated diseases, the effect of inhalation of a high concentration of H2 on AR remains unknown. In the current study, female BALB/c mice were sensitized with ovalbumin (OVA) followed by intranasal OVA challenge to establish an animal model of AR. Mice were subjected to exposure to H2 and the inert gas helium at different frequencies and durations. The frequencies of sneezing/scratching and the body weights of mice were recorded. Histological analysis and multiplex cytokine assays were performed to evaluate the effects of H2 on AR. Challenge with OVA induced significant nasal mucosa inflammation. H2 inhalation reduced the infiltration of inflammatory cells into mucosa and lowered the levels of interleukin (IL)-5, IL-13 and monocyte chemoattractant protein-1 in serum. H2 inhalation slightly increased the level of interferon-γ, however the difference was not statistically significant. Treatment with H2 limited the weight increase in healthy mice and reversed the weight loss in mice with AR. Furthermore, H2 inhalation induced a therapeutic effect on AR in a dose-dependent manner. The current results demonstrate that H2 may demonstrate a therapeutic value for allergic diseases.

Construction of Epidermal Growth Factor Receptor Peptide Magnetic Nanovesicles with Lipid Bilayers for Enhanced Capture of Liver Cancer Circulating Tumor Cells.

Highly effective targeted tumor recognition via vectors is crucial for cancer detection. In contrast to antibodies and proteins, peptides are direct targeting ligands with a low molecular weight. In the present study, a peptide magnetic nanovector platform containing a lipid bilayer was designed using a peptide amphiphile (PA) as a skeleton material in a controlled manner without surface modification. Fluorescein isothiocyanate-labeled epidermal growth factor receptor (EGFR) peptide nanoparticles (NPs) could specifically bind to EGFR-positive liver tumor cells. EGFR peptide magnetic vesicles (EPMVs) could efficiently recognize and separate hepatoma carcinoma cells from cell solutions and treated blood samples (ratio of magnetic EPMVs versus anti-EpCAM NPs: 3.5 ± 0.29). Analysis of the circulating tumor cell (CTC) count in blood samples from 32 patients with liver cancer showed that EPMVs could be effectively applied for CTC capture. Thus, this nanoscale, targeted cargo-packaging technology may be useful for designing cancer diagnostic systems.

Electrophoretic Deposition of Dexamethasone-Loaded Mesoporous Silica Nanoparticles onto Poly(L-Lactic Acid)/Poly(ε-Caprolactone) Composite Scaffold for Bone Tissue Engineering.

The incorporation of microcarriers as drug delivery vehicles into polymeric scaffold for bone regeneration has aroused increasing interest. In this study, the aminated mesoporous silica nanoparticles (MSNs-NH2) were prepared and used as microcarriers for dexamethasone (DEX) loading. Poly(l-lactic acid)/poly(ε-caprolactone) (PLLA/PCL) nanofibrous scaffold was fabricated via thermally induced phase separation (TIPS) and served as template, onto which the drug-loaded MSNs-NH2 nanoparticles were deposited by electrophoretic deposition (EPD). The physicochemical and release properties of the prepared scaffolds (DEX@MSNs-NH2/PLLA/PCL) were examined, and their osteogenic activities were also evaluated through in vitro and in vivo studies. The release of DEX from the scaffolds revealed an initial rapid release followed by a slower and sustained one. The in vitro results indicated that the DEX@MSNs-NH2/PLLA/PCL scaffold exhibited good biocompatibility to rat bone marrow-derived mesenchymal stem cells (BMSCs). Also, BMSCs cultured on the DEX@MSNs-NH2/PLLA/PCL scaffold exhibited a higher degree of osteogenic differentiation than those cultured on PLLA/PCL and MSNs-NH2/PLLA/PCL scaffolds, in terms of alkaline phosphatase (ALP) activity, mineralized matrix formation, and osteocalcin (OCN) expression. Furthermore, the in vivo results in a calvarial defect model of Sprague-Dawley (SD) rats demonstrated that the DEX@MSNs-NH2/PLLA/PCL scaffold could significantly promote calvarial defect healing compared with the PLLA/PCL scaffold. Thus, the EPD technique provides a convenient way to incorporate osteogenic agents-containing microcarriers to polymer scaffold, and thus, prepared composite scaffold could be a potential candidate for bone tissue engineering application due to its capacity for delivery of osteogenic agents.

GSK3ß expression and phosphorylation during neuronal maturation in the rat dorsal root ganglion.

Glycogen synthase kinase 3ß (GSK-3ß) protein is a key regulator of neurogenesis, neuronal differentiation and polarisation during neurodevelopment. Sensory neurons in dorsal root ganglion (DRG) undergo a series of development stages during its maturation. In this study, we investigated the dynamic changes in GSK-3ß expression and phosphorylation of its N-terminal serine-9 residue (p-GSK-3ß (S9)) during DRG development. Sprague-Dawley (SD) rats were divided according to the following ages: Embryonic 13(th) (E13), E15, E19, Postnatal 1(st) (P1), P3, P7, P14, P21 and P60 days. GSK-3ß was detected by immunohistochemistry and double immunofluorescence on DRGs. Western blotting was used to determine the quantity of GSK-3ß and p-GSK-3ß (S9) expression. It was found that GSK-3ß immunopositive cells in the DRG appeared as early as E13 development phase, and gradually increased to a peak level at P3, at which almost all neurons were GSK-3ß positive, and then stayed at a high level to the experiment day 60. GSK3ß expression was cell-type-specific during DRG maturation and exhibited cytoplasmic staining in the neuronal cell body and the axon. Glial cells consistently remained negative in DRGs at all stages. Western blot analysis revealed that GSK3ß expression stayed the same during DRG maturation. In contrast, p-GSK-3ß (S9) expression was stage-specific and decreased from E13 to P60 (P < 0.01). Taken together, these results suggest that GSK-3ß expression is stage-specific and cell-type-specific during DRG maturation.

Role of RHOT1 on migration and proliferation of pancreatic cancer.

Pancreatic cancer (PC) is one of the most malignant tumors. Rho GTPases can affect several types of human cancers, including PC. In this study, we investigated the role of Ras homolog family member T1 (RHOT1), a new member of Rho GTPases in PC. IHC results showed that RHOT1 was expressed significantly higher in PC tissues than paracancerous tissues (P<0.01) and SMAD family member 4 (SMAD4) was expressed lower in PC tissues (P<0.01). RHOT1 was widely expressed in PC cell lines analyzed by reverse transcription PCR (RT-PCR), real-time quantitative PCR (RT-qPCR) and western blotting (WB). SiRNA-RHOT1 significantly suppressed the proliferation and migration of SW1990 cells. Moreover, SMAD4 was identified as an effector of RHOT1. Our findings suggest that RHOT1 can regulate cell migration and proliferation by suppressing the expression of SMAD4 in PC, which may provide a novel sight to explore the mechanism and therapeutic strategy for PC.

A rat model for chronic spinal nerve root compression.

OBJECTIVES: The pathophysiology of radiculopathy associated with lumbar spinal stenosis and lumbar disc herniation is incompletely understood. The goal of the present study was to establish a chronic spinal nerve root compression model that can mimic lumbar disc herniation or spinal stenosis using silicone tube compression. We also try to link the pathology changes of damaged nerve root with the reaction of microglia in spinal cord in same rat at different time points. METHODS: Thirty rats were used in this study. The L5 nerve roots (dorsal and ventral) were exposed by hemilaminectomy; the diameter of the L5 nerve root was measured at the 2 mm proximal from the dorsal root ganglia. The dorsal and ventral nerve roots of L5 were compressed using a silicone tube, and the sham group was only exposed dorsal and ventral roots of L5. Five rats from the sham group were perfused at 8 days after surgery, and 25 rats from the model groups were perfused at 3, 8, 12, 45 days, and 5 months after surgery, each model group was composed of 5 rats according to the time point. The L5 spinal cord segments and nerve root that compressed by silicone tube were harvested from the same rat. Microglia and neuron in the spinal cord were stained by immunohistochemistry, and the nerve root was shown by electron microscope. RESULTS: In sham-operated rat, the arrangement of axon and myelin sheath is normal, the ventral root is mainly composed of large axon (>6 µm) and it is composed of 46.3 % of all the axons of the ventral root; the average myelin thickness of large axon is 1.86 µm; the dorsal root is mainly composed of medium (2-3.9 or 4-5.9 µm) axons and they are composed of 79.1 % of all the axons of the dorsal root; the average myelin thickness of this category is 0.94 or 1.55 µm. The average myelin thickness of large axon in ventral root reduced to 0.97 and 1.19 µm from more than 1.86 µm after compression for 3 and 8 days separately. Most of myelin sheath disappeared after 12 days of compression; the myelin sheath was partly restored at 45 days after compression which the myelin sheath thickness of large axons in ventral root was 0.47 µm. The medium category in dorsal root reduced to 0.59 or 0.72 µm from 0.94 µm, and 1.55 µm after compression for 3 days (p < 0.05 to p < 0.0001). The medium category axon in dorsal root is also 0.47 µm after compression for 45 days (p ≤ 0.0001). The myelin sheath was almost totally restored at the 5 months of compression; the myelin sheath thickness returned to normal and the axons were intact in structure under EM. The number of Iba1-positive microglia increased by 18.69, 40.44, and 18.49 % after compression for 3, 8, and 12 days separately in the ipsilateral dorsal horn and 21.26, 32.15, 22.87 % in ventral horns, and the activation of microglia was also prominent in contralateral sides of the dorsal and ventral horn at 8 days time point. The microglia cell reconverted to resting status after compression for 45 days or 5 months. CONCLUSION: The chronic spinal nerve root compression with silicone tube produces a recoverable damage to nerve root, which produces recoverable microglial activation in the spinal cord. These results demonstrated that the chronic spinal nerve root compression with silicone tube could mimic the pathological changes of lumbar spinal stenosis or lumbar disc herniation.

Modulation of MAPK and Akt signaling pathways in proximal segment of injured sciatic nerves.

Mitogen-activated protein kinases (MAPKs) and phosphatidylinositol-3-kinase (PI3K)/Akt-mediated signaling pathways play critical roles in peripheral nerve injury. However, the mechanism by which activate these signaling is unclear. We examined the activation of MAPK and Akt pathways in the proximal segments of crushed rat sciatic nerve after 1-30 days injury. We found that the phosphorylation level of Erk was attenuated in protein level. Phosphorylation of JNK and p38 increased from day 1 to day 15 following injury. In addition, activation of Akt was up-regulated predominantly in the ipsilateral proximal nerves and located in Schwann cells. Furthermore, phosphorylated GSK3ß (Ser9) and GSK3ß (Tyr216) were highly augmented from the third day to the 30th day and from 3 to 7 days after injury, respectively. Moreover, mTOR/p70S6 were activated within 7 days injury. Taken together, our studies suggest that the PI3K/Akt signaling is required for the regulation of axon regeneration in Schwann cells in the proximal nerve segments after injury. Furthermore, the contralateral nerves have some compensable response to the injury, at least, including the changes of signaling molecules.