Isoquercitrin attenuates the osteoclast-mediated bone loss in rheumatoid arthritis via the Nrf2/ROS/NF-κB pathway.

An excess of osteoclastogenesis significantly contributes to the development of rheumatoid arthritis (RA). Activation of the nuclear factor erythroid-2 related factor 2 (Nrf2) and nuclear factor kappa B (NF-κB) ligand (RANKL)-induced reactive oxygen species (ROS)-to-NF-κB signaling cascade are important mechanisms regulating osteoclastogenesis; however, whether Nrf2 is involved in RANKL-induced NF-κB activation is controversial. Isoquercitrin, a natural flavonoid compound, has been shown to have Nrf2-dependent antioxidant effects inprevious studies. We sought to verify whether isoquercitrin could modulate RANKL-induced NF-κB activation by activating Nrf2, thereby affecting osteoclastogenesis. Tartrate-resistant acid phosphatase staining, F-actin ring staining and resorption pit assay suggested that isoquercitrin significantly inhibited osteoclastogenesis and osteolytic function. Mitosox staining showed that RANKL-induced ROS generation was significantly inhibited by isoquercitrin from day 3 of the osteoclast differentiation cycle. Quantitative real-time PCR, Western blot, and immunofluorescence indicated that isoquercitrin activated the Nrf2 signaling pathway and inhibited NF-κB expression. And when we used the Nrf2-specific inhibitor ML385, the inhibition of NF-κB by isoquercitrin disappeared. Moreover, we found that Nrf2 is not uninvolved in RANKL-induced NF-κB activation and may be related to the timing of ROS regulation. When we limited isoquercitrin administration to 2 days, Nrf2 remained activated and the inhibition of NF-κB disappeared. In vivo experiments suggested that isoquercitrin attenuated RA modeling-induced bone loss. Overall, isoquercitrin-activated Nrf2 blocked the RANKL-induced ROS-to-NF-κB signaling cascade response, thereby inhibiting osteoclastogenesis and bone loss. These findings provide new ideas for the treatment of RA.

Correction to "Rapamycin Inhibits Osteoclastogenesis and Prevents LPS-Induced Alveolar Bone Loss by Oxidative Stress Suppression".

[This corrects the article DOI: 10.1021/acsomega.3c01289.].

Rapamycin Inhibits Osteoclastogenesis and Prevents LPS-Induced Alveolar Bone Loss by Oxidative Stress Suppression.

Periodontitis is a progressive inflammatory skeletal disease characterized by periodontal tissue destruction, alveolar bone resorption, and tooth loss. Chronic inflammatory response and excessive osteoclastogenesis play essential roles in periodontitis progression. Unfortunately, the pathogenesis that contributes to periodontitis remains unclear. As a specific inhibitor of the mTOR (mammalian/mechanistic target of rapamycin) signaling pathway and the most common autophagy activator, rapamycin plays a vital role in regulating various cellular processes. The present study investigated the effects of rapamycin on osteoclast (OC) formation in vitro and its effects on the rat periodontitis model. The results showed that rapamycin inhibited OC formation in a dose-dependent manner by up-regulating the Nrf2/GCLC signaling pathway, thus suppressing the intracellular redox status, as measured by 2',7'-dichlorofluorescein diacetate and MitoSOX. In addition, rather than simply increasing the autophagosome formation, rapamycin increased the autophagy flux during OC formation. Importantly, the anti-oxidative effect of rapamycin was regulated by an increase in autophagy flux, which could be attenuated by blocking autophagy with bafilomycin A1. In line with the in vitro results, rapamycin treatment attenuated alveolar bone resorption in rats with lipopolysaccharide-induced periodontitis in a dose-dependent manner, as assessed by micro-computed tomography, hematoxylin-eosin staining, and tartrate-resistant acid phosphatase staining. Besides, high-dose rapamycin treatment could reduce the serum levels of proinflammatory factors and oxidative stress in periodontitis rats. In conclusion, this study expanded our understanding of rapamycin's role in OC formation and protection from inflammatory bone diseases.

[Construction and evaluation of a gradient stress model of PC12 cells induced by corticosterone].

Objective: A gradient stress model of PC12 cells induced by corticosterone was established to provide a basis for the evaluation and regulation of cell stress. Methods: The effect of corticosterone on cell viability was observed by measuring PC12 cell viability at different concentrations of corticosterone (0~1 000 µmol/L) after different intervention times (8~48 h) to screen the cell models for optimal intervention conditions. Key stress indicators (MDA, SOD, NADH, LDH) were measured spectrophotometrically and microscopically to evaluate the models. Results: When the concentration of corticosterone was below 200 µmol/L and the intervention time was 12 h, the cell viability was below half inactivation rate, which could reduce the confounding factors due to the decrease of cell viability in each group. Compared with the blank control group, corticosterone increased the levels of MDA, NADH and LDH,and decreased the levels of SOD in the model group in a concentration-dependent manner (P＜0.01), which was consistent with the construction of the gradient stress model. Conclusion: A gradient stress injury model of PC12 cells was successfully established, with intervention concentrations of 0 µmol/L, 25 µmol/L, 50 µmol/L, 100 µmol/L, 150 µmol/L and 200 µmol/L corticosterone at an intervention time of 12 h. The degree of stress injury of the cell model was increased gradually, which could be used as a basis and object for conducting cell stress injury assessment and regulation experiments.

[Effects of three Polyphenolic compounds on the intestinal flora of mice exposed simulated intermittent plateau hypoxia].

OBJECTIVE: To investigate the protective effects of three Polyphenolic compounds on intestinal microbial communities in mice exposed intermittent plateau hypoxia. METHODS: In this study, 60 healthy male Balb/c mice were randomly divided into plain control group, plateau control group, primary anthocyanin intervention group, quercetin intervention group and resveratrol intervention group, 12 mice in each group. Primary anthocyanin, quercetin and resveratrol were administrated by gavage at the doses of 50, 100 and 20 mg/kg in pharmacological intervention group, respectively. After exposure of the mice to simulation plateau-condition for 30 days, the serum samples were collected for DAO testing, sterile feces were collected in mice, and the diversity and genus level of the mouse gut bacteria were detected by using 16S rRNA technology. Ileum tissue was fixed and stained with HE. RESULTS: HE staining showed that the plateau control group had significant damage to the intestinal tissue structure compared to the plain control group, and the serum DAO concentration was increased (P＜0.05), but there was no statistical difference in the abundance and diversity of intestinal flora species. Contrast to simulated intermittent plateau hypoxia group, the structure of the intestine tissue and the level of DAO in the quercetin intervention group and resveratrol intervention group were improved(P＜0.05), the abundance and α diversity of the intestinal flora were decreased, the relative abundance of Bacteroidetes was reduced(P＜0.05), and the Firmicutes was increased. Concomitantly, significant decreases in relative abundance were observed for Corynebacterium glutamicum and Lactobacillus reuteri(P＜ 0.05). CONCLUSION: Quercetin and resveratrol showed some degree of protection to mice intestinal microbial communities, and increased the diversity and the abundance of the dominant flora and inhibited the growth of conditional pathogenic bacteria.

[Resveratrol attenuates cardiac function impairment in plateau hypobaric hypoxia rats].

Objective: To study the protective effects of resveratrol (RSV) on cardiac function in rats with high altitude hypobaric hypoxia and its mechanisms. Methods: Thirty-six rats were randomly divided into control group, hypobaric hypoxia group (HH) and hypobaric hypoxia + RSV group (HH+RSV) according to the random number, 12 rats in each group. Rats in the HH and HH+RSV groups were subjected to chronic long-term high altitude hypobaric hypoxia intervention for 8 weeks in a hypobaric chamber at a simulated altitude of 6 000 m for 20 h / d. The rats of HH + RSV were fed with RSV at a dose of 400 mg/(kg·d). The rats were tested once a week for body weight and twice a week for food intake. Before execution, the rats were tested by blood cell analyzer for routine blood parameters and echocardiogram for cardiac function parameters in each group. The routine blood indexes of each group were measured by blood cell analyzer, the cardiac function indexes of each group were measured by echocardiography, myocardial hypertrophy was evaluated by HE staining, myocardial tissue reactive oxygen levels were evaluated by dihydroethidium (DHE) staining. Oxidative stress was evaluated by serum and myocardial tissue total antioxidant capacity (T-AOC), superoxide dismutase activity (SOD) and malondialdehyde (MDA) content. Results: Compared with the C group, the body mass and food intake of rats were decreased significantly (P＜0.05) in HH group, while compared with the C group, RSV had no significant effects on the body mass and food intake of rats in the HH+RSV group (P＞0.05). Compared with the C group, the levels of erythrocytes and hemoglobin of rats in the HH group were increased significantly (P＜0.05), while the platelet concentration was decreased significantly(P＜0.05); compared with the HH group, the erythrocyte and hemoglobin levels were decreased significantly (P＜0.05) and platelet concentration was increased significantly(P＜0.05) in rats of the HH+RSV group. Compared with the C group, the cardiac coefficient, myocardial fiber diameter and thickness were significantly increased in the HH group (P＜0.05); compared with the HH group, the cardiac coefficient and myocardial fiber thickness were significantly decreased in the HH+RSV group (P＜0.05). Echocardiographic analysis showed a significant increase in ventricular wall thickness (P＜0.05) and a significant decrease in ejection fraction and cardiac output (P＜0.05) in the HH group compared with the C group, and a significant decrease in ventricular wall thickness and a significant improvement in cardiac function (P＜0.05) in the HH+RSV group compared with the HH group. The results of DHE staining showed that myocardial tissue reactive oxygen levels were increased significantly in the HH group compared with the C group (P＜0.05); myocardial tissue reactive oxygen levels were significantly restored in the HH+RSV group compared with the HH group (P＜0.05). The oxidative/antioxidant results showed that the serum and myocardial T-AOC and SOD activities were decreased significantly (P＜0.05) and the MDA level was increased significantly (P＜0.05) in the HH group compared with the C group; the serum and myocardial T-AOC and SOD activities were increased significantly (P＜0.05) and the MDA level was decreased significantly(P＜0.05) in the HH+RSV group compared with the HH group. Conclusion: Long-term plateau hypobaric hypoxia exposure leads to myocardial hypertrophy and reduced cardiac function in rats. Resveratrol intervention significantly improves myocardial hypertrophy and cardiac function in rats caused by altitude hypobaric hypoxia exposure, which is closely related to reducing of reactive oxygen species and improving myocardial oxidative stress levels.

Multiplex-Heterogeneous Network-Based Capturing Potential SNP "Switches" of Pathways Associating With Diverse Disease Characteristics of Asthma.

Asthma is a complex heterogeneous respiratory disorder. In recent years nubbly regions of the role of genetic variants and transcriptome including mRNAs, microRNAs, and long non-coding RNAs in the pathogenesis of asthma have been separately excavated and reported. However, how to systematically integrate and decode this scattered information remains unclear. Further exploration would improve understanding of the internal communication of asthma. To excavate new insights into the pathogenesis of asthma, we ascertained three asthma characteristics according to reviews, airway inflammation, airway hyperresponsiveness, and airway remodeling. We manually created a contemporary catalog of corresponding risk transcriptome, including mRNAs, miRNAs, and lncRNAs. MIMP is a multiplex-heterogeneous networks-based approach, measuring the relevance of disease characteristics to the pathway by examining the similarity between the determined vectors of risk transcriptome and pathways in the same low-dimensional vector space. It was developed to enable a more concentrated and in-depth exploration of potential pathways. We integrated experimentally validated competing endogenous RNA regulatory information and the SNPs with significant pathways into the ceRNA-mediated SNP switching pathway network (CSSPN) to analyze ceRNA regulation of pathways and the role of SNP in these dysfunctions. We discovered 11 crucial ceRNA regulations concerning asthma disease feature pathway and propose a potential mechanism of ceRNA regulatory SNP → gene → pathway → disease feature effecting asthma pathogenesis, especially for MALAT1 (rs765499057/rs764699354/rs189435941) → hsa-miR-155 → IL13 (rs201185816/rs1000978586/rs202101165) → Interleukin-4 and Interleukin-13 signaling → inflammation/airway remodeling and MALAT1 (rs765499057/rs764699354/rs189435941) → hsa-miR-155 → IL17RB (rs948046241) → Interleukin-17 signaling (airway remodeling)/Cytokine-cytokine receptor interaction (inflammation). This study showed a systematic and propagable workflow for capturing the potential SNP "switch" of asthma through text and database mining and provides further information on the pathogenesis of asthma.

Influence of progestational stress on BDNF and NMDARs in the hippocampus of male offspring and amelioration by Chaihu Shugan San.

BACKGROUND: Progestational stress has been proven to be a risk for the neural development of offspring, especially in the hippocampus. However, whether Chaihu Shugan San (CSS) can ameliorate hippocampal neural development via the regulation of brain-derived neurotrophic factor (BDNF), and N-methyl-D-aspartate receptors (NMDAR) 2A (NR2A) and 2B (NR2B), and the mechanism of such action remains unclear. METHODS: Thirty-six female rats were randomly allocated into control, chronic immobilization stress (CIS) and CSS groups according to the random number table, respectively. The male offspring were fed for 21 days after birth then randomly divided into the same three groups (6 rats/group) as the female rats. Female rats, except for the control group, underwent 21-day CIS to established a progestational stress anxiety-like model which was evaluated by body weight, the elevated plus-maze (EPM) test and serum dopamine (DA) measured using an enzyme-linked immunosorbent assay (ELISA). The expression levels of estrogen receptors (ERα/ERß) and progesterone receptor (PR) in female rat ovaries were quantified by real-time fluorescence quantitative polymerase chain reaction (RT-qPCR) and Western blot analysis. The hippocampal tissue in the 21-day offspring was observed by hematoxylin-eosin (HE) staining. The concentration of BDNF, NR2A, and NR2B were measured by RT-qPCR and immunohistochemistry in the CA3 and dentate gyrus (DG) regions of offsprings' hippocampus. RESULTS: Compared with the female control group, significant differences in body weight, EPM test and DA concentration were observed in the CIS group, meanwhile, the concentration of ERα (P < 0.05), PR (P < 0.05) and ERß in the ovaries were decreased. In the offsprings' hippocampus of the CIS group, the chromatin of the nucleus was edge set and with condensed and irregular morphology nucleus, and the cytoplasm was unevenly stained with spaces around the cells, moreover, the expression levels of BDNF, NR2A, and NR2B were also declined (P < 0.05). However, Chaihu Shugan San reversed these changes, especially the BDNF in the DG region (P < 0.05), and NR2A and NR2B in the CA3 and DG region (P < 0.05). CONCLUSIONS: CSS could ameliorate the neural development of the hippocampus in offspring damaged by anxiety-like progestational stress in female rats via regulating the expression levels of ERα, ERß, and PR in female rat ovaries and BDNF, NR2A, and NR2B in the hippocampus of their offspring.

Predicting Postherpetic Neuralgia in Patients with Herpes Zoster by Machine Learning: A Retrospective Study.

INTRODUCTION: Postherpetic neuralgia (PHN) is a neuropathic pain secondary to shingles. Studies have shown that early pain intervention can reduce the incidence or intensity of PHN. The aim of this study was to predict whether a patient with acute herpetic neuralgia will develop PHN and to help clinicians make better decisions. METHOD: Five hundred two patients with shingles were reviewed and classified according to whether they had PHN. The risk factors associated with PHN were determined by univariate analysis. Logistic regression and random forest algorithms were used to do machine learning, and then the prediction accuracies of the two algorithms were compared, choosing the superior one to predict the next 60 new cases. RESULTS: Age, NRS score, rash site, Charlson comorbidity index (CCI) score, antiviral therapy and immunosuppression were found related to the occurrence of PHN. The NRS score was the most closely related factor with an importance of 0.31. As for accuracy, the random forest was 96.24%, better than that of logistic regression in which the accuracy was 92.83%. Then, the random forest model was used to predict 60 newly diagnosed patients with herpes zoster, and the accuracy rate was 88.33% with a 95% confidence interval (CI) of 77.43-95.18%. CONCLUSIONS: This study provides an idea and a method in which, by analyzing the data of previous cases, we can develop a predictive model to predict whether patients with shingles will develop PHN.

TWIST1 transcriptionally regulates glycolytic genes to promote the Warburg metabolism in pancreatic cancer.

Reprogrammed glucose metabolism is essential for tumor initiation and development, especially for pancreatic ductal adenocarcinoma (PDAC). Most cancer cells rely on aerobic glycolysis, a phenomenon termed "the Warburg effect", to support uncontrolled proliferation and evade apoptosis. However, the direct regulators of the Warburg effect remain areas of active investigation. In this study, we found that the highly conserved transcription factor, TWIST1, is a crucial regulator of aerobic glycolysis in PDAC. Genetic silencing of TWIST1 significantly inhibited the glycolytic phenotypes of PDAC cells as revealed by reduced glucose uptake, lactate production, and extracellular acidification rate, which can be restored by re-expression of siRNA-resistant TWIST1. Moreover, tamoxifen-inducible expression of TWIST1 promoted the Warburg metabolism of PDAC cells. Mechanistically, by luciferase reporter assay and chromatin immunoprecipitation experiment, we showed that TWIST1 can directly increase the expression of several glycolytic genes, including SLC2A1, HK2, ENO1, and PKM2. Of note, the transcriptional regulation by TWIST1 was not dependent on HIF1α or c-Myc. In The Cancer Genome Atlas and Gene Expression Omnibus accession GSE15471, we confirmed that TWIST1 was closely associated with the glycolysis pathway. Collectively, our findings indicate that TWIST1 is likely to act as important regulator of the Warburg effect in PDAC.

[The effects of Sestrin2 on apoptosis of heat-exposed lung epithelial cells and its mechanism].

OBJECTIVE: To investigate the protective effects of Sestrin2 protein on lung epithelial Beas-2B cells in the heat-exposure environment and its mechanism. METHODS: Lung epithelial Beas-2B cells were cultured at 37℃, 39℃, 40℃ and 41℃ respectively. Cells were harvested at different times (0, 3, 6 and 12 h) after pancreatin digestion. The expressions of Sestrin2, superoxide dismutase(SOD), reactive oxygen species(ROS), cell mitochondrial membrane potential and apoptosis rate of cells were detected by Western blot, fluorescence spectrophotometer and flow cytometry, respectively. Gene expression sequence was cloned into high expression plasmid pcDNA3.1+. Beas-2B cells were transfected by Lipfectamine 2000 to construct Sestrin2 and SOD high expression cells. The changes of mitochondrial membrane potential and cell apoptosis were observed in the Sestrin2 and SOD high expression cells. RESULTS: With the increase of temperature, the expression level of Sestrin2 protein in heat treatment group was decreased compared with the control group. When Beas-2B cells were exposed to 41℃, the ROS level was increased, mitochondrial membrane potential was decreased significantly and apoptosis rate was increased at different time points. After high expression of Sestrin2 and SOD in the Beas-2B cells, the expression level of ROS was decreased and the change tendency of mitochondrial membrane potential was decreased, and the apoptosis rate was reduced at 41℃ exposure. CONCLUSION: Sestrin2 can alleviate the apoptosis of lung epithelial cells induced by heat exposure through mitochondrial membrane potential and SOD, which has protective effect on lung epithelial Beas-2B cells.

Xiaoyaosan exerts antidepressant-like effects by regulating the functions of astrocytes and EAATs in the prefrontal cortex of mice.

BACKGROUND: Mounting evidence indicates that the cerebral cortex is an important physiological system of emotional activity, and its dysfunction may be the main cause of stress. Glutamate is the primary excitatory neurotransmitter in the central nervous system (CNS), which initiates rapid signal transmission in the synapse before its reuptake into the surrounding glia, specifically astrocytes (ASTs). The astrocytic excitatory amino acid transporters 1 (EAAT1) and 2 (EAAT2) are the major transporters that take up synaptic glutamate to maintain optimal extracellular glutamic levels, thus preventing accumulation in the synaptic cleft and ensuing excitotoxicity. Growing evidence has shown that excitotoxicity is associated with depression. Therefore, we hypothesized that the underlying antidepressant-like mechanism of Xiaoyaosan (XYS), a Chinese herbal formula, may be related to the regulation of astrocytic EAATs. Therefore, we studied the antidepressant mechanism of XYS on the basis of EAAT dysfunction in ASTs. METHODS: Eighty adult C57BL/6 J mice were randomly divided into 4 groups: a control group, a chronic unpredictable mild stress (CUMS) group, a Xiaoyaosan (XYS) treatment group and a fluoxetine hydrochloride (Flu) treatment group. Except for the control group, mice in the other groups all received chronic unpredictable mild stress for 21 days. Mice in the control and CUMS groups received gavage administration with 0.5 mL of normal saline (NS) for 21 days, and mice in the XYS and Flu treatment groups were administered dosages of 0.25 g/kg/d and 2.6 mg/kg/d by gavage. The effects of XYS on the depressive-like behavioral tests, including the open field test (OFT), forced swimming test (FST) and sucrose preference test (SPT), were examined. The glutamate (Glu) concentrations of the prefrontal cortex (PFC) were detected with colorimetry. The morphology of neurons in the PFC was observed by Nissl staining. The expression of glial fibrillary acidic protein (GFAP), NeuN, EAAT1 and EAAT2 proteins in the PFC of mice was detected by using Western blotting and immunohistochemistry. Quantitative real-time PCR (qPCR) was used to detect the expression of the GFAP, NeuN, EAAT1 and EAAT2 genes in the PFC of mice. RESULTS: The results of behavioral tests showed that CUMS-induced mice exhibited depressive-like behavior, which could be improved in some tests with XYS and Flu treatment. Immunohistochemistry and Western blot analysis showed that the protein levels of GFAP, NeuN, EAAT1 and EAAT2 in the PFC of CUMS mice were significantly lower than those in the control group, and these changes could be reversed by XYS and Flu. The results of qPCR analysis showed that the expression of GFAP, NeuN, EAAT1 and EAAT2 mRNAs in the PFC of CUMS mice was not significantly changed, with the exception of EAAT2, compared with that of the control group, while the expression of the above mRNAs was significantly higher in the XYS and Flu groups than that in the CUMS group. CONCLUSION: XYS may exert antidepressant-like effects by improving the functions of AST and EAATs and attenuating glutamate-induced neuronal damage in the frontal cortex.

miR-506 attenuates methylation of lncRNA MEG3 to inhibit migration and invasion of breast cancer cell lines via targeting SP1 and SP3.

BACKGROUND: Breast cancer has been the first death cause of cancer in women all over the world. Metastasis is believed to be the most important process for treating breast cancer. There is evidence that lncRNA MEG3 functions as a tumor suppressor in breast cancer metastasis. However, upstream regulation of MEG3 in breast cancer remain elusive. Therefore, it is critical to elucidate the underlying mechanism upstream MEG3 to regulate breast cancer metastasis. METHODS: We employed RT-qPCR and Western blot to examine expression level of miR-506, DNMT1, SP1, SP3 and MEG3. Besides, methylation-specific PCR was used to determine the methylation level of MEG3 promoter. Wound healing assay and transwell invasion assay were utilized to measure migration and invasion ability of breast cancer cells, respectively. RESULTS: SP was upregulated while miR-506 and MEG3 were downregulated in breast tumor tissue compared to adjacent normal breast tissues. In addition, we found that miR-506 regulated DNMT1 expression in an SP1/SP3-dependent manner, which reduced methylation level of MEG3 promoter and upregulated MEG3 expression. SP3 knockdown or miR-506 mimic suppressed migration and invasion of MCF-7 and MDA-MB-231 cells whereas overexpression of SP3 compromised miR-506-inhibited migration and invasion. CONCLUSIONS: Our data reveal a novel axis of miR-506/SP3/SP1/DNMT1/MEG3 in regulating migration and invasion of breast cancer cell lines, which provide rationales for developing effective therapies to treating metastatic breast cancers.

Prognostic significance of the expression of HER family members in primary osteosarcoma.

The prognosis of patients with metastatic osteosarcoma is poor and has shown no significant improvement in nearly 20 years. The human epidermal growth factor (EGF) receptor (HER) family is frequently overexpressed in the majority of human carcinomas, and is involved in promoting the proliferation and survival of cancer cells. However, the role of EGFR and HER-2 expression in osteosarcoma survival remains controversial and no previous study has simultaneously investigated the association of the expression of all the four HER family members with the prognostic significance of osteosarcoma. Therefore, the present study investigated the expression levels of the complete members of the HER family in osteosarcoma specimens, as well as their associations with the clinicopathological parameters, progression-free survival (PFS) and overall survival (OS) time of patients with osteosarcoma. The expression of HER family members was detected in osteosarcoma tumor specimens from 60 patients using immunohistochemistry. The association of the expression of HER receptors in osteosarcoma with clinicopathological parameters was analyzed using χ2 test and Fishers exact test. Survival analyses were evaluated by Kaplan-Meier method and Cox proportional hazards regression model. Overall, 18 (30%), 13 (22%), 23 (38%) and 19 (32%) patients presented with high expression of EGFR, HER-2, HER-3 and HER-4, respectively, and the co-expression of 2, 3 and all 4 members of the HER family was observed. High expression of EGFR and HER-4 was associated with distant metastasis. High HER-3 expression was significantly associated with an advanced Enneking stage and distant metastasis. Multivariate analysis demonstrated that the expression of EGFR, HER-3, HER-4, EGFR/HER-3, EGFR/HER-4 and HER-3/HER-4 was an independent predictor of poor PFS and OS time in osteosarcoma patients with stage I-IIB disease. In patients with stage IIB osteosarcoma, the expression of HER-4 and EGFR/HER-4 demonstrated a more significant effect on PFS and OS time. In conclusion, therapies targeting EGFR, HER-3 and HER-4 may provide promising strategies for primary osteosarcoma.

Suppression of synaptic plasticity by fullerenol in rat hippocampus in vitro.

Fullerenol, a water-soluble fullerene derivative, has attracted much attention due to its bioactive properties, including the antioxidative properties and free radical scavenging ability. Due to its superior nature, fullerenol represents a promising diagnostic, therapeutic, and protective agent. Therefore, elucidation of the possible side effects of fullerenol is important in determining its potential role. In the present study, we investigated the acute effects of 5 µM fullerenol on synaptic plasticity in hippocampal brain slices of rats. Incubation with fullerenol for 20 minutes significantly decreased the peak of paired-pulse facilitation and long-term potentiation, indicating that fullerenol suppresses the short- and long-term synaptic plasticity of region I of hippocampus. We found that fullerenol depressed the activity and the expression of nitric oxide (NO) synthase in hippocampus. In view of the important role of NO in synaptic plasticity, the inhibition of fullerenol on NO synthase may contribute to the suppression of synaptic plasticity. These findings may facilitate the evaluation of the side effects of fullerenol.

Foxp3 promoter methylation impairs suppressive function of regulatory T cells in biliary atresia.

Biliary atresia (BA) is characterized by progressive inflammation of the biliary system leading to liver cirrhosis, necessitating liver transplantation in pediatric patients. Various cell types have been reported to participate in the proinflammatory response in rhesus rotavirus (RRV)-induced BA mouse models, including T helper (Th) 1, Th2, Th17, CD8+ T cells, and natural killer cells. The immune suppressive regulatory T (Treg) cells, on the contrary, were reported not to function properly. The underlying mechanism is largely unknown. Focusing on the impaired suppressive function of Treg, we found methylation status of CpG islands within the Foxp3 promoter region of Treg cells in BA patients and murine models were both increased. Moreover, by injecting 5-aza-2'-deoxycytidine (Aza) as DNA-methylation inhibitor to RRV-infected mice, BA phenotypes were alleviated. Furthermore, Treg cells isolated from "RRV+Aza"-injected mice had better suppressive function than Treg cells from mice injected with RRV only, both in vivo and ex vivo. Thus we concluded that aberrant increased methylation status of "Foxp3 promoter" in Treg cells leads to impaired Treg suppressive function, exacerbating inflammatory injury in BA.

Effects of sphincter of Oddi motility on the formation of cholesterol gallstones.

AIM: To investigate the mechanisms and effects of sphincter of Oddi (SO) motility on cholesterol gallbladder stone formation in guinea pigs. METHODS: Thirty-four adult male Hartley guinea pigs were divided randomly into two groups, the control group (n = 10) and the cholesterol gallstone group (n = 24), which was sequentially divided into four subgroups with six guinea pigs each according to time of sacrifice. The guinea pigs in the cholesterol gallstone group were fed a cholesterol lithogenic diet and sacrificed after 3, 6, 9, and 12 wk. SO manometry and recording of myoelectric activity were obtained by a multifunctional physiograph at each stage. Cholecystokinin-A receptor (CCKAR) expression levels in SO smooth muscle were detected by quantitative real-time PCR (qRT-PCR) and serum vasoactive intestinal peptide (VIP), gastrin, and cholecystokinin octapeptide (CCK-8) were detected by enzyme-linked immunosorbent assay at each stage in the process of cholesterol gallstone formation. RESULTS: The gallstone formation rate was 0%, 0%, 16.7%, and 83.3% in the 3, 6, 9, and 12 wk groups, respectively. The frequency of myoelectric activity in the 9 wk group, the amplitude of myoelectric activity in the 9 and 12 wk groups, and the amplitude and the frequency of SO in the 9 wk group were all significantly decreased compared to the control group. The SO basal pressure and common bile duct pressure increased markedly in the 12 wk group, and the CCKAR expression levels increased in the 6 and 12 wk groups compared to the control group. Serum VIP was elevated significantly in the 9 and 12 wk groups and gastrin decreased significantly in the 3 and 9 wk groups. There was no difference in serum CCK-8 between the groups. CONCLUSION: A cholesterol gallstone-causing diet can induce SO dysfunction. The increasing tension of the SO along with its decreasing activity may play an important role in cholesterol gallstone formation. Expression changes of CCKAR in SO smooth muscle and serum VIP and CCK-8 may be important causes of SO dysfunction.

Characterization of Rebound Depolarization in Neurons of the Rat Medial Geniculate Body In Vitro.

Rebound depolarization (RD) is a response to the offset from hyperpolarization of the neuronal membrane potential and is an important mechanism for the synaptic processing of inhibitory signals. In the present study, we characterized RD in neurons of the rat medial geniculate body (MGB), a nucleus of the auditory thalamus, using whole-cell patch-clamp and brain slices. RD was proportional in strength to the duration and magnitude of the hyperpolarization; was effectively blocked by Ni(2+) or Mibefradil; and was depressed when the resting membrane potential was hyperpolarized by blocking hyperpolarization-activated cyclic nucleotide-gated (HCN) channels with ZD7288 or by activating G-protein-gated inwardly-rectifying K(+) (GIRK) channels with baclofen. Our results demonstrated that RD in MGB neurons, which is carried by T-type Ca(2+) channels, is critically regulated by HCN channels and likely by GIRK channels.

Sodium salicylate potentiates the GABAB-GIRK pathway to suppress rebound depolarization in neurons of the rat's medial geniculate body.

Rebound depolarization (RD) is a voltage response to the offset from pre-hyperpolarization of neuronal membrane potential, which manifests a particular form of the postsynaptic membrane potential response to inhibitory presynaptic inputs. We previously demonstrated that sodium salicylate (NaSal), a tinnitus inducer, can drastically suppress the RD in neurons of rat medial geniculate body (MGB) (Su et al, 2012; PLoS ONE 7, e46969). The purpose of the present study was to investigate the underlying cellular mechanism by using whole-cell patch-clamp recordings in rat MGB slices. NaSal (1.4 mM) had no effects on the current mediated by T-type Ca(2+) channels, indicating that it does not target these channels to suppress the RD. Instead, NaSal was shown to hyperpolarize the resting membrane potential to suppress the RD. NaSal had no effects on the current mediated by hyperpolarization-activated cyclic nucleotide-gated (HCN) channels, indicating that it does not target these channels to hyperpolarize the resting membrane potential. NaSal induced an outward leak current that could be abolished by CGP55845, a GABAB receptor blocker, or respectively by Ba(2+) and Tertiapin-Q, blockers for G-protein-gated inwardly rectifying potassium (GIRK) channels, indicating that NaSal potentiates the GABAB-GIRK pathway to hyperpolarize the resting membrane potential. Our study demonstrates that NaSal targets GABAB receptors to alter functional behaviors of MGB neurons, which may be implicated in NaSal-induced tinnitus.