Bone Morphogenetic Protein Signaling Agonist SB4 (BMPSB4) Inhibits Corticotroph Pituitary Neuroendocrine Tumors by Activation of Autophagy via a BMP4/SMADs-Dependent Pathway.

Corticotroph pituitary neuroendocrine tumors (PitNETs), associated with Cushing's disease (CD), have limited treatment options other than surgical resection. Bone morphogenetic protein 4 (BMP4), a potential therapeutic target, is decreased in patients with CD. Previous studies have identified BMPSB4 as a potent agonist of the BMP4 signaling pathway. Here, we investigated the effect of BMPSB4 on the corticotroph PitNET cell line AtT20/D16v-F2 and explored the underlying mechanisms and therapeutic potential. We verified the low expression patterns of BMP4 and downstream p-SMAD1/5/9 in CD samples at the transcriptional and protein levels. In addition, BMPSB4 activated SMAD1/5/9 in a time- and concentration-dependent manner, with concomitant inhibitory effects on AtT20/D16v-F2 cells. Further RNA sequencing, transmission electron microscopy (TEM), and transfection with the mRFP-EGFP-LC3 adenoviral vector revealed that BMPSB4 induced cellular autophagy, which was the basis for the inhibitory effect of BMPSB4. Moreover, we demonstrated that autophagy induced by BMPSB4 was achieved through the SMADs-dependent pathway. In vivo, BMPSB4 inhibited tumor growth and significantly reduced adrenocorticotrophin (ACTH) and corticosterone (CORT) secretion, thereby alleviating the CD phenotype. In conclusion, this study identified BMPSB4 as an effective therapeutic agent for CD. BMPSB4 activates autophagy through a SMADs-dependent pathway, which in turn promotes autophagy-mediated cell death. Our work further elucidates the mechanism of the BMP4 signaling pathway in CD and suggests broad prospects for the development and application of BMPSB4 in CD therapy.

Dynamic and static angry faces influence time perception differently-Evidence from ERPs.

The dynamic characteristics of facial expressions might affect time perception. Compared with static emotional faces, dynamic emotional faces are more intense, have higher ecological validity, and contain time series information, which may lead to time overestimation. In the present study, we aimed at investigating how dynamic characteristics of angry facial expressions affect time perception, as measured using event-related potentials (ERPs). Dynamic and static angry and neutral faces with different durations (400, 600, 800, 1000, 1200, 1400, and 1600 ms) were presented in the classical temporal bisection paradigm. Participants were asked to judge whether the duration of the presented face was closer to 400 or 1600 ms. The behavioral results showed a significant overestimation effect for dynamic angry faces compared with static faces, both in terms of proportion of long and Bisection Point. The ERP results indicated that the processing mechanisms are significantly different between judging the duration of dynamic and static angry faces. Dynamic angry faces evoked a larger N2 and Late Positive Potential than did static faces, while the static angry faces evoked a larger P2 and Early Posterior Negativity. The Contingent Negative Variation showed a complex change pattern over time. Our results indicate that dynamic angry facial expressions influence time perception differently than do static faces. Static angry faces were processed earlier and were considered to cause an overestimation of time through early emotional arousal and attentional bias, while dynamic angry faces may have caused the overestimation of time through response inhibition and late sustained attention.

Magnetic Resonance Neurography for Evaluation of Dorsal Root Ganglion Morphology.

BACKGROUND: In this study, the morphologic characteristics and anatomic position of the dorsal root ganglion (DRG) were measured and analyzed in healthy people using magnetic resonance neurography (MRN), which provided an anatomical reference for minimally invasive spinal surgery. METHODS: From January 2018 to December 2019, 20 healthy adult volunteers (10 male and 10 female volunteers between 20 and 65 years old) were scanned and imaged by 3.0 T magnetic resonance imaging combined with neuroimaging technology. Here, the position of the DRG was located, and the shape and size of the DRG, as well as its distance to the upper pedicle, were measured. RESULTS: All volunteers provided satisfactory MRN scans of the L1-S1 lumbar DRG. According to the spatial position of the DRG, the morphology of the DRG can be divided into the intervertebral foramen type (81.01%), intraspinal type (16.01%), extraforaminal type (0.8%), and mixed type (2.0%). CONCLUSIONS: The intervertebral foramen type and Intraspinal type were observed to be the main distribution forms of lumbar DRG. Due to the downward movement of lumbar segments, the position of the DRG was noted to gradually move to the spinal canal while its volume gradually increased. In addition, the distance from the upper pedicle was found to decrease gradually. MRN imaging can clearly show the shape, location, and adjacent relationship of the DRG, providing effective imaging guidance for the minimally invasive lumbar techniques.

Development of Young Children's Time Perception: Effect of Age and Emotional Localization.

Time perception is a fundamental aspect of young children's daily lives and is affected by a number of factors. The present study aimed to investigate the precise developmental course of young children's time perception from 3 to 5 years old and the effects of emotion localization on their time perception ability. A total of 120 children were tested using an adapted time perception task with black squares (Experiment 1) and emotional facial expressions (Experiment 2). Results suggested that children's time perception was influenced by stimulus duration and improved gradually with increasing age. Both accuracy and reaction time were affected by the presentation sequence of emotional faces, indicating an effect of emotion localization. To summarize, young children's time perception showed effects of age, stimulus duration, and emotion localization.

Knockdown of Tripartite Motif 8 Protects H9C2 Cells Against Hypoxia/Reoxygenation-Induced Injury Through the Activation of PI3K/Akt Signaling Pathway.

Tripartite motif 8 (TRIM8) is a member of the TRIM protein family that has been found to be implicated in cardiovascular disease. However, the role of TRIM8 in myocardial ischemia/reperfusion (I/R) has not been investigated. We aimed to explore the effect of TRIM8 on cardiomyocyte H9c2 cells exposed to hypoxia/reoxygenation (H/R). We found that TRIM8 expression was markedly upregulated in H9c2 cells after stimulation with H/R. Gain- and loss-of-function assays proved that TRIM8 knockdown improved cell viability of H/R-stimulated H9c2 cells. In addition, TRIM8 knockdown suppressed reactive oxygen species production and elevated the levels of superoxide dismutase and glutathione peroxidase. Knockdown of TRIM8 suppressed the caspase-3 activity, as well as caused significant increase in bcl-2 expression and decrease in bax expression. Furthermore, TRIM8 overexpression exhibited apposite effects with knockdown of TRIM8. Finally, knockdown of TRIM8 enhanced the activation of PI3K/Akt signaling pathway in H/R-stimulated H9c2 cells. Inhibition of PI3K/Akt by LY294002 reversed the effects of TRIM8 knockdown on cell viability, oxidative stress, and apoptosis of H9c2 cells. These present findings defined TRIM8 as a therapeutic target for attenuating and preventing myocardial I/R injury.

miR-425-5p improves inflammation and septic liver damage through negatively regulating the RIP1-mediated necroptosis.

OBJECTIVE AND DESIGN: Sepsis, a systemic inflammatory response syndrome, is still a common cause of death even the patients who are in the intensive care unit. Alleviating septic liver damage may be effective in improving sepsis. Necroptosis and miRNAs have been regarded as a potential target in sepsis. MATERIAL OR SUBJECTS: The aim of this work is to explain the potential role of miR-425-5p in septic liver damage. LPS was intraperitoneal-injection to C57BL/6 mice for sepsis, and hepatocytes treated with septic serum in vitro. H&E staining for histological evaluation, luciferase reporter assay for target validation, and qRT-PCR, WB, and ELISA analysis for assessment of miR-425-5p, RIP1, inflammatory factors, and LDH levels. RESULTS: Down-regulated miR-425-5p and up-regulated RIP1/RIP3 were in LPS-induced sepsis mice. Liver damage, RIP1-mediated necroptosis, IL-1ß, and TNF-α were suppressed by miR-425-5p agomiR, but further aggravated by miR-425-5p antagomiR. Furthermore, we demonstrated miR-425-5p targeted the 3'UTR of RIP1 mRNA to inhibit RIP1 expression and activated RIP1 reversed miR-425-5p-induced suppression of necroptosis and inflammation in septic hepatocytes. CONCLUSIONS: The data suggest miR-425-5p negatively controls the RIP1-mediated necroptotic signaling cascades and inflammation, and sepsis-related liver damage. miR-425-5p/RIP1 axis is a potential therapeutic strategy for sepsis-related liver damage through necroptosis and inflammation.

Sevoflurane Post-treatment Upregulated miR-203 Expression to Attenuate Cerebral Ischemia-Reperfusion-Induced Neuroinflammation by Targeting MyD88.

To investigate the expression of miR-203 by sevoflurane treatment and its effect on neuroinflammation induced by cerebral ischemia-reperfusion. Rats were randomly divided into sham operation group (C), cerebral ischemia-reperfusion group (I/R), and sevoflurane treatment group (S). The neurological function score was evaluated. The area of cerebral infarction was evaluated by TTC staining. The expression of inflammatory factor in brain tissue was detected by ELISA. The apoptosis of neurons was detected by TUNEL. A miR-203 agonist and inhibitor treated the cerebral ischemia-reperfusion model. The luciferase assay verified whether miR-203 targeted MyD88. To further verify the relationship between miR-203 and MyD88, the I/R group was treated with MyD88 activator and inhibitor, and the mRNA expressions of miR-203 and MyD88 in brain tissue were detected by RT-PCR. Western blot was used to detect the expression of MyD88 protein in brain tissue, and the above experiment was repeated. Compared with the I/R group, miR-203 mRNA was significantly increased in brain tissue and the neurological function score, the area of cerebral infarction, the expression of inflammatory factor, and MyD88 mRNA were decreased in the S group (P < 0.05). After treatment of miR-203 agonist and inhibitor in the I/R group, overexpression of miR-203 could alleviate cerebral ischemia-reperfusion injury, and miR-203 inhibitor could aggravate cerebral ischemia-reperfusion injury. The miR-203 agonist could enhance the action of sevoflurane, and the miR-203 inhibitor could reverse the action of sevoflurane. miR-203 agonist treatment could inhibit the expression of MyD88 gene and protein and reduce the neuroinflammation induced by cerebral ischemia-reperfusion. The treatment of sevoflurane upregulated miR-203 expression, which targeted MyD88 and attenuate neuroinflammation induced by cerebral ischemia-reperfusion.

MicroRNA-301b promotes the proliferation and invasion of glioma cells through enhancing activation of Wnt/ß-catenin signaling via targeting Glypican-5.

Accumulating evidence has suggested that Glypican-5 (GPC5) is a tumor suppressor gene in many types of cancers. However, whether GPC5 is involved in glioma remains unknown. This study was designed to explore the expression, biological function and regulatory mechanism of GPC5 in glioma. Our results demonstrated that GPC5 expression was significantly decreased in multiple glioma cell lines. Gain-of-function experiments showed that the ectopic expression of GPC5 markedly inhibited the proliferation, invasion and Wnt/ß-catenin signaling of glioma cell lines. GPC5 was identified as a target gene of microRNA-301b (miR-301b). Further data showed that miR-301b expression was significantly up-regulated in glioma tissues and cell lines. In addition, miR-301b expression was inversely correlated with GPC5 expression in clinical glioma tissues. The overexpression of miR-301b promoted the proliferation, invasion and Wnt/ß-catenin signaling of glioma cell lines, whereas the inhibition of miR-301b showed the opposite effect. However, the silencing of GPC5 significantly reversed the antitumor effect of miR-301b inhibition. Overall, our results revealed a tumor suppressive role of GPC5 in glioma and suggested that GPC5 expression was regulated by miR-301b. Our study indicates that the inhibition of miR-301b represses the proliferation and invasion of glioma cells by up-regulating GPC5 expression.

TSP-1 is downregulated and inversely correlates with miR-449c expression in Cushing's disease.

The pathogenesis of Cushing's disease, which is caused by pituitary corticotroph adenoma, remains to be studied. Secreted angioinhibitory factor thrombospondin-1 (TSP-1) is an adhesive glycoprotein that mediates cell-to-cell and cell-to-matrix interactions and is associated with platelet aggregation, angiogenesis and tumorigenesis. We have found that the expression of TSP-1 is significantly lower in human pituitary corticotroph tumours compared with normal adenohypophysis. This study aims to elucidate the role of TSP-1 in regulating the tumour function of pituitary adenomas. Forced overexpression of TSP-1 in a murine AtT20 pituitary corticotroph tumour cell line decreased corticotroph precursor hormone proopiomelanocortin (POMC) transcription and adrenocorticotropic hormone (ACTH) secretion. Functional studies showed that TSP-1 overexpression in pituitary adenoma cells suppressed proliferation, migration and invasion. We have demonstrated that TSP-1 is a direct target of miR-449c. Further study showed that miR-449c activity enhanced tumorigenesis by directly inhibiting TSP-1 expression. Low expression of lncTHBS1, along with low expression of TSP-1, was associated with the high expression of miR-449c in Cushing's disease patients. Furthermore, RNA-immunoprecipitation associates miR-449c with lncTHBS1 suggesting that lncTHBS1 might be a negative regulator of miR-449c. Taken together, this study has demonstrated that lncTHBS1 might function as competing endogenous RNA for miR-449c, which could suppress the development of Cushing's disease.

Identification of genes and pathways associated with multiple organ dysfunction syndrome by microarray analysis.

Multiple organ dysfunction syndrome (MODS) is characterized by the development of progressive physiological dysfunction of ≥2 organs or organ systems and is responsible for the majority of the morbidity and mortality among patients in intensive care units. The aim of the present study was to investigate the potential genes and pathways associated with MODS. The microarray dataset GSE60088 was downloaded from the Gene Expression Omnibus and used to identify differentially expressed genes (DEGs) between organ tissues (lung, liver and kidney) obtained from a murine model of MODS and healthy controls. The interactions between DEGs in lungs, liver and kidneys were revealed by Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis. Furthermore, protein­protein interaction (PPI) data for DEGs were obtained from the Search Tool for the Retrieval of Interacting Genes and a PPI network was constructed. Additionally, DEGs that were common among the three organs were screened and transcription factors that regulated them were predicted using the iRegulon plugin. A total of 943, 267 and 227 DEGs were identified in lung, liver and kidney samples, respectively, between mice with MODS and healthy controls. In lung and liver samples, two pathways that were enriched with DEGs were identified and were common between lung and liver samples, including 'cytokine­cytokine receptor interaction' and 'Jnk­STAT signaling pathway', and examples of DEGs associated with these pathways include C­X­C motif chemokine ligand (Cxcl)1 and Cxcl10, and signal transducer and activator of transcription (Stat)1, respectively. Furthermore, two common pathways were identified in liver and kidney samples, which included 'MAPK signaling pathway' and 'p53 signaling pathway', and DEGs associated with these pathways included growth arrest and DNA damage­inducible α. A total of 18 DEGs were common among lung, liver and kidney tissues, including CCAAT/enhancer binding protein ß (Cebpb) and olfactomedin­like 1 (Olfml1). Cebpb modulated various other DEGs, such as Cxcl1, and Olfml1 was regulated by Stat5A. These genes and pathways may serve roles in the progression of MODS and may be considered to be potential therapy targets for MODS.

Decreased expression of SFRP2 promotes development of the pituitary corticotroph adenoma by upregulating Wnt signaling.

Cushing's disease is primarily caused by pituitary adrenocorticotropin­secreting adenoma. However, its pathogenesis has remained obscure. In the present study, whole transcriptome analysis was performed by RNA sequencing (RNA­Seq) and expression of secreted frizzled­related protein 2 (SFRP2) was decreased in corticotroph tumors compared with normal pituitary glands. Furthermore, the RNA­Seq results were validated and the expression of SFRP2 in tumor tissues was analyzed by comparing another cohort of 23 patients with Cushing's disease and 3 normal human pituitary samples using reverse transcription­quantitative polymerase chain reaction, western blot and immunohistochemistry staining. Clinically, there was an association between lower SFRP2 expression and aggressive adenoma characteristics, including larger size and invasiveness. Conversely, SFRP2 overexpression reduced the ability of AtT20 cells to proliferate and migrate, and reduced production of the adrenocorticotrophic hormone in vitro. Mechanistically, overexpressed SFRP2 reduced the level of ß­catenin in the cytoplasm and nucleus, and decreased Wnt signaling activity in AtT20 cells. Therefore, SFRP2 appears to act as a tumor suppressor in Cushing's disease by regulating the activity of the Wnt signaling pathway.

Effect of Hydrogen-Rich Saline on Postoperative Intra-Abdominal Adhesion Bands Formation in Mice.

BACKGROUND Postsurgical peritoneal adhesions (PPAs) are pathologic fibrous bands within the peritoneal cavity. The aim of this study was to investigate the protective effect of hydrogen-rich saline (HRS) on PPAs formation in mice. MATERIAL AND METHODS Adhesions were induced in mice using the cecum rubbing model. The mice were allocated into 4 groups: control sham group without cecum rubbing; PPA group with saline applied intraperitoneally (i.p.) daily after cecum rubbing; PPA+HRS (5) group with 5 ml/kg of HRS applied i.p. daily after cecum rubbing; and PPA+HRS (10) group with 10 ml/kg of HRS applied i.p. daily after cecum rubbing. On the 1st, 3rd, and 7th days after the operation, mice were killed and pathological adhesion bands were quantified to detect the effect of HRS on PPAs formation. RESULTS HRS did not affect PPAs formation on the 1st day, but did make a significant reduction on the 3rd and 7th days. A significant increase of t-PA and decrease of TGF-b1 and PAI-1 in the peritoneal fluids were observed in the HRS-treated groups. The levels of MDA and MPO in the HRS-treated groups were significantly lower than those in the PPA group. TNF-α and IL-6 levels in HRS-treated groups significantly decreased compared with those in the PPA group on postoperative day 3 and 7. Moreover, HRS decreased the mRNA levels of pro-inflammatory cytokines and TGF-ß1 expression in the postsurgical adhesion bands. CONCLUSIONS These results showed that HRS had therapeutic potential for preventing PPAs formation, possibly through balancing the expression of TGF-ß1, t-PA, and PAI-1, and inhibiting oxidative stress and inflammation.

Knockdown of mTOR by lentivirus­mediated RNA interference suppresses atherosclerosis and stabilizes plaques via a decrease of macrophages by autophagy in apolipoprotein E­deficient mice.

Atherosclerotic plaque destabilization and rupture leads to acute coronary syndromes which cause serious damage to human health worldwide. However, there is currently a lack of efficient therapeutic methods. Mammalian target of rapamycin (mTOR) has been suggested to be involved in the development of atherosclerotic plaques and serves as a therapeutic target. The present study was performed to determine whether RNA interference (RNAi) of mTOR in vivo by LV­mediated small hairpin RNA (shRNA) was capable of inhibiting the progression of atherosclerotic plaques. LV­mediated shRNA against mTOR (LV­shmTOR) was designed and obtained. Male apolipoprotein E­deficient mice were fed a high­fat diet and a constrictive collar was placed around the right carotid arteries of these mice to induce plaque formation. Eight weeks after surgery, mice were randomly divided into the mTOR RNA interference (LV­shmTOR) group, receiving treatment with LV­mTOR­shRNA; the LV­shCON group, receiving treatment with LV­non­specific­shRNA; and the control group, receiving treatment with phosphate­buffered saline. Following transfection, the mice were sacrificed to evaluate the effects of mTOR expression silencing on atherosclerosis. Transfection of LV­mTOR­shRNA markedly inhibited the mRNA and protein expression levels. Knockdown of mTOR ameliorated dysregulated blood lipid metabolism and stabilized aortic atherosclerotic plaques by decreasing the plaque area and increasing the fibrous cap and cap­to­core ratio. Furthermore, macrophages were decreased by silencing mTOR in atherosclerotic plaques. In addition, western blot analysis revealed that the knockdown of mTOR increased autophagy­related protein 13 (Atg13) dephosphorylation and light chain 3­I/light chain 3­II (LC3­I/LC3­II) ratios, both of which were associated with a high activity of autophagy, suggesting an increase of autophagy in atherosclerotic plaques. Moreover, genes including matrix metalloproteinase 2, monocyte chemoattractant protein 1 and tissue factor, which promote plaque instability, were downregulated by silencing mTOR. These results demonstrate that LV­mediated mTOR silencing by RNAi treatment induces macrophage autophagy and is a potential strategy for the treatment of atherosclerotic plaques.