Silent Information Regulator 1 (SIRT1) Promotes Pancreatic β-Cell Mitochondrial Autophagy and Pyrin Domains-Containing Protein 3 (NLRP3) Activation Under High Glucose Environment

Mitochondrial autophagy and inflammatory response involves in diabetes. This study mainly explores the role of Silent Information Regulator (SIRT1) in pancreatic β-cells under high glucose conditions and related mechanism. Pancreatic β cells was cultured in a high-glucose environment with SRT1720 and EX527 respectively to define activation group and inhibition group followed by analysis of SIRT1, P-FOXO1, FOXO1, LC3, ATG5, PINK, Parkin, Mfn1, Mfn2, Fis1, IL-6, TNF-α, NLRP3 protein and mRNA expression by qRT-PCR, Western blot and fluorescent probe technology. Compared with control group, SIRT1 protein and mRNA expression in the high glucose group was significantly reduced. Activation group had highest protein and mRNA expression of SIRT1 P-FOXO1, FOXO1, Mfn1, Mfn2, Fis1, PINK, Parkin and mitochondrial membrane potential followed by blank group and inhibition group.SIRT1 secretion by pancreatic β-cells under high glucose environment is reduced. After activating SIRT1, mitochondrial autophagy decreased significantly and inflammatory response is significantly alleviated, indicating that SIRT1 might be used as a therapeutic target.

Stromal Cell-Derived Factor-1α (SDF-1α) Promotes Growth and Migration of Bone Marrow Stromal Cells (BMSCs) and Gastric Cancer Cells Through Phosphatidylinositol 3-Kinase/AKT (PI3K/Akt) Pathway

SDF-1α activity is closely related to information transmission and cell migration when contributing to lymphatic metastasis in various tumors. Herein, we explored the interaction among SDF-1α, CXCR4 and PI3K/Akt signaling pathway in gastric cancer (GC) and their roles in this disorder. Human GC cells KATO-III and BMSCs were co-cultured without contact. GC cells were transfected with SDF-1α, CXCR4 inhibitor, and PI3K inhibitor. After examining the efficiency of transfection, cell migration was evaluated using Transwell chamber, and expression SDF-1α, CD133, and CXCR4 was determined by RT-qPCR. With transfection rate of 98%, the number of migrated cells reduced upon inhibition of CXCR4 and PI3K. Luciferase activity in 565 nm are high than CXCR4 inhibition group. (p < 0.05). Likewise, up-regulation of SDF-1α increased the expression of SDF-1 (0.825±0.061), CD133 (0.875±0.058), CXCR4 (0.801±0.052), and Akt (0.852±0.062), compared to the blank group, CXCR4 inhibition group and PI3K inhibition group (p < 0.05). Down-regulation of CXCR4 and PI3K, however, decreased the expression insignificantly (p > 0.05). Collectively, up-regulation of SDF-1α activates CXCR4 signaling pathway of BMSCs and stimulates its downstream PI3K/Akt signaling pathway and and increases the expression of CD133, thereby promoting malignant behaviors of GC cells.

MiR-26b-5p Promotes Osteogenesis of Bone Mesenchymal Stem Cells via Suppressing FGF21

Introduction: It has been established that miR-26b-5p actively participate in the osteogenic differentiation of bone mesenchymal stem cells (BMSCs), which is of great value in osteoporosis treatment. Database showed that Fibroblast growth factor(FGF)21 is a potential binding site of miR-26b-5p. This study aimed to investigate the molecular osteogenic mechanisms of miR-26b-5p targeting FGF21 in postmenopausal osteoporosis (PMOP). Methods: 5ml of bone marrow was aspirated from the anterior superior iliac spine in 10 PMOP women during bone marrow puncture. BMSCs were used to establish an in vitro cell model, and BMSCs markers were analyzed by flow cytometry. miR-26b-5p and FGF21 were overexpressed for 48h, and then placed in an osteogenic induction medium for osteogenic induction culture, the expression of RNA was detect using RT-qPCR. Cells from miR-26b-5p group were collected on days 7, 14 and 21 of induction for ALP and alizarin red S staining. On day 7 of induction, RT-qPCR was used to measure Runx2, Osterix (Osx), and target gene FGF21 expression levels in each group. The dual-luciferase reporter gene system was used to verify that FGF21 was a direct target of miR-26b-5p. FGF21 was measured by western blotting in the miR-26b-5p overexpression group and in the miR-26b-5p inhibition group. Results: BMSCs were identified according with the antigenic characteristics. miR-26b-5p expression was significantly upregulated after the expression of miR-26b-5p mimics; however, FGF21 expression was downregulated after FGF21 mimics. After overexpression of miR-26b-5p, the alkaline phosphatase activity and nodules of alizarin red S in the culture medium gradually increased as the induction time increased. RT-qPCR showed that the expressions of master osteogenic factors Runx2 and Osx in the BMSC+ osteogenic differentiation medium group was significantly higher than in the BMSC group, the expressions of the factors in the BMSC+ miR-26b-5p overexpression group was significantly higher than in the control group. Target gene FGF21 expression was significantly lower in the BMSC+ osteogenic differentiation medium group than in the BMSC group, and was significantly lower in the BMSC+ miR-26b-5p overexpression group than in the control group. Luciferase reporter assays demonstrated that FGF21 was a direct target of miR-26b-5p. Finally, western blotting analysis showed that FGF21 expression was significantly downregulated in the miR-26b-5p overexpressed group and upregulated in the miR-26b-5p inhibition group. Conclusion: miR-26b-5p can regulate the osteogenic differentiation of BMSCs and participate in PMOP pathogenesis via suppressing FGF21. The present study provides the basis for further studies on PMOP.

Wnt/β-Catenin Pathway Balances Scaffold Degradation and Bone Formation in Tissue-Engineered Laminae

Tissue engineering provides a promising way for the regeneration of artificial vertebral laminae. Previous studies have confirmed the feasibility of reconstructing vertebral laminae via hydroxyapatite-collagen I scaffolds and mesenchymal stromal cells. However, there were no studies exploring the degradation of hydroxyapatite-collagen I scaffolds and the function of Wnt/β-catenin pathway in the process. In this study, tissue-engineered laminae (TEL) were constructed by nanohydroxyapatite/collagen I scaffolds and umbilical cord Wharton’s Jelly mesenchymal stromal cells (WJ-MSCs). Cell attachment was observed by scanning electron microscopy, and cell viability was confirmed by Live/Dead staining. The rat models were randomly divided into control and β-catenin inhibition groups. Vertebral lamina defect rat models were made on the fifth lumbar vertebrate, and TEL was implanted into the defect site. After 14 weeks, the newborn laminae were harvested for microcomputed tomography, histology, or transcriptional profile analysis. We found that, for the control group, the newborn lamina formation matched with the scaffold degradation and complete newborn laminae formed at the 14th week; for the β-catenin inhibition group, the scaffold degradation rate overrated the lamina formation and no complete artificial laminae were formed at the 14th week. In addition, the osteoclastic genes, such as Cathepsin K or RANKL, in the control groups were significantly lower than the β-catenin inhibition group, and the antiosteoclastic gene, OPG, in the control group was significantly higher than the β-catenin inhibition group. In conclusion, inhibition of Wnt/β-catenin pathway led to speedy scaffold degradation and deferred artificial lamina formation. Wnt/β-catenin pathway played a critical role in maintaining the balance between scaffold degradation and bone formation in the process of vertebral lamina reconstruction.

Training self-other distinction facilitates perspective taking in young children

Adults and children sometimes commit ‘egocentric errors’, failing to ignore their own perspective, when interpreting others’ communication. Training imitation-inhibition reduces these errors in adults, facilitating perspective-taking. This study tested whether imitation-inhibition training may also facilitate perspective-taking in 3-6-year-olds, an age where the egocentric perspective may be particularly influential. Children participated in a 10-minute imitation-inhibition, imitation, or non-social-inhibition training (white, n=25 per condition, 33 female), and subsequently the communicative-perspective-taking Director task. Training had a significant effect (F(2, 71)= 3.268, p= .044, η2= .084): on critical trials the imitation-inhibition group selected the correct object more often than the imitation and non-social-inhibition training groups. The imitation-inhibition training thus specifically enhanced the perspective-taking process, indicating that perspective-taking from childhood onwards involves managing self-other representations.

In Vivo Inhibition of MicroRNA-326 in a NOD.H-2h4 Mouse Model of Autoimmune Thyroiditis

BackgroundPrevious studies reported that various miRNAs participate in autoimmune diseases, but the potential regulatory mechanism of miRNAs in autoimmune thyroiditis (AIT) needs further exploration.ObjectiveThis study aimed to further verify that miR-326 contributes to AIT by regulating Th17/Treg balance through Ets-1 using lentiviral gene delivery through tail vein and thyroid injection in NOD.H-2h4 mice.Materials and MethodsFive-week-old NOD.H-2h4 mice were divided randomly into tail vein and thyroid injection groups, and each received either mmu-miR-326 sponge (LV-sponge) or lentiviral vector control. Mice were divided for tail vein injection: the therapeutic LV-ctrl, therapeutic LV-sponge, prophylactic LV-ctrl, and prophylactic LV-sponge groups. The control group was fed high-iodine water without vein injection. The thyroid infiltration of lymphocytes and serum TgAb value were investigated by thyroid hematoxylin and eosin (HE) staining and ELISA, respectively. Ets-1 and lymphocyte counts were measured by RT-PCR, western blotting, and flow cytometry. The thyroid CD4+IL-17a+ cells and CD4+Ets-1+ cells were detected by immunofluorescence, and the serum cytokines were tested by ELISA.ResultsIn the tail vein injection groups, the thyroid inflammatory score and serum TgAb titer were significantly lower in the LV-sponge groups than in the control and LV-ctrl groups while Ets-1 protein expression in mouse spleens was increased in the LV-sponge groups. Moreover, Th17/Treg ratio declined in the LV-sponge group and decreased significantly in the prophylactic LV-sponge group (P = 0.036) tested by flow cytometry. Immunofluorescence showed that, in LV-sponge groups, CD4+IL-17a+ cells were decreased significantly (P = 0.001), while CD4+Ets-1+ cells were increased significantly in the LV-sponge group (P = 0.029). The serum IL-17/IL-10 was decreased significantly in the LV-sponge group (P &lt; 0.05). In the thyroid injection groups, the thyroid inflammatory score and serum TgAb titer in the LV-sponge group decreased significantly compared with those in the LV-ctrl group (P &lt; 0.05). In addition, in LV-sponge groups, CD4+IL-17a+ cells were decreased, while CD4+Ets-1+ cells were increased significantly in the inhibition group evaluated by immunofluorescence. Moreover, tail vein injection of LV-sponge resulted in much lower TgAb levels in thyroiditis compared with thyroid injection.ConclusionMiR-326 targeted therapy may be a promising approach for AIT. In addition, tail vein injection may achieve a better intervention effect than thyroid injection.

PSD-95 protects the pancreas against pathological damage through p38 MAPK signaling pathway in acute pancreatitis

Acute pancreatitis is one of the leading causes of gastrointestinal disorder-related hospitalizations, yet its pathogenesis remains to be fully elucidated. Postsynaptic density protein-95 (PSD-95) is closely associated with tissue inflammation and injury. We aimed to investigate the expression of PSD-95 in pancreatic acinar cells, and its function in regulating the inflammatory response and pancreatic pathological damage in acute pancreatitis. A mouse model of edematous acute pancreatitis was induced with caerulein and lipopolysaccharide in C57BL/6 mice. Tat-N-dimer was injected to inhibit the PSD-95 activity separately, or simultaneously with SB203580, inhibitor of p38 MAPK phosphorylation. Rat pancreatic acinar cells AR42J were cultured with 1 μM caerulein to build a cell model of acute pancreatitis. PSD-95-knockdown and negative control cell lines were constructed by lentiviral transfection of AR42J cells. Paraffin-embedded pancreatic tissue samples were processed for routine HE staining to evaluate the pathological changes of human and mouse pancreatic tissues. Serum amylase and inflammatory cytokine levels were detected with specific ELISA kits. Immunofluorescence, immunohistochemical, Western-blot, and qRT-PCR were used to detect the expression levels of PSD-95, p38, and phosphorylated p38. Our findings showed that PSD-95 is expressed in the pancreatic tissues of humans, C57BL/6 mice, and AR42J cells, primarily in the cytoplasm. PSD-95 expression increased at 2 h, reaching the peak at 6 h in mice and 12 h in AR42J cells. IL-6, IL-8, and TNF-α increased within 2 h of disease induction. The pancreatic histopathologic score was greater in the PSD-95 inhibition group compared with the control ( P < 0.05), while it was lesser when phosphorylation of p38 MAPK was inhibited compared with the PSD-95 inhibition group ( P < 0.05). Moreover, phosphorylation of p38 MAPK increased statistically after PSD-95 knocked-down. In conclusion, PSD-95 effectively influences the pathological damage of the pancreas in acute pancreatitis by affecting the phosphorylation of p38 MAPK.

Mir-23b down-regulates the expression of target gene of acetaldehyde dehydrogenase 1a1 and increases the sensitivity of cervical cancer stem cells to cisplatin

Purpose: To study the effect of miR-23b on the expression of the target gene of acetaldehyde dehydrogenase 1A1 (ALDH1A1), and cisplatin (CDDP) susceptibility of cervical carcinoma stem cells. Methods: Human cervical cancer cell line Hela cells were cultured in vitro, and miR-23b mimic and negative control were transfected into the cells using lipofectamine method. The growth of the two groups of cells was determined using growth curve method, and their proliferation measured using plate clone formation. The influence of treatments on the sensitivity of the cells to CDDP was assayed using MTT method. The mRNA expression of ALDH1A1 in Hela cells was assayed using real-time quantitative polymerase hain reation (PCR), while its protein expression was assayed by Western blot. Results: The levels of expressions of ALDH1A1 protein and mRNA in the miR-23b overexpression group were significantly lower than those in the control group (p < 0.05). The sensitivities of Hela cells to CDDP in the ALDH1A1 inhibition group and the control group were dose-dependent to some extent, but cell inhibition in ALDH1A1 inhibition group markedly increased, relative to control when the CDDP dose was 0.1 ppc (p < 0.01). Conclusion: Up-regulating the expression of miR-23b significantly inhibits the growth and proliferation of cervical cancer cells, and increases their sensitivity to CDDP via down-regulation of the expression of the target gene for ALDH1A1. Therefore, during cervical carcinoma treatment, increasing the level of miR-23b may produce a chemotherapeutic effect. Keywords: MiR-23b, Acetaldehyde dehydrogenase 1A1, Cervical cancer, Cisplatin, Sensitivity

Not context monitoring but inhibition plays a privileged role in childhood cognitive control

Childhood cognitive control is an important predictor for positive development, yet interventions seeking to improve it have provided mixed results. This is partly due to lack of clarity surrounding mechanisms of cognitive control, notably the role of inhibition and context monitoring. Here we use a randomized controlled trial to causally test the contributions of inhibition and context monitoring to cognitive control in childhood. Sixty children aged 6 to 9-years were assigned to three groups training either inhibition, context monitoring group or response speed using a gamified, highly variable and maximally adaptive training protocol. Whereas all children improved in the targeted cognitive functions over the course of training, pre-post data show that only the inhibition group improved on cognitive control. These data support a privileged role of inhibition in cognitive control during childhood. Further, gamified and maximally adaptive interventions hold promise for improving cognitive control at developmental periods of heightened plasticity.

Ascorbic acid improves extrapyramidal syndromes and corpus striatal degeneration induced by dopamine-2 receptor inhibition in Wistar rats

ObjectivesThe prolonged uses of fourth-generation antipsychotics have been implicated in inducing extrapyramidal syndromes characterized by the motor deficit. This was attributed to the loss of dopamine-2 receptor (D2R) signaling. However, ascorbic acid (SVCT2R stimulation) in the brain is proposed to modulate D2R activity. We, therefore, investigated the beneficial roles of ascorbic acid in improving the extrapyramidal symptoms seen in D2R loss.MethodsTwenty adult male Wistar rats of average weight 200 g were distributed randomly into four groups. The control (NS) received normal saline for 28 days, Untreated D2R inhibition group (−D2R) received normal saline for seven days and then subsequently received chlorpromazine for 21 days, D2R inhibition group treated with ascorbic acid (−D2R+SVCT2R) received chlorpromazine for 21 days and was subsequently treated with ascorbate for seven days while the withdrawal group (WG) received chlorpromazine for 21 days and subsequently received normal saline for seven days. Motor deficits were assessed using a rotarod and cylinder test. The corpus striatum was harvested, processed, and stained using H&E and Nissl stains. Cellular density was analyzed using Image J software 1.8.0.ResultsMotor deficit was observed in −D2R animals administered chlorpromazine with less improvement in WG compared to control (p<0.05) in both rotarod and cylinder test. Ascorbic acid (SVCT2R stimulation) significantly (p<0.001) improved the latency of fall and climbing attempts observed in −D2R animals. The density of basophilic trigoid bodies was significantly (p<0.001) restored in −D2R+SVCT2R group, suggesting recovery of neural activity in the corpus striatum. Moreover, the hallmarks of neuronal degeneration were less expressed in the ascorbic acid treatment groups.ConclusionsAscorbic acid putatively ameliorates extrapyramidal symptoms observed in D2R blockage by chlorpromazine in Wistar rats.