Effect of Oxygen-Glucose Deprivation of Microglia-Derived Exosomes on Hippocampal Neurons: A Study on miR-124 and Inflammatory Cytokines.

Stroke is a cerebrovascular disease that threatens human health. Developing safe and effective drugs and finding therapeutic targets has become an urgent scientific problem. The aim of this study was to investigate the effect of oxygen-glucose deprivation of the microglia-derived exosome on hippocampal neurons and its relationship to miR-124 in the exosome. We incubated hippocampal neurons with exosomes secreted by oxygen-glucose deprivation/ reoxygenation (OGD/R) microglia. The levels of glutamic acid (GLU) and gamma-aminobutyric acid (GABA) in the culture supernatant were detected by ELISA. CCK-8 was used to measure neuronal survival rates. The mRNA levels of TNF-α and IL-6 were detected by RT-qPCR to evaluate the effect of exosomes on neurons. RT-qPCR was then used to detect miR-124 in microglia and their secreted exosomes. Finally, potential targets of miR-124 were analyzed through database retrieval, gene detection with dual luciferase reporters, and western blotting experiments. The results showed that the contents of GLU, TNF-α and IL-6 mRNA increased in the supernatant of cultured hippocampal neurons, the content of GABA decreased, and the survival rate of neurons decreased. Oxygen-glucose deprivation increases miR-124 levels in microglia and their released exosomes. miR-124 acts as a target gene on cytokine signaling suppressor molecule 1(SOCS1), while miR-124 inhibitors reduce the expression of TNF-α and IL-6 mRNA in neurons. These results suggest that oxygen- and glucose-deprived microglia regulate inflammatory cytokines leading to reduced neuronal survival, which may be achieved by miR-124 using SOCS1 as a potential target.

VEGF overexpression in transplanted NSCs promote recovery of neurological function in rats with cerebral ischemia by modulating the Wnt signal transduction pathway.

Neural stem cell transplantation is a good method to treat stroke, but the mechanism is still unclear. Therefore, this study aims to explore the regulatory mechanism of VEGF overexpression in transplanted NSCs to promote the recovery of neural function in ischemic rats by regulating Wnt signal transduction pathways. We amplified VEGF gene fragments by PCR and transfected them into NSCs with Ad5 adenovirus. Rat brain IRI model was established by MCAO method, and VEGF transfected NSCs (VEGF-NSCs) were transplanted 24 h after successful IRI model. One week after the transplant, cognitive function was assessed using a neurological deficit score; Brain injury was assessed by histopathology; Photochemical and ELISA methods were used to detect oxidative stress markers and inflammatory factors, respectively. Western blotting has been detected in molecules of the Wnt signaling pathway. The results showed that the transduced NSCs express VEGF at least for 14 days. VEGF-NSCs transplantation (VNT) improved spatial learning and memory in rats, and inhibited oxidative stress injury, inflammatory response, and histopathological injury. VNT also resulted in significant changes in the phosphorylation levels of ß-catenin and GSK-3ß proteins, ultimately triggering activation of the Wnt signal transduction pathway. These results suggest that the neuroprotective effects of VNT may be related to the regulation of the Wnt signal transduction pathway.

Neural Stem Cell-Derived Exosomes Improve Neurological Function in Rats with Cerebral Ischemia-Reperfusion Injury by Regulating Microglia-Mediated Inflammatory Response.

Purpose: To investigate the effect of neural stem cell-derived exosomes (NSC-Exos) on neural function after rat cerebral ischemia-reperfusion injury by regulating microglia-mediated inflammatory response. Methods: SD rats were randomly divided into Sham group, IRI group, PBS group and NSC-Exos group. Each group was divided into 1d, 3d, 7d and 14d subgroups. In the Sham group, only cervical vessels were isolated without blockage. MCAO model was constructed in the other three groups by blocking middle cerebral artery with thread embolism. PBS group and NSC-Exos group were, respectively, injected into the lateral ventricle of PBS and Exos. Neurobehavioral deficit scores were performed for each subgroup at relative time points, then brains were taken for TTC staining, parietal cortex histopathology and microglia-mediated inflammatory response-related factors were detected. Results: Compared with Sham group, neurological defect score and infarction volume in both the IRI and PBS groups were significantly increased. The exploration target quadrant time and escape latency time of maze test were increased. The number of CD86+/Iba1+ double-positive cells increased, while CD206+/Iba1+ double-positive cells decreased. The expressions of IL-6 and CD86 in parietal cortex were increased, while the expressions of Arg1 and CD206 were decreased. Compared with the IRI group and PBS group, neurological defect score and infarction volume in NSC-Exos group were decreased. The exploration target quadrant time and escape latency time of water maze test were decreased. The number of CD206+/Iba1+ double-positive cells increased, while CD86+/Iba1+ double-positive cells decreased. The expressions of Arg1 and CD206 in parietal cortex were increased, while the expressions of IL-6 and CD86 were decreased. Conclusion: NSC-Exos can promote the polarization of microglia, that is, inhibit the polarization of M1 and promote polarization of M2, reduce microglia-mediated neuroinflammation, suggesting that NSC-Exos may be a strategy for the treatment of microglia-mediated neuroinflammation after ischemic brain injury.

Alpha-ketoglutarate up-regulates autophagic activity in peri-implant environment and enhances dental implant osseointegration in osteoporotic mice.

AIM: The osseointegration of dental implants is impaired in patients with osteoporosis, leading to significantly higher failure rates. This study set out to investigate the potential effects of alpha-ketoglutarate (α-KG) on implant osseointegration in an osteoporotic mouse model. MATERIALS AND METHODS: Female C57BL/6 mice received ovariectomy and bilateral first maxillary molar extraction at the age of 7 weeks. Dental implants were inserted 8 weeks after tooth extraction. In one of the groups, α-KG was administered via drinking water throughout the experimental period. Specimens were collected on post-implant days (PIDs) 3, 7, 14, and 21 for micro-CT, histological, and immunohistochemical analyses. At the same time, bone-marrow-derived mesenchymal stem cells (BMMSCs) treated with α-KG were interrogated for osteogenic differentiation, autophagic activity, and apoptosis. RESULTS: α-KG supplementation in drinking water resulted in enhanced dental implant osseointegration in ovariectomized mice, with up-regulated osteogenic and autophagic activity and down-regulated osteoclast differentiation and cell apoptosis. α-KG-treated BMMSCs showed enhanced activity in proliferation, survival, colony formation, and osteogenic differentiation, as well as autophagic activity. CONCLUSIONS: Systemic α-KG supplementation effectively prevents the failure of dental implant osseointegration in mice under an osteoporotic state.

Gavage-administered lactoferrin promotes palatal expansion stability in a dose-dependent manner.

OBJECTIVE: To investigate the effects of different lactoferrin concentrations on mid-palatal suture bone remodeling during palatal expansion and relapse in rats. MATERIALS AND METHODS: Thirty-two 5-week-old male Wistar rats were randomly divided into four groups: EO (expansion only), E+LF1 (expansion plus 10 mg/kg/day daily LF), E+LF2 (expansion plus 100 mg/kg/day daily LF), and E+LF3 (expansion plus 1 g/kg/day daily LF). Thereafter, micro-computed tomography and micro-morphology of the mid-palatal suture were analyzed on day 7 and day 14, respectively. RESULTS: The arch widths were increased in all the four groups after expansion, and there was no significant difference among them on day 7. After relapse, however, the arch width in the E+LF3 group was significantly larger compared with EO group. In E+LF3 group and E+LF2 group, new bone formation and osteoblast number were enhanced with up-regulated expression of osteocalcin and collagen type I, while the expression of cathepsin K-positive cells was downregulated in E+LF3 group. CONCLUSION: Lactoferrin gavage administration might increase the stability of palatal expansion and reduce relapse in a concentration-dependent manner by enhancing bone formation and inhibiting resorption. LF administration may be promising for optimizing the maxillary expansion outcome.

Lithium chloride promotes osteogenesis and suppresses apoptosis during orthodontic tooth movement in osteoporotic model via regulating autophagy.

Osteoporosis is a widely distributed disease that may cause complications such as accelerated tooth movement, bone resorption, and tooth loss during orthodontic treatment. Promoting bone formation and reducing bone resorption are strategies for controlling these complications. For several decades, the autophagy inducer lithium chloride (LiCl) has been explored for bipolar . In this study, we investigated the autophagy-promoting effect of LiCl on bone remodeling under osteoporotic conditions during tooth movement. Ovariectomy was used to induce osteoporosis status in vivo. The results showed that LiCl rejuvenated autophagy, decreased apoptosis, and promoted bone formation, thus protecting tooth movement in osteoporotic mice. Furthermore, in vitro experiments showed that LiCl reversed the effects of ovariectomy on bone marrow-derived mesenchymal stem cells (BMSCs) extracted from ovariectomized mice, promoting osteogenesis and suppressing apoptosis by positively regulating autophagy. These findings suggest that LiCl can significantly decrease adverse effects of osteoporosis on bone remodeling, and that it has great potential significance in the field of bone formation during tooth movement in osteoporosis patients.

The role of EphB4/ephrinB2 signaling in root repair after orthodontically-induced root resorption.

INTRODUCTION: This study aimed to investigate the effect of EphB4/ephrinB2 signaling on orthodontically-induced root resorption repair and the possible molecular mechanism behind it. METHODS: Seventy-two 6-week-old male Wistar rats were randomly divided into 3 groups: blank control group, physiological regeneration group (PHY), and EphB4 inhibitor local injection group (INH). A root repair model was built on experimental rats of the PHY and INH groups. The animals in the INH groups received a daily periodontal local injection of EphB4 inhibitor NVP-BHG712, whereas the blank control group and PHY groups received only the vehicle. RESULTS: Histologic staining and microcomputed tomography analysis showed that root regeneration was inhibited in the INH group compared with the PHY group with a greater number of osteoclasts. Immunohistochemical staining showed active EphB4/ephrinB2 signaling activities during root regeneration. The cementogenesis-related factors cementum attachment protein, alkaline phosphatase, osteopontin, and runt-related transcription factor 2, and osteoclastic-related factors RANKL and osteoprotegerin were affected by regulated EphB4/ephrinB2 signaling. CONCLUSIONS: These findings demonstrated that the EphB4/ephrinB2 signaling might be a promising therapeutic target for novel therapeutic approaches to reduce orthodontically-induced root resorption through enhancement of cementogenesis.

Lactoferrin promotes osteogenesis of MC3T3-E1 cells induced by mechanical strain in an extracellular signal-regulated kinase 1/2-dependent manner.

INTRODUCTION: This study aimed to investigate the role of lactoferrin (LF) in the mechanical strain-induced osteogenesis of nontransformed osteoblastic cells (MC3T3-E1 cells) and related mechanism. METHODS: MC3T3-E1 cells were cultured in vitro and treated with 100 µg/mL LF, followed by a 2000 µ mechanical strain load. U0126 was used to determine the role of extracellular signal-regulated kinase 1/2 (Erk1/2). Alizarin red S staining was performed to observe the cell mineralization potential. The osteogenic results were analyzed by reverse transcription-polymerase chain reaction and western blotting. RESULTS: The expression of Col1, Alp, Ocn, Bsp, and Opn mRNA and p-Erk1/2 proteins was significantly upregulated under mechanical strain load. In addition, mineralized nodule formation was increased. After adding LF, the expression of the biomarkers and the formation of mineralized nodules were further promoted. On treatment with the Erk1/2 inhibitor U0126, the expression of Col1, Alp, and p-Erk1/2 mRNA and protein was significantly downregulated. CONCLUSIONS: These findings demonstrate that LF promotes osteogenic activity by activating osteogenesis-related biomarkers, corroborating that the effects of mechanical strain depend on Erk1/2 signaling pathway.

Long noncoding RNA expression profiles in intermittent parathyroid hormone induced cementogenesis.

The aim of this study was to explore the involvement of long noncoding RNAs (lncRNAs) during intermittent parathyroid hormone (PTH) induced cementogenesis. Expression profiles of lncRNAs and mRNAs were obtained using high-throughput microarray. Gene Ontology enrichment analysis, Kyoto Encyclopedia of Genes and Genomes pathway analysis, and coding-noncoding gene coexpression networks construction were performed. We identified 190 lncRNAs and 135 mRNAs that were differentially expressed during intermittent PTH-induced cementogenesis. In this process, the Wnt signaling pathway was negatively regulated, and eight lncRNAs were identified as possible core regulators of Wnt signaling. Based on the results of microarrray analysis, we further verified the repressed expression of Wnt signaling crucial components ß-catenin, APC and Axin2. Above all, we speculated that lncRNAs may play important roles in PTH-induced cementogenesis via the negative regulation of Wnt pathway.

Glucose-induced inhibition of seed germination in Lotus japonicus is alleviated by nitric oxide and spermine.

Seed germination is sensitive to glucose (Glc), nitric oxide (NO) and polyamine (PA). To elucidate whether cross-talk among Glc, NO and PAs occurs in mediation of seed germination, effects of Glc, NO and spermine on seed germination of Lotus japonicus were studied. Glc retarded seed germination in a concentration-dependent manner. NO donor sodium nitroprusside (SNP) alleviated Glc-induced inhibition of seed germination, whereas the NO scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethyl-imidazoline-1-oxyl-3-oxide (cPTIO) diminished the SNP-dependent alleviation of seed germination. These observations indicate that Glc may inhibit seed germination by interacting with NO signaling pathways. Exogenous spermine enhanced and the inhibitor of the spermine synthase, methylglyoxal-bis-guanyl hydrazone (MGBG), inhibited seed germination, respectively. Like SNP, spermine alleviated the Glc-induced inhibition of seed germination, whereas MGBG exaggerated the Glc-induced inhibition of seed germination. These results suggest that Glc may inhibit the spermine synthesis, leading to reductions in seed germination. NO scavenger and spermine synthase inhibitor diminished the SNP-induced alleviation of Glc-induced inhibition of seed germination. These findings reveal that both NO and spermine participate in the Glc-induced inhibition of seed germination in L. japonicus.