Exosomes derived from M2-type microglia ameliorate oxygen-glucose deprivation/reoxygenation-induced HT22 cell injury by regulating miR-124-3p/NCOA4-mediated ferroptosis.

Background: Although it has been reported that miRNA carried by M2 microglial exosomes protects neurons from ischemia-reperfusion brain injury, the mechanism of action remains poorly understood. This study aimed to explore the miRNA signaling pathway by which M2-type microglia-derived exosomes (M2-exosomes) ameliorate oxygen-glucose deprivation/reoxygenation (OGD/R)-induced cytotoxicity in HT22 cells. Methods: BV2 microglia were induced by M2 polarization. Then, M2-exosomes were identified via transmission electron microscopy and special biomarker detection and co-cultured with HT22 cells. Cell proliferation was evaluated using the Cell Counting Kit-8 (CCK-8) assay. Intracellular concentrations of reactive oxygen species (ROS), Fe2+, glutathione (GSH), and malondialdehyde (MDA) were determined using dichlorofluorescein fluorescence and biochemical determination. miR-124-3p levels were determined using qRT-PCR, and protein expressions were examined via western blotting. Results: OGD/R suppressed the proliferation and induced the accumulation of Fe2+, ROS, and MDA and reduction of GSH in mouse HT22 cells, suggesting ferroptosis of HT22 cells. OGD/R-induced changes in the above mentioned indexes was ameliorated by M2-exosomes but restored by the exosome inhibitor GW4869. M2-exosomes with (mimic-exo) or without miR-124-3p (inhibitor-exo) promoted and suppressed proliferation and ferroptosis-associated indexes of HT22 cells, respectively. Moreover, mimic-exo and inhibitor-exo inhibited and enhanced NCOA4 expression in HT22 cells, respectively. NCOA4 overexpression reversed the protective effects of miR-124-3p mimic-exo in OGD/R-conditioned cells. NCOA4 was targeted and regulated by miR-124-3p. Conclusions: M2-exosome protects HT22 cells against OGD/R-induced ferroptosis injury by transferring miR-124-3p and NCOA4 into HT22 cells, with the latter being a target gene for miR-124-3p.

MicroRNA-379-5p targets MAP3K2 to reduce autophagy and alleviate neuronal injury following cerebral ischemia via the JNK/c-Jun signaling pathway.

MicroRNAs (miRNAs) are abundant in neurons and play key roles in the function and development of the nervous system. This study focuses on the function of miR-379-5p in neurological function recovery during ischemic stroke. The expression of miR-379-5p in the serum of patients with ischemic stroke was determined. Human cerebral cortical neuron cells (HCN-2) were subjected to oxygen/glucose deprivation (OGD) to mimic an ischemic stroke in vitro, whereas mice subjected to middle cerebral artery occlusion (MCAO) were used as an animal model. The serum of patients with ischemic stroke and OGD-treated HCN-2 cells displayed a poor expression of miR-379-5p. Upregulation of miR-379-5p reduced the OGD-induced cell damage and decreased the expression of the autophagy marker protein Beclin1 in cells. Rapamycin, an autophagy activator, blocked the protective functions of miR-379-5p. Further, miR-379-5p directly bound to MAP3K2. MAP3K2 activated the JNK/c-Jun signaling pathway and suppressed the neuroprotective events mediated by miR-379-5p. The in vitro results were reproduced in vivo, where upregulation of miR-379-5p reduced neurological impairment and infarct size in MCAO-induced mice. This study suggested that miR-379-5p showed a neuroprotective effect on ischemic stroke and reduced autophagy of neurons through the suppression of MAP3K2 and the JNK/c-Jun axis.

Ibrutinib ameliorates cerebral ischemia/reperfusion injury through autophagy activation and PI3K/Akt/mTOR signaling pathway in diabetic mice.

Bruton's tyrosine kinase (BTK) is involved in the diabetogenic process and cerebral ischemic injury. However, it remained unclear whether BTK inhibition has remedial effects on ischemia/reperfusion (I/R) injury complicated with diabetes. We aim to investigate the regulatory role and potential mechanism of ibrutinib, a selective inhibitor of BTK, in cerebral I/R injured diabetic mice. The cytotoxicity and cell vitality tests were performed to evaluate the toxic and protective effects of ibrutinib at different incubating concentrations on normal PC12 cells or which were exposed to high glucose for 24 h, followed by hypoxia and reoxygenation (H/R), respectively. Streptozotocin (STZ) stimulation-induced diabetic mice were subjected to 1 h ischemia and then reperfusion. Then the diabetic mice received different dosages of ibrutinib or vehicle immediately and 24 h after the middle cerebral artery occlusion (MCAO). The behavioral, histopathological, and molecular biological tests were then performed to demonstrate the neuroprotective effects and mechanism in I/R injured diabetic mice. Consequently, Ibrutinib improved the decreased cell viability and attenuated oxidative stress in the high glucose incubated PC12 cells which subjected to H/R injury. In the I/R injured diabetic mice, ibrutinib reduced the cerebral infarct volume, improved neurological deficits, ameliorated pathological changes, and improved autophagy in a slightly dose-dependent manner. Furthermore, the expression of PI3K/AKT/mTOR pathway-related proteins were significantly upregulated by ibrutinib treatment. In summary, our finding collectively demonstrated that Ibrutinib could effectively ameliorate cerebral ischemia/reperfusion injury via ameliorating inflammatory response, oxidative stress, and improving autophagy through PI3K/Akt/mTOR signaling pathway in diabetic mice.

Human Saliva Metabolome for Oral Lichen Planus Biomarker Identification.

BACKGROUND: Oral Lichen Planus (OLP) is one of the most common oral mucosal diseases. However, the current diagnostic method for OLP has limitations, and sometimes it is easy to be misdiagnosed. Salivary metabolomics may provide new ideas for the diagnosis of OLP. OBJECTIVE: To identify the biomarkers for the early detection of OLP. METHODS: A non-targeted metabolomic analysis method was established based on UHPLC-Q-Orbitrap HRMS (Ultra-performance liquid chromatography-quadrupole/orbitrap high resolution mass spectrometry) to analyze the differential metabolites in saliva samples of patients with OLP and healthy subjects. Saliva samples were collected from 120 OLP patients and 125 healthy subjects. RESULTS: A total of 19 differential metabolites were identified, including 6 amino acid metabolites, 2 carnitines, 2 lipid metabolites and 9 other metabolites. The integrated biomarkers were constructed by 3 metabolites according to Receiver Operating Characteristic (ROC). Meanwhile, multiple metabolic pathways were found to be involved in the occurrence and development of OLP. CONCLUSION: Metabolomics can be used to characterize the characteristics of metabolic disorders in patients with OLP, which is also helpful to the early diagnosis of OLP and reveal the pathological process of OLP.

The protective effects of curcumin in cerebral ischemia and reperfusion injury through PKC-θ signaling.

Ischemic stroke is a common cerebrovascular disease with the main cause considered to be cerebral ischemia and reperfusion (I/R), which exerts irreparable injury on nerve cells. Thus, the development of neuroprotective drugs is an urgent concern. Curcumin, a known antioxidant, has been found to have neuroprotective effects. To determine the protective mechanism of curcumin in ischemic stroke, oxygen and glucose deprivation/reoxygenation (OGD/R) was used to treat PC12 cells to mimic the cerebral I/R cell model. Curcumin (20 µM) was applied to OGD/R PC12 cells, followed by Ca2+ concentration, transepithelial electrical resistance (TEER), and cell permeability measurements. The results showed that OGD/R injury induced a decrease in TEER and increases in Ca2+ concentration and cell permeability. In contrast, curcumin alleviated these effects. The protein kinase C θ (PKC-θ) was associated with the protective function of curcumin in the OGD/R cell model. Moreover, the middle cerebral artery occlusion and reperfusion model (MCAO/R) was applied to simulate the I/R rat model. Our results demonstrated that curcumin could reverse the MCAO/R-induced increase in Ca2+ concentration and blood-brain barrier (BBB) disruption. Our study demonstrates the mechanisms by which curcumin exhibited a protective function against cerebral I/R through PKC-θ signaling by reducing BBB dysfunction.

Gingival crevicular fluid levels of SLIT3 are increased in periodontal disease.

This study aims to investigate the levels of SLIT3 in gingival crevicular fluid (GCF) of healthy and periodontal disease subjects, and their correlations to periodontal disease. A total of 45 periodontal patients and 45 periodontally healthy volunteers were enrolled. The clinical parameters, radiographic bone loss and the levels of SLIT3, receptor activator of NF-κB ligand (RANKL) and osteoprotegerin (OPG) in GCF were measured. The prevalences of Porphyromonas gingivalis, Treponema denticola, and Tannerella forsythia in subgingival plaque were also analyzed. The expression of SLIT3 and RANKL was detected in the periodontium of experimental periodontitis in rats and lipopolysaccharide (LPS)-induced mouse macrophage. The total amounts and concentrations of SLIT3 and RANKL were significantly higher in periodontitis than those in healthy, while the level of OPG was significantly lower (p < .05). Significant positive correlations were observed between the level of GCF SLIT3 and clinical attachment level and radiographic bone loss (p < .05). There existed a significant positive correlation between SLIT3 and RANKL (p < .05). Increased expression of SLIT3 and RANKL was observed in the periodontium of periodontal rats. SLIT3 expression was induced by LPS stimulation in macrophages. These results suggest that SLIT3 may act as a diagnostic indicator of periodontal disease and should be further investigated.

Effect of dyslipidemia on intima-media thickness of intra- and extracranial atherosclerosis by regulating the expression of hsp70 in rabbits.

The aim of this study was to explore the effect of dyslipidemia on intima-media thickness (IMT) of Intra- and extracranial atherosclerosis by regulating the expression of heat shock protein 70 (HSP70) in rabbits. Twenty-seven male white rabbits were randomly divided into normal control group A, high fat group B and high fat + endothelial injury operation group C (each group was 9), we measured lipids and obtained tissues from different cerebral arteries including Bilateral common carotid artery (CCA), Internal carotid artery (ICA), middle cerebral artery (MCA) and vertebral artery (VA). Pathological analysis were done, western blot analysis was used to detect the expression of HSP70 in CCA and MCA. The Serum lipid levels were overall significantly increased at 12(th) week in Group B and Group C compared to normal control (P < 0.05); at 12(th) week, the IMT of CCA and MCA in group B and C were showed significant increment compared with Group A; the correlation between HDL/CHOL/LDL and IMT of different cerebral arteries are as follows: MCA > ICA > CCA > VA; between TG and IMT of different cerebral arteries: VA > ICA > MCA > CCA; the expression of HSP70 from MCA were increased compared with CCA in group B and group C (P < 0.05). Significant positive correlations were observed between hyperlipidemia and different cerebral arteries. Hyperlipidemia has more impact on IMT of intracranial cerebral arteries. The expression of HSP70 from intracranial cerebral arteries is significantly increased. The mechanisms underlied was speculated that might be involved in inhibiting the inflammatory via HSP70.

Focal adhesion kinase activation is required for TNF-α-induced production of matrix metalloproteinase-2 and proinflammatory cytokines in cultured human periodontal ligament fibroblasts.

Since focal adhesion kinase (FAK) was proposed as a mediator of the inflammatory response, we have investigated the role of this molecule in the release of inflammatory cytokines by cultured human periodontal ligament fibroblasts (HPDLFs), cells that are thought to be important in the patient's response to periodontal infection. Human periodontal ligament fibroblasts were stimulated by tumor necrosis factor alpha (TNF-α) and its effects on interleukin (IL)-6 and IL-8 release were measured by ELISA. Expression of matrix metalloproteinase 2 (MMP-2) protein was analysed by western blotting. The levels of IL6, IL8, and MMP2 mRNA were evaluated by real-time PCR. Tumor necrosis factor alpha dose-dependently induced the phosphorylation of FAK, whereas small interfering FAK (siFAK) inhibited TNF-α-induced FAK phosphorylation. Tumor necrosis factor alpha also stimulated the production of IL-6, IL-8, and MMP-2 in a dose-dependent manner. Knockdown of FAK significantly suppressed TNF-α-induced expression of IL6 and IL8 mRNA and release of IL-6 and IL-8 protein in HPDLFs. Similarly, MMP-2 down-regulation was significantly prevented by siFAK. Our results strongly suggest that knockdown of FAK can decrease the production of TNF-α-induced IL-6, IL-8, and MMP-2 in HPDLFs. These effects may help in understanding the mechanisms that control expression of inflammatory cytokines in the pathogenesis of periodontitis.