Regulation of TREM2 on BV2 inflammation through PI3K/AKT/mTOR pathway.

This work sought to determine how lipopolysaccharide (LPS)-induced pro-inflammatory factor production in BV2 microglia was influenced by myeloid cell 2 (TREM2) expressions. LPS (0.1, 1, and 10 µg/mL) induced inflammation in BV2 cells, MTT and QPCR were used to detect the occurrence of inflammation; TREM2 activation and inhibition vectors were used to activate and inhibit TREM2; Cell Proliferation was detected using CCK-8 and cell cloning experiments. LY294002 was used to inhibit the activity of PI3K/AKT signal pathway; Western blot and ELISA were used to detect cell polarization and signal pathway changes. CCK-8 and cell clone experiments found that the activation of TERM2 can promote the proliferation of BV2 cells; and the activation of TERM2 can promote the expression of IL6, IL1ß, TNFα and the expression of M2 cell phenotype molecules Arg-1 and CD206. The effect of adding LY294002 signaling pathway by TERM2 activation was inhibited, indicating that TERM2 can affect the occurrence of inflammation by regulating the activity of PI3K/AKT signaling pathway. Finally, Western blotting and ELISA showed that activation of TERM2 can promote the expression of Arg-1 and CD206 in BV2 cells, and promote the transformation of BV2 cells to M2 polarization. TERM2 can affect the inflammatory response in microglia through the PI3K/AKT signaling pathway, suggesting that TERM2 may be a target for the treatment of inflammatory response in glial cells. This study provides a treatment plan for alleviating the impact of inflammation on central nervous system.

miRNA-192-5p targets Dyrk1a to attenuate cerebral injury in MCAO mice by suppressing neuronal apoptosis and neuroinflammation.

INTRODUCTION: Ischemic stroke (IS) is a leading cause of disability and mortality worldwide. Several studies have demonstrated the involvement of microRNAs (miRNAs) in brain diseases. miRNA-192-5p is a regulatory molecule in neurodegenerative diseases and its expression was found to be significantly downregulated in the whole blood samples of IS patients, but the specific role of miRNA-192-5p in IS not fully understood. Here, we investigated the role of miRNA-192-5p in a murine model of acute cerebral injury after IS. MATERIAL AND METHODS: Male C57BL/6J mice received an intracerebroventricular (i.c.v.) injection of agomir-192-5p or antagomir-192-5p 2 h before middle cerebral artery occlusion (MCAO). Infarct volume was assessed by 2,3,5 triphenyltetrazolium chloride (TTC) staining. Brain slices were subjected to Fluoro-Jade B, TUNEL, and immunofluorescence stainings. Contents of pro-inflammatory cytokines (TNF-α, IL-1ß, and IL-6) were measured using enzyme-linked immunosorbent assay (ELISA) kits. In vitro, murine microglial BV-2 cells were subjected to oxygen-glucose deprivation (OGD), and the contents of pro-inflammatory cytokines were measured in cell lysates. RESULTS: miRNA-192-5p was downregulated in the ischemic penumbra of the cerebral cortex. Pretreatment with agomir-192-5p attenuated neurological deficits and reduced cerebral edema and infarct volume in MCAO mice. Agomir-192-5p-treated animals had fewer degenerating and apoptotic neurons in the ischemic penumbra. Additionally, agomir-192-5p significantly suppressed neuroinflammation as evidenced by decreased immunostaining for GFAP and Iba1 and decreased levels of pro-inflammatory cytokines. Antagomir-192-5p pretreatment showed the opposite effect. Furthermore, dual specificity tyrosine phosphorylation regulated kinase 1A (Dyrk1a) was identified as a target gene of miRNA-192-5p, and the elevated Dyrk1a expression in the ischemic penumbra was markedly reduced by agomir-192-5p. Dyrk1a overexpression in BV-2 microglial cells impaired miRNA-192-5p-mediated inhibition of OGD-induced activation of BV-2 microglial cells. Opposite results were obtained using miRNA-192-5p inhibitor and Dyrk1a siRNA. CONCLUSIONS: We found that intracerebroventricular administration of miRNA-192-5p before MCAO attenuatedacute cerebral injury by suppressing neuronal apoptosis and neuroinflammation in mice, and these protective effects might be mediated by downregulation of Dyrk1a. This study would help identify novel therapeutic targets for IS.

Genipin protects against mitochondrial damage of the retinal pigment epithelium under hyperglycemia through the AKT pathway mediated by the miR-4429/JAK2 signaling axis.

Background: To investigate the protective effect and mechanism of genipin (GE) on mitochondrial damage in retinal pigment epithelial (RPE) cells induced by high glucose. Methods: Differential genes of GE in the treatment of diabetic retinopathy (DR) were screened by the Gene Expression Omnibus (GEO) database. Differential genes located in the AKT pathway were obtained. TargetScan, miRDB, and DIANA databases were used to predict the targeted microRNAs (miRNAs) of differential genes. A high-fat diet combined with streptomycin (STZ) intraperitoneal injection were used to establish a diabetic mouse model. Diabetic mice were treated with GE by intragastric administration. The functional and molecular changes of the retina were detected by electroretinogram (ERG) and reverse transcription-polymerase chain reaction (RT-PCR). ARPE-19 cells were cultured under hyperglycemic conditions with AKT and JAK2 inhibitors. MiR-4429 was knocked down/overexpressed to detect changes in cell function, activity, and mitochondrial function. The dual luciferase reporter assay confirmed the targeted binding of miR-4429 with JAK2. Results: Bioinformatics analysis finally yielded JAK2 as the research target gene. miR-4429 was predicted to be the targeted miRNA of JAK2 by online databases. The results of animal experiments showed that the retinal function of mice recovered after GE administration (P<0.05), the expression of AKT and miR-4429 in RPE cells was significantly increased (P<0.05), and the expression of JAK2 was significantly decreased (P<0.05). The results of cell experiments showed that the functions of cells and mitochondria recovered after the addition of GE under hyperglycemia (P<0.05). Cell and mitochondrial functions were decreased after the addition of AKT inhibitor (P<0.05). Overexpression of miR-4429 or inhibition of JAK2 increased cell activity and mitochondrial function (P<0.05). The results of the dual luciferase reporter assay showed that miR-4429 had a targeted binding site with JAK2. Conclusions: GE protects ARPE-19 cell functional activity, inflammatory responses, and mitochondrial damage by promoting the AKT signaling pathway and regulating the expression of the miR-4429/JAK2 signaling axis.

Ferrostatin-1 Polarizes Microglial Cells Toward M2 Phenotype to Alleviate Inflammation After Intracerebral Hemorrhage.

BACKGROUND: Intracerebral hemorrhage (ICH) is one of the most lethal stroke types and lacks effective therapeutic regimens. Recently, evidence has suggested the involvement of the ferroptosis inhibitor ferrostatin-1 (Fer-1) in the pathophysiological process of ICH. In this study, we examined the underlying mechanism. METHODS: We induced an in vitro apoptosis model in organotypic hippocampal slice (OHS) using hemoglobin (Hb) and an in vivo ICH model using collagenase. OHSs were treated with MK-801, Fer-1, glutamate, and Hb to assess the impacts of Fer-1 on neuron apoptosis, glutathione peroxidase-4 activity, reactive oxygen species production, inflammation-related factors, expression of M1 markers and M2 markers, and the phagocytic function of microglial cells in vitro. Then, ICH mice were treated with Fer-1 and ruxolitinib to evaluate the effects of Fer-1-orchestrating janus kinase 1/signal transducer and activator of transcription 6 pathway on neurological function, brain water content, hematoma volume, the anti-inflammatory factor, M1 and M2 markers, and the phagocytic function of microglial cells in vivo. RESULTS: Hb or glutamate facilitated glutathione peroxidase dysfunction, reactive oxygen species production, and neuronal apoptosis in OHSs, which was nullified by Fer-1. Fer-1 polarized microglial cells to the M2 phenotype, enhanced their phagocytic function, and prevented inflammation in Hb-induced OHSs. In the ICH mouse model, Fer-1 was found to improve neurological function and promote hematoma absorption. In addition, Fer-1 activated the Fer-1-orchestrating janus kinase 1/signal transducer and activator of transcription 6 pathway, which accelerated microglial M2 polarization, enhanced the phagocytic function of microglial cells, and restrained inflammation in ICH mice. CONCLUSIONS: Overall, our findings suggest that Fer-1 may be a novel mechanism underlying microglial M2 polarization and inflammation after ICH.

Milrinone Ameliorates the Neuroinflammation and Memory Function of Alzheimer's Disease in an APP/PS1 Mouse Model.

PURPOSE: Alzheimer's disease (AD) is a complex neurodegenerative disorder, which is characterized by memory loss and cognitive deficits. The neuroprotective role of milrinone on the injury of spinal cord or cerebral ischemia-reperfusion has been confirmed. However, the accurate function of milrinone on AD pathogeny is still unclear. METHODS: APP/PS1 transgenic mouse was used to explore the role of milrinone in behaviour tests, and the effects on histopathologic features of AD such as the formation of neuronal amyloid-ß (Aß) plaque, microglial activation, tau protein hyperphosphorylation, oxidative stress, and neuroinflammation. Lipopolysaccharide (LPS)/Aß-treated BV-2 cells were used to understand the anti-inflammation mechanism of milrinone on AD in vitro. RESULTS: Our in vivo results showed that milrinone ameliorates the memory functions of AD mice. Meanwhile, milrinone reduced Aß deposits, repressed microglial activation and tau protein hyperphosphorylation, attenuated the oxidative stress, and decreased the levels of inflammatory cytokines. The in vitro results demonstrated that milrinone could inhibit the secretion of interleukin (IL)-1ß, IL-6, and tumor necrosis factor (TNF)-α via regulation of NLRP3 inflammasomes and TLR4/MyD88/NF-κB signalling pathway. CONCLUSION: Overall, milrinone could ameliorate the memory loss and cognitive deficits through repressing the multiple pathological processes of AD, suggesting that milrinone may be an underlying and effective drug for treating AD clinically.