MSCs­derived exosomes attenuate ischemia-reperfusion brain injury and inhibit microglia apoptosis might via exosomal miR-26a-5p mediated suppression of CDK6.

BACKGROUND: This study aimed to explore the role of mesenchymal stromal cells (MSCs)-derived exosomes (MSCs-Exo) in the cerebral ischemia-reperfusion (I/R) injury. METHODS: Exosomes were isolated from MSCs of adult C57BL/6J mice by the gradient centrifugation method. The expression of miR-26a-5p and CDK6 in MSCs-Exo and mice brain tissues were evaluated by qRT-PCR and western blot. miR-26a-5p mimics and miR-NC were transfected into MSCs, and exosomes were isolated from the MSCs stably expressing miR-26a-5p. Then MSCs-Exo-miR-26a-5p mimics or MSCs-Exo-miR-NC was injected into mice through the tail vein, or added into medium to stimulate BV-2 cells. Cell viability was evaluated by CCK-8 assay. Cell apoptosis was detected by flow cytometry. The apoptosis in brain tissues was evaluated by TUNEL staining assay. Bioinformatics analysis and luciferase reporter assay were performed to determine the binding relationship between miR-26a-5p and CDK6. RESULTS: miR-26a-5p was downregulated and CDK6 was upregulated in MSCs-Exo of MCAO-mice and OGD-induced MSCs. MSCs-Exo-miR-26a-5p mimics significantly reduced cell apoptosis of OGD-injured BV-2 cells. MSCs-Exo-miR-26a-5p mimics significantly reduced the infarct volume of MCAO-induced mice. Luciferase reporter assay revealed that CDK-6 was a target of miR-26a-5p. In addition, MSCs-Exo-miR-26a-5p mimics significantly decreased the expression of CDK6 in both OGD-induced BV-2 cells and the brain tissues of MCAO-treated mice. CONCLUSION: Our results indicated that MSCs­Exo attenuated I/R injury in mice by inhibiting microglia apoptosis might via exosomal miR-26a-5p mediated suppression of CDK6. Our study shed light on the application of MSC-Exo as a potential therapeutic tool for cerebral I/R injury.

Recombinant protein TRAIL-Mu3 enhances the antitumor effects in pancreatic cancer cells by strengthening the apoptotic signaling pathway.

Pancreatic cancer is a highly malignant type of cancer and its treatment remains a major challenge. The novel recombinant protein TNF-related apoptosis-inducing ligand (TRAIL)-Mu3 has been shown to exert stronger tumor inhibitory effects in colon cancer in vitro and in vivo compared with TRAIL. The present study investigated the antitumor effects of TRAIL-Mu3 on pancreatic cancer cells, and the possible mechanisms were further examined. Compared with TRAIL, TRAIL-Mu3 exhibited significantly higher cytotoxic effects on pancreatic cancer cell lines. The inhibitory effect of TRAIL-Mu3 on the viability of PANC-1 cells was shown to be a caspase-dependent process. The affinity of TRAIL-Mu3 to PANC-1 cell membranes was significantly enhanced compared with TRAIL. In addition, TRAIL-Mu3 upregulated death receptor (DR) expression in PANC-1 cells and promoted the redistribution of DR5 in lipid rafts. Western blotting results demonstrated that TRAIL-Mu3 activated the caspase cascade in a faster and more efficient manner compared with TRAIL in PANC-1 cells. Therefore, TRAIL-Mu3 enhanced the antitumor effects in pancreatic cancer cells by strengthening the apoptotic signaling pathway. The present study indicated the potential of TRAIL-Mu3 for the treatment of pancreatic cancer.

Different Responses to Identical Trauma Between BALB/C and C57BL/6 Mice.

BACKGROUND Different responses to identical trauma may be related to the genetic background of individuals, but the molecular mechanism is unclear. In this study we investigated the heterogeneity of trauma in mice and the potential biological explanations for the differences. MATERIAL AND METHODS Compared with other organs, the pathological response of the lung after injury is the earliest and most serious. We used C57BL/6 and BALB/C mice to explore the genetic background of different responses to trauma in the lung. We measured mortality rate, pulmonary microvascular permeability, and Cxcl15 gene expression in BALB/C and C57BL/6 mice before and after blast-wave injury. Microvascular permeability was measured using a fluorescent tracer, and Cxcl15 gene expression level and expression distribution were measured using fluorogenic probe quantitative polymerase chain reaction and northern blot. RESULTS C57BL/6 mice showed lower mortality rates and pulmonary microvascular permeability than BALB/C mice after blast-wave injury; there was no significant difference in the permeability before blast-wave injury. The Cxcl15 gene was expressed specifically in the lung tissue of mice. The level of Cxcl15 expression in BALB/C mice was higher than in C57BL/6 mice before and after injury, and the variation trend of Cxcl15 expression level after injury was significantly different between BALB/C and C57BL/6 mice. CONCLUSIONS Our results indicated that BALB/C and C57BL/6 mice had significant heterogeneity in posttraumatic response in terms of mortality and degree of lung damage. The differences in genetic factors such as Cxcl15 may have played a role in this heterogeneity.

An adenosine A1R-A2aR imbalance regulates low glucose/hypoxia-induced microglial activation, thereby contributing to oligodendrocyte damage through NF-κB and CREB phosphorylation.

Microglial activation-mediated inflammatory damage to oligodendrocytes is a key step in the etiology of ischemic white matter lesions. The adenosine A1 receptor (A1R) and adenosine A2a receptor (A2aR) have been reported to regulate the activation of microglia, however, the underlying mechanisms remain elusive. Thus, the present study used a microglia/oligodendrocyte co­culture model exposed to low glucose/hypoxia, and treated with agonists/antagonists of A1R and A2aR to investigate the role of A1R and A2aR. Changes in A1R and A2aR expression and inflammatory cytokine secretion by the microglia, and oligodendrocyte damage, after exposure were examined. Low glucose/hypoxia induced a higher elevation of A1R than A2aR. In addition, activation of A1R inhibited A2aR protein expression and vice versa. The A1R antagonist DPCPX (100 nM) and A2aR agonist CGS 21680 (100 nM) inhibited microglial activation, reduced the production of inflammatory cytokines and attenuated oligodendrocyte damage, along with elevating the levels of phosphorylated nuclear factor (NF)­κB and cyclic adenosine monophosphate response element binding protein (CREB). These data indicate that an A1R­A2aR imbalance is able to modulate low glucose­induced microglial activation and the cellular immune response through altering NF­κB and CREB phosphorylation. This suggests that rebalancing A1R­A2aR is a promising approach for treating white matter injury.

Adenosine A1-Receptors Modulate mTOR Signaling to Regulate White Matter Inflammatory Lesions Induced by Chronic Cerebral Hypoperfusion.

We sought to investigate the role of the adenosine A1 receptors (A1ARs) in white matter lesions under chronic cerebral hypoperfusion (CCH) and explore the potential repair mechanisms by activation of the receptors. A right unilateral common carotid artery occlusion (rUCCAO) method was used to construct a CCH model. 2-chloro-N6-cyclopentyladenosine (CCPA), a specific agonist of A1ARs, was used to explore the biological mechanisms of repair in white matter lesions under CCH. The expression of mammalian target of rapamycin (mTOR), phosphorylation of mTOR (P-mTOR), myelin basic protein (MBP, a marker of white matter myelination) were detected by Western-blot. Pro-inflammatory cytokine tumor necrosis factor-α (TNF-α) and anti-inflammatory cytokine interleukin-10 (IL-10) levels were determined by ELISA. Compared with the control groups on week 2, 4 and 6, in CCPA-treated groups, the ratio of P-mTOR/mTOR, expression of MBP and IL-10 increased markedly, while the expression of TNF-α reduced at week 6. In conclusion, A1ARs appears to reduce inflammation in white matter via the mTOR signaling pathway in the rUCCAO mice. Therefore, A1ARs may serve as a therapeutic target during the repair of white matter lesions under CCH.

The Effect of Pre-Condition Cerebella Fastigial Nucleus Electrical Stimulation within and beyond the Time Window of Thrombolytic on Ischemic Stroke in the Rats.

OBJECTIVE: To investigate the effect of neurogenic neuroprotection conferred by cerebellar fastigial nucleus stimulation (FNS) and the role of PPARγ-mediated inflammation in a rat model of cerebral ischemia reperfusion. METHODS: After a continuous 1 hour fastigial nucleus electric stimulation, the male Sprague Dawley (SD) rats were given middle cerebral artery occlusion (MCAO) for 1, 3, 6, 9, 12 and 15 hours undergoing reperfusion with intravenous recombinant tissue plasminogen activator (rt-PA), while the control group received without FNS. After 72 h of reperfusion, the neurological deficits, infarct volume and brain edema were evaluated. The brain tissue in ischemic penumbra was determined the myeloperoxidase (MPO) activity by a spectrophotometer and expression of PPARγ was measured by Rt-PCR and Western blotting. RESULTS: Our findings showed that FNS group had significantly reduced infarct volume and brain edema, and improved neurological deficits compared with the control group, especially in 6 h and 9 h reperfusion subgroups (p<0.05). The expression levels of PPARγ increased gradually and the peak may be before and after 9 h reperfusion, the 3 h, 6 h, 9 h, 12 h and 15 h reperfusion subgroups were higher than each control group (p<0.05). The MPO activity of 6 h, 12 h and 15 h reperfusion subgroups were higher than each control group (p<0.05). CONCLUSIONS: The neuroprotective effects of FNS have been shown to prolong the therapeutic window in cerebral ischemia/reperfusion, which might be related to the PPARγ mediated-inflammation in penumbral region.

Chronic Cerebral Ischemia Induces Downregulation of A1 Adenosine Receptors During White Matter Damage in Adult Mice.

The role of A1 adenosine receptors (A1ARs) in the white matter under chronic cerebral ischemic conditions remains unclear. Here, we used right unilateral common carotid artery occlusion (rUCCAO) to construct a chronic cerebral ischemic mouse model. A1AR expression and proteolipid protein (PLP, a marker of white matter myelination) in the corpus callosum were observed by immunoreaction and immunohistochemistry, respectively. Pro-inflammatory interleukin-1ß (IL-1ß) and anti-inflammatory interleukin-10 (IL-10) levels were determined by ELISA. The Morris water maze test was employed to detect cognitive impairment. A1AR expression significantly decreased in the rUCCAO group as compared with the sham control group on weeks 2, 4, and 6, respectively. IL-10 levels in the rUCCAO group significantly declined on week 6, while there was no significant change in IL-1ß expression. PLP expression significantly decreased in the rUCCAO group on weeks 2, 4, and 6. Moreover, latency time for the Morris water maze test significantly increased in the rUCCAO group on weeks 4 and 6, while the number of platform location crossing significantly decreased in the rUCCAO group on weeks 2, 4, and 6. In conclusion, this study provides the first evidence that chronic cerebral ischemia appears to induce A1AR downregulation and inhibition of IL-10 production, which may play key roles in the neuropathological mechanisms of ischemic white matter lesions. These data will facilitate future studies in formulating effective therapeutic strategies for ischemic white matter lesions.