Background: Neuroinflammation plays an important role in cerebral ischemia/reperfusion (I/R) injury. The P2X7 receptor (P2X7R) has been reported to be involved in the inflammatory response of many central nervous system diseases. However, the role of P2X7Rs in transient global cerebral I/R injury remains unclear. The purpose of this study is to determine the effects of inhibiting the P2X7R in a rat model of transient global cerebral I/R injury, and then to explore the association between the P2X7R and neuroinflammation after transient global cerebral I/R injury.Methods: Immediately after infusion with the P2X7R antagonists Brilliant blue G (BBG), adenosine 5′-triphosphate-2′,3′-dialdehyde (OxATP) or A-438079, 20 minutes of transient global cerebral I/R was induced using the four-vessel occlusion (4-VO) method in rats. Survival rate was calculated, neuronal death in the hippocampal CA1 region was observed using H & E staining, and DNA cleavage was observed by deoxynucleotidyl transferase-mediated UTP nick end labeling TUNEL). In addition, behavioral deficits were measured using the Morris water maze, and RT-PCR and immunohistochemical staining were performed to measure the expression of IL-1β, TNF-α and IL-6, and to identify activated microglia and astrocytes.Results: The P2X7R antagonists protected against transient global cerebral I/R injury in a dosage-dependent manner. A high dosage of BBG (10 μg) and A-0438079 (3 μg), and a low dosage of OxATP (1 μg) significantly increased survival rates, reduced I/R-induced learning memory deficit, and reduced I/R-induced neuronal death, DNA cleavage, and glial activation and inflammatory cytokine overexpression in the hippocampus.Conclusions: Our study indicates that inhibiting P2X7Rs protects against transient global cerebral I/R injury by reducing the I/R-induced inflammatory response, which suggests inhibition of P2X7Rs may be a promising therapeutic strategy for clinical treatment of transient global cerebral I/R injury.

In the clinic selective serotonin reuptake inhibitors (SSRIs), like Fluoxetine, remain the primary treatment for major depression. It has been suggested that miR-16 regulates serotonin transporters (SERT) via raphe nuclei and hippocampal responses to antidepressants. However, the underlying mechanism and regulatory pathways are still obtuse. Here, a chronic unpredicted mild stress (CUMS) depression model in rats was established, and then raphe nuclei miR-16 and intragastric Fluoxetine injections were administered for a duration of three weeks. An open field test and sucrose preference quantification displayed a significant decrease in the CUMS groups when compare to the control groups, however these changes were attenuated by both miR-16 and Fluoxetine treatments. A dual-luciferase reporter assay system verified that hsa-miR-16 inhibitory effects involve the targeting of 3'UTR on the 5-HTT gene. Expression levels of miR-16 and BDNF in the hippocampus were examined with RT-PCR, and it was found that increased 5-HT2a receptor expression induced by CUMS can be decreased by miR-16 and Fluoxetine administration. Immunofluorescence showed that expression levels of neuron NeuN and MAP-2 in CUMS rats were lower. Apoptosis and autophagy levels were evaluated separately through relative expression of Bcl-2, Caspase-3, Beclin-1 and LC3II. Furthermore, CUMS was found to decrease levels of hippocampal mTOR, PI3K and AKT. These findings indicate that apoptosis and autophagy related pathways could be involved in the effectiveness of antidepressants, in which miR-16 participates in the regulation of, and is likely to help integrate rapid therapeutic strategies to alleviate depression clinically. These findings indicate that miR-16 participates in the regulation of apoptosis and autophagy and could account for some part of the therapeutic effect of SSRIs. This discovery has the potential to further the understanding of SSRIs and accelerate the development of new treatments for depression.

Triptolide (TP), one of the major active components of the traditional Chinese herb Tripterygium wilfordii Hook F, and 2, 4-diamino-6-hydroxypyrimidine (DAHP), an inhibitor of tetrahydrobiopterin (BH4) synthesis, have been reported to have potent anti-inflammatory and immunosuppressive properties. However, the protective effects of TP and DAHP on cerebral ischemia have not been reported yet. In this study, we investigated the neuroprotective effects of TP and DAHP in a middle cerebral artery occlusion (MCAO) rat model. Furthermore, we examined whether the neuroprotective effects of TP and DAHP were associated with the inhibition of apoptosis through suppressing BH4 and inducible NOS (iNOS) synthesis or the activation of the phosphoinositide-3-kinase/serine-threonine kinase Akt/ mammalian target of rapamycin (PI3K/Akt/mTOR) pathway. Our results showed that pretreatments with Triptolide (0.2 mg/kg) and DAHP (0.5g/kg) significantly reduced ischemic lesion volume, water content and neuronal cell death compared with the vehicle MCAO rats. In addition, compared with the MCAO group, TP and DAHP pretreatment groups significantly reduced astrocyte numbers, caspase-3, cleaved caspase-3 and NF-κB up-regulation, while increased Bcl-2 expression. Moreover, protein expressions of PI3K, Akt and mTOR increased, while extracellular signal-regulated protein kinases 1 and 2 (ERK1and 2) phosphorylation decreased in both the TP-treated rats and DAHP-treated rats. These results demonstrate that TP and DAHP can decrease cell apoptosis in focal cerebral ischemia rat brains and that the mechanism may be related to the activation of the PI3K/Akt/mTOR pathway and inactivation of the ERK1/2 pathway. Thus our hypothesis was reached PI3K/Akt/mTOR and ERK1/2 pathways may provide distinct cellular targets for a new generation of therapeutic agents for the treatment of stroke, and TP and DAHP may be potential neuroprotective agents for cerebral ischemia/reperfusion injury.

Postpartum depression (PPD) is associated with mood disorders and elevated inflammation. Studies have evidenced the activation/inhibition of autophagy and excessive activation of microglia to have a close relationship with depression. C57 and microglia-specific autophagy-deficient mice (Cx3Cr1^Cre/+ATG5^loxp/loxp) were employed to establish the chronic unpredicted mild stress depression mice model from embryonic day 7 (E7) to embryonic day 16 (E16). Fluoxetine was administered for 3 weeks (commencing from 1 week after birth). Behavioral tests (open field, forced swimming, and sucrose preference tests) were implemented. Western blot and immunofluorescence staining were employed to assess the brain-derived neurotrophic factor (BDNF) expression level, autophagy-associated proteins, and inflammatory factors. Depressive behavior was reversed following fluoxetine treatment; this was evidenced via open field, sucrose preference, and forced swimming tests. Both BDNF and autophagy-associated proteins (ATG5, Beclin-1, and LC3II) were upregulated following fluoxetine treatment. Inflammatory factors including nuclear factor kappa B and inducible nitric oxide synthase were reduced while anti-inflammatory factor interleukin-10 (IL-10) was increased after fluoxetine treatment. Microglia-specific autophagy-deficient mice (Cx3Cr1^Cre/+ATG5^loxp/loxp) showed a curtailed autophagy level, higher inflammatory level, and reduced BDNF expression when compared with C57 mice. Autophagy inhibition in microglia contributes to inflammation, which further instigates PPD. Fluoxetine might mediate its antidepressant effect in PPD through the autophagic pathway while upregulating BDNF expression. In view of this, regulating BDNF in microglia is a potential novel therapy target for PPD.

Ischemia cerebral stroke is one of the common neurological diseases with severe inflammatory response and neuron death. The inhibition of colony-stimulating factor 1 receptor (CSF1R) which especially expressed in microglia/macrophage exerted neuroprotection in stroke. However, the underlying neuroinflammatory regulation effects of CSF1R in ischemia stroke are not clear. In this study, cerebral ischemia stroke mice model was established. The C57/B6J mice were administered with Ki20227, a CSF1R inhibitor, by gavage for 7 consecutive days (0.002 mg/kg/day) before modeling. The Rota-Rod test and neurobehavioral score test were investigated to assess neurobehavioral functions. The area of infarction was assessed by 2, 3, 5-triphenyltetrazolium chloride (TTC) staining. The mRNA expressions of M1/M2 microglia markers were evaluated by real-time PCR. Immunofluorescence and Western blot were utilized to detect the changes of Iba1 and NLRP3 pathway proteins. Results showed that neurobehavioral function improvement was demonstrated by an increased stay time on the Rota-Rod test and a decreased neurobehavioral score in the Ki20227 treatment group. The area of infarction reduced in Ki20227 group when compared to the stroke group. Moreover, the mRNA expression of M1 microglia markers (TNF-α and iNOS) decreased while M2 microglia markers (IL-10 and Arg-1) increased. Meanwhile, compared to the stroke and stroke+PBS group, Ki20227 administration downregulated the expression of NLRP3, active caspase 1, and NF-κB protein in the ischemia penumbra of Ki20227 treatment group mice. In short, the CSF1R inhibitor, Ki20227, played vital neuroprotective roles in ischemia cerebral stroke mice, and the mechanisms may be via inhibiting microglia M1 polarization and NLRP3 inflammasome pathway activation. Our study provides a potential new target for the treatment of ischemic stroke injury.

Stroke is the leading cause of neurological disability in humans. Middle cerebral artery occlusion (MCAO) followed by reperfusion is widely accepted to mimic stroke in basic medical research. Triptolide is one of the major active components of the traditional Chinese herb Tripterygium wilfordii Hook F, and has been reported to have potent anti‐inflammatory and immunosuppressive properties. Since its preclinical effects on stroke were still unclear, we decided to study the effects of Triptolide on focal cerebral ischemia/reperfusion injury in this study. The results showed that Triptolide treatment significantly attenuates brain infarction volume, water content, neurological deficits, and neuronal cell death rate, which were increased in the MCAO model rats. Immunohistochemistry was used to analyze the expression of glial fibrillary acidic protein (GFAP), Cyclooxygenase‐2 (COX‐2), inducible nitric oxide (iNOS), and NF‐κB in the ischemic brains. The administration of Triptolide showed down‐regulation of the iNOS, COX‐2, GFAP, and NF‐κB expression in MCAO rats. It also increased the expression of bcl‐2, and suppressed levels of bax and caspase‐3 compared with the MCAO group. Our findings revealed that Triptolide exerts its neuroprotective effects against inflammation with the involvement of inhibition of NF‐κB activation. Anat Rec, 299:256–266, 2016.