MicroRNA-132 attenuates LPS-induced inflammatory injury by targeting TRAF6 in neuronal cell line HT-22.

Epilepsy is a common neurological disorder in the central nervous system. Inflammation disrupts the blood-brain barrier (BBB), which is responsible for maintaining brain homeostasis. This study was aimed to investigate the functional role of microRNA (miR)-132 in hippocampal HT-22 cells under lipopolysaccharide (LPS) stimulation. In vitro cell inflammatory model was constructed by LPS stimulation. Inflammatory cell injury was evaluated according to the alterations of cell viability, apoptosis, and expression of inflammatory cytokines. Then, miR-132 level after LPS treatment was assessed. Subsequently, miR-132 was abnormally expressed after cell transfection, and the effects of miR-132 on LPS-induced cell inflammatory injury as well as phosphorylated levels of key kinases in the NF-κB and MAPK kinase (MEK)/ERK pathways were determined. The target gene of miR-132 was virtually screened and verified, and whether miR-132 affected HT-22 cells under LPS stimulation through regulating the target gene was verified. The results showed that the level of miR-132 was down-regulated by LPS in HT-22 cells, and the LPS-induced inflammatory injury could be reduced by miR-132 overexpression. Then, the phosphorylated levels of kinases in the NF-κB and MEK/ERK pathways were decreased by miR-132 overexpression. Tumor necrosis factor receptor-associated factor 6 (TRAF6) was predicted and verified to be a target of miR-132. Moreover, the alterations induced by miR-132 overexpression in the LPS-treated HT-22 cells were abrogated by TRAF6 overexpression. Therefore, we drew the conclusion that LPS down-regulated miR-132 and miR-132 attenuated LPS-induced inflammatory cell injury by targeting TRAF6, along with the inhibition of the NF-κB and MEK/ERK pathways.

Effects of catalpol on ATPase and amino acids in gerbils with cerebral ischemia/reperfusion injury.

This study aimed to investigate the effects of catalpol on ATPase and amino acids in gerbils following cerebral ischemia/reperfusion (CI/R) injury. Gerbil model of CI/R was prepared by bilateral common carotid occlusion for 10 min followed by 6 h of reperfusion. Catalpol (5, 10 or 20 mg/kg per day) was injected intraperitoneally for 3 days before the carotid occlusion. Stroke index was measured during the reperfusion. ATPase activity, glutamate (Glu) and aspartate contents in brain tissue homogenate were examined. The results showed that catalpol significantly improved the stroke index compared with sham group (P < 0.05 or P < 0.01). Catalpol markedly increased the activities of Na(+)-K(+)-ATPase and Ca(2+)-ATPase (P < 0.05 or P < 0.01), and significantly decreased the content of Glu in brain tissue (P < 0.05 or P < 0.01). These data suggest that the efficacy of catalpol pretreatment on CI/R injury is associated with the enhancement of ATPase activity and the inhibition of excitatory amino acid toxicity.

Effects of resveratrol on apoptosis in a rat model of vascular dementia.

Resveratrol is a natural polyphenol widely present in plants, particularly in the skin of red grapes and in wine. It possesses a wide range of biological effects and exhibits neuroprotective effects in numerous diseases. However, data evaluating the effects of resveratrol in vascular dementia (VaD) are lacking. In the present study, the permanent, bilateral common carotid artery occlusion rat model was used to study the effects of resveratrol on VaD. The Morris water maze was used to test the spatial learning and memory performance of the rats. The expression levels of Bax, Bcl-2, cleaved caspase-3 and cleaved poly(ADP-ribose) polymerase (PARP) in the hippocampus were measured. The results showed that resveratrol inhibited memory impairment in the VaD rat model, and attenuated the increases in the expression levels of Bax, cleaved caspase-3 and cleaved PARP and the reductions in the expression levels of Bcl-2 that were induced by VaD. These results provide a novel insight into the neuroprotective effects of resveratrol and its possible therapeutic role in VaD.

[Protective effect of musk extract on rat's cerebral cortical neurons with inflammatory injury].

OBJECTIVE: To investigate the protective effects of musk extract (ME) and its possible mechanism on rat's cerebral cortical neurons with inflammatory injury induced by lipopolysaccharide (LPS). METHODS: Neurons and astrocytes from newborn rat cerebral cortex were cultured in vitro respectively, and the astrocyte conditioned medium (ACM), obtained by treating astrocytes with 10 mg/L LPS and different concentrations of ME for 24 h, was added in the culture fluid of neurons. The survival rate and apoptotic rate of neurons were measured by MTT method and AO/EB stain; and the changes of inflammatory factors in the ACM were determined by ELISA. RESULTS: The survival rate (%) of neurons treated by ACM with ME in concentrations of 18 mg/L, 36 mg/L, 72 mg/L and 144 mg/L was 52.55 +/- 3.52, 55.77 +/- 2.36, 64.89 +/- 3.45 and 73.67 +/- 1.80, respectively, significantly higher than that in the model neurons (43.62 +/- 4. 51, P < 0.05), while the apoptotic rate (%) in them, 68.11 +/- 2.16, 44.27 +/- 3.68, 32.56 +/- 2.14 and 21.89 +/- 2.46, respectively, was significantly lower than that in model neurons (71.33 +/- 3.25, P < 0.05 or P < 0.01). Level of IL-6 was decreasing along with the raising of ME concentration in the ACM, showing a concentration-dependent state. CONCLUSION: ME shows apparent protective effect on neurons against inflammatory injury, especially in a high concentration (144 mg/L), which may be associated with the reduction of IL-6 secreted by astrocytes.

[Effects of DSCAM on differentiation of rat marrow mesenchymal stem cells into neurons in vitro].

OBJECTIVE: To study the effects of Down syndrome cellular adhesion molecule (DSCAM) on differentiation of rat marrow mesenchymal stem cells (MSCs) into neurons in vitro. METHODS: MSCs from Sprague-Dawley rats were induced into neurons by baicalin. The expression of DSCAM before and after induction was evaluated by immunocytochemical staining and Western blot assay. After knockdown of DSCAM by siRNA transfection, the differentiation rate of neurons derived from MSCs was measured. RESULTS: Before induction, the expression of DSCAM was not detectable in MSCs. After bFGF preinduction for 24 hrs, DSCAM was slightly expressed in MSCs (1.71+/- 0.67%). The DSCAM expression increased 6 hrs after baicalin induction (15.79+/- 4.24%), reached a peak at 3 days (53.16+/- 5.94%) and then decreased gradually. The DSCAM expression 6 days after baicalin induction (28.99+/- 6.72%) was significantly lower than that at 3 days (P<0.01). However, after DSCAM-siRNA transfection, the DSCAM expression in MSCs was significantly reduced. MSCs did not express neuron-specific beta-III-tubulin before induction. After baicalin induction for 6 hrs, 3 days and 6 days, the expression of beta-III-tubulin was 1.40+/- 0.79%, 41.59+/- 3.17% and 59.11+/- 4.76% respectively. But the beta-III-tubulin expression significantly decreased 3 and 6 days after DSCAM-siRNA transfection (28.57+/- 2.91% and 43.90+/- 12.31% respectively). CONCLUSIONS: DSCAM may play an important role in MSCs differentiation into neural cells.

[Effects of Notch-1 signaling in bone marrow mesenchymal stem cells differentiating into neurons: experiment in vitro].

OBJECTIVE: To investigate the role of Notch-1 signaling in bone marrow mesenchymal stem cells (MSCs) differentiating into neurons. METHODS: Mice Notch-1 small hairpin RNA (mNotch-1 shRNA) was constructed and transfected into the MSCs obtained from the tibiae of BALB/c mice. MSCs transfected with glyceraldehyde-3-phosphate hydoxygenase (GADPH) shRNA and untransfected MSCs were used as controls. The cell survival rate was detected by ELISA. The MSCs of different groups were cultured in Neurobasal-A medium so as to be induced to differentiate into neurons. Apoptosis of the MSCs was detected by TUNEL. RESULTS: After induction of 6 days the MSCs transfected with mNotch-1 shRNA displayed typical neuronal morphology and high expression of neuron-specific markers: nestin, neuron-specific enolase (NSE), neurofilament 200 (NF 200), and Notch-1 protein, however, gilal fibrillary acidic protein (GFAP), the glia-specific marker, was not detected. The percentage of apoptotic cells in the MSCs transfected with mNotch-1 shRNA was 13.3% +/- 2.3%, significantly higher than those of the MSCs transfected with mGAPH shRNA and untransfected MSCs (4.7% +/- 0.5% and 4.5% +/- 0.4%, both P < 0.01). CONCLUSION: Block of the Notch signal pathway may increase the differentiation of MSCs into neurons.