Melittin protects against neural cell damage in rats following ischemic stroke.

In this study, we explored the neuroprotective effect of melittin (MEL) after brain ischemia using a rat model. The rats underwent middle cerebral artery occlusion (MCAO) for 60 min and were randomly divided into the control group, saline group, and MEL group. Rats in each group were injected intraperitoneally with MEL one day before MCAO until sacrificed. Morris water maze and rotation test were used to assess locomotor function and cognitive ability. The 9.4 Tesla MRI was used to scan and assess the infarct volume of the rat brains. Immunohistochemistry was used to detect the sites of action of MEL on microglia. Western blot and ELISA were used to measure the effect of MEL on the production of pro-inflammatory cytokines. The effect of MEL on neuronal cell apoptosis was observed by flow cytometry. Compared with the saline group, MEL treatment significantly increased the density of neurons in the cerebral cortical and reduced the cerebral infarct size after MCAO (33.9 ± 8.8% vs. 15.8 ± 3.9%, P < 0.05). Meanwhile, the time for MEL-treated rats to complete the water maze task on the 11th day after MCAO was significantly shorter than that of rats in the saline group (P < 0.05). MEL treatment also prolonged the rotarod retention time on day 14 after MCAO. Immunohistochemistry analysis showed that MEL inhibited the activation of microglia and suppressed the expression of TNF-α, IL-6, and IL-1β in the brain after ischemia. MEL treatment resulted in a significant decrease in TLR4, MyD88, and NF-κB p65 levels in extracts from the ischemic cerebral cortex. Finally, MEL reduced neuronal apoptosis induced by ischemic stroke (P < 0.05). MEL treatment promotes neurological function recovery after cerebral ischemia in rats. These effects are potentially mediated through anti-inflammatory and anti-apoptotic mechanisms.

Yao X ，Kang J ，Li Y ，Zhang H ，Zhang H ，Chen E ... -  《-》

Anti-inflammatory effects of quinolinyl analog of resveratrol targeting TLR4 in MCAO/R ischemic stroke rat model.

Among adults, stroke is the main causes of mortality and permanent disability. Neuroinflammation is one of the main causes of stoke-mediated neuronal death. Our previous study revealed that (E)-5-(2-(Quinolin-4-yl) vinyl) benzene-1, 3-diol (RV01), a quinolinyl analog of resveratrol, inhibits microglia-induced neuroinflammation and safeguards neurons from inflammatory harm. The preventive role of RV01 in ischemic stroke and its underlying cellular mechanisms and molecular targets remain poorly understood. To investigate whether RV01 alleviates ischemia-reperfusion (I/R) injury by inhibiting microglia-mediated neuroinflammation and determine the potential molecular mechanisms and targets by which RV01 inhibits the I/R-mediated microglia activation. Rat middle cerebral artery occlusion and reperfusion (MCAO/R) and BV-2 or primary microglial cells oxygen-glucose deprivation and reperfusion (OGD/R) models were established. The neurological behavior scores, 2, 3, 5-triphenyl tetrazolium chloride staining and immunofluorescence were used to detect the neuroprotective effect of RV01 in the MCAO/R rats. In addition, the mRNA expression levels of IL-6, TNF-α, and IL-1β were detected to reveal the antineuroinflammatory effect of RV01. Moreover, a western blot assay was performed to explore the protein expression changes in NF-κB-mediated neuroinflammation. Finally, we identified TLR4 as an RV01 target through molecular docking, drug sensitivity target stability analysis, cellular thermal shift analysis, and surface plasmon resonance techniques. RV01 reduced the infarct volume and neurological deficits, increased the rotarod duration, and decreased the number of rightward deflections in the MCAO/R rats. RV01 inhibited the NF-κB signaling pathway in vitro and in vivo, as demonstrated by the reduction in the transcription factor p65-mediated expression of several inflammatory factors including IL-6, TNF-α, and IL-1β. Further studies showed that its protective effect was associated with targeting the TLR4 protein. Notably, the anti-inflammatory effect of RV01 was markedly reinforced by the TLR4 knockdown, but inhibited by the overexpression of TLR4. Results revealed that the conditioned medium derived from the RV01-treated BV-2 cells significantly decreased the OGD/R-mediated neuronal damage. Our results are the first to reveal the protective effects of RV01 on cerebral ischemia, depending on its inhibitory effect on the NF-κB pathway by targeting TLR4. RV01 could be a potential protective agent in ischemic stroke treatment.

Xu L ，Mi Y ，Meng Q ，Liu Y ，Wang F ，Zhang G ，Liu Y ，Chen G ，Hou Y ... -  《-》

Kellerin from Ferula sinkiangensis exerts neuroprotective effects after focal cerebral ischemia in rats by inhibiting microglia-mediated inflammatory responses.

Ferula sinkiangensis K. M. Shen is a traditional Chinese medicine that has a variety of pharmacological properties relevant to neurological disorders and inflammations. Kellerin, a novel compound extracted from Ferula sinkiangensis, exerts a strong anti-neuroinflammatory effect by inhibiting microglial activation. Microglial activation plays a vital role in ischemia-induced brain injury. However, the potential therapeutic effect of kellerin on focal cerebral ischemia is still unknown. To explore the effect of kellerin on cerebral ischemia and clarify its possible mechanisms, we applied the middle cerebral artery occlusion (MCAO) model and the LPS-activated microglia model in our study. Neurological outcome was examined according to a 4-tiered grading system. Brain infarct size was measured using TTC staining. Brain edema was calculated using the wet weight minus dry weight method. Neuron damage and microglial activation were observed by immunofluorescence in MCAO model in rats. In in vitro studies, microglial activation was examined by flow cytometry and the viability of neuronal cells cultured in microglia-conditioned medium was measured using MTT assay. The levels of pro-inflammatory cytokines were measured by qRT-PCR and ELISA. The proteins involved in NF-κB signaling pathway were determined by western blot. Intracellular ROS was examined using DCFH-DA method and NADPH oxidase activity was measured using the NBT assay. We found that kellerin improved neurological outcome, reduced brain infarct size and decreased brain edema in MCAO model in rats. Under the pathologic conditions of focal cerebral ischemia, kellerin alleviated neuron damage and inhibited microglial activation. Moreover, in in vitro studies of LPS-stimulated BV2 cells kellerin protected neuronal cells from being damaged by inhibiting microglial activation. Kellerin also reduced the levels of pro-inflammatory cytokines, suppressed the NF-κB signaling pathway, and decreased ROS generation and NADPH oxidase activity. Our discoveries reveal that the neuroprotective effects of kellerin may largely depend on its inhibitory effect on microglial activation. This suggests that kellerin could serve as a novel anti-inflammatory agent which may have therapeutic effects in ischemic stroke.

Mi Y ，Jiao K ，Xu JK ，Wei K ，Liu JY ，Meng QQ ，Guo TT ，Zhang XN ，Zhou D ，Qing DG ，Sun Y ，Li N ，Hou Y ... -  《-》

Neuroprotective and Angiogenesis Effects of Levetiracetam Following Ischemic Stroke in Rats.

Objective: The present study explored whether levetiracetam (LEV) could protect against experimental brain ischemia and enhance angiogenesis in rats, and investigated the potential mechanisms in vivo and in vitro. Methods: The middle cerebral artery was occluded for 60 min to induce middle cerebral artery occlusion (MCAO). The Morris water maze was used to measure cognitive ability. The rotation test was used to assess locomotor function. T2-weighted MRI was used to assess infarct volume. The neuronal cells in the cortex area were stained with cresyl purple. The anti-inflammatory effects of LEV on microglia were observed by immunohistochemistry. Enzyme-linked immunosorbent assays (ELISA) were used to measure the production of pro-inflammatory cytokines. Western blotting was used to detect the levels of heat shock protein 70 (HSP70), vascular endothelial growth factor (VEGF), and hypoxia-inducible factor-1α (HIF-1α) in extracts from the ischemic cortex. Flow cytometry was used to observe the effect of LEV on neuronal cell apoptosis. Results: LEV treatment significantly increased the density of the surviving neurons in the cerebral cortex and reduced the infarct size (17.8 ± 3.3% vs. 12.9 ± 1.4%, p < 0.01) after MCAO. Concurrently, the time required to reach the platform for LEV-treated rats was shorter than that in the saline group on day 11 after MCAO (p < 0.01). LEV treatment prolonged the rotarod retention time on day 14 after MCAO (84.5 ± 6.7 s vs. 59.1 ± 6.2 s on day 14 compared with the saline-treated groups, p < 0.01). It also suppressed the activation of microglia and inhibited TNF-α and Il-1β in the ischemic brain (135.6 ± 5.2 pg/ml vs. 255.3 ± 12.5 pg/ml, 18.5 ± 1.3 pg/ml vs. 38.9 ± 2.3 pg/ml on day 14 compared with the saline-treated groups, p < 0.01). LEV treatment resulted in a significant increase in HIF-1α, VEGF, and HSP70 levels in extracts from the ischemic cerebral cortex. At the same time, LEV reduced neuronal cell cytotoxicity and apoptosis induced by an ischemic stroke (p < 0.01). Conclusion: LEV treatment promoted angiogenesis and functional recovery after cerebral ischemia in rats. These effects seem to be mediated through anti-inflammatory and antiapoptotic activities, as well as inducing the expression of HSP70, VEGF, and HIF-1α.

Yao X ，Yang W ，Ren Z ，Zhang H ，Shi D ，Li Y ，Yu Z ，Guo Q ，Yang G ，Gu Y ，Zhao H ，Ren K ... -  《Frontiers in Pharmacology》

Propofol protects against focal cerebral ischemia via inhibition of microglia-mediated proinflammatory cytokines in a rat model of experimental stroke.

Zhou R ，Yang Z ，Tang X ，Tan Y ，Wu X ，Liu F ... -  《PLoS One》