Tanshinone IIA, a fat-soluble diterpenoid isolated from Salvia miltiorrhiza Bunge, has been shown to attenuate the cerebral ischemic injury. The aim of this study was to examine the effects on neuroprotection and microglia activation of Tanshinone IIA. Male Sprague-Dawley rats were subjected to middle cerebral artery occlusion (MCAO). We found that Tanshinone IIA significantly reduced infarction volume, alleviated neuronal injuries, reduced the release of TNF-α, IL-1β, and IL-6, increased SOD activity, and decrease the content of MDA in MCAO rats. Hematoxylin and eosin staining, Nissl staining, TUNEL staining and immunofluorescence staining showed that Tanshinone IIA improved the distribution and morphology of neurons in brain tissues and reduced apoptosis. In addition, Co-immunofluorescence staining of rat brain tissues and the mRNA expression levels of CD11b, CD32, iNOS, and Arg-1, CD206, IL-10 in BV2 cells indicated that Tanshinone IIA can downregulate M1 microglia and upregulate M2 microglia in MCAO rats. Further, BV2 microglial cells were subjected to oxygen-glucose deprivation, the protein expression levels were detected by western blot. Tanshinone IIA inhibited the expression levels of NF-κB signaling pathway related proteins. Taken together, this study suggested that Tanshinone IIA modulated microglial M1/M2 polarization via the NF-κB signaling pathway to confer anti-neuroinflammatory effects.

Monocyte locomotion inhibitory factor (MLIF) is a heat-stable pentapeptide from Entamoeba histolytica. Our previous study found that MLIF protects against ischemic stroke in rats and mice and exerts a neuroprotection effect in human neuroblastoma SH-SY5Y cells. Microglia/macrophage polarization has been proven to be vital in the pathology of ischemic stroke. Nevertheless, whether MLIF is able to modulate microglia/macrophage polarization remains unclear. We performed middle cerebral artery occlusion (MCAO) on C57BL/6J male mice and induced cultured BV2 microglia by oxygen-glucose deprivation (OGD), respectively. Immunfluorescence was utilized to detect the M1/2 markers, such as CD206 and CD16/32. qPCR and ELISA were used to detect the signature gene change of M1/2. The MAPK and NF-κB pathway associated proteins were measured by Western blot. To identify the protein target of MLIF, a pull-down assay was performed. We found that MLIF promoted microglia transferring from a “sick” M1 phenotype to a “healthy” M2 phenotype in vivo or in vitro. Furthermore, we proved that eukaryotic elongation factor 1A1 (eEF1A1) was involved in the modulation of microglia/macrophage polarization. Knocking down eEF1A1 by siRNA exhibited the M1 promotion effect and M2 inhibition effect. Taken together, our results demonstrated MLIF modulated microglia/macrophage polarization by targeting eEF1A1 in ischemic stroke.

MQ (l-methionyl-l-glutamic acid), anti-inflammatory dipeptide, is one of the metabolites of monocyte locomotion inhibitory factor, a thermostable pentapeptide secreted by Entamoeba histolytica. Monocyte locomotion inhibitory factor injection has been approved as an investigational drug for the potential neural protection in acute ischemic stroke. This study further investigated the neuroprotective effect of MQ in ischemic brain damage. Ischemia-reperfusion injury of the brain was induced in the rat model by middle cerebral artery occlusion. 2,3,5-triphenyltetrazolium chloride staining assay was used to measure cerebral infarction areas in rats. Laser Doppler measurement instrument was used to detect blood flow changes in the rat model. Nissl staining and NeuN staining were utilized to observe the numbers and structures of neuron cells, and the pathological changes in the brain tissues were examined by hematoxylin–eosin staining. Terminal deoxynucleotidyl transferase deoxyuridine triphosphate nick end labeling (TUNEL) staining was used to assess cell apoptosis. The changes in oxidative stress indexes, superoxide dismutase and malondialdehyde (MDA), were measured in serum. Methyl thiazolyl tetrazolium was used to measure the survival rates of PC12 cells. Flow cytometry assessed the apoptosis rates and the levels of reactive oxygen species. Real-time PCR was used to evaluate the mRNA expression levels, and Western blotting was used to analyze the changes in protein levels of p-JNK, Bax, cleaved Caspase3. We revealed that MQ improved neurobehavior, decreased cerebral infarction areas, altered blood flow volume, and the morphology of the cortex and hippocampus. On the other hand, it decreased the apoptosis of cortical neurons and the levels of MDA, and increased the levels of superoxide dismutase. In vitro studies demonstrated that MQ enhanced the cell survival rates and decreased the levels of reactive oxygen species. Compared to the oxygen-glucose deprivation/reperfusion group, the protein and mRNA expressions of p-JNK, Bax, cleaved Caspase3 was decreased significantly. These findings suggested that MQ exerts a neuroprotective effect in cerebral ischemia by blocking apoptosis via the p-JNK/Bax pathway.

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