Inhibition of the myeloid differentiation primary response protein 88 reducres neuron injury in the early stages of subarachnoid hemorrhage in an in vitro experimental model.

Subarachnoid hemorrhage (SAH) is a life-threatening cerebrovascular disease with high rates of morbidity and mortality and a paucity of effective therapies. The development of early brain injury (EBI) is closely related to prognosis in SAH, and inflammation plays an important role in its pathophysiology. A previous experiment showed that ST2825, a selective inhibitor of MyD88, could alleviate EBI in vivo. However, this protective effect in vivo is affected by a variety of pathophysiology processes making the result to some extent uncertain. whether there is a coincident result in vitro ruling out the effect of other factors remains unknown, and further investigation using cultured neurons is necessary. Primary neuronal cells were cultured to construct an in vitro model of SAH. The cells were cultured and then divided into three groups: (1) a blank control group, (2) an oxygenated hemoglobin + vehicle group, and (3) an oxygenated hemoglobin + ST2825 group. In each group, apoptosis of neuronal cells along with changes in the expression of proteins including MyD88, p-JNK, p-Erk, p-p38, NFκB, Bcl-2, and P53 were measured. Results showed that after stimulating neurons with oxygenated hemoglobin, the expression of the MyD88 protein in the vehicle group increased significantly. The quantity of p-JNK, p-p38, and p-Erk also increased significantly, as did the quantity of p65 in the nucleus. Expression of the anti-apoptotic protein Bcl-2 was markedly reduced, while that of the cleaved caspase-3 protein was significantly increased. In addition, in this group, the apoptosis rate of neurons was significantly increased. In the ST2825 group, the expression of p-JNK, p-p38, p-Erk, cleaved caspase-3, and p65 in the nucleus was significantly decreased, the expression of Bcl-2 was significantly increased, and the apoptosis rate of neurons was significantly reduced. The results of this study suggest that in an experimental in vitro SAH model, ST2825, a selective inhibitor of MyD88, can have a neuroprotective effect by inhibiting neuronal apoptosis mediated by the MAPK and NFκB signaling pathways, and this has a certain protective effect on EBI after SAH.

MicroRNA-370-3p inhibits human vascular smooth muscle cell proliferation via targeting KDR/AKT signaling pathway in cerebral aneurysm.

OBJECTIVE: Cerebral aneurysm is a common vascular disease with high morbidity and mortality. Vascular smooth muscle deletion or dysplasia is an important reason for the development of cerebral aneurysm. MiRNAs participate in a variety of biological functions through inhibiting target gene translation. The aim of the present study was to evaluate the role of miRNAs in the regulation of vascular smooth muscle cell proliferation. MATERIALS AND METHODS: MiRNA and mRNA expressions were tested by Real-time PCR. Cell cycle was detected by flow cytometry. Cell viability was evaluated by MTT assay. HUASMC cell proliferation was determined by BrdU assay. Protein expressions were determined using Western blot. MiRNA target gene was confirmed by luciferase assay. RESULTS: MiR-370-3p expression was increased in cerebral aneurysm tissues. Ectopic expression of miR-370-3p suppressed proliferation of vascular smooth muscle cells and blocked cell cycle. Numerous cell proliferation and apoptosis-related factors were down-regulated by miR-370-3p. Results of target prediction database and dual-luciferase assay revealed that KDR is a direct target of miR-370-3p. Importantly, FOXO1 activity and AKT and FOXO1 phosphorylation were inhibited by miR-370-3p. We suggest that miR-370-3p directly targets KDR, resulting in the activation of AKT signaling pathway. CONCLUSIONS: MiR-370-3p was involved in the development of cerebral aneurysm by targeting KDR and blocking AKT/FOXO1 signaling pathway. The results provide theoretical basis for further investigation of potential clinical prevention and treatment of cerebral aneurysm.

Expression of Toll-like receptor 4 in the brain in a rabbit experimental subarachnoid haemorrhage model.

OBJECTIVE: To investigate the expression of the Toll-like receptor (TLR) 4 in the brain after experimental subarachnoid haemorrhage (SAH) in rabbits. METHODS: A total of 52 rabbits were randomly divided into four groups: control group; day 3, day 5, and day 7 groups. Day 3, day 5, and day 7 groups were all SAH groups in which the rabbits were killed on day 3, 5, and 7, respectively. In SAH groups, autologous arterial blood was injected into cisterna magna twice on day 0 and day 2. Immunostaining and immunoblotting experiments were performed to detect the expression of TLR4 protein. Reverse-transcriptase polymerase chain reaction was used to analyze the presence and quantity of TLR4 mRNA. RESULTS: The expressions of TLR4 protein and mRNA were increased remarkably in SAH groups compared with the control group. The immunohistochemical staining demonstrated high level expression of TLR4 was present mainly in the endothelial cells of capillaries in the brain. CONCLUSION: Our results indicate that TLR4 expression is upregulated in the brain after experimental SAH.