Dexmedetomidine attenuates propofol-induced apoptosis of neonatal hippocampal astrocytes by inhibiting the Bcl2l1 signalling pathway.

Apoptosis shapes brain structure and function during early life. However, aberrant apoptosis, including that associated with the general anaesthetic propofol, is undesirable. Dexmedetomidine (DEX) provides potential neuroprotection against apoptosis, but the underlying mechanism remains unknown. We exposed neonatal rodent hippocampal astrocytes to propofol alone and in combination with DEX and yohimbine (an α2 -adrenergic receptor antagonist), then evaluated cell viability using the MTT assay. The underlying regulatory mechanism associated with apoptosis-related genes was detected using a combinational strategy including double immunofluorescent staining, real-time reverse transcription polymerase chain reaction (RT-PCR), western blot, and flow cytometry. Propofol reduced matrix metallopeptidase 9 (MMP9) in cultured astrocytes, and activated the rno-miR-665/Bcl2-like 1 (Bcl2l1)/cleaved caspase 9 (CC9)/cleaved caspase 3 (CC3) pathway. Combinations incorporating propofol with A-1155463 (a selective Bcl2l1 inhibitor) or MMP9 antagomir reduced Bcl2l1 and promoted apoptosis. Co-culture of propofol with Bcl2l1 or with MMP9 agomir was sufficient to decrease the pro-apoptotic effects of propofol. Interestingly, DEX alone had no significant effect on apoptosis. When combined with propofol, however, the negative effects of propofol on the MMP9 and apoptosis-related genes (Bcl2l1, CC9, and CC3) were reduced. Furthermore, yohimbine pretreatment blocked the neuroprotective effects of DEX. Rno-miR-665 was also found to reduce MMP9 expression in propofol-treated hippocampal astrocytes. Taken together, the results indicate that DEX pretreatment reduces propofol-associated pro-apoptosis in developing astrocytes via downregulation of anti-apoptotic signalling mediated by Bcl2l1.

rno-miR-665 targets BCL2L1 (Bcl-xl) and increases vulnerability to propofol in developing astrocytes.

Propofol exerts a cytotoxic influence over immature neurocytes. Our previous study revealed that clinically relevant doses of propofol accelerated apoptosis of primary cultured astrocytes of developing rodent brains via rno-miR-665 regulation. However, the role of rno-miR-665 during the growth spurt of neonatal rodent brains in vivo is still uncertain. Post-natal day 7 (P7) rats received a single injection of propofol 30 mg/kg intraperitoneally (i.p.), and neuroapoptosis of hippocampal astrocytes was analyzed by immunofluorescence and scanning electron microscopy. The differential expression of rno-miR-665, BCL2L1 (Bcl-xl), and cleaved caspase 3 (CC3) was surveyed by qRT-PCR and western blotting. In addition, the utility of A-1155463, a highly potent and BCL2L1-selective antagonist, was aimed to assess the contribution of BCL2L1 for neuroglial survival. Following the intraventricular injection of lentivirus rno-miR-665, neuroprotection was detected by 5-point scale measurement. The single dose of propofol 30 mg/kg triggered dose-dependent apoptosis of developing hippocampal astrocytes. Meanwhile, propofol triggered both rno-miR-665 and CC3, and depressed BCL2L1, which was predicted as one target gene of rno-miR-665. Combination treatment with A-1155463 and propofol induced lower mRNA and protein levels of BCL2L1 and more CC3 activation than propofol treatment alone in vivo. The lentivirus-mediated knockdown of rno-miR-665 elevated BCL2L1 and attenuated CC3 levels, whereas up-regulation of rno-miR-665 suppressed BCL2L1 and induced CC3 expression in vivo. More importantly, rno-miR-665 antagomir infusion improved neurological outcomes of pups receiving propofol during the brain growth spurt. Rno-miR-665, providing a potential target for alternative therapeutics for pediatric anesthesia, is susceptible to propofol by negatively targeting antiapoptotic BCL2L1. Relatively little is known about the association between exposure of astrocytes to brief propofol anaesthesia and risk for impairment. Here, it revealed that propofol-related neurotoxicity of neonatal astrocytes was under rno-miR-665 regulation during the brain growth spurt. Rno-miR-665 might act as a clinically alternative therapeutic target for treatment of neurological disorders in peadiatric anesthesia or sedation with propofol in future.

LncRNA SNHG4 Attenuates Inflammatory Responses by Sponging miR-449c-5p and Up-Regulating STAT6 in Microglial During Cerebral Ischemia-Reperfusion Injury.

BACKGROUND: Inflammatory response mediated by microglia plays a key role in cerebral ischemia-reperfusion injury. This study intends to probe the role of lncRNA SNHG4 in regulating the inflammatory response of the microglia during cerebral ischemia reperfusion. MATERIALS AND METHODS: Blood samples and cerebrospinal fluid samples were collected from acute cerebral infarction (ACI) patients and healthy controls. The middle cerebral artery occlusion (MCAO) models were constructed with rats. LPS induction and oxygen-glucose deprivation methods were respectively applied to simulate the activation of microglia in vitro. qRT-PCR was employed to determine the expressions of SNHG4, miR-449c-5p and related inflammatory factors in vivo and in vitro. The inflammatory responses of the microglia subject to the varied expressions of SNHG4 and miR-449c-5p were detected. Luciferase assays were conducted to verify the crosstalk involving SNHG4, miR-449c-5p and STAT6. RESULTS: Compared with the control group, the expression of SNHG4 derived from the samples of ACI patients and the microglia of MCAO group were remarkably down-regulated, but the expression of miR-449c-5p was dramatically up-regulated. Overexpression of SNHG4 and knock-down of miR-449c-5p could inhibit the expression of pro-inflammatory cytokine in the microglia and promote the expression of anti-inflammatory factors. Meanwhile, the phospho-STAT6 was up-regulated, whereas the knock-down of SNHG4 and over-expression of miR-449c-5p in microglia had the opposite effects. Luciferase assay confirmed that SNHG4 could target miR-449c-5p, while miR-449c-5p could target STAT6. CONCLUSION: SNHG4 can regulate STAT6 and repress inflammation by adsorbing miR-449c-5p in microglia during cerebral ischemia-reperfusion injury.

Propofol-induced rno-miR-665 targets BCL2L1 and influences apoptosis in rodent developing hippocampal astrocytes.

Propofol exerts neurotoxic effects on the developing mammalian brains, but the underlying molecular mechanism remains unclear. MicroRNAs (miRNAs) are a class of small noncoding RNAs that modulate gene expression at the post-transcriptional level. However, in specific types of neurocytes, the detailed functions of miRNAs were not entirely understood. We investigated the potential role of miRNAs in astrocyte pathogenesis caused by propofol. We performed genome-wide microRNA expression profiling in immature cultured hippocampal astrocytes by microarray analysis and predicted their targets and functions using bioinformatics tools. The functional effects of one differentially expressed miRNA were examined experimentally in relation to astrocyte viability. The results showed that 13 miRNAs were significantly differentially expressed after both short-term exposure to high-concentration propofol (10 µg/ml for 1h) and long-term exposure to low-concentration propofol (0.9 µg/ml for 48 h), including rno-miR-665, differing significantly between the 2. Bioinformatics predicted putative binding sites for rno-miR-665 existing in the 3'-untranslated region of Bcl-2-like protein 1 BCL2L1 (Bcl-xl) mRNA. Moreover, such relationship was assessed by luciferase reporter assay, qRT-PCR and western blot. Rno-miR-665 which was significantly up-regulated by propofol can suppress BCL2L1 and elevate cleaved caspase-3 expression in immature astrocytes in vitro. Apoptosis of developing hippocampal astrocytes was thus significantly influenced by propofol or rno-miR-665, or both. Taken together, rno-miR-665 is involved in the neurotoxicity induced by propofol via a caspase-3 mediated mechanism by negatively regulating BCL2L1. It might act as an alternative therapeutic target for treatment of neurological disorders in peadiatric prolonged anesthesia or sedation with propofol clinically.