Scutellarin protects oxygen/glucose-deprived astrocytes and reduces focal cerebral ischemic injury.
Scutellarin protects oxygen/glucose-deprived astrocytes and reduces focal cerebral ischemic injury.
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灯盏乙素保护缺氧/缺糖的星形胶质细胞并减少局灶性脑缺血损伤
DOI：
10.4103/1673-5374.235293
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发表时间：
2018-08
影响因子：
6.1
通讯作者：
Cheng X
中科院分区：
文献类型：
--
作者：
Sun JB;Li Y;Cai YF;Huang Y;Liu S;Yeung PK;Deng MZ;Sun GS;Zilundu PL;Hu QS;An RX;Zhou LH;Wang LX;Cheng X
灯盏乙素是从黄芩中分离得到的一种生物活性黄酮，具有抗炎、抗神经毒性、抗凋亡和抗氧化等作用，在我国已被用于治疗心脑血管疾病。然而，灯盏乙素介导脑缺血神经保护的机制尚不清楚。通过分子对接研究了野黄芩苷与烟酰胺腺嘌呤二核苷酸磷酸氧化酶2（NOX 2）的相互作用，结果表明野黄芩苷与NOX 2具有高亲和力的选择性结合。用2、10或50 μM野黄芩苷预处理从新生Sprague-Dawley大鼠大脑皮层分离的原代星形胶质细胞培养物30分钟。然后通过在95%N2/5%CO2培养箱中在无葡萄糖的Dulbecco改良Eagle培养基中孵育2小时，使星形胶质细胞经受氧/葡萄糖剥夺，随后模拟再灌注22小时。通过细胞计数试剂盒-8测定法评估细胞活力。Western blot检测NO X2、连接蛋白43和caspase-3的表达水平。活性氧物种进行了测定。10或50 μM野黄芩苷预处理显著增加缺血再灌注后星形胶质细胞的存活率，降低NOX 2和caspase-3的表达，增加连接蛋白43的表达，降低活性氧水平。我们还评估了灯盏乙素在大鼠短暂性大脑中动脉闭塞脑缺血再灌注损伤模型中的作用。术前2 h腹腔注射灯盏乙素100 mg/kg。采用Bederson量表评估神经功能缺损，采用氯化2，3，5-三苯基四氮唑染色测量梗死面积。Western blot检测脑组织中NOX 2和连接蛋白43的表达。采用酶联免疫吸附法检测脑组织中8-羟基脱氧鸟苷（8-OHdG）、4-羟基-2-壬烯醛（4-HNE）和3-硝基酪氨酸（3-NT）。免疫荧光双染法检测caspase-3和NeuN的共表达。灯盏乙素预处理可改善局灶性脑缺血大鼠的神经功能，缩小梗死面积，减少损伤脑组织中NOX 2的表达，降低8-OHdG、4-HNE和3-NT的水平，减少caspase-3和NeuN共表达细胞的数量。此外，我们还研究了NOX 2抑制剂apocynin的作用。夹竹桃麻素在体内和体外显著增加连接蛋白43的表达。总的来说，我们的研究结果表明，灯盏乙素在体外和体内通过下调NOX 2，上调连接蛋白43，减少氧化损伤和减少凋亡细胞死亡来保护缺血性损伤。
Scutellarin, a bioactive flavone isolated from Scutellaria baicalensis, has anti-inflammatory, anti-neurotoxic, anti-apoptotic and anti-oxidative effects and has been used to treat cardiovascular and cerebrovascular diseases in China. However, the mechanisms by which scutellarin mediates neuroprotection in cerebral ischemia remain unclear. The interaction between scutellarin and nicotinamide adenine dinucleotide phosphate oxidase 2 (NOX2) was assessed by molecular docking study, which showed that scutellarin selectively binds to NOX2 with high affinity. Cultures of primary astrocytes isolated from the cerebral cortex of neonatal Sprague-Dawley rats were pretreated with 2, 10 or 50 μM scutellarin for 30 minutes. The astrocytes were then subjected to oxygen/glucose deprivation by incubation for 2 hours in glucose-free Dulbecco's modified Eagle's medium in a 95% N2/5% CO2 incubator, followed by simulated reperfusion for 22 hours. Cell viability was assessed by cell counting kit-8 assay. Expression levels of NOX2, connexin 43 and caspase-3 were assessed by western blot assay. Reactive oxygen species were measured spectrophotometrically. Pretreatment with 10 or 50 μM scutellarin substantially increased viability, reduced the expression of NOX2 and caspase-3, increased the expression of connexin 43, and diminished the levels of reactive oxygen species in astrocytes subjected to ischemia-reperfusion. We also assessed the effects of scutellarin in vivo in the rat transient middle cerebral artery occlusion model of cerebral ischemia-reperfusion injury. Rats were given intraperitoneal injection of 100 mg/kg scutellarin 2 hours before surgery. The Bederson scale was used to assess neurological deficit, and 2,3,5-triphenyltetrazolium chloride staining was used to measure infarct size. Western blot assay was used to assess expression of NOX2 and connexin 43 in brain tissue. Enzyme-linked immunosorbent assay was used to detect 8-hydroxydeoxyguanosine (8-OHdG), 4-hydroxy-2-nonenal (4-HNE) and 3-nitrotyrosin (3-NT) in brain tissue. Immunofluorescence double staining was used to determine the co-expression of caspase-3 and NeuN. Pretreatment with scutellarin improved the neurological function of rats with focal cerebral ischemia, reduced infarct size, diminished the expression of NOX2, reduced levels of 8-OHdG, 4-HNE and 3-NT, and reduced the number of cells co-expressing caspase-3 and NeuN in the injured brain tissue. Furthermore, we examined the effect of the NOX2 inhibitor apocynin. Apocynin substantially increased connexin 43 expression in vivo and in vitro. Collectively, our findings suggest that scutellarin protects against ischemic injury in vitro and in vivo by downregulating NOX2, upregulating connexin 43, decreasing oxidative damage, and reducing apoptotic cell death.
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影响因子：
5.4
作者：
Cheng, Xiao;Hou, Zijun;Cai, Yefeng
通讯作者：
Cai, Yefeng
一种基于 TXNIP 的新机制，用于 Cx43 介导的氧化药物损伤调节。
影响因子：
5.3
作者：
Gao K;Chi Y;Zhang X;Zhang H;Li G;Sun W;Takeda M;Yao J
通讯作者：
Yao J
影响因子：
8.3
作者：
BEDERSON, JB;PITTS, LH;BARTKOWSKI, H
通讯作者：
BARTKOWSKI, H
影响因子：
6.2
作者：
Farahani, R;Pina-Benabou, MH;Rozental, R
通讯作者：
Rozental, R
DOI：
10.1083/jcb.145.4.795
发表时间：
1999-05-17
期刊：
The Journal of cell biology
影响因子：
--
作者：
Boitier E;Rea R;Duchen MR
通讯作者：
Duchen MR