Recombinant milk fat globule-EGF factor-8 reduces apoptosis via integrin β3/FAK/PI3K/AKT signaling pathway in rats after traumatic brain injury.
Recombinant milk fat globule-EGF factor-8 reduces apoptosis via integrin β3/FAK/PI3K/AKT signaling pathway in rats after traumatic brain injury.
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DOI：
10.1038/s41419-018-0939-5
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发表时间：
2018-08-28
影响因子：
9
通讯作者：
Hang CH
中科院分区：
文献类型：
--
作者：
Gao YY;Zhang ZH;Zhuang Z;Lu Y;Wu LY;Ye ZN;Zhang XS;Chen CL;Li W;Hang CH
越来越多的证据表明，创伤性脑损伤（TBI）后神经元凋亡有可能导致更有害的病理过程。先前的研究已经确定，乳脂球-EGF因子-8（MFG-E8）通过调节炎症、氧化应激，尤其是脑缺血和神经退行性疾病中的细胞凋亡，提供神经保护。然而，MFG-E8对TBI中神经元凋亡的影响尚未研究。因此，我们探讨了MFG-E8在TBI后抗凋亡的作用及其可能的机制。在第一组实验中，将成年雄性Sprague-Dawley（SD）大鼠随机分为假手术组和TBI组，每组进一步分为代表不同时间点（6小时、24小时、72小时和7天）的五组（每组n = 9）。通过Western印迹、实时荧光定量PCR和免疫荧光染色鉴定MFG-E8的表达和细胞定位。在第二组实验中，随机分配四组：假手术组、TBI +媒介物组和TBI + rhMFG-E8（1和3 μg）（n = 15）。重组人MFGE 8（rhMFG-E8）以两种浓度通过侧脑室（i. c. v.）在TBI诱导后1小时注射。在TBI后24和72 h测量脑含水量、神经系统严重程度评分、蛋白质印迹法和免疫荧光染色。在最后一组实验中，i. c. v.注射MFG-E8 siRNA（500 pmol/3 µl）、整联蛋白β3 siRNA（500 pmol/3 µl）和PI 3 K抑制剂LY 294002（5和20 µM），然后将大鼠暴露于TBI。采用Western blotting、免疫荧光染色、脑组织含水量、神经功能缺损评分和Fluoro-Jade C（FJC）染色等方法研究整合素β3/FAK/PI 3 K/AKT信号通路对MFG-E8介导的TBI后抗凋亡作用的影响。MFG-E8主要表达于小胶质细胞，伤后24 h达高峰。在TBI后24和72 h，rhMFG-E8（3 μg）治疗显著降低脑含水量，改善神经功能缺损，并减少神经元凋亡。rhMFG-E8可显著增强整合素β3/FAK/PI 3 K/AKT通路相关组分的表达。给予整合素-β3 siRNA和LY 294002（5和20 μM）消除了rhMFG-E8对TBI后抗凋亡和神经保护的作用。本研究首次证明rhMFG-E8抑制神经元凋亡并提供神经保护。这表明通过调节整合素-β3/FAK/PI 3 K/AKT信号通路发生，突出了rhMFG-E8作为TBI患者的潜在有希望的治疗策略。
Accumulating evidence suggests neuronal apoptosis has the potential to lead to more harmful effects in the pathological processes following traumatic brain injury (TBI). Previous studies have established that milk fat globule-EGF factor-8 (MFG-E8) provides neuroprotection through modulation of inflammation, oxidative stress, and especially apoptosis in cerebral ischemia and neurodegenerative disease. However, the effects of MFG-E8 on neuronal apoptosis in TBI have not yet been investigated. Therefore, we explored the role of MFG-E8 on anti-apoptosis and its potential mechanism following TBI. In the first set of experiments, adult male Sprague–Dawley (SD) rats were randomly divided into Sham and TBI groups that were each further divided into five groups representing different time points (6 h, 24 h, 72 h, and 7 days) (n = 9 each). Western blotting, quantitative real-time PCR, and immunofluorescence staining were performed to identify the expression and cellular localization of MFG-E8. In the second set of experiments, four groups were randomly assigned: Sham group, TBI + Vehicle group, and TBI + rhMFG-E8 (1 and 3 µg) (n = 15). Recombinant human MFGE8 (rhMFG-E8) was administrated as two concentrations through intracerebroventricular (i.c.v.) injection at 1 h after TBI induction. Brain water content, neurological severity score, western blotting, and immunofluorescence staining were measured at 24 and 72 h following TBI. In the final set of experiments, MFG-E8 siRNA (500 pmol/3 µl), integrin β3 siRNA (500 pmol/3 µl), and PI3K inhibitor LY294002 (5 and 20 µM) were injected i.c.v. and thereafter rats exposed to TBI. Western blotting, immunofluorescence staining, brain water content, neurological severity score, and Fluoro-Jade C (FJC) staining were used to investigate the effect of the integrin-β3/FAK/PI3K/AKT signaling pathway on MFG-E8-mediated anti-apoptosis after TBI. The expression of MFG-E8 was mainly located in microglial cells and increased to peak at 24 h after TBI. Treatment with rhMFG-E8 (3 µg) markedly decreased brain water content, improved neurological deficits, and reduced neuronal apoptosis at 24 and 72 h after TBI. rhMFG-E8 significantly enhanced the expression of integrin-β3/FAK/PI3K/AKT pathway-related components. Administration of integrin-β3 siRNA and LY294002 (5 and 20 µM) abolished the effect of rhMFG-E8 on anti-apoptosis and neuroprotection after TBI. This study demonstrated for the first time that rhMFG-E8 inhibits neuronal apoptosis and offers neuroprotection. This is suggested to occur through the modulation of the integrin-β3/FAK/PI3K/AKT signaling pathway, highlighting rhMFG-E8 as a potentially promising therapeutic strategy for TBI patients.
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DOI：
10.1083/jcb.144.6.1337
发表时间：
1999-03-22
期刊：
The Journal of cell biology
影响因子：
--
作者：
Farrelly N;Lee YJ;Oliver J;Dive C;Streuli CH
通讯作者：
Streuli CH
影响因子：
82.9
作者：
Silvestre, JS;Théry, C;Mallat, Z
通讯作者：
Mallat, Z
影响因子：
64.5
作者：
Ensslin, MA;Shur, BD
通讯作者：
Shur, BD
影响因子：
4
作者：
Raymond, Adam;Ensslin, Michael A.;Shur, Barry D.
通讯作者：
Shur, Barry D.
影响因子：
9.3
作者：
Li E;Noda M;Doi Y;Parajuli B;Kawanokuchi J;Sonobe Y;Takeuchi H;Mizuno T;Suzumura A
通讯作者：
Suzumura A