Epidermal growth factor-like domain 7 promotes the repair and reconstruction of cerebral microcirculation in rat models of craniocerebral injury

doi:10.3969/j.issn.2095-4344.1491

Abstract

Abstract:

BACKGROUND: Vascular endothelial cell damage is shown to be closely related to blood brain barrier function disorder after traumatic brain injury. Epidermal growth factor-like domain 7 (EGFL7) is an important factor to promote angiogenesis. Whether EGFL7 can promote traumatic brain injury repair by activating angiogenesis remains unknown.
OBJECTIVE: To explore the mechanisms by which EGFL7 promotes angiogenesis after traumatic brain injury in rats.
METHODS: Forty-two healthy adult male rats were randomly divided into craniocerebral injury group (n=36, craniocerebral injury model was established by impacting rat dura mater with modified Feeney method) and sham group (n=6, only complete dura mater was exposed) and the rats were divided into six subgroups according to the sample time after injury: 1 hour, 6 hours, 24 hours, 3 days, 7 days and 14 days, 6 rats in each subgroup. The contusion lesion and the surrounding brain tissue were obtained after the neurological injury score was available. Another 18 rats were selected and randomized into three groups (n=6/group) to establish craniocerebral injury model. rAd-vector, rAd-EGFL7 and combined PI3K/Akt pathway inhibitor rAd-EGFL7+LY294002 was respectively injected into the ventricles of rats at 1 hour after injury. Samples were obtained at 12 hours later.
RESULTS AND CONCLUSION: (1) RT-PCR, western blot assay and immumohistochemical staining results showed that the EGFL7 and CD34 were significantly up-regulated in rat brain after craniocerebral injury (P < 0.05). The expression of EGFL7 peaked on day 3 (P < 0.05), and the expression of CD34 had the same trend with EGFL7. The number of angiogenesis peaked on day 7. (2) After 12 hours of the rAd-EGFL7 microinjection in rat brain at 1 hour post-injury, the expression levels of EGFL7, CD34, pAkt and cyclin D1 were significantly up-regulated and the FOXO1 expression was significantly down-regulated (all P < 0.05). (3) Microinjection of rAd-EGFL7 and PI3K/Akt inhibitor in rat brain at 1 hour post-injury, the EGFL7 expression was not significantly changed, but the CD34 expression was significantly inhibited (P < 0.05), and the number of angiogenesis was significantly decreased (P < 0.05). (4) To conclude, up-regulated EGFL7 in the injured brain tissue may be related to angiogenesis after traumatic brain injury. Overexpression of the EGFL7 promotes the angiogenesis in the injured brain tissue through activating PI3K/Akt signaling.

Key words: EGF-like domain 7, CD34, traumatic brain injury, PI3K/Akt signaling, angiogenesis, modified Feeney method, cerebral injury repair, neurological dysfunction

CLC Number:

Xu Pengxiang1, Li Qiang1, Xu Qiongguan1, Luo Mengyanan2, Cheng Kai1, Xie Zhenming1, Fu Zhoufeng1 . Epidermal growth factor-like domain 7 promotes the repair and reconstruction of cerebral microcirculation in rat models of craniocerebral injury[J]. Chinese Journal of Tissue Engineering Research, 2019, 23(35): 5619-5625.

Figures/Tables 2

免疫组织化学染色结果显示，与假手术组相比，TBI模型组损伤脑组织中CD34蛋白表达水平及分布逐渐增加，且在TBI-7 d达到最大，而在TBI-14 d则减少到TBI 0-6 h水平，见图1D。免疫组织化学染色结果以胞浆/核呈棕黄色或棕褐色染色为阳性细胞，每只大鼠随机选取5张非连续切片，并于200倍光学显微镜下随机选择3个视野观察计数，最终结果取平均值，结果显示TBI模型组损伤脑组织中MVD数目逐渐增多，在TBI-7 d到达最大，较TBI-1 h显著增加(P < 0.05)，在TBI-14 d又恢复到TBI-6 h水平，较TBI- 7 d时显著减少(P < 0.05)，见图1E。结果表明，EGFL7在TBI大鼠颅脑损伤后脑组织中表达上调，其表达趋势与血管生成因子CD34的表达一致，提示TBI大鼠颅脑损伤后脑组织中高表达的EGFL7可能与血管再生过程有关。 2.4 EGFL7过表达活化PI3K/Akt信号据文献报道，EGFL家族成员均属于血管生成因子，在体外实验中，EGFL可通过激活PI3K信号通路诱导内皮细胞迁徙，促进血管形成[15]。为了验证在颅脑损伤模型中过表达EGFL7是否激活了PI3K/Akt信号通路，在注射rAd-EGFL7的颅脑损伤大鼠的损伤脑组织中检测PI3K/Akt信号关键基因pAkt、cyclin D1、FOXO1的表达。RT-PCR及Western blot结果显示，与对照rAd-vector相比，在大鼠脑损伤1 h，过表达EGFL7后损伤脑组织中的Akt及cyclin D1 mRNA转录水平及pAkt、cyclin D1蛋白水平显著增加(P < 0.05)，同时FOXO1的转录及蛋白翻译水平显著减少(P < 0.05)，见图2。结果表明过表达EGFL7后，颅脑损伤大鼠损伤脑组织中的PI3K/Akt信号通路被激活。 2.5 EGFL7激活PI3K/Akt信号促损伤后脑组织中血管新生前面的实验结果表明，EGFL7可激活颅脑损伤大鼠脑组织中的PI3K/Akt信号，因此实验猜测：颅脑损伤大鼠脑组织中上调的EGFL7能够通过激活PI3K/Akt信号促进损伤脑组织中的血管生成。为了验证这个猜测，在TBI-1 h脑损 "

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