The Basic Research On The Function And Mechanism Of MiR-21-5p In The Restoration Of Neuro-vascular Unit After Traumatic Brain Injury

Objective: Traumatic brain injury(TBI) results in an extremely high rate of mortality and disability. The severity of secondary brain damage is closely related to the prognosis of TBI. The neurovascular unit(NVU) is a functional unit composed of neurons, gliocytes, vascular endothelial cells and extracellular matrix. The interactions of these components are capable of maintaining homeostasis in brain. Promoting the restoration of injured NVU is crucial for the treatment of secondary brain injury and the management of TBI. The research on mi RNAs in TBI has not been widely reported. In the previous study of our research group, we found that the expression level of mi R-21-5p in the injured cerebral cortex increased apparently after TBI. In addition, up-regulation of mi R-21-5p level can alleviate cellular apoptosis in brain and improve the neurological outcome after TBI. This research will employ TBI rats to further elucidate the function and mechanism of mi R-21-5p in the restoration of injured NVU. It will also provide insights into developing a novel therapeutic strategy of mi RNAs therapy for TBI.Methods: 1. Detect the expression level of mi R-21-5p in the traumatic foci, and its in-situ expression in neurons, astrocytes, microglias and brain microvascular endothelial cells(BMVECs) using q RT-PCR and combined mi RNA in-situ hybridization(ISH) and immunoflorescent(IF) staining. 2. Evaluate the neurological outcome of TBI rats using modified Neurological Severity Score and the Morris Water Maze. Evaluate the impact of mi R-21-5p on cellular apoptosis in brain after TBI using TUNEL assay. Evaluate the impact of mi R-21-5p on the angiogenesis in brain after TBI by measuring the microvascular density(MVD) using IF staining. Evaluate the impact of mi R-21-5p on the permeability of BBB after TBI by measuring the wet/dry weight of traumatic hemisphere, the amount of Evans Blue dye extravasation, and the expression level of tight junction proteins(including Occludin and Claudin-5). 3. Detecting the expression of possible target gene(PTEN), signaling pathway and downstream proteins(apoptosis-related proteins, including Bax, Bcl-2 and Caspase-3; angiogenesis-related proteins, including Ang-1 and Tie-2) under the regulation of mi R-21-5p in TBI using Western Blot and q RT-PCR.Results: 1.(1) The mi R-21-5p level in the traumatic foci is increased at 6h post-injury, reached the peak level at 3d post-injury, then gradually declined to baseline at 14 d post-injury. And its expressions in neurons, astrocytes, microglias and BMVECs are all increased in various degree after TBI.(2) In the nerve tissue of brain, mi R-21-5p are mainly expressed in neurons and astrocytes.(3) The intracerebroventricular infusion of liposome-transfected mi R-21-5p agomir/antagomir can up-/down-regulate the mi R-21-5p level in the traumatic foci at 6h, 1d and 3d post-injury. This method can also up-/down-regulate the in-situ expression of mi R-21-5p in above cells at the same time points. 2.(1) Increased mi R-21-5p in brain after TBI contributes to improve the neurological outcome after TBI.(2) mi R-21-5p can inhibit cellular apoptosis in brain.(3) mi R-21-5p can promote angiogenesis in brain.(4) mi R-21-5p can alleviate BBB leakage. 3.(1) mi R-21-5p can impact the expression level of apoptosis-related proteins in brain after TBI.(2) mi R-21-5p can target PTEN and inhibit its expression at the post-transcriptional level, and promote the phosphorylation of Akt in brain.(3) mi R-21-5p can promote the expression of angiogenesis-related proteins(Ang-1 and Tie-2) in brain.Conclusion: 1. The mi R-21-5p level in the traumatic foci is increased after TBI, and its expressions in neurons, astrocytes, microglias and BMVECs are all increased in various degree. 2. mi R-21-5p inhibits cellular apoptosis in brain after TBI by regulating the expression of apoptosis-related proteins through targeting PTEN(inhibiting its expression at the post-transcriptional level) and activating the Akt signaling. 3. BMVECs-expressed mi R-21-5p promotes angiogenesis and reduces BBB leakage in brain after TBI by activating the Ang-1/Tie-2 signaling. 4. mi R-21-5p promotes the restoration of neuro-vascular unit through inhibiting cellular apoptosis, promoting angiogenesis and reducing BBB leakage in brain after TBI. Thus, it can alleviate the secondary brain damage and improve the neurological outcome after TBI. 5. mi R-21-5p could be a potential therapeutic target for interventions of secondary brain damage after TBI.