Effect of miR-20b on the motor dysfunction after traumatic brain injury in mice and the underlying mechanism / 中华创伤杂志

ABSTRACT

Objective:To investigate effect of miR-20b on the motor dysfunction after traumatic brain injury (TBI) in mice and the underlying mechanism.Methods:Sixty C57BL/6J mice were divided into sham group, TBI group and TBI+miR-20b Agomir (Agomir-20b) group according to the random number table, with 20 mice per group. A model of severe TBI was induced by controlled cortical impact. After injury, the mice in TBI group were subjected to tail-vein injection of 200 μl Agomir-negative control at dosage of 50 μmol/L and the mice in TBI+Agomir-20b group were subjected to tail-vein injection of 200 μl Agomir-20b at dosage of 50 μmol/L. At days 3 and 7 postinjury, the rate of neuronal apoptosis in the pericontusional region was detected by TUNEL assay, expression levels of apoptosis-related proteins in the pericontusional region were detected by Western blot analysis, including cleaved caspase-3, cleaved poly adenosine diphosphate-ribose polymerases (PARP), B-cell lymphoma-2 (Bcl-2) and Bcl-2-associated X protein (Bax), motor function was evaluated by beam walking test, and expression levels of cytokine mRNAs in the pericontusional region were detected by real-time quantitative PCR (RT-qPCR), including interleukin (IL)-1β, IL-6, IL-10, inducible nitric oxide synthase (iNOS), arginase (Arg) and macrophage mannose receptor 1 (CD206).Results:In TUNEL assay, the rate of neuronal apoptosis in sham group was significantly lower than that in TBI group and TBI+Agomir-20b group at days 3 and 7 postinjury (all P<0.01), and there was a significantly lower rate of neuronal apoptosis in TBI+Agomir-20b group as compared with TBI group (all P<0.01). In Western blot analysis, significantly increased levels of cleaved caspase-3, cleaved PARP and Bax proteins and lowered level of Bcl-2 protein were observed in TBI group at days 3 and 7 postinjury as compared with sham group (all P<0.01); similar levels of cleaved caspase-3, cleaved PARP and Bax proteins were found in TBI+Agomir-20b group at days 3 and 7 postinjury as compared with sham group (all P>0.05), and level of Bcl-2 protein in TBI+Agomir-20b group also showed no obvious variation at day 7 postinjury as compared with sham group ( P>0.05) in regardless of a significant reduction at day 3 postinjury ( P<0.01). Significantly increased levels of cleaved caspase-3, cleaved PARP and Bax proteins as well as a significantly reduced level of Bcl-2 protein were found in TBI group at days 3 and 7 postinjury as compared with TBI+Agomir-20b group (all P<0.05 or 0.01). In beam walking test, the latency and foot slip rate in TBI group were significantly higher than those in sham group and TBI+Agomir-20b group at days 3 and 7 postinjury (all P<0.01). In RT-qPCR analysis, levels of IL-1β, IL-6 and iNOS mRNA in TBI group were significantly higher than those in TBI+Agomir-20b group at days 3 and 7 postinjury (all P<0.01), but the two groups were similar in levels of IL-10, Arg and CD206 mRNA (all P>0.05). In comparison with sham group, levels of IL-1β, IL-6, iNOS and IL-10 mRNA in TBI+Agomir-20b group had no obvious change at days 3 and 7 postinjury (all P>0.05); level of Arg mRNA in TBI+Agomir-20b group was significantly increased at days 3 and 7 postinjury (all P<0.01); level of CD206 mRNA in TBI+Agomir-20b group did not change significantly at day 3 postinjury ( P>0.05), but was significantly increased at day 7 postinjury ( P<0.01). Conclusions:miR-20b can obviously inhibit neuronal apoptosis to improve motor function after TBI in mice, for which the underlying mechanism is related to Agomir-20b inhibiting the transformation of microglia to pro-inflammatory M1 type after TBI.