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    Urolithin A alleviates blood-brain barrier disruption and attenuates neuronal apoptosis following traumatic brain injury in mice
  • Figure 1|Urolithin A attenuates blood-brain barrier leakage and tight junction protein disruption 72 hours following traumatic brain injury. 

    The results from the brain water content assay, a pilot study, showed that TBI significantly increased brain water content (P < 0.001, compared with sham) and was reversed by UA administration of 2.5 mg/kg (P = 0.016, compared with TBI + vehicle). Compared with TBI + UA (2.5 mg/kg), 5 and 10 mg/kg UA did not further reduce brain water content (P = 0.395). No significant difference was found between sham and sham + vehicle (P = 0.227; Figure 1A). Therefore, we used 2.5 mg/kg as the dosage for subsequent experiments to reduce unnecessary sacrifice of experimental animals.
    To investigate whether UA alleviates TBI-induced BBB disruption, Evans blue dye extravasation was performed to evaluate BBB permeability. The TBI + vehicle group exhibited more extravasation compared with the sham group, and UA administration after TBI alleviated the extravasation (Figure 1B). Quantification of the extravasation showed a significant increase in the EB concentration in brain tissue in the TBI + vehicle group compared with the sham group (P < 0.001), and UA administration after TBI significantly reduced EB concentration compared with TBI + vehicle (P = 0.005; Figure 1C), indicating that UA administration can alleviate TBI-induced BBB leakage.
    Tight junction proteins are closely associated with BBB function (Obermeier et al., 2013), and therefore we investigated the changes in expression of ZO-1 and occludin in injured cortex. Immunofluorescence staining showed that tight junction proteins accompany the vascular marker CD31. Following TBI, the integrity of tight junction proteins was disrupted and multiple gaps were observed (white arrows; Figure 1D and E), but there was no disruption of CD31 integrity. The TBI + UA group exhibited fewer gaps compared with the TBI + vehicle group (Figure 1D and E). Western blot was used to assess the expression of tight junction proteins (Figure 1F and G). Consistent with immunofluorescence staining, the expression of tight junction proteins was significantly reduced after TBI (P < 0.001, compared with sham), and UA administration after TBI alleviated the reduction of tight junction proteins compared with TBI + vehicle (P < 0.001).

    Figure 2|Urolithin A attenuates neuronal apoptosis 72 hours following traumatic brain injury. 

    To assess whether UA could attenuate neuronal apoptosis following TBI, we performed TUNEL/NeuN double immunostaining of cerebral tissue ipsilateral to the injury site (Figure 2A). Extensive neuronal apoptosis was observed in the cortex following TBI. The number of apoptotic neurons was significantly decreased in the TBI + UA group compared with that in the TBI + vehicle group (P < 0.001), indicating that UA attenuated neuronal apoptosis after TBI (Figure 2B).
    Caspase-3 and bcl-2 are key regulators of the apoptotic pathway (Franklin, 2011). Therefore, we investigated the cortical expression of cleaved caspase-3, caspase-3, and bcl-2 by western blot. Consistent with the TUNEL assay, our results showed that UA administration after TBI significantly reduced the protein expression of cleaved caspase-3 and increased the protein expression of bcl-2 compared with TBI + vehicle (P < 0.001 and P = 0.008, respectively; Figure 2C and D). Caspase-3 levels did not significantly change among the three groups (P = 0.488).

    Figure 3|Urolithin A reinforces neuronal autophagy in injured cortex activated by traumatic brain injury.

    UA administration activates autophagy (Ahsan et al., 2019; Andreux et al., 2019; Lin et al., 2020). To investigate autophagy levels in cortical neurons, we used immunofluorescence to identify LC3 and p62, two autophagy markers, and the neuron marker NeuN in injured cortex (Figure 3A and B). We found that LC3 fluorescence intensity significantly increased following TBI, and the TBI + UA group had higher LC3 fluorescence intensity than the TBI + vehicle group (P < 0.001). In comparison, p62 fluorescence intensity significantly decreased following TBI, and the TBI + UA group had lower p62 fluorescence intensity than the TBI + vehicle group (P = 0.010; Figure 3C). Both LC3 and p62 immunostaining were surrounded by or merged with NeuN immunostaining in injured cortex. Consistent with our immunostaining results, western blot of injured cortex also showed increased LC3-II expression and decreased p62 expression following TBI (P < 0.001 for both LC3-II and p62, compared with sham), and the TBI + UA group had higher LC3-II expression (P = 0.006) and lower p62 expression (P = 0.003; Figure 3D and E) than the TBI + vehicle group. These results indicate that neuronal autophagy is activated following TBI, and UA administration could reinforce neuronal autophagy.

    Figure 4|PI3K/Akt/mTOR and Akt/IKK/NFκB signaling pathways may be involved in the neuroprotective effect of urolithin A.

    Many publications have reported that UA acts as an inhibitor of Akt phosphorylation, thus reinforcing autophagy via the phosphatidylinositol 3-kinase (PI3K)/Akt/mTOR signaling pathway (Komatsu et al., 2018; Xu et al., 2018; Totiger et al., 2019). Akt is also a key regulator in the Akt/IKK/NFκB signaling pathway, which regulates neuroinflammation (Ozes et al., 1999). Therefore, we speculate that the neuroprotective effects of UA may be partially achieved by inhibiting Akt phosphorylation, thus reinforcing autophagy and attenuating neuroinflammation via the PI3K/Akt/mTOR and Akt/IKK/NFκB signaling pathways, respectively (Figure 4A). Western blot analysis of injured cortex samples showed that the phosphorylation levels of Akt and mTOR decreased after TBI (P < 0.001, compared with sham) and were further decreased by UA administration after TBI (P = 0.010 and P = 0.001, for Akt and mTOR, respectively, compared with TBI + vehicle). The phosphorylation levels of IKKα and NFκB increased after TBI (P < 0.001, compared with sham) and were reduced by UA administration after TBI (P < 0.001; Figure 4B and C). These results indicate the involvement of the PI3K/Akt/mTOR and Akt/IKK/NFκB signaling pathways in the neuroprotective effects of UA to reinforce autophagy and attenuate neuroinflammation.

    Figure 5|Urolithin A ameliorates neurological deficits following traumatic brain injury. 

    We evaluated the neurological deficits in mice by the mNSS score and the rotarod test. Our results showed that the mNSS score of the TBI + UA group was significantly reduced 72 hours after TBI; this reduction continued to 14 days following TBI (P = 0.658, P = 0.002, P = 0.007, and P = 0.003, for 1, 3, 7, and 14 days after TBI, respectively; Figure 5A). The rotarod test showed that the TBI + UA group had a longer latency to fall than the TBI + vehicle group at 3, 7, and 14 days following TBI (P = 0.226, P = 0.008, P < 0.001, and P < 0.001, for 1, 3, 7, and 14 days after TBI, respectively; Figure 5B).


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  • 发布日期: 2022-03-10  浏览: 298
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