miR-210 Regulates Autophagy Through the AMPK/mTOR Signaling Pathway, Reduces Neuronal Cell Death and Inflammatory Responses, and Enhances Functional Recovery Following Cerebral Hemorrhage in Mice

Recently, a growing body of research has shown that MicroRNAs (miRNAs) are crucial in the pathophysiological mechanisms of brain disorders, miR-210 is one of the significant miRNAs implicated in these disorders, and its function in intracerebral hemorrhage (ICH) is not yet fully understood. Research the impact of miR-210 on intracerebral hemorrhage and probe into its working mechanism. The ICH model was established by injecting collagenase into the basal ganglia of male C57/BL6 mice (n = 142). Firstly, the mice were divided into sham group (n = 6) and ICH group (n = 30) (3 h, 6 h, 12 h, 24 h, 72 h), the samples of the sham group were collected at 48 h after operation, the brain tissues of the left and right basal ganglia were collected in each groupand. qPCR was used to detect the level of miR-210 in each group. Then, LV-miR-210 was injected into the lateral ventricle to establish a model of miR-210 overexpression, and NS injection was set as a comparison, which was divided into sham group (n = 15), ICH group (n = 15), ICH + NS group (n = 15), and ICH + LV-miR-210 group (n = 15). Water maze training was started on the 2 d after surgery. qPCR was used to detect the levels of miR-210, iNOS, IL-1β, IL-6, TNF-α, and IL-10 in each group at 3 d after operation. Western blotting was used to detect the levels of p-AMPK/AMPK, p-mTOR/mTOR, Beclin 1, Bax, Bcl-2, and LC3 II/I in each group. Immunofluorescence was used to detect the expression of lentivirus-mediated miR-210 in mouse brain. Water maze was used to evaluate the learning and memory function of the mice. The dry-wet method was used to evaluate brain edema, TUNEL was used to detect the Apoptosis of brain cells in each group. Then, Rapamycin and AICAR were used to intervene p-AMPK/AMPK and p-mTOR/mTOR, and they were divided into sham group (n = 6), ICH group (n = 6), ICH + LV-miR-210 group (n = 6), ICH + LV-miR-210 + AICAR group (n = 6), and ICH + LV-miR-210 + Rapamycin group (n = 6). The levels of miR-210 in each group were detected by qPCR at 3 d after operation, and the levels of p-AMPK/AMPK, p-mTOR/mTOR, Beclin 1, Bax, Bcl-2, and LC3 II/I in each group were detected by WB. Finally, HT22 cells were stimulated with Hemin to construct an in vitro intracerebral hemorrhage model, and the time gradient was set (control group, 3 h, 6 h, 12 h, and 24 h). qPCR was used to detect the expression of miR-210 in each group. Then HT22 cells were transfected with lentivirus, and rapamycin and AICAR were used to interfere with p-AMPK/AMPK and p-mTOR/mTOR. Control group, Hemin group, Hemin + LV-miR-210 group, Hemin + LV-miR-210 + AICAR group, and Hemin + LV-miR-210 + Rapamycin group. qPCR was used to detect the level of miR-210 in each group. The levels of p-AMPK/AMPK, p-mTOR/mTOR, Beclin 1, Bax, Bcl-2, and LC3 II/I in each group were detected by Western blotting. miR-210 is significantly increased in a short time after intracerebral hemorrhage in mice. miR-210 can alleviate secondary injury of ICH by improving neurological deficit and reducing brain edema. In addition, upregulation of miR-210 expression inhibited Autophagy and alleviated Apoptosis and inflammation. In our study, we found that miR-210 significantly inhibited the activation of AMPK/ mTOR pathway triggered by ICH, and the neuroprotective effect of miR-210 was partially reversed when Rapamycin and AICAR reversed this inhibition. At the mechanistic level, miR-210 exerts its function by regulating AMPK/mTOR signaling pathway, thereby inhibiting Autophagy and reducing Apoptosis and inflammation. Further studies at the cellular level were basically consistent with the above results. miR-210 is up-regulated after ICH and can play a neuroprotective role by regulating the AMPK/mTOR signaling pathway mediated by Autophagy, suggesting that it may become a therapeutic target for reducing nerve injury after ICH.

miR-210; Apoptosis; Autophagy; Cerebral hemorrhage; Neuroinflammation.