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

Abstract

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.

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

Fig. 1.1

In Vivo experimental workflow and cohort stratification

Fig. 1.2

In vitro experimental workflow and cohort stratification

Fig. 2

Levels of miR-210 in mouse brain tissue post-intracerebral hemorrhage at various time points

Fig. 3

Lentivirus-mediated Expression of miR-210 in Mouse Brain. (A) Coronal section of the mouse brain following induction of intracerebral hemorrhage (ICH); the designated boxes delineate the regions where immunofluorescence and TUNEL staining assessments were conducted. (B) Microscopic images demonstrating dual immunofluorescence staining for copGFP/NeuN, copGFP/GFAP, and copGFP/Iba-1 post lentiviral injection; scale bar equals 25 micrometers. (C) PCR analysis was conducted five days post-administration of LV-miR-210 and normal saline (NS) to quantify miR-210 expression in ipsilateral brain tissue. Each experiment was replicated six times, and the results are presented as mean ± SEM. ####Statistical significance: P < 0.0001; &&& denotes P < 0.001 when compared to the sham group; && denotes P < 0.01 when compared to the sham group

Fig. 4

Depicts the impact of miR-210 on neurological deficiencies and brain edema in mice following induced by ICH. (A) Typical swimming patterns of mice from each group were noted during the Morris water maze testing phase. (B) The study explored the effect of miR-210 on the escape latency times observed in the Morris water maze test. (C) The instances of platform crossings encountered during the probe trial were documented. (D) The duration mice spent in the correct quadrant during the probe trial was measured.(E) Brain water content was individually measured for each group, albeit this information did not directly correspond with the results of the probe trial. (F) Seven days post-ICH induction, modified neurological severity scores (mNSS) were assessed for mice in each group. All experiments were conducted six, yielding results presented as mean ± SEM. Statistical significance: ^##P < 0.01, ^#P < 0.05 indicate significant differences compared to the sham group, with ^&&&P < 0.001 and ^&&P < 0.01 denoting highly significant and significant differences, respectively, when compared to the sham group’s performance

Fig. 5

miR-210 inhibited ICH-induced autophagy and attenuated apoptosis and neuroinflammation. (A) Western blot analysis was employed to measure Beclin1, Bax, and Bcl-2 levels, as well as the LC3II/I, p-AMPK/AMPK, and p-mTOR/mTOR ratios in the brain tissue of mice from each group. (B) A quantitative assessment was conducted to determine the protein levels of Beclin1, Bax, and Bcl-2, and the ratios of LC3II/I, p-AMPK/AMPK, and p-mTOR/mTOR. (C) Real-time PCR was used to investigate the expression of iNOS, IL-1β, IL-6, TNF-α, and IL-10 in the brain tissue of mice from each group. (D) Brain sections were stained with TUNEL (green) to detect apoptotic cells and counterstained with DAPI (blue) to visualize nuclei; the scale bar is 100 μm. (E) The percentage of TUNEL-positive cells was calculated. All qPCR experiments were performed six times, WB experiments were performed three times. and the results are presented as mean ± SEM. Significance levels: ^####P < 0.0001, ^###P < 0.001, ^##P < 0.01, ^#P < 0.05; ^&&&P < 0.001, ^&&P < 0.01, ^&P < 0.05 compared to the sham group

Fig. 6

Promoted activation of AMPK/mTOR signaling reversed the neuroprotective effects of miR-210. (A) Western blot analysis was performed to assess the levels of Beclin 1, Bax, Bcl-2, and the ratios of LC3 II/I, p-AMPK/AMPK, and p-mTOR/mTOR in mouse brain tissue from each group. Subsequently, a quantitative evaluation was conducted specifically on the ratios of p-AMPK/AMPK, p-mTOR/mTOR, and LC3II/I, as well as the protein abundance of Bax, Bcl-2, and Beclin1, as detailed in (B), (C), (D), (E), (F), (G), (H). All experiments were performed three times. and the results are presented as mean ± SEM. Significance levels: ^####P < 0.0001, ^###P < 0.001, ^##P < 0.01, ^#P < 0.05; ^&&&P < 0.001, ^&&P < 0.01, ^&P < 0.05 compared to the sham group

Fig. 7

Role of miR-210 in Hemin-treated HT22 cells (A), (B) miR-210 level in each group; (C) Western blot analysis was performed to assess the levels of Beclin 1, Bax, Bcl-2, and the ratios of LC3 II/I, p-AMPK/AMPK, and p-mTOR/mTOR in HT22 from each group. Subsequently, a quantitative evaluation was conducted specifically on the ratios of p-AMPK/AMPK, p-mTOR/mTOR, and LC3II/I, as well as the protein abundance of Bax, Bcl-2, and Beclin1, as detailed in (D). All qPCR experiments were performed six times, WB experiments were performed three times, and the results are presented as mean ± SEM. Significance levels: ^####P < 0.0001, ^###P < 0.001, ^##P < 0.01, ^#P < 0.05; ^&&&P < 0.001, ^&&P < 0.01, ^&P < 0.05 compared to the sham group

Fig. 8

miR-210 regulates autophagy and apoptosis through AMPK/mTOR