Figure legends
Figure 1. H19, miR-29b, and C1QTNF6 expression in patients with acute stroke. A . Quantitative PCR of H19, miR-29b, and C1QTNF6 levels in neutrophils of patients with acute stroke. Acute ischemic stroke group (N = 50) and control group (N = 42). B . Correlation between miR-29b and C1QTNF6 expression at admission (N = 50). C . Correlation of H19 and miR-29b expression with the NIHSS score at admission (N = 50). D . The predicted binding sites between miR-29b and C1QTNF6 and between miR-29b and H19.E. Plasma TNF-α, IL-1β, and MMP-9 levels in patients with acute ischemic stroke (N = 50). *P < 0.05, ***P < 0.001 vs. control group.
Figure 2. Changes in H19 and miR-29b levels, as well as subsequent effects on cerebral injury and neurological deficits in MCAO rats. A . H19 and miR-29b expression in leukocytes and ischemic brain tissue after MCAO injury (N = 9). B . Quantitative PCR was performed to confirm the efficacy of H19 siRNA and miR-29b antagomir transfection (N = 9). C . Effects of intravenous injections of H19 siRNA and miR-29b antagomir on cerebral injury and neurological function deficits at 24 h after stroke (N = 12). D andE . Neuronal apoptosis in the ipsilateral cortex as detected by NeuN/TUNEL immunofluorescence double staining (N = 6). *P < 0.01, ***P < 0.001 vs. sham group. #P < 0.05, ###P < 0.001 vs. MCAO group. Scale bar = 20 μm.
Figure 3. H19 and miR-29b regulated C1QTNF6 expression in the leukocytes of MCAO rats A. C1QTNF6, IL-1β, and TNF-α protein levels in leukocytes were assessed by western blotting (N = 6).B. C1QTNF6, IL-1β, and TNF-α protein levels in the ipsilateral brain tissue were assessed by western blotting (N = 6). *P < 0.05 vs. sham group; #P < 0.05, MCAO group.
Figure 4. H19 siRNA reversed miR-29b antagomir-aggravated acute cerebral ischemic injury. A. Cerebral infarct volume and brain oedema evaluated by TTC staining of coronal brain sections (N = 12).B. Neuronal apoptosis in the ipsilateral cortex as detected by NeuN/TUNEL immunofluorescence double staining (N = 6). C.C1QTNF6 mRNA, IL-1β, and TNF-α protein levels in the leukocytes were assessed by qRT-PCR and western blotting (N = 6). D. C1QTNF6, IL-1β, and TNF-α protein levels in the ipsilateral brain tissue were assessed by western blotting (N = 6). &P < 0.05, &&&P < 0.001 vs. MCAO + miR-29b antagomir + H19 siRNA control group. Scale bar = 20 μm.
Figure 5. MiR-29b antagomir and H19 siRNA regulated C1QTNF6 levels in leukocytes. A. Immunofluorescence detection of C1QTNF6 in leukocytes (CD11b+) at 24 hours after MCAO (N = 6).B. Leukocytes (CD11b+) levels in brain tissue (N = 6).C. C1QTNF6 expression levels in leukocytes were assessed by immunofluorescence (N = 6). ***P < 0.001 vs. Sham group; #P < 0.05 vs. MCAO group. &P < 0.05 vs. MCAO+miR-29b antagomir + H19 siRNA control group. Scale bar = 20 μm.
Figure 6. H19 and miR-29b regulated ZO-1 and occludin expression in hCMEC/D3 cells A and B. hCMEC/D3 cell apoptosis induced by H19 siRNA and miR-29b antagomir treatment (N = 6).C. C1QTNF6, IL-1β, and TNF-α protein levels in HL-60 cells were assessed by western blotting (N = 6). D. ZO-1 and occludin protein levels in hCMEC/D3 cells were assessed by western blotting (N = 6). * P < 0.05 vs. Sham group; *** P < 0.001 vs. Sham group; # P < 0.05 vs. OGD group; ## P < 0.01 vs. OGD group; &P < 0.05 vs. OGD+miR-29b antagomir group; &&&P < 0.001 vs. OGD+miR-29b antagomir group.
Figure 7. Schematic diagram depicting the possible involvement of the H19/miR-29b/C1QTNF6 axis in cerebral ischemic stroke. The ischemia-dependent activation of the H19/miR-29b/C1QTNF6 axis enhances IL-1β and TNF-α expression in leukocytes; moreover, it causes further BBB disruption to aggravate neuron injury.