Protective effect of microRNA-181c on the nerves of rats with cerebral hemorrhage by targeting cysteinyl aspartate-specific protease-3 signaling pathway
Objective To investigate the effect of microRNA(miR)-181c on the nerves of rats with cerebral hemorrhage(ICH)and the molecular mechanism of targeting cysteinyl aspartate-specific pro-tease(Caspase)-3 signaling pathway to regulate ICH.Methods Totally,60 SD male rats were divided in-to sham operation group(sham group),ICH group,miR-181c agonist group(ICH+agomir group)and miR-181c inhibitor group(ICH+antagomir group)by the random number table method.There were 15 rats in each group.RNA was extracted from brain tissue of rats in each group,and the expression level of miR-181c was detected by real-time fluorescent quantitative polymerase chain reaction(RT-qPCR).He-matoxylin-eosin staining,Nissl staining and Zea Longa scoring criteria were used to evaluate the brain inju-ry and neurological function of rats in each group.The levels of tumor necrosis factor-α(TNF-α),interleu-kin-1 β(IL-1 β),3-nitrotyrosine(3-NT)and 4-hydroxynonenal acid(4-HNE)in cerebrospinal fluid(CSF)were detected by enzyme-linked immunosorbent assay(ELISA).Western blotting was used to detect the expression of cleaved Caspase-3/pro-Caspase-3.Bioinformatics analysis and luciferase reporter gene experi-ment were used to analyze the targeted regulation relationship between miR-181c and Caspase-3.Independ-ent sample t test was used for comparison between the two groups.Results RT-qPCR results showed that the expression level of miR-181c in ICH group was lower than that in sham group(0.56±0.09 vs.1.00± 0.13,t=7.230,P＜0.05).HE staining showed that the number of neurons in the ICH group decreased,the volume became smaller,the arrangement was irregular,and the neurons displayed vacuole-like chan-ges,nuclear pyknosis and rupture,which were alleviated in the ICH+agomir group.Nissl staining showed that the number of Nissl bodies in the neuronal cytoplasm of ICH+agomir group was more than that of ICH group(98.30±14.12 vs.48.90±9.20,t=9.337,P＜0.01),and there was no significant difference between ICH+antagomir group and ICH group.ELISA results showed that the levels of TNF-α,IL-1β,3-NT and 4-HNE in brain tissue of ICH+agomir group were significantly lower than those of ICH group[TNF-α:(3.30±1.25)vs.(8.22±1.66)ng/ml,t=9.120,P＜0.01;IL-1β:(9.87±3.02)vs.(15.14±2.93)ng/ml,t=8.183,P＜0.01;3-NT:(23.39±3.44)vs.(45.61±2.65)pg/ml,t=7.902,P＜0.01;4-HNE:(10.10±1.75)vs.(14.93±2.63)pg/ml,t=5.778,P＜0.05].Western blotting showed that the expression level of leaved Caspase-3/pro-Caspase-3 in ICH group was significantly higher than that in sham group(2.63±0.58 vs.1.00±0.10,t=8.190,P＜0.01).The levels of leaved Caspase-3/pro-Caspase-3 in ICH+agomir group were significantly lower than those in ICH group(1.52± 0.37 vs.2.63±0.58,t=5.398,P＜0.05).Bioinformatics analysis showed that miR-181c and Caspase-3 had complementary nucleotide sequences.The luciferase activity in miR-181c mimics+WT-Caspase-3 group was significantly lower than that in NC mimics+WT-Caspase-3 group(0.52±0.07 vs.1.03±0.08,t=6.334,P＜0.01).Conclusion MiR-181c can reduce neurological damage and sec-ondary brain edema after ICH by targeting Caspase-3 signaling pathway,and reduce oxidative stress and in-flammatory response in brain tissue,which has a protective effect on ICH rats.
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