ObjectiveTo explore the feasibility and mechanism of inhibiting miR-429 to improve the permeability of the blood spinal cord barrier (BSCB) in vitro, and provide a new gene therapy target for enhancing the spinal cord microenvironment.MethodsFirst, the immortalized human brain microvascular endothelial cell line (hCMEC/D3) was transfected with the anti-miR-429 antagonist (antagomiR-429) and its negative control (antagomiR-429-NC), respectively. The miR-429 expression of hCMEC/D3 cells was observed by fluorescence microscopy and real-time fluorescence quantitative PCR to verify the transfection efficiency of antagomiR-429. Then the effect of miR-429 on BSCB permeability was observed in vitro. The experiment was divided into 4 groups. The blank control group (group A) was constructed of normal hCMEC/D3 cells and Ha-sc cells to prepare the BSCB model, the hypoxia-induced group (group B), the hypoxia-induced+antagomiR-429-NC group (group C), and the hypoxia-induced+antagomiR-429 group (group D) were constructed of normal, antagomiR-429-NC transfected, and antagomiR-429 transfected hCMEC/D3 cells and Ha-sc cells to prepare the BSCB models and hypoxia treatment for 12 hours. The permeability of BSCB in vitro was measured by horseradish peroxidase (HRP) permeability. Real-time fluorescence quantitative PCR, Western blot, and immunofluorescence staining were used to observe the expressions of ZO-1, Occludin, and Claudin-5.ResultsThe antagomiR-429 and antagomiR-429-NC were successfully transfected into hCMEC/D3 cells under a fluorescence microscope, and the transfection efficiency was about 90%. Real-time fluorescence quantitative PCR results showed that the relative expression of miR-429 in antagomiR-429 group was 0.109±0.013, which was significantly lower than that of antagomiR-429-NC group (0.956±0.004, P<0.05). HRP permeability measurement, real-time fluorescence quantitative PCR, and Western blot results showed that the HRP permeability of groups B and C were significantly higher than those of groups A and D (P<0.05), and the relative expressions of ZO-1, Occludin, and Claudin-5 proteins and mRNAs were significantly lower in groups B and C than in groups A and D (P<0.05) and in group D than in group A (P<0.05); there was no significant difference between groups B and C (P>0.05). Immunofluorescence staining showed that the immunofluorescence of ZO-1, Occudin, and Claudin-5 at the cell membrane boundary in group D were stronger than those in groups B and C, but not as strong as that in group A.ConclusionInhibition of miR-429 expression can promote the expressions of ZO-1, Occludin, and Claudin-5 proteins in microvascular endothelial cells, thereby improving the increased permeability of BSCB due to hypoxia.
Objective To investigate the mechanism of bone morphogenetic protein-4 (BMP4) in promoting the recovery of small intestinal mucosal barrier function during the recovery period of small intestine ischemia-reperfusion (I/R) injury. Methods Twenty-eight C57BL/6J male mice aged 6–8 weeks were randomly selected and assigned to small intestine I/R group (n=24) and sham operation (SO) group (n=4) by random number table method. Small intestine I/R injury models of 24 mice were established, then 4 mice were randomly selected at 6, 12, 24 and 48 h after I/R established modeling and killed to observe the morphological changes of small intestinal mucosa and detect the expression of BMP4 mRNA in the jejunal epithelial cells, the other 8 mice were allocated for the experimental observation at the recovery period of small intestine I/R injury (24 h after I/R was selected as the observation time point of recovery period of small intestine I/R injury according to the pre-experimental results). Twelve mice were randomly divided into I/R-24 h-BMP4 group (recombinant human BMP4 protein was injected intraperitoneally), I/R-24 h-NS (normal saline) group (NS was injected intraperitoneally), and I/R-24 h-blank group (did nothing), 4 mice in each group. Then the small intestinal transmembrane electrical impedance (TER) was measured by Ussing chamber. The expressions of BMP4 protein and tight junction proteins (occludin and ZO-1), Notch signaling pathway proteins (Notch1 and Jagged1), and Smad6 protein were detected by Western blot. Results At 24 h after I/R injury, the injuries of villous epithelium, edema, and a small part of villi were alleviated. The BMP4 mRNA expressions at 6, 12, 24 and 48 h after I/R injury in the small intestinal epithelial cells were increased as compared with the SO group. Compared with the I/R-24 h-NS group and the I/R-24 h-blank group, the TER was increased, and the expression levels of occludin, ZO-1, p-Smad6, Notch1, Jagged1 were increased in the I/R-24 h-BMP4 group. Conclusion From the preliminary results of this study, during recovery period of small intestine I/R injury, the expression of BMP4 in small intestinal epithelial cells is increased, permeability of jejunal mucosal barrier is increased, which might promote the recovery of small intestinal mucosal barrier function by activating the Notch signaling pathway (Notch1 and Jagged1), Smad classic signaling pathway, and promoting the increase of tight junction protein expression (occludin and ZO-1).