Western转膜缓冲液(10×)

Objective Chronic insomnia can easily lead to clinical distress or cause mental, social, physical, educational, occupational, or other functional impairments. Considering the role of circadian rhythm in insomnia, we focused on exploring the action of miR-29a in regulating the PER2 gene in improving chronic insomnia. Methods LPS induces the expression of miRNAs targeting PER2 in HMC3 and PRM cells, which was verified by RT-qPCR. Poly-lactic- co -glycolic acid (PLGA) nanoparticles (NP) were used to encapsulate short hairpin (sh)-miR-29a to construct sh-miR-29a-NP. Morris water environment method was employed to establish a sleep deprivation rat model to investigate the therapeutic effects of sh-miR-29a-NP. Cell viability and levels of cell polarization factors were evaluated using CCK8 and ELISA, respectively. The Morris water maze test was applied to assess the learning and memory capabilities of the rats. Immunohistochemistry, immunofluorescence, and western blot were applied to test the expression of glial cell polarization, neuronal cell activation, apoptosis, and Period2 (PER2) /nuclear factor kappa B (NF-κB) axis proteins. Results miR-29a was significantly upregulated in LPS-induced HMC3 and PRM cells, with the most significantly altered miRNAs/PER2 interaction. In LPS-induced HMC3 and PRM cells, sh-miR-29a promoted the PER2 and CD206 expressions, and inhibited the expression of ionized calcium-binding adaptor molecule 1 (IBA-1), NF-κB, and CD86, while this effect was blocked by small interfering-PER2. Further in vivo experiments confirmed that PER2 and CD206 expression was reduced, while NF-κB, CD86, and IBA-1 expression were up-regulated in the hippocampal tissue of CSD rats. However, this effect was reversed by treatment with sh-miR-29a-NP. Treatment with sh-miR-29a-NP in CSD rats shortened the escape latency and increased the number of crossings over the original platform, while inhibited the expression of NLR Family Pyrin Domain Containing 3, caspase-1, Gasdermin D (GSDMD), and TUNEL signal in the hippocampal tissue. Conclusion The regulation of PER2/NF-κB pathway by sh-miR-29a-NP promoted M2 polarization of microglial cells and inhibited neuronal cell pyroptosis, thereby improving cognitive dysfunction in chronic insomnia.

Objective Stem cell therapy is expected to become a new treatment for central nervous system damage associated with perinatal hypoxic-ischemic encephalopathy (HIE), but the specific effects are unknown. This study explores the effects of human umbilical cord blood (HUCB) cells-secreted exosomal (HUCB-ex) MFG-E8 in neonatal rats with hypoxic-ischemic brain damage (HIBD), aiming to gain a theoretical foundation for the cure of perinatal HIE. Methods HIBD model was constructed in the Sprague Dawley rats (7-day-old). Rats were then intervened with 1×10 6 HUCB cells, HUCB-ex, or HUCB-ex oe-MFG-E8 , and HUCB-ex si-MFG-E8 . Primary microglia from rats were induced with oxygen-glucose deprivation and re-oxygenation (OGD/R), then co-cultured with either HUCB or primary neuronal cells, and subjected to treatment with HUCB-ex, HUCB-ex oe-MFG-E8 , HUCB-ex si-MFG-E8 , or Stattic. The expression of polarization factors and secreted factors in the microglia was measured using RT-qPCR, immunofluorescence, and western blot. Neuronal cell damage was assessed using MTT assays and flow cytometry. Behavioral impairments and brain tissue damage in the rats were evaluated using assays including the geotaxis reflex, cliff avoidance response, grip strength test, hematoxylin-eosin staining, TTC staining, and immunofluorescence. Results Early intervention with HUCB cells in HIBD rats increased test scores, decreased brain tissue weight, infarct area, as well as the IL-6, TNF-α, and IL-1β levels, and increased MFG-E8 levels. HUCB cells also decreased the levels of CD11b/c + CD45hi cells in HIBD rat brain tissue, and increased the levels of CD206 + CD11b/c + cells. In vitro experiments confirmed high expression of MFG-E8 in HUCB-ex. HUCB-ex si-MFG-E8 inhibited M2 polarization and induced neuronal cell injury through the SOCS3/STAT3 pathway. HUCB-ex and HUCB cells have equivalent therapeutic effects in HIBD rats. The treatment effectiveness of HUCB-ex was improved after delivering HUCB-ex oe-MFG-E8 , while was blocked after delivering HUCB-ex si-MFG-E8 . Conclusions HUCB-ex oe-MFG-E8 promoted M2 polarization of microglial and inhibited neuronal cell apoptosis through the SOCS3/STAT3 pathway, to alleviate behavioral disorders and brain tissue damage in HIBD rats.