ABSTRACT
Objective To investigate the regulatory effects of activated endoplasmic reticulum stress（ERS） and unfolded protein response（UPR） on the immune behavior of the stressed muscle fibers in inflammatory environments induced by interferon-γ（IFN-γ）. Methods The myogenic precursor cells（MPCs） of C57 BL/6 mice cultured in vitro were differentiated into multinucleated myogenic tubes by horse serum and then to set up: 1. Control group; 2. IFN-γ group; 3. Tunicamycin group; 4. Thapsigargin group; 5. IFN-γ and 4-phenylbutyrate（4-PBA） combined treatment group; 6. IFN-γ, TG and 4-PBA combined treatment group; 7. IFN-γ and 4μ8 c combined treatment group; 8. IFN-γ, TG and 4μ8 c combined treatment group; 9. IFN-γ and GSK2606414 combined treatment group; 10. IFN-γ, TG and GSK2606414 combined treatment group. The level of myokines gene was detected by Real-time PCR. The expression of UPR key molecules including eukaryotic intiatio factor 2α（eIF2α）, inositol requrring enzyme 1α（IRE1α） and activating transcription factor 6（ATF6） in muscle fibers was observed by immunofluorescence. Western blotting was used to detect immune molecules related to muscle cells, myokines and key molecules of UPR. Luminex analyzed the levels of pro-inflammatory myokines in muscle fibers. Results The expression of H-2 Kb, H2-Ea, Toll like receptor 3（TLR3）, p-eIF2α and p-IRE1α were up-regulated in IFN-γ induced inflammatory environment. The expression of H-2 Kb, H2-Ea, TLR3 and myokines in the group with UPR inhibitor 4-PBA was down-regulated compared with IFN-γ group, and the expression of these molecules in the group with IRE1α specific inhibitor 4μ8 c was down-regulated compared with the IFN-γ group. The addition of protein kinase R-like endoplasmic eticulum（PERK） specific inhibitor GSK2606414 showed no significant change. Conclusion In IFN-γ induced inflammatory environment, the UPR-IRE1α pathway activates and inhibits the synthesis of muscle fiber immune-related molecules, which further inhibits the muscle fiber mediated immune response and facilitates muscle regeneration.
ABSTRACT
<p><b>OBJECTIVE</b>To investigate the effects of neuropeptide Y (NPY) Y1 receptor antagonist PD160170 in promoting osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) and accelerating healing of femoral defect in rats.</p><p><b>METHODS</b>The third generation of rat BMSCs were treated with PBS (control) or 10, 10, or 10 mol/L NPY Y1 receptor antagonist PD160170. After 7 and 14 days of treatment, the cells were examined for osteogenic differentiation with alkaline phosphatase (ALP) and alizarin red staining. At 7 and 21 days of treatment, the mRNA and protein expressions of collagen type I (COLI), osteocalcin (OCN) and Runt-related transcription factor 2 (Runx2) in the cells were detected using q-PCR and Westem Blotting. In a male SD rat model (body weight 300∓20 g) of bilateral femoral condyle defects (2.5 mm in diameter), the effect of daily local injection of 0.2 mL PD160170 (10 and 10 mol/L, for 28 consecutive days) in promoting bone defect repair was evaluated with micro-CT scans.</p><p><b>RESULTS</b>ALP and alizarin red staining showed that the BMSCs treated with PD160170, at the optimal concentration of 10 mol/L, contained more intracellular cytoplasmic brown particles and mineralized nodules in extracellular matrix than PBS-treated cells. PD160170 (10 mol/L) significantly up-regulated the mRNA and protein expressions of COLI at day 7 and those of OCN and Runx2 at day 21 (P<0.05). In the rat models of femoral bone defect, the volume/tissue volume ratio, bone mineral density and the number of bone trabeculae were significantly greater in 10 mol/L PD160170 group than in the control group (P<0.05), but the bone trabecular thickness (P=0.07) and bone volume (P=0.35) were similar between the two groups.</p><p><b>CONCLUSION</b>NPY Y1 receptor antagonist PD160170 can promote osteogenic differentiation of BMSCs and healing of femoral defects in rats, suggesting the potential of therapeutic strategies targeting NPY Y1 receptor signaling in the prevention and treatment of bone fracture and osteoporosis.</p>