GLP-2 ameliorates D-galactose induced muscle aging by IGF-1/Pi3k/Akt/FoxO3a signaling pathway in C2C12 cells and mice.

BACKGROUND: The study aimed to investigate the effect of Glucagon-like peptide-2 (GLP-2) on muscle aging in vivo and in vitro. METHODS: Six-week-old C57BL/6J mice were administered with D-galactose (200 mg/kg/day, intraperitoneally) for 8weeks, followed by daily subcutaneous injections of GLP-2 (300 or 600 µg/kg/day) for 4weeks. Skeletal muscle function and mass were evaluated using relative grip strength and muscle weight. The sizes and types of muscle fibers and apoptosis were assessed through histological analysis, immunofluorescence staining, and TUNEL staining, respectively. C2C12 myotubes were treated with D-galactose (40 mg/mL) and GLP-2. Protein expression of differentiation-related myogenic differentiation factor D (MyoD), myogenin (MyoG), and myosin heavy chain (Myhc), degradation-related Muscle RING finger 1 (MuRF-1), and muscle atrophy F-box (MAFbx)/Atrogin-1, and apoptosis-related B-cell leukemia/lymphoma 2 (Bcl-2) and Bax, were assessed using western blots. The Pi3k inhibitor LY294002 was applied to investigate whether GLP-2 regulated myogenesis and myotube aging via IGF-1/Pi3k/Akt/FoxO3a signaling pathway. RESULTS: The results demonstrated that GLP-2 significantly reversed the decline in muscles weight, relative grip strength, diameter, and cross-sectional area of muscle fibers induced by D-galactose in mice. Apart from suppressing the expressions of MuRF-1 and Atrogin-1 in the muscles and C2C12 myotubes, GLP-2 significantly increased the expressions of MyoD, MyoG, and Myhc compared to the D-galactose. GLP-2 significantly suppressed cell apoptosis. Western blot analysis indicated that the regulation of GLP-2 may be attributed to the activation of theIGF-1/Pi3k/Akt/FoxO3a phosphorylation pathway. CONCLUSIONS: This study suggested that GLP-2 ameliorated D-galactose induced muscle aging by IGF-1/Pi3k/Akt/FoxO3a pathway.

SGLT2 inhibitor empagliflozin alleviates cardiac remodeling and contractile anomalies in a FUNDC1-dependent manner in experimental Parkinson's disease.

Recent evidence shows a close link between Parkinson's disease (PD) and cardiac dysfunction with limited treatment options. Mitophagy plays a crucial role in the control of mitochondrial quantity, metabolic reprogramming and cell differentiation. Mutation of the mitophagy protein Parkin is directly associated with the onset of PD. Parkin-independent receptor-mediated mitophagy is also documented such as BCL2/adenovirus E1B 19 kDa protein-interacting protein 3 (BNIP3) and FUN14 domain containing 1 (FUNDC1) for receptor-mediated mitophagy. In this study we investigated cardiac function and mitophagy including FUNDC1 in PD patients and mouse models, and evaluated the therapeutic potential of a SGLT2 inhibitor empagliflozin. MPTP-induced PD model was established. PD patients and MPTP mice not only displayed pronounced motor defects, but also low plasma FUNDC1 levels, as well as cardiac ultrastructural and geometric anomalies (cardiac atrophy, interstitial fibrosis), functional anomalies (reduced E/A ratio, fractional shortening, ejection fraction, cardiomyocyte contraction) and mitochondrial injury (ultrastructural damage, UCP2, PGC1α, elevated mitochondrial Ca2+ uptake proteins MCU and VDAC1, and mitochondrial apoptotic protein calpain), dampened autophagy, FUNDC1 mitophagy and apoptosis. By Gene set enrichment analysis (GSEA), we found overtly altered glucose transmembrane transport in the midbrains of MPTP-treated mice. Intriguingly, administration of SGLT2 inhibitor empagliflozin (10 mg/kg, i.p., twice per week for 2 weeks) in MPTP-treated mice significantly ameliorated myocardial anomalies (with exception of VDAC1), but did not reconcile the motor defects or plasma FUNDC1. FUNDC1 global knockout (FUNDC1-/- mice) did not elicit any phenotype on cardiac geometry or function in the absence or presence of MPTP insult, but it nullified empagliflozin-caused cardioprotection against MPTP-induced cardiac anomalies including remodeling (atrophy and fibrosis), contractile dysfunction, Ca2+ homeostasis, mitochondrial (including MCU, mitochondrial Ca2+ overload, calpain, PARP1) and apoptotic anomalies. In neonatal and adult cardiomyocytes, treatment with PD neurotoxin preformed fibrils of α-synuclein (PFF) caused cytochrome c release and cardiomyocyte mechanical defects. These effects were mitigated by empagliflozin (10 µM) or MCU inhibitor Ru360 (10 µM). MCU activator kaempferol (10 µM) or calpain activator dibucaine (500 µM) nullified the empagliflozin-induced beneficial effects. These results suggest that empagliflozin protects against PD-induced cardiac anomalies, likely through FUNDC1-mediated regulation of mitochondrial integrity.

Plasminogen activator inhibitor-1 regulates LPS induced inflammation in rat macrophages through autophagy activation.

BACKGROUND: The mechanisms by which plasminogen activator inhibitor-1 (PAI-1) regulates inflammation, especially in acute respiratory distress syndrome (ARDS), are largely unknown. OBJECTIVE: To assess the relationship between PAI-1 and autophagy in inflammatory reactions induced by LPS in rat NR8383 cells. METHODS: ELISA was used to assess the amounts of TNF-α, IL-1ß, and PAI-1 in cell culture supernatants; TLR4, MyD88, PAI-1, LC3, Beclin1, and mTOR protein and mRNA levels were determined by western blot and quantitative RT-PCR, respectively; western blot was used to determine NF-κB protein levels. To further evaluate the role of PAI-1, the PAI-1 gene was downregulated and overexpressed using the siRNA transfection technology and the pCDH-PAI-1, respectively. Finally, the GFP Positive Expression Rate Method was used to determine the rate of GFP-LC3 positive NR8383 cells. RESULTS: In LPS-induced NR8383 cells, TNF-α, IL-1ß, and PAI-1 expression levels increased remarkably. Upon PAI-1 knockdown, TNF-α, IL-1ß, PAI-1, TLR4, MyD88, NF-κB, LC3, and Beclin1 levels were decreased, while mTOR increased. Conversely, overexpression of PAI-1 resulted in increased amounts of TNF-α, IL-1ß, PAI-1, TLR4, MyD88, NF-κB, LC3, and Beclin1. However, no significant change was observed in mTOR expression. CONCLUSIONS: In NR8383 cells, PAI-1 contributes in the regulation of LPS-induced inflammation, likely by promoting autophagy.

[The effect of lipopolysaccharide on gene expression of TLR4 and MD-2 in rat alveolar macrophage and its secretion of inflammation cytokines].

OBJECTIVE: To investigate the effect of lipopolysaccharide (LPS) on gene expression of Toll-Like receptor 4 (TLR4) and myeloid differentiation protein-2 (MD-2) in rat alveolar macrophage NR8383 cell and its secretion of inflammation cytokines with or without small interference RNA target MD-2 (MD2-siRNA) treatment. METHODS: NR8383 cell was cultured with F-12K medium and stimulated with LPS (0.01 approximately 10 mg/L) for 2 h or stimulated with 1 microg/ml LPS for 2-24 h. MD2-siRNA oligo was transfected into NR8383 cell by Lipofectamine 2000. The expression of TLR4 mRNA and MD-2 mRNA in cell were detected by semi-quantitative revels transcription polymerase (RT-PCR). The contents of TNF-alpha, IL-6 and IL-1beta in the cell cultured supernatant were tested by ELISA. One way ANOVA was used for difference comparison during groups and Pearson correlation was used for correlation analysis. RESULTS: (1) The mRNA expression of TLR4 and MD-2 in control NR8383 cell were 0.52+/-0.05 and 0.44+/-0.09, respectively. There was no obviously change after 0.01 mg/L LPS stimulation. The increase occurred in a dose dependent manner from 0.1 mg/L to 10 mg/L. The highest TLR4 and MD-2 mRNA expression were 0.72+/-0.06 and 0.65+/-0.10 (F=17.255 and 6.045, P<0.01). The changes of TNF-alpha, IL-6 and IL-1beta contents in cell cultured supernatant were similar with that of TLR4 and MD-2 gene expression. TNF-alpha, IL-6 and IL-1beta content were (25.8+/-3.4) ng/L, (62.4+/-4.7) ng/L and (31.6+/-1.7) ng/L in control group, while the corresponding contents were (58.9+/-5.3) ng/L, (96.5+/-3.9) ng/L and (55.4+/-5.4) ng/L after 10 mg/L LPS simulation (F=29.55, 54. 47 and 31.45, P<0. 01). (2) When stimulated with 1 microg/ml LPS, the mRNA expressions of TLR4 and MD-2 in NR8383 cell were increased from hour 2. The highest mRNA expressions of TLR4 and MD-2 occurred at hour 6, and then decreased slowly from hour 8. The mRNA expressions at hour 24 were still higher than those in cells without LPS stimulation (F=5.279 and 4.106, P<0.01). The changes of TNF-alpha, IL-6 and IL-1beta content in cell cultured supernatant were also similar with that of gene expression (F=10.64, 11.23 and 17.58, P<0. 01). The secretion peaks of TNF-alpha and IL-1beta occurred from hour 6 to hour 8, and the secretion peak of IL-6 occurred from hour 8 to hour12. (3) The mRNA expression of MD-2 was related with that of TLR4 positively (r=0.513, P<0.01). (4) The interfere efficiency of MD-2 siRNA was 67%. There was no obvious increase of TNF-alpha, Il-1beta and IL-6 in MD-2 siRNA treated group after LPS stimulation. CONCLUSION: Higher dose LPS can up-regulate the gene expression of TLR4 and MD-2 in rat alveolar macrophage NR8383 cell with a long time and promote the secretion of TNF-alpha, IL-6 and IL-1beta. MD-2 siRNA can inhibit NR8383 cell secrete TNF-alpha, IL-6 and IL-1beta induced by LPS.