Angiopoietin-1 alleviates LPS-induced inflammatory injury by up-regulation of miR-126 in pancreas cell line HPDE6-C7.

BACKGROUND

Acute pancreatitis is an inflammatory disorder of the pancreas, leading to multiple organ dysfunction syndrome in severe cases. Angiopoietin-1 (Ang-1), a Tie-2 receptor agonist, and microRNA-126 (miR-126) have been reported to be involved in angiogenesis and anti-inflammatory functions. In the present study, we explored the effects of Ang-1 and miR-126 on lipopolysaccharide (LPS)-induced inflammatory injury in pancreatic cells, HPDE6-C7.

METHODS

The immortalized human pancreatic duct epithelial cell line, HPDE6-C7, was treated with LPS (10 µg/mL) to induce cell injury. Ang-1 was used at 300 ng/mL concentration. Cell viability was measured using CCK-8 assay and apoptosis was assessed using flow cytometry. Quantitative real time polymerase chain reaction (RT-PCR) was used to measure the mRNA expressions of different proteins. Enzyme linked immunosorbent assay (ELISA) was used to measure the concentrations of the pro-inflammatory cytokines. Luciferase activity assay was done to identify the target of miR-126. Western blot was used to measure the expressions of different proteins.

RESULTS

Ang-1 promoted LPS-suppressed cell viability and inhibited LPS-induced cell apoptosis, pro-inflammatory cytokine (TNF-α, IL-1β, IL-6, and IL-8) production, and activation of NF-κB and JNK pathways in HPDE6-C7 cells. Furthermore, Ang-1 promoted the expression of miR-126, which in turn protected PDE6-C7 cells against LPS-induced injury. PDCD4 was identified as a direct target of miR-126 and was negatively regulated by miR-126. Mechanistic study revealed that overexpression of PDCD4 reversed miR-126-mediated inhibition of LPS-induced activation of NF-κB and JNK pathways.

CONCLUSION

Ang-1 alleviates LPS-induced inflammatory injury by up-regulation of miR-126 and down-regulation of PDCD4 in pancreatic cell line HPDE6-C7.