Nrf2 activation and down-regulation of HMGB1 and MyD88 expression by amnion membrane extracts in response to the hypoxia-induced injury in cardiac H9c2 cells

Biomed Pharmacother. 2019 Jan:109:360-368. doi: 10.1016/j.biopha.2018.10.035. Epub 2018 Nov 3.

Abstract

Background: human Amniotic Membrane (hAM) extracts contain bioactive molecules such as growth factors and cytokines. Studies have confirmed the ability of hAM in reduction of post-operative dysfunction in patients with cardiac surgery. However, the function of Amniotic Membrane Proteins (AMPs), extracted from hAM, against hypoxia-induced H9c2 cells injury have never been investigated. In this study, we aimed to appraise the protective impact of AMPs on H9c2 cells under hypoxia condition.

Methods: Cardiomyocyte cells were pre-incubated with AMPs and subjected to 24 h hypoxia to elucidate its effects on expression of Nuclear factor erythroid 2-related factor 2 (Nrf2) and heme oxygenase-1(HO-1). Furthermore, the high mobility group box-1 (HMGB1) and Myeloid differentiation primary response 88 (MyD88) expressions were detected by qPCR and western-blotting. The mitochondrial membrane potential (ΔΨm) was estimated by JC-1 using fluorescent microscopy and fluorimetry. Moreover, the cell apoptosis and intracellular calcium levels were measured by flow cytometry.

Results: Pre-treatment of AMPs resulted in significant induction in cell viability and decreased the LDH release under hypoxic condition in H9c2 cells. Accordingly, these protective effects of AMPs were associated with a reduction in apoptosis rates and intracellular Ca2+, meanwhile, ΔΨm was increased. Pre-treatment with AMPs resulted in degradation of HMGB1 and MyD88 levels and depicted pro-survival efficacy of AMPs against hypoxia-induced cell damage through induction of HO-1 and Nrf2.

Conclusion: The data indicated that AMPs mediated HO-1 regulation by Nrf2 activation and plays critical protective effects in hypoxia-induced H9c2 injury in vitro by the inhibition of myocardial HMGB1 and MyD88 inflammatory cascade.

Keywords: Amniotic membrane proteins; H9c2 cardiomyocytes; HMGB1; HO-1; Hypoxia; Nrf2.

MeSH terms

  • Amnion / metabolism*
  • Animals
  • Cell Hypoxia / drug effects
  • Cell Hypoxia / physiology
  • Cell Line
  • Down-Regulation / drug effects
  • Down-Regulation / physiology*
  • Female
  • Gene Expression
  • HMGB1 Protein / antagonists & inhibitors
  • HMGB1 Protein / metabolism*
  • Humans
  • Intercellular Signaling Peptides and Proteins / isolation & purification
  • Intercellular Signaling Peptides and Proteins / pharmacology
  • Myeloid Differentiation Factor 88 / antagonists & inhibitors
  • Myeloid Differentiation Factor 88 / metabolism*
  • NF-E2-Related Factor 2 / metabolism*
  • Rats

Substances

  • HMGB1 Protein
  • HMGB1 protein, human
  • Intercellular Signaling Peptides and Proteins
  • MYD88 protein, human
  • Myeloid Differentiation Factor 88
  • NF-E2-Related Factor 2
  • NFE2L2 protein, human