Yousef Faridvand1, Samira Nozari2, Vahid Vahedian3, Nasser Safaie4, Masoud Pezeshkian4, Parinaz Haddadi5, Mina Mamipour6, Arezoo Rezaie-Nezhad6, Ahmadreza Jodati7, Mohammad Nouri8. 1. Stem Cell and Regenerative Medicine (SCARM), Tabriz University of Medical Sciences, Tabriz, Iran; Department of Biochemistry and Clinical Laboratories, Faculty of Medicine, Tabriz University of Medical Sciences, Tabriz, Iran. 2. Stem Cell and Regenerative Medicine (SCARM), Tabriz University of Medical Sciences, Tabriz, Iran. 3. Rofeydeh Rehabilitation Hospital, University of Social Welfare and Rehabilitation Science (USWR), Tehran, Iran. 4. Cardiovascular Research Center, Tabriz University of Medical Sciences, Tabriz, Iran. 5. Department of Biochemistry, Faculty of Sciences, Tabriz University, Tabriz, Iran. 6. Stem Cell Research Center, Tabriz University of Medical Sciences, Tabriz, Iran. 7. Cardiovascular Research Center, Tabriz University of Medical Sciences, Tabriz, Iran. Electronic address: Jodati@tbzmed.ac.ir. 8. Stem Cell and Regenerative Medicine (SCARM), Tabriz University of Medical Sciences, Tabriz, Iran; Stem Cell Research Center, Tabriz University of Medical Sciences, Tabriz, Iran; Medical Biochemistry Biochemist & Embryologist Infertility Center Alzahra Hospital, Tabriz University of Medical Sciences, Tabriz, Iran. Electronic address: nourimd@yahoo.com.
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.
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.
Authors: Agnieszka Klama-Baryła; Ewa Rojczyk; Diana Kitala; Wojciech Łabuś; Wojciech Smętek; Katarzyna Wilemska-Kucharzewska; Marek Kucharzewski Journal: Int Wound J Date: 2020-01-15 Impact factor: 3.315