Mesenchymal stem cell conditioned medium alleviates oxidative stress injury induced by hydrogen peroxide via regulating miR143 and its target protein in hepatocytes

Xuejing Xu,Xiuli Ju,Xue Li,Qing Shi,Dong Li

doi:10.1186/s12865-017-0232-x

Abstract

BackgroundTo investigate the impact of miRNA (microRNA) on hepatic oxidative stress damage under the human mesenchymal stem cell conditioned medium (MSC-CM) and explore the roles of the beta-1 adrenergic receptor (ADRB1) and hexokinase 2 (HK2) in this process.MethodsHydrogen peroxide was used to induce oxidative stress injury in the human normal liver cell line L02. MSC-CM was separately prepared. After treatment with MSC-CM, the protective effects of MSC-CM on oxidative stress injury were assessed by changes in apoptosis, cell viability, cell cycle, and mitochondrial membrane potential. According to the microarray analysis, 19 disparately expressed miRNAs were selected for RT-PCR and miR143 identified as having significant differential expression in MSC-CM against oxidative stress injury. Subsequently, the predicted target proteins of miR143 were selected by bioinformatics software, and verified by western blot. In addition, down-regulation and up-regulation of miR143 expression and hydrogen peroxide induced hypoxia injury were carried out on L02 cells to study the role of miR143.ResultsMSC-CM significantly attenuated H2O2 induced oxidative stress injury. The expression of miR143 was increased following oxidative stress injury whereas it decreased after MSC-CM treatment. The expression levels of HK2 and ADRB1 regulated by miR143 and Bcl-2 decreased under H2O2 treatment but were restored following MSC-CM treatment. However the expression levels of Bax and BMF increased after H2O2 injury and decreased after MSC-CM treatment. Moreover over-expression or down-regulation of miR143 aggravated or alleviated hepatocyte apoptosis respectively.ConclusionsMSC-CM may alleviate H2O2 induced oxidative stress injury by inhibiting apoptosis and adjusting miRNA expression. Moreover down-regulation of miR143 protects L02 cells from apoptosis and initiates an adaptive process by adjusting the expression of HK2 ADRB1 and apoptosis-related proteins.

Highlights

To investigate the impact of miRNA on hepatic oxidative stress damage under the human mesenchymal stem cell conditioned medium (MSC-CM) and explore the roles of the beta-1 adrenergic receptor (ADRB1) and hexokinase 2 (HK2) in this process
Protective effect of Mesenchymal stem cell (MSC)-CM on apoptosis of L02 cells exposed to Hydrogen peroxide (H2O2) To quantify the effects of MSC-CM (20% MSC-CM for 24 h) on H2O2-induced (1 mM H2O2 for 3 h) cell apoptosis and necrosis, we performed flow cytometry with AnnexinV/Propidium iodide (PI) double staining, which was used to discriminate apoptosis from necrotic cells, JC-1 was employed to detect membrane potential (MMP) (Δψ), PI single staining was applied to detect cell cycle and Cell Counting Kit-8 (CCK-8) analysis was applied to demonstrate that the MSC-CM could prevent L02 cell from undergoing apoptosis
Compared with the H2O2 group, the apoptosis rate of normal cells increased by 16.31% ± 3.26% (65.69% ± 2.91% vs. 82.00% ± 3.11%, Fig. 2a–b, P < 0.05) in the H2O2 + MSC-CM group

Summary

Introduction

To investigate the impact of miRNA (microRNA) on hepatic oxidative stress damage under the human mesenchymal stem cell conditioned medium (MSC-CM) and explore the roles of the beta-1 adrenergic receptor (ADRB1) and hexokinase 2 (HK2) in this process. The mechanisms of hypoxia are mainly derived from cellular damage [5, 6]. No effective therapeutic protocol has been proven to modify the course of oxidative stress injury. Substantial previous work has demonstrated that mesenchymal stem cells (MSCs) have notable capability to repair ischemic organ tissue [7]. Studies have shown that MSC conditioned medium (MSC-CM) is a promising novel therapeutic agent to promote proliferation and antiapoptotic and anti-inflammatory responses after acute organ injury [8,9,10,11,12,13]. The rationale for MSC therapy is increasingly recognized to act by a secretion (paracrine) rather than differentiation mechanism [14,15,16]

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Discussion

Conclusion