ABSTRACT
BACKGROUND: Myocardial ischemia-reperfusion (I/R) is a serious and irreversible injury. Bone marrow-derived mesenchymal stem cells (MSCs) is considered to be a potential therapy for I/R injury due to the paracrine effects. High-mobility group box 1 (HMGB1) is a novel mediator in MSC and regulates the response of inflammation injury. Signal Transduction and Transcription Activator 3 (STAT3) is a critical transcription factor and important for release of paracrine factors. However, the relationship between HMGB1 and STAT3 in paracrine effect of MSC remains unknown. METHODS: In vitro, hypoxia/reoxygenation injury model was established by AnaeroPack System and examined by Annexin V flow cytometry, CCK8 assay and morphology observation. Detection of apoptotic proteins and protein expression of HMGB1 and STAT3 by Western blot. RESULTS: The conditioned medium of MSCs with or without LPS pretreatment was cocultured with H9C2 cells for 24 h before hypoxia treatment and MSC showed obvious cardiomyocytes protect role, as evidence by decreased apoptosis rate and improved cells viability, and LPS pretreated MSC exhibited better protect role than untreated MSC. However, such effect was abolished in HMGB1 deficiency group, silencing HMGB1 decreased the secretion of vascular endothelial growth factor (VEGF), hepatocyte growth factor (HGF), insulin growth factor (IGF), cell viability, and the expression of STAT3. Furthermore, STAT3 silence attenuated the protective effect of LPS in MSC. CONCLUSIONS: These findings suggested that LPS improved MSC-mediated cardiomyocytes protection by HMGB1/STAT3 signaling.
ABSTRACT
Mesenchymal stem cell-derived conditioned medium (MSC-CM) improves cardiac function after myocardial infarction; however, this cardioprotective effect is moderate and transient. Lipopolysaccharide (LPS) pretreatment partially improves MSC-CM-mediated cardioprotective effects owing to the presence of paracrine factors. However, the mechanism underlying these improved effects remains unknown. To study the effect of LPS-pretreated MSC-CM on hypoxia/reoxygenation (H/R)-induced injury, MSCs were treated with or without LPS (400 ng/mL) for 48 h, and the supernatant was collected (MSC-CM). Subsequently, H9c2 cells were co-cultured with Nor-CM (CM derived from LPS-untreated MSCs) and LPS-CM (CM derived from LPS-pretreated MSCs) for 24 h and subjected to H/R. MSC-CM inhibited the progression of H/R-induced injury in H9c2 cells, and this protective effect was enhanced via LPS pretreatment as evidenced by the improved apoptosis assessment index (i.e. caspase-3 and B-cell lymphoma-2 [Bcl-2] expression) and decreased levels of lactic dehydrogenase (LDH) and cardiac troponin (cTn). In addition, the results of haematoxylin-eosin staining (H&E), transmission electron microscopy (TEM) and TdT-mediated dUTP nick-end labelling (TUNEL) validated that MSC-CM inhibited H/R-induced injury in H9c2 cardiomyocytes. LPS pretreatment downregulated the expression of high mobility group box-1 (HMGB1) and BTB and CNC homology-1 (Bach1) proteins in MSCs but upregulated the expression of vascular endothelial growth factor (VEGF), hepatocyte growth factor (HGF) and insulin-like growth factor (IGF). HMGB1 knockdown (MSC/siHMGB1-CM) significantly decreased the expression of Bach1 and increased the expression of VEGF, HGF and IGF. Bach1 knockdown (MSC/siBach1-CM) did not alter the production of HMGB1 but increased the expression of VEGF and IGF. LPS pretreatment did not alter the expression of the paracrine factors VEGF and HGF in the MSC/siHMGB1 group but increased their expression in the MSC/siBach1 group. The myocyte anti-apoptotic effects of MSCs/siBach1-CM were similar to those of untreated MSCs, which were not enhanced by LPS. LPS-pretreated MSC-CM protects H9c2 cells against H/R-induced injury partly through the HMGB1/Bach1 signalling pathway.
Subject(s)
HMGB1 Protein , Lipopolysaccharides , Humans , Apoptosis , Basic-Leucine Zipper Transcription Factors/metabolism , Basic-Leucine Zipper Transcription Factors/pharmacology , HMGB1 Protein/metabolism , Hypoxia , Lipopolysaccharides/pharmacology , Myocytes, Cardiac , Signal Transduction , Vascular Endothelial Growth Factor A/pharmacology , Animals , Rats , Cell LineABSTRACT
Mesenchymal stem cell-derived conditioned medium (MSC-CM) improves cardiac function, which is partly attributed to the released paracrine factors. Since such cardioprotection is moderate and transient, it is essential that MSC-CM's effective components are optimized to alleviate myocardial injury. To optimize MSC-CM, MSCs were treated with or without lipopolysaccharides (LPSs) for 48 h (serum-free), and the supernatant was collected. Then, LPS-CM (MSC stimulated by LPS) was further treated with LPS remover (LPS Re-CM) or was concentrated with a 10 kDa cutoff filter (10 kDa-CM). Enzyme-linked immunosorbent assay showed that all the pretreatments increased the levels of vascular endothelial growth factor (VEGF), hepatocyte growth factor (HGF), and insulin growth factor (IGF) except LPS Re-CM; 10 kDa-CM was superior to the other CMs. Cell Counting Kit-8 displayed that the viability of injured H9c2 cells was enhanced with the increase in the MSC-CM concentration. We also found that the 10 kDa-CM significantly alleviated H9c2 hypoxia/reoxygenation (H/R) injury, as evidenced by the increased Bcl-2/Bax ratio, and decreased the levels of lactate dehydrogenase and cardiac troponin. Transmission electron microscopy (TEM), TdT-mediated dUTP nick-end labelling (TUNEL), and hematoxylin and eosin staining (H&E) confirmed that 10 kDa-CM inhibited H/R-induced H9c2 morphological changes. Proteomic analysis identified 41 differentially expressed proteins in 10 kDa-CM, among which anti-inflammation, proangiogenesis, and antiapoptosis were related to cardiac protection. This study indicates that 10 kDa-CM protects H9c2 cardiomyocytes from H/R injury by preserving most of the protective factors, such as VEGF, HGF, and IGF, in MSC-CM.