Role and related mechanism of Mst-1 on regulating hypoxic reoxygenation induced autophagy and apoptosis in cardiomyocytes of mouse / 中华心血管病杂志

Objective: To explore the role and related mechanism of mammalian sterile 20-like kinase 1(Mst-1)in regulating hypoxia reoxygenation (HR) induced myocardial cell autophagy and apoptosis. Methods: Enzyme digestion method combined with differential adherent method was used to culture neonatal mouse myocardial cells. HR model was established by hypoxia for 24 hours and reoxygenation for 6 hours. The experimental groups including control group (normal cultured cardiomyocytes), Mst-1 empty virus group (cardiomyocytes transfected with recombinant lentiviral empty vector for 48 hours), Mst-1 knockdown group (recombinant lentivirus carrying Mst-1small interfering RNA (siRNA) was transfected into cardiomyocytes for 48 hours), Mst-1 overexpression group (cardiomyocytes were transfected with recombinant lentivirus carrying Mst-1 gene for 48 hours), HR group (cardiomyocytes exposed to HR), Mst-1 knockdown+HR group (HR model of cardiomyocyte was established 48 hours after transfection with recombinant lentivirus carrying Mst-1siRNA) and Mst-1 overexpression+HR group (HR model of cardiomyocyte was established 48 hours after transfection with recombinant lentivirus carrying Mst-1 gene). Real-time fluorescence quantitative RCR (qPCR) and Western blot were used to detect the relative expression of Mst-1 mRNA and protein in the cells, immunofluorescence staining was used to detect cardiomyocyte troponin T (cTnT), and autophagosomes and autophagy enzyme changes. TUNEL method was used to detect myocardial cell apoptosis, Western blot was adopted to detect autophagy-related protein microtubule-related protein 1 light chain 3 (LC3) Ⅱ/LC3 Ⅰ, P62 and apoptosis-related protein cleaved-caspase 9, pro-caspase 9, cleaved-caspase-3, pro-caspase-3, and myeloid leukemia 1 (MCL-1) expression. MCL-1 inhibitor A1210477 was used to validate the signaling pathway of Mst-1 on regulating cardiomyocyte apoptosis and autophagy. Results: Immunofluorescence detection revealed that the cultured cells expressed cardiomyocyte-specific marker cTnT. The expression of Mst-1 in cardiomyocytes increased in HR model. Lentiviral transfection could effectively inhibit or overexpress Mst-1 in treated cells. The levels of autophagosomes and autophagolysosomes in cardiomyocytes undergoing HR and in Mst-1 overexpression+HR group were lower than those of control group, while autophagosomes and autophagolysosomes in cardiomyocytes of Mst-1 knockdown+HR group was significantly higher than in the HR group (all P<0.05). The TUNEL results showed that the proportion of TUNEL positive cells was significantly increased in the HR group and Mst-1 overexpression+HR group than in the control group, while the proportion of TUNEL positive cells was significantly decreased in the Mst-1 knockdown group+HR group as compared to the HR group (all P<0.05). Western blot results showed that the LC3 Ⅱ/LC3 Ⅰ levels were significantly lower, while the expression levels of P62, cleaved-caspase-9 and cleaved-caspase-3 were significantly higher in the HR group and Mst-1 overexpression+HR group than in control group (all P<0.05). The LC3 Ⅱ/LC3 Ⅰ value was significantly higher, and the expression levels of P62, cleaved-caspase-9 and cleaved-caspase-3 were significantly lower in the Mst-1 knockdown+HR group than in the HR group (P both<0.05). The expression level of P-MCL-1 protein was significantly lower in cardiomyocytes of HR and Mst-1 overexpression+HR group than in control group, and the expression level of P-MCL-1 protein was higher in Mst-1 knockdown+HR group than in HR group (P both<0.05). The recovery experiment showed that inhibiting MCL-1 in cells can block the regulatory effect of Mst-1 siRNA on cell autophagy and apoptosis. Conclusion: Inhibiting Mst-1 expression in cardiomyocytes can promote the autophagy of cardiomyocytes induced by hypoxic reoxygenation and reduce the apoptosis of cardiomyocytes via activating McL-1.

Effect of gene modified mesenchymal stem cells overexpression human receptor activity modified protein 1 on inflammation and cardiac repair in a rabbit model of myocardial infarction / 中华心血管病杂志

<p><b>OBJECTIVE</b>To investigate the effect of mesenchymal stem cells (MSCs) overexpressing human receptor activity modified protein 1 (hRAMP1) by adenovirus vector on infarction related inflammation and cardiac repair in a rabbit model of myocardial infarction (MI).</p><p><b>METHODS</b>Thirty rabbits underwent coronary artery ligation for 60 minutes followed by 24 hours reperfusion and divided into MSC(hRAMP1) group (intravenously injection of MSCs transfected with adenovirus vector encoding hRAMP1 gene enhanced green fluorescent protein, EGFP, n = 10), MSC(null) group (MSCs transfected with adenovirus vector encoding only EGFP without hRAMP1 gene, n = 10) and control group (equally volume of phosphate buffered saline, PBS, n = 10). The plasma level of tumor necrosis factor-α (TNF-α) and interleukin-10 (IL-10) were quantified by ELISA assay at before and 1, 3, 7, 28 days after induction of MI. The expression of nuclear factor-κB (NF-κB) and hRAMP1 in infracted myocardium were measured by Western blot at 1, 3, 7, 28 day following MI. The area of MI and collagen deposition and fibrosis were evaluated by TTC staining and Masson staining, respectively.</p><p><b>RESULTS</b>Area of MI and collagen content were significantly reduced in MSC(hRAMP1) group compared those in MSC(null) and control group [(10.1 ± 2.9)% vs. (30.6 ± 2.7)% and (22.5 ± 3.2)%, P < 0.05]. Myocardial expression of NF-κB and plasma TNF-α[7 days after transplantation: (97.2 ± 6.7) pg/ml vs. (207.6 ± 12.7) pg/ml and (153.2 ± 9.9) pg/ml, P < 0.05] were also lower while plasma level of IL-10 [7 days after transplantation: (238.5 ± 17.5) pg/ml vs. (177.3 ± 19.8) pg/ml and (244.6 ± 27.3) pg/ml, P < 0.05] was significantly higher in MSC(hRAMP1) group than in MSC(null) and control group.</p><p><b>CONCLUSION</b>MSCs overexpression hRAMP1 could further reduce area of MI possibly through inhibiting the myocardial expression of NF-κB and reducing the plasma TNF-α level and raising plasma IL-10 level.</p>

rhG-CSF promotes re-endothelialization and attenuates intima hyperplasia in carotid artery of rabbits post balloon catheter injury / 中华心血管病杂志

<p><b>OBJECTIVE</b>To investigate the effect of rhG-CSF on mobilizing bone marrow-MSCs, re-endothelialization and intima hyperplasia in carotid artery of rabbits post balloon catheter injury.</p><p><b>METHODS</b>Rabbits were treated with rhG-CSF (25 microg/kg, twice daily, i.p, n = 35) or saline (n = 32) for 5 days, then, carotid arteries of rabbits were injured by balloon catheter. The number of peripheral MSCs was detected with FACS. The morphology of injured artery was examined with hematoxylin and eosin stain, PCNA was determined with immunohistochemistry.</p><p><b>RESULTS</b>(1) Number of peripheral MSCs was similar at baseline and significantly increased at 24 hours and peaked at 7 days and remained increased till 14 days post rhG-CSF. (2) Significant endothelial cell deletion was evidenced in the control group, while scatter endothelial cells was observed in the rhG-CSF group at 1 week post injury. Two weeks after injury, new endothelial area was significantly higher in rhG-CSF group compared to control group. At 4 weeks post injury, endothelial connection was evidenced and regularly displayed in rhG-CSF treated group. (3) PCNA-positive cells in the tunica intima were significantly lower in rhG-CSF treated rabbits at 7, 14 and 28 days compared that in control rabbits (all P < 0.01).</p><p><b>CONCLUSION</b>rhG-CSF could mobilize the bone marrow-MSCs and promote re-endothelialization and attenuate intima hyperplasia post balloon catheter injury in carotid arteries of rabbits.</p>