Henning Robert J, Sanberg Paul, Jimenez Ernesto
Center for Cardiovascular Research, James A. Haley VA Hospital and the University of South Florida College of Medicine, Tampa, Florida, USA.
Center for Cardiovascular Research, James A. Haley VA Hospital and the University of South Florida College of Medicine, Tampa, Florida, USA.
Cytotherapy. 2014 Aug;16(8):1158-68. doi: 10.1016/j.jcyt.2014.01.415. Epub 2014 May 10.
We hypothesized that paracrine factors from human umbilical cord blood mononuclear cells (hUCBC) activate in injured cardiomyocytes the survival protein kinase Akt and limit activation of death protein kinases JNK and p38.
We treated hUCBC with H2O2 and measured growth factors and cytokines secreted by hUCBC. We then treated cardiomyocytes with H2O2 for 24 h and measured Akt, JNK and p38 activation by means of Western blots. We also measured myocyte viability and apoptosis with the use of fluorescence-activated cell-sorting cytometry. We then investigated myocytes treated for 24 h with H2O2 plus hUCBC and myocytes without hUCBC or H2O2. Four million hUCBC were placed in transwells permeable only to hUCBC paracrine factors, and the transwells were placed in flasks with H2O2 + Dulbecco's modified Eagle's medium or in flasks with myocytes plus H2O2+Dulbecco's modified Eagle's medium.
hUCBC increased secretion during H2O2 of hepatocyte growth factor by 338%, insulin-like growth factor by 200%, interleukin-4 by 200%, vascular endothelial cell growth factor by 192%, placental growth factor by 150%, interleukin-10 by 150% and angiogenin by 121%. H2O2 increased myocyte JNK activation by 237% and p38 activation by 60%, decreased myocyte viability by 38% and increased necrosis by 34% (all P < 0.01). hUCBC paracrine factors increased in myocytes with H2O2 Akt activation by ≥ 25%, decreased JNK and p38 activation by > 35%, increased viability by > 22% and decreased apoptosis by > 33% (all P < 0.05). Akt inhibitor API-1 prevented the effects of hUCBC and enhanced H2O2 decrease of myocyte viability. Addition of JNK inhibitor SP600125 or p38 inhibitor SB203580 to myocytes plus H2O2 prevented H2O2 decrease in viability and increased hUCBC beneficial effects.
During free radical stress, hUCBC paracrine factors activate myocyte Akt, which increases myocyte viability by decreasing activation of death-promoting protein kinases JNK and p38.
我们推测人脐带血单个核细胞（hUCBC）分泌的旁分泌因子可激活受损心肌细胞中的存活蛋白激酶Akt，并限制死亡蛋白激酶JNK和p38的激活。
我们用H2O2处理hUCBC，并检测hUCBC分泌的生长因子和细胞因子。然后用H2O2处理心肌细胞24小时，通过蛋白质免疫印迹法检测Akt、JNK和p38的激活情况。我们还用荧光激活细胞分选流式细胞术检测心肌细胞活力和凋亡情况。然后我们研究了用H2O2加hUCBC处理24小时的心肌细胞以及未用hUCBC或H2O2处理的心肌细胞。将400万个hUCBC置于仅对hUCBC旁分泌因子具有通透性的Transwell小室中，然后将这些Transwell小室置于含有H2O2+杜氏改良 Eagle培养基的培养瓶中，或置于含有心肌细胞加H2O2+杜氏改良 Eagle培养基的培养瓶中。
hUCBC使H2O2处理期间肝细胞生长因子的分泌增加338%，胰岛素样生长因子增加200%，白细胞介素-4增加200%，血管内皮细胞生长因子增加192%，胎盘生长因子增加150%，白细胞介素-10增加150%，血管生成素增加121%。H2O2使心肌细胞JNK激活增加237%，p38激活增加60%，使心肌细胞活力降低38%，坏死增加34%（均P<0.01）。hUCBC旁分泌因子使H2O2处理的心肌细胞中Akt激活增加≥25%，JNK和p38激活降低>35%，活力增加>22%，凋亡降低>33%（均P<0.05）。Akt抑制剂API-1可阻断hUCBC的作用，并增强H2O2对心肌细胞活力的降低作用。向加有H2O2的心肌细胞中添加JNK抑制剂SP600125或p38抑制剂SB203580可防止H2O2降低细胞活力，并增强hUCBC的有益作用。
在自由基应激期间，hUCBC旁分泌因子激活心肌细胞Akt，通过降低促死亡蛋白激酶JNK和p38的激活来增加心肌细胞活力。
