| Part1:The mechanism of cyclophosphamide protects myocardial ischemia-reperfusion injuryObject:Cyclophosphamide has been used in the treatment of autoimmune disorders, including Systemic Lupus Erythematosus (SLE), Chronic Glomerulonephritis, Rheumatoid Arthritis, et al. There is inflammation in myocardial ischemia-reperfusion injury similar to that in autoimmune disorders. We hypothesized that myocardial ischemia-reperfusion injury could be partially ameliorated with cyclophosphamide treatment.Methods:Open chest rats were submitted to30min of ischemia followed by3h,12h or24h of reperfusion. Rats were divided into sham group, I/R group and cyclophosphamide group, and each group included3time-point subgroups (3h,12h and24h; n=8for each subgroup).750mg/m2cyclophosphamide or saline was intraperitoneally administrated in cyclophosphamide group or I/R group. A polyethylene tube was inserted into the left ventricular cavity to detect left ventricular systolic pressure (LVSP), left ventricular end diastolic pressure (LVEDP) and±dp/dt max. At the end of experiment, hearts were harvested for histopathological assessment and infarct size determination and blood was collected for hs-CRP and TNF-adetection. Serum TNF-a was measured by cytometric bead array (CBA) and immunohistochemistry was used to detect TNF-a in myocardium.Results:Compared with I/R group, rats treated with low dose cyclophosphamide showed a significant recovery in myocardial function with improved left ventricular systolic pressure (LVSP)(88.3±3.8vs.68.6±3.8mmHg of3h reperfusion;92.0±3.8vs.63.7±4.9mmHg of12h;90.4±4.0vs.64.2±4.9mmHg of24h; P<0.01respectively). The left ventricular end diastolic pressure (LVEDP) and±dp/dt max also had the similar trends.(for LVEDP,14.57±0.94vs14.45±0.48mmHg of3h reperfusion, P>0.05;13.00±0.88vs15.13±0.88mmHg of12h, P<0.05;13.24±0.79vs15.50±0.81mmHg of24h, P<0.05). The myocardial infarct size was reduced after low dose cyclophosphamide treatment compared with I/R group (26.1±0.4%vs40.4±0.4%of3h reperfusion;21.6±0.4%vs49.9±0.4%of12h;21.6±0.4%vs40.0±0.4%of24h, P<0.01respectively.); Histopathological damage score was attenuated (1.83±0.14vs.2.17±0.14of3h reperfusion;2.33±0.14vs.3.17±0.14of12h;2.83±0.14vs.3.83±0.14of24h; P<0.01respectively); Polymorph nuclear leukocytes (PMNs) infiltration in myocardium also decreased after low dose cyclophosphamide treatment (6.9±2.7vs20.4±1.5/HPF of3h reperfusion;18.6±2.8vs37.5±3.6/HPF of12h;16.8±2.5vs58.8±5.0/HPF of24h, P<0.01respectively.).High sensitive C-reactive protein (hs-CRP) was elevated pronouncedly in I/R group at3h,12h and24h after reperfusion. The increase of hs-CRP was prevented after cyclophosphamide administration at three time-points (29.3±0.5vs32.3±0.5ng/ml of3h reperfusion;29.1±0.5vs31.8±0.5ng/ml of12h;28.6±0.5vs31.9±0.5ng/ml of24h, P<0.01respectively). Compared with I/R group, rats treated with cyclophosphamide showed a significant difference with decreased serum TNF-α (13.3±2.6vs14.1±6.0pg/mL at3h reperfusion,10.1±2.7vs12.5±5.0pg/mL at12h,10.4±5.6vs13.0±3.6pg/mL at24h reperfusion, P<0.05respectively). And also the immunostaining was less intense with cyclophosphamide injection at each reperfusion time. The score of the intensity of myocardial TNF-α staining was down regulated with cyclophosphamide injection in each time-point between cyclophosphamide and I/R group (2.42±0.38vs3.33±0.36at3h reperfusion,3.75±0.52vs5.08±0.58at12h reperfusion,6.50±0.45vs8.08±0.8at24h reperfusion, P<0.01respectively).Conclusions:Low dose cyclophosphamide might improve cardiac function and alleviate histopathological damage in myocardial ischemia-reperfusion rats. Cyclophosphamide was a feasible strategy for anti-inflammation to protect myocardial ischemia-reperfusion injury.Part2:The study of endoplasmic reticulum stress-inflammation response of myocardial ischemia/reperfusion injuryObject:Myocardial ischemia/reperfusion activates endoplasmic reticulum stress and inflammatory responses. The stress-response pathways, however, are still unclear. Studies in other areas have suggested that endoplasmic reticulum stress induced by a variety of appropriate stimulations, mediated by two signaling molecule NF-Kb and JNK, cause the expression of inflammatory transcription factors and a variety of inflammatory cytokines, start the inflammatory response, thereby increase cell damage. We speculate that NF-Kb plays an important role in the process of endoplasmic reticulum stress-inflammation responses induced by myocardial ischemia/reperfusion.Methods:Primary cultured neonatal rat cardiomyocytes were exposed to hypoxia for12hours and subsequently reoxygenation for12hours. Five groups were studied,(1) Control group;(2) Tunicamycin group (Tm group, containing tunicamycin lmg/L) was the positive control of this study;(3) hypoxia/reoxygenation group (H/R group,12h hypoxia followed by12h reoxygenation);(4) tunicamycin+SN50group (Tm+SN50group, containing tunicamycin1mg/L and SN5050μg/ml);(5) hypoxia/reoxygenation+SN50group (H/R+SN50group, containing SN5050μg/ml). Western blot and RT-PCR were applied to monitor the expression of GRP78(glucose regulated protein78) and GRP78mRNA. TNF-a was measured by cytometric bead array (CBA). The cell viability of primary cultured neonatal rat cardiomyocytes was explored by CCK-8(Cell Counting Kit-8) assay. Myocardial activity was observed by inverted phase contrast microscope. Hematoxylin-eosin (HE) staining was appiled to observe myocardial cell morphology and pulsation.Results:Compared with the control group, the hypoxia/reoxygenation group significantly increase the expression of GRP78and GRP78mRNA. The concentration of TNF-a is increased significantly, too. CCK-8and HR are decreased. Histopathological damage score shows myocardial injury is more serious. SN50, however, significantly decrease the changes which induced by Hypoxia/reoxygenation. Compared with the H/R group, the H/R+SN50group significantly decrease the expression of GRP78(indicated by GRP78/GAPDH,1.16±0.12vs1.44±0.02) and GRP78mRNA(1.95±0.86vs6.91±0.81), the concentration of TNF-a is decreased (8.10±1.12vs11.70±1.31pg/ml), too. CCK-8(1.21±0.05vs0.81±0.06) and HR (97.3±1.2vs58.7±5.5) is also increased. And histopathological damage score is decreased (1.66±0.58vs1.33±0.58).Conclusions:These findings suggest that hypoxia/reoxygenation can induce endoplasmic reticulum stress and inflammation. And more importently, our results show NF-κb plays an important role in ERS-imfammation pathway. |
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