| BackgroundMechanical trauma, such as that induced by motor vehicle crashes, athletic sports contretemps, war, and natural disaster, represents a major medical and economic problem in modern society. Trauma can not only cause direct organ damage, but also be the role of injury factor in the human body, resulting in multiple sites and multiple organ damage. Nowadays, trauma is the leading cause of mortality in the young aged population.Thus trauma treatment has important social meaning.A number of studies report that mechanical trauma can cause direct heart damage, such as coronary artery dissection and cardiac contusion.As a result of advanced prehospital care and regional trauma systems development, fewer critically injured patients are dying at the scene of the accident. However, recently published clinical reports have indicated that mechanical trauma may cause cardiac death even in the absence of direct cardiomyocyte injury during the first24h. These results suggest that nonlethal mechanical trauma can induce delayed cardiac injury. However, the mechanisms responsible for nonlethal mechanical trauma-induced delayed cardiac injury have not yet been identified.Studies have shown that body damage can cause disorders of the extracellular and intracellular environment. Under the regulation of the nervous and endocrine system, the environment changes will be adjusted accordingly, thus maintaining the body’s steady-state balance. Homeostasis maintenance is crucial to ensure the function of body organs, and homeostatic dysregulation may cause multiple organ dysfunctions. There is compelling evidence that autophagy is important for the maintenance of homeostasis under basal conditions. Autophagy is an important cellular function that enables the recycling of long-lived proteins or damaged organelles. Autolysosomal degradation of membrane lipids and proteins generates free fatty acids and amino acids, which can be reused to maintain mitochondrial ATP production and protein synthesis and promote cell survival. Disruption of this pathway prevents cell survival in diverse organisms.Clearing the damaged mitochondria is the major mechanism for autophagy to maintain the homeostasis. Mitochondria are the major source of cellular energy, providing more than90%of the ATP required for cell work. Mitochondria are most sensitive to a variety of damage. When mitochondria are rendered dysfunctional, a bioenergetic limitation of cell and cardiac function is expected. Although substantial evidence now exists that autophagy plays a critical role in homeostasis maintenance and organ function, whether or not autophagy is changed and contributes to delayed cardiac injury after mechanical trauma remains largely unknown.In the present study, we observed the possible mechanisms of myocardial delayed dysfunction after trauma through the use of in vitro and in vivo experimental models and pharmacological drug treatment, which would supply experimental evidence for seeking the best treatment to prevent multiple organ failure after trauma.Objective1To investigate whether nonlethal mechanical trauma may result in the change of cardiomyocyte autophagy.2To determine whether myocardial autophagy may contribute to delayed cardiac dysfunction.3Looking for the main serum proteins which play a role in delayed heart damage caused by non-lethal trauma.Materials and methods1Materials1.1Experimental animalsHealthy male SD rats weighing about180g-220g, provided by the Experimental Animal Center of Shanxi Medical University. The use of experimental animals is follow the People’s Republic of China Ministry of Health Decree (No.55)----Rules for the Implementation of medical management of laboratory animals.1.2Cell linesH9c2(2-1) cell lines:purchased from the Shanghai Cell Bank, Chinese Academy of Sciences.2The experimental groups2.1Experiment groups in vivoTrauma group (Trauma):Male SD rats were anesthetized with chloral hydrate (3ml/kg). The rats were then placed in a Noble-Collip drum, a plastic wheel with internal shelves on which a rat is traumatized as the wheel is rotated(40rpm,5min);Pseudo-trauma group (Sham):Sham trauma rats were subjected to the same revolution but the animals were taped on the inner wall of drum, thus avoiding traumatic injury. Drug treated group:Given autophagy agonist rapamycin (RAPA)8mg/kg half hours before trauma.2.2Experiment groups in vitroNormal cell group:given the normal DMEM medium and10%fetal bovine serum, and placed in5%CO2incubator;Intervention group:normal DMEM medium, added to15%rat serum subjected trauma, replaced fetal bovine serum;SiRNA group:Imported SiRNA plasmid and silenced H9c2cells autophagy gene Beclinl then added to15%rat serum subjected trauma, replaced fetal bovine serum;.3Method3.1Sacrificing experimental specimensAt the end of the experiment, bloods were extracted from the abdominal aora and the rat hearts were removed to detect.3.2Sacrificing experimental specimens of H9c2(2-1) cellsAt the end of the experiment, the cell culture supernatant and the cells were set aside to detect.3.3Determination of indicators3.3.1The detection of rat ECG, MABP and cardiac function in vivo;3.3.2The detection of rat cardiac function in vitro;3.3.3The detection of myocardial structure and protein Immunofluorescence by immunohistochemical;3.3.4Determination of creatine kinase isoenzyme MB (CK.-MB) and cardiac troponin l (cTnl) content in serum;3.3.5Determination of Free three iodine armour gland original acid (FT3), free thyroxine (FT4) and thyroid-stimulating hormone (TSH) in serum by radiation immune assay;3.3.6Determination of the myocardial tissue LC3Ⅱ, Beclinl in the protein content by Western blot:3.3.7Determination of the myocardial tissue LC311, Beclinl in the mRNA content by real-time PCR;3.3.8The detection of mitochondrial membrane potential by JC-1method; 3.3.9The detection of mitochondrial function by99mTc-MIBI myocardial imaging;3.3.10The detection of cell survival by CCK8kit;3.3.11Determination of creatine kinase (CK.) and Lactate Dehydrogenase (LDH) content in cell culture supernatant;3.3.12Determination of the myocardial cell LC3Ⅱ, Beclinl in the protein content by Western blot;3.3.13To determination the myocardial cell LC3Ⅱ, Beclinl in the mRNA content by real-time PCR;3.3.14shRNA expression vector and identification;3.3.15The cell transfection and screening;3.3.16The detection of cytokines by protein chip in rat serum after trauma;3.3.17The detection of cytokines in serum after trauma by kit.Results1The nonlethal mechanical trauma rat models were set up successfully.1.1There was no obvious change in ECG,respiratory, heart rates within24h after mechanic traumaCompared with the sham group, there was no obvious change in T wave and ST-segment of ECG (P>0.05) within24h after trauma. And there was no obvious change in heart rate and respiratory rate (P>0.05). Anesthetized rats generally waked about1.5hours after trauma, and the survival rate within24h was100%.1.2There was no obvious change in mean artery blood pressure (MABP), cardiac function in vivo and cardiac structure within24h after traumaCompared with sham trauma group (103.20±12.42mmHg), the MABP decreased significantly (79.92±12.08mmHg)(P<0.01) after trauma, then returned to normal after1h. And the MABP didn’t recur abnormal within24h.LVD P,+dP/dTmax and-dP/dTmax reflect the left ventricular systolic and diastolic function, respectively. Compared with the sham group, the cardiac function in vivo was no significant difference at I h,3h,6h,12h and24h after trauma (P>0.05).Compared with sham trauma group,there was no obvious change in cardiac structure at1h,3h,6h,12h and24h after trauma. And there was also no inflammatory cell infiltration. 1.3There was no significant change of creatine kinase isoenzyme (CK-MB) and troponin I (cTnI) in traumatic serum.Compared with the sham group, CK-MB and cTnl did not significantly change at1h,3h,6h,12h and24h after trauma (P>0.05).1.4There was no significant change of thyroid hormone (FT3, FT4and TSH) in traumatic serum.Compared with the sham group, thyroid hormone (FT3, FT4, TSH) does not significantly change at1h,3h,6h,12h and24h after trauma (P>0.05).1.5The isolated cardiac function decreased24h after trauma in ratsCompared with the sham group, LVDP,+dP/dTmax and-dP/dTmax at24h after trauma was significant decreased [LVDP:(87.5±11.6) mmHg, vs.(103.0±8.9) mmHg, P<0.05;+dP/dTmax:(2060.0±266.6) mmHg/sec, vs.(2830.16±395.3) mmHg/sec, P<0.01], and-dP/dTmax was also markedly decreased [(-1666.5±243.4) mmHg/sec vs.(-2048.3±282.9) mmHg/sec, P<0.05]24h after trauma (Fig.9). While compared with the sham group, the isolated cardiac function was no significant difference at1h,3h,6h, and12h aftere trauma (P>0.05).2The decline of myocardial tissue autophagy level may be the important cause of cardiac dysfunction after trauma2.1The level of autophagy in myocardial tissue declined after trauma within24h.2.1.1Detecting the change of Beclin1in protein and mRNA levels by western blot and real-time PCR at different time points after traumaCompared with the sham group (0.94±0.23), the protein level of Beclin1decreased significantly at1h (0.59±0.10, P<0.05), and the expression reduced to the lowest (0.30±0.15, P<0.01) at3h after trauma.The expression was recovery gradual to normal at24h.Compared with the sham group (1.00±0.15), the mRNA level of Beclin1decreased significantly at1h (0.49±0.11, P<0.05), and the level reduced to the lowest (0.32±0.22, P<0.01) at3h after mechanic trauma. The level was recovery gradual to normal at24h.2.1.2Detecting the change of LC3Ⅱ in protein and mRNA levels by western blot and real-time PCR at different time points after traumaCompared with the sham group (1.01±0.12). the expression of LC311protein decreased significantly at1h (0.62±0.16, P<0.05vs. Sham), and the expression reduced to the lowest (0.30±0.1, P<0.01vs. Sham) at6h after trauma. The expression was recovery gradual to normal at24h(1.08±0.32, P>0.05). The results showed that the mRNA level of LC3Ⅱ decreased significantly at1h (0.46±0.16, P<0.05vs. Sham) compared with the sham trauma group (0.98±0.13), and the level reduced to the lowest (O.34±0.15, P<0.01vs. Sham) at6h after mechanic trauma. The level was recovery gradual to normal at24h (0.98±0.40, P>0.05).2.2Increasing the level of autophagy could improve the cardiac function in vitro after mechanic trauma2.2.1The agonist of autophagy Rapamycin (RAPA) could upregulate autophagy2.2.1.1Rapamycin (RAPA) could increase the Beclin1expression in protein and mRNA levelsCompared with the sham group (1.14±0.33), the expression of Beclin1protein decreased significantly (0.52±0.15, P<0.05) after mechanic trauma. However, the level of Beclin1could be upregulated when administered with RAPA30min before mechanic trauma (1.18±0.26, P>0.05).Compared with the sham group (1.18±0.29), the mRNA level of Beclin1decreased significantly (0.74±0.15, P<0.05) after mechanic trauma. However, the mRNA level of Beclin1could be upregulated when administered RAPA at30min before mechanic trauma (1.03±0.26, P <0.05).2.2.1.2Rapamycin (RAPA) could increase the LC3Ⅱ in protein and mRNA levelsCompared with the sham group (1.28±0.29), the protein expression of LC3Ⅱ decreased significantly (0.69±0.15, P<0.05) after rauma. However, the level of LC3Ⅱ could be upregulated when administered with RAPA30min before mechanic trauma (1.13±0.26, P<0.05).Compared with the sham trauma group (1.00±0.28), the mRNA level of LC3Ⅱ decreased significantly (0.55±0.13, P<0.01) after mechanic trauma. However, the mRNA level of LC3Ⅱ could be upregulated when administered RAPA at30min before mechanic trauma (0.86±0.31, P<0.05).2.2.1.3PAPR could increase the fluorescence intensity which represented the Beclin1and LC3ⅡTo observe the expression of LC3Ⅱ and Beclin I by laser scanning confocal. Compared with the sham group, the expression of LC3Ⅱ and Beclin1proteins decreased after trauma (P <0.05). However, the expression of LC3Ⅱ and Beclin1proteins increased after administered with RAPA (P<0.05).2.2.2Upregulated autophagy could improve the cardiac function in vitro after trauma Compared with the sham group, LVDP,+dP/dTmax and-dP/dTmax decreased significantly [LVDP:(87.5±11.6) mmHg, vs.(103.0±8.9)mmHg, P<0.05;+dP/dTmax:(2060.0±266.6) mmHg/sec, vs.(2830.16±395.3) mmHg/sec, P<0.01;-dP/dTmax:(-1666.5±243.4) mmHg/sec vs.(-2048.3±282.9) mmHg/sec, P<0.05] at24h after trauma. Given RAPA30min before trauma, then detected the cardiac function in vitro and the results showed that LVDP,+dP/dTmax and-dP/dTmax all increased significantly [LVDP:(94.3±9.8) mmHg, vs.(87.5±11.6) mmHg, P <0.05;+dP/dTmax:(2663.0±332.2) mmHg/sec, vs.(2060.0±266.6) mmHg/sec, P<0.01;-dP/dTmax:(-1914.8±215) mmHg/sec, vs.(-1666.5±243.4) mmHg/sec, P<0.01].3Upregulation of autophagy could improve the mitochondrial function after mechanic trauma3.1Mitochondrial membrane potential decreased in cardiac tissue after traumaMitochondrial membrane potential was detected by mitochondrial membrane potential detection kit (JC-1method). When the mitochondrial membrane potential was normal, JC-1aggregated in the mitochondrial matrix, and formed the polymer which showed red fluorescence; however, when the mitochondrial membrane potential decreased, JC-1couldn’t be gathered in the mitochondrial matrix, and existed as monomer form which resulted in green fluorescence. The results showed that the mitochondrial membrane potential of sham group displayed the red and the mitochondrial membrane potential of trauma group was green.3.2Upregulated autophagy could improve the mitochondrial function after mechanic traumaThe results of heart perfusion imaging which dectected by the radionuclide99mTc-MIBI indicated that compared with the sham trauma group (1.96±0.13), the uptake of99mTc-MIBI decreased in trauma myocardial mitochondrial (1.68±0.19, P<0.05vs. Sham), and administered with RAPA before trauma the uptake of99mTc-MIBI increased obviously (1.94±0.20, P<0.01vs. Trauma).4The role of autophagy in myocardial cell injury led by post-traumatic serum4.1The myocardial cell injury model led by post-traumatic serum was set up successfullyThere was no obvious difference of cell viability between normal fetal bovine serum (control group) and sham post-traumatic serum (Sham group) cultured H9c2cells at3h,6h,12h,24h and48h (P>0.05). Compared with the sham group, cell viability decreased significantly which was cultured three hours with3h post-traumatic serum (P<0.05) and cultured12h, the viability decreased to the lowest (P<0.01). Compared with sham group, cell viability was significantly decreased which was cultured three hours with6h post-traumatic serum (P<0.05) and cultured12h, the viability decreased to the lowest (P<0.01).4.2The changes of CK and LDH in supernatant of cell cultured with different time points post-traumatic serumCompared with the sham group [CK:(138.33±17.18U/L); LDH:(112.40±12.13U/L)], the level of CK and LDH were significantly increased [CK:(160.16±18.85U/L, P<0.05); LDH:(141.80±15.19U/L, P<0.05)] in supernatant of cell cultured with12h post-traumatic serum.4.3The change of autophagy in H9c2cells cultured with different time points post-traumatic serum4.3.1The protein and mRNA level of Beclin1were elevatedCompared with sham group (1.08±0.32), protein level of Beclin1rose to the highest (1.76±0.55, P<0.01vs. Sham) cultured with3h post-traumatic serum, then gradually restored to normal within24after trauma (1.18±0.28, P>0.05).Compared with sham trauma group (1.00±0.54), mRNA level of Beclin1rose to the highest (1.63±0.24, P<0.01) cultured with3h post-traumatic serum, then the level gradually restored to normal.4.3.2The protein and mRNA level of LC3Ⅱ were elevatedCompared with sham group (1.11±0.27), protein level of LC3Ⅱ rose to the highest (1.82±0.64, P<0.01vs. Sham) cultured with6h post-traumatic serum, then the level gradually restored to normal within24after trauma (1.25±0.46, P>0.05).Compared with sham group (1.01±0.17), mRNA level of LC311rose to the highest (1.85±0.43, P<0.01) cultured with12h post-traumatic serum, then the level gradually restored to normal within24h after trauma (1.08±0.18, P>0.05).4.4The change of autophagy in H9c2cells transfected wiht pSUPER-Beclin14.4.1The change of the protein and mRNA level of Beclin1Compared with normal cells, protein level of Beclinl decreased significantly in H9c2cells transfected wiht pSUPER-Beclin1. However, the protein level of Beclin1unchanged in H9c2cells transfected with empty plasm id pSUPER-non.Compared with normal cells, mRNA level of Beclin1decreased in H9c2cells transfected wiht pSUPER-Beclin1(0.39±0.08vs.0.95±0.24, P<0.05). However, the mRNA level of Beclin1unchanged in H9c2cells transfected with empty plasmid pSUPER-non (0.89±0.19vs.0.95±0.24P>0.05). 4.4.1The change of the protein and mRNA level of LC3ⅡCompared with normal cells, protein level of LC3Ⅱ decreased significantly in H9c2cells transfected wiht pSUPER-Beclin1. However, the protein level of LC3Ⅱ unchanged in H9c2cells transfected with empty plasmid pSUPER-non.Compared with normal cells, mRNA level of LC3Ⅱ decreased in H9c2cells transfected wiht pSUPER-Beclin1(0.48±0.08vs.0.93±0.20P<0.05). However, the mRNA level of LC3Ⅱ unchanged in H9c2cells transfected with empty plasmid pSUPER-non (0.97±0.24v.s0.93±0.20P>0.05).4.5The H9c2cells transfected with pSUPER-Beclin1were damaged when when cultured with post-traumatic serum4.5.1The cells survival rate decreasedCompared with sham group (104.90±16.92), the cells survival rate dropped to the lowest cultured with6h post-traumatic serum (51.30±10.70, P<0.01).4.5.2The CK and LDH level increasedCompared with sham group (143.00±15.32) U/L. the level of CK rose to the highest cultured with12h post-traumatic serum (243.50±35.05, P<0.01) U/L.Compared with the sham group (112.40±12.03) U/L, the level of LDH rose to the highest cultured with12h post-traumatic serum (201.80±16.30, P<0.01) U/L.5Detecting stress-related cytokine in post-traumatic serum by protein chip5.1Compared with sham group, the levels of IL-1β,TNF-α, IFN-γ, CINC-3and CNTF increased significantly (P<0.05).5.2The level of TNF-a increased at different time points in post-traumatic serumCompared with the sham trauma group [(66.57±14.18) pg/ml], the TNF-α level rose to the highest immediately after trauma [(178.00±39.81) pg/ml,P<0.01]. Then, the level gradually restored normal at24h after trauma [(77.33±19.45) pg/ml, P>0.05].5.3The level of IL-1β increased at different time points in post-traumatic serumCompared with the sham group [(29.48±11.07) pg/ml], the IL-1β level rose to the highest at1h after trauma [(29.48±11.07) pg/ml. P<0.01]. Then, the level gradually restored normal within6h after trauma [(27.30±9.72) pg/ml, P>0.05].5.4The level of IFN-y increased at different time points in post-traumatic serum Compared with the sham group [(1.12±0.14) pg/ml], the IFN-y level rose to the highest at6h after trauma [(19.87±1.67) pg/ml, P<0.01]. Then, the level gradually restored normal, but the level remained at a high level at24h after trauma.5.5The level of CINC-3increased at different time points in post-traumatic serumCompared with the sham group [(67.62±8.57) pg/ml], the CINC-3level rose to the highest at3h after trauma [(375.10±52.13) pg/ml, P<0.01]. Then, the level gradually restored normal within12h after trauma [(59.31±12.61) pg/ml, P>0.05].5.6The level of CNTF increased at different time points in post-traumatic serumCompared with the sham group [(0.16±0.03) pg/ml], the CNTF level began to rise [(1.22±0.22) pg/ml, P<0.01] immediately after trauma. And the level rose to the top at6h after trauma. Then, the level gradually restored normal, but the level remained at a high level at24h after trauma [(1.16±0.20) pg/ml, P<0.01].Conclusion1Non-lethal mechanical trauma can lead to the decrease of myocardial autophagy.2The decreased cardiomyocyte autophagy after trauma is a critical contributor to posttraumatic cardiac mitochondrial damage and the followed cardiac dysfunction in vitro.3Serum after trauma can result in H9c2myocardial cell injury.Inhibition of myocardial autophagy can increase the injury of myocardial cells by the serum of post-traumatic.4The pathological media in serum which lead to myocardial cell injury may be one of the IL-1β、TNF-α、IFN-γ、CINC-3and CNTF. |
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