| Lung is one of the most vulnerable target organ occurs in trauma,blood loss and other factors which led to severe shock. Acute lung injury(ALI) is characterized by interstitial edema, inflammatory cells infiltration,disruption of respiratory membrane and confused gas exchange, which is a key part of the inflammation spreading. However, understanding of the integrated mechanism of lung follows shock remains incomplete. ?-3polyunsaturated fatty acids(?-3PUFAs) is commonly used as a parenteral nutrition in clinical treatment, which have various role, such as relieve systemic inflammatory response, enhance immune function, reduce the rate of infection in critically ill patients, improve intestinal barrier function,and it has not been reported in hemorrhagic shock. Autophagy, which resides in almost all cells that have mitochondria, is a traditional conserved homeostasis process. And plays an important role in various disease, that has a certain correlate with the ALI caused by hemorrhagic shock, nevertheless we need some further study to explore the mechanism.In this study, we firstly observed the role of ?-3PUFAs in hemorrhagic shock rats, realized the role in the survival time in rats following hemorrhagic shock and on the basis of observation, we may discuss the intervention of ?-3PUFAs to ALI in hemorrhagic shock in rats and make a further study in the role of autophagy in ?-3PUFAs reduce ALI after hemorrhagic shock.In this study, hemorrhagic shock model in rats was developed by a one-time bleeding(the amount of blood volume 1/4), and fluid resuscitation(the full amount plus the same amount of blood Ringer),which after 90 minutes. Firstly, 12 male Wistar rats were randomized tohemorrhagic shock group and hemorrhagic shock plus ?-3PuFAs group.We Use this part to observe ?-3PuFAs influence on survival time and rate of rats. Our study indicates that the six rats in shock group all died within48 h, and the other six rats in ?-3PuFAs group were alive in 72 h.?-3PuFAs treatment significantly prolonged survival time in rats following hemorrhagic shock. The results confirmed that ?-3PuFAs has a good intervention of hemorrhagic shock at the overall level.Secondly we make a further study to observe ?-3PuFAs protect ALI of rats from hemorrhagic shock and autophagy make a difference in this process, We then use autophagy activator rapamycin(mTOR protein target of rapamycin inhibitor, RAPA) and autophagy inhibitor 3- methyladenine(3-MA) to activate and inhibit autophagy in rats following hemorrhagic shock. We put 54 rats in 9 groups as fellows, sham group, sham plus?-3PuFAs group, shock group, shock plus ?-3PuFAs group, sham plus RAPA group, shock plus RAPA group, sham plus 3-MA group, shock plus 3-MA group, shock plus RAPA and ?-3PUFAs group. We made a one-time bleeding(the amount of blood volume 1/4) and a fluid resuscitation after 90 minutes in the rats of the shock, shock+?-3PuFAs,shock+RAPA, shock+3-MA, shock+RAPA+?-3PUFAs groups. Then, the shock and shock+RAPA groups were resuscitated by the whole blood and the same amount of Ringer's solution. shock+?-3PuFAs group and shock+RAPA+?-3PUFAs group were resuscitated by the liquid which contained the whole blood, ?-3PuFAs(0.2 g / kg) and the same amount of Ringer's solution, Shock+3-MA group was resuscitated by the whole blood, 3-MA(30 mg / kg) of the same amount of Ringer's solution; at the same time, Shock group, Shock+?-3PuFAs group, Shock+3-MA group were injected with vehicle, shock+RAPA group,shock+RAPA+?-3PUFAs group were injected RAPA(10 mg / kg).Besides we made surgery only in sham group, sham+?-3PuFAs group, sham+RAPA group and sham+3-MA group without bleeding, atthe point of the resuscitation began of shock groups we injected with vehicle to those sham group including sham group, sham+?-3PuFAs group and Sham+3-MA group and sham+RAPA group were injected with RAPA(10 mg / kg),at the same time, Sham group and Sham+RAPA group were injected by Ringer's solution which at the amount of 1/4whole blood, besides Sham+?-3PuFAs group injected with same amount of Ringer's solution contained ?-3PuFAs(0.2 g / kg), identically Sham+3-MA group injected with same amount of Ringer's solution comprise intravenous 3-MA(30 mg / kg).Lung tissue were measured at 4h or equivalent time points after fluid resuscitation, the left lung tissue for wet to dry ratio(W/D) was measured and the Deputy right lung lobe fixed in 4% paraformaldehyde for tissue morphological and immunohistochemical staining which was detected for the expression of light chain 3II(LC3-II), autophagy labeled protein, the other lung tissue stored at low temperature freezers for extracting LC3-II protein by western blot and the expression of protein in autophagy-related pathways class III PI3K-AKt-mTOR.As indicated by increased lung wet/dry ratio(W/D) in shock group compared with sham group, and the same as Shock+?-3PUFAs Group.Histological examination showed that the sham group and Sham+?-3PUFAs group has structure normal lung tissue and Shock group had a series of change such as, lung septal thickening, alveolar hemorrhage, and airway epithelial damage, we found that the performance, and histological scores were significantly higher than both sham and sham+?-3PuFAs groups, besides Shock +?-3PUFAs group has a small amount of alveolar hemorrhage and pulmonary septal thickening phenomenon, we found that the histological scores was significantly lower than Shock group; LC3-II protein levels in the shock group was significantly higher than sham group and sham+?-3PUFAs group, andLC3-II protein expression in shock +?-3PUFAs group was significantly lower than Shock group.Together, the results demonstrated that activate autophagy may cause more severe lung injury, on the contrary inhibited autophagy make alleviated lung injury and investigate that it may has relationship with PI3K/Akt pathway. And ?-3PUFAs may aggravate ALI by activate PI3K/Akt pathway.In summary, the present results revealed that ?-3PUFAs treatment enhanced the survival time of rats following hemorrhagic shock, alleviated the tissue injury and autophagy level of lung, and its mechanism is related to the signaling pathway of PI3K-AKt-mTOR. These results suggested that hyper-autophagy plays a certain role in acute lung injury following hemorrhagic shock, and ?-3PUFAs may represent a novel approach for prevention and therapy of ALI in severe hemorrhagic shock. |
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