Role Of Stellate Ganglion Blockage Alleviating Post-hemorrhagic Shock Mesenteric Lymph-mediated Acute Lung Injury Through Inhibiting Autophagy

Acute lung injury(ALI)is one of the earliest organ injuries after hemorrhagic shock,with high incidence and morbidity.Therefore,it is essential to explore the therapies for ALI prevention and treatment.Post-hemorrhagic shock mesenteric lymph(PHSML)return is one of the key mechanisms and links for ALI.Hence,it is of active and practical significance exploring the therapies for ALI targeting the PHSML.The recent studies indicate that stellate ganglion block(SGB)has favorable effects on reducing the myocardial injury and dysfunction,alleviating the intestinal hyper-permeability and intestinal villi morphological injury,relieving the excessive oxidative stress response and restoring the gastrointestinal function.Autophagy has essential roles in growth,development and homeostasis of cells through the regulation of protein and organelle degradation.More and more studies indicate that autophagy plays a complex role in the pathogenesis and development of ALI induced by various pathogenic factors.However,whether the autophagy is involved in the process of hemorrhagic shock-induced ALI and PHSML-mediated ALI,it remains unknown.In addition,whether SGB alleviates PHSML-meditated ALI following hemorrhagic shock,its mechanism is or not related to the regulation of autophagy,it needs further verification.In view of above analysis we hypothyzed that SGB alleviates PHSML-mediated ALI through the inhabitation of autophagy.To test the hypothesis,the current study investigated the role of mechanism by which SGB alleviating PHSML-mediated ALI from the animal and cellular experiments.On the one hand,the conscious rats undergoed hemorrhagic shock were used to observe the effect of SGB and autophagy inhibitor 3-MA on hemorrhagic shock induced ALI,and further the effect of autophagy agonist RAPA and intravenous infusion of PHSML on SGB-alleviated ALI were investigated.On the other hand,the primary cultured rat pulmonary microvascular endothelial cells(PMVECs)were treated with PHSML from SGB and non-SGB rats to dissect the role of SGB through the observation of cellular viability and autophagy.At first,male Wistar rats were randomly divided into the sham group(saline was injected into the stellate ganglion),shock group(saline was injected into the stellate ganglion,kept blood pressure at 40 mm Hg for 60 min),sham-SGB group(SGB,the other operation was the same as the sham group),shock-SGB group(SGB,the other operation was the same as the shock group).After resuscitation of the groups with hemorrhage,mesenteric lymph drainage was operated for three hours,and mesenteric lymph drainage was operated for 30 min in the groups without hemorrhage at the corresponded time.Then we got the mesenteric lymph from different groups,named as normal mesenteric lymph(NML),NML-SGB,PHSML,PHSML-SGB.Then kept them in the-80℃ refrigerator for the following studies.Next,rats were randomly divided into seven groups,as follows,sham,shock,sham-SGB,shock-SGB,shock+3-MA(intravenous infusion of 3-MA solution during resuscitation,the other operation was the same as the shock group),shock-SGB+RAPA(intraperitoneal injection of RAPA solution at the same time of resuscitation,the other operation was the same as the shock-SGB group),shock-SGB+PHSML(intravenous infusion of PHSML after resuscitation,the other operation was the same as the shock-SGB group).The water content,the morphological observation of lung tissue and the expressions of LC3 Ⅱ and Beclin 1 were detected at three hours after resuscitation or indicated time points.Our data demonstrated that the lung tissue in the shock group showed increased structural damage,water content and the expressions of LC3 Ⅱ and Beclin 1 compared to the sham group.These results suggested that lung injury and autophagy were exacerbated after hemorrhagic shock.While SGB relieve these phenomena,indicating alleviated acute lung injury and reduced autophagy.After the application of autophagy inhibitor 3-MA,the lung morphological injury,water content and the expressions of LC3 Ⅱ and Beclin 1 were decreased significantly than that in the shock group.Furthermore,after the application of autophagy agonist RAPA abolished the therapeutic effects of SGB on acute lung injury and autophagy after shock.These results suggested that SGB may function through reducing autophagy level.Likewise,intravenous infusion of PHSML abolished the protective effects of SGB on acute lung injury and autophagy after shock.These results indicated PHSML induced acute lung injury through promoting autophagy,and inhibition of autophagy represent a therapeutic strategy for PHSML-induced lung injury.PMVECs were the first cell type injuried by PHSML.Cell viability was tested using 8% mesenteric lymph from control group,NML group,PHSML group,NML-SGB group,PHSML-SGB group.Meanwhile,the levels of LC3-Ⅱ protein and Beclin-1 protein were detected.Compared to control group,cell viability was decreased and the expressions of LC3-Ⅱ and Beclin-1 protein were increased significantly in PHSML group.Especially,compared to PHSML group,cell viability was increased and the levels of LC3-Ⅱ and Beclin-1 were decreased significantly in PHSML-SGB group.These results suggested that SGB alleviated PMVECs injury and autophagy induced by PHSML.Collectively,SGB and autophagy inhibition alleviated ALI after hemorrhagic shock,while intravenous infusion of PHSML and autophagy activation inhibited the protective effect of SGB.The current results suggest that the role of SGB alleviating ALI is through the inhibition of autophagy,and is relate to the PHSML return.In summary,the inhibition of excessive autophagy is involved in the mechanism by which SGB alleviated PHSML-mediated ALI.