| Maintaining the dynamic balance of the intestinal mucosal barrier is essential for maintaining the stability of the intestinal environment,and its dysfunction is a key link in causing structural damage and dysfunction of remote organs after hemorrhagic shock.The treatment of stellate ganglion block（SGB）is widely used in clinical treatment.Our previous studies have found that preventive administration of SGB treatment can increase the survival rate of rats after hemorrhagic shock and help improve the intestinal barrier Features.However,whether SGB treatment（ie,SGB post-treatment）after shock occurs can also play an overall intervention effect on hemorrhagic shock and reduce intestinal damage after hemorrhagic shock.There is no relevant research report.In addition,the role of autophagy in intestinal ischemic injury has attracted increasing attention.Many studies have reported that excessive autophagy is involved in the process of ischemic intestinal barrier injury and is an important factor in intestinal injury.However,it is unclear whether autophagy is involved in the mechanism of SGB post-processing to improve intestinal injury after hemorrhagic shock.For this reason,this study hypothesized that SGB post-treatment can reduce the intestinal barrier damage in hemorrhagic shock rats,and its mechanism of action is related to the inhibition of autophagy.Animal experiments have been used to verify and reveal the autophagy mechanism of SGB post-treatment.Healthy male Wistar rats（SPF grade,weighing 300±20g）were used in this study.All animals were randomly divided into four groups,as follows:sham operation（Sham）group,sham operation+SGB（Sham+SGB）group,shock（Shock）group and shock+SGB（Shock+SGB）group,with 13rats in each group.In the awake state of the rat,the hemorrhagic shock model of the conscious rat was established by the method of losing 40%of the whole blood volume.Rats were treated with SGB at 1 hour after bloodletting or corresponding time point.The control group was injected with an equal volume of normal saline.The appearance of drooping eyelids is a sign of successful treatment of SGB.Subsequently,the drawn whole blood was diluted with Ringer’s solution in equal proportions for fluid resuscitation.After resuscitation,the rats were sent back to the animal room for breeding.The effect of SGB post-treatment on the survival rate of rats after hemorrhagic shock was observed within 72 hours after resuscitation.The results showed that the rats in the Sham group and Sham+SGB group survived for a long time,and the survival rate was 100%.In Shock group,3rats survived within 72 hours,the median survival time was 17.5 hours,and the survival rate was 23.1%.In Shock+SGB group,6 rats survived within72 hours,the median survival time was 76.9 hours,and the survival rate was 53.8%.The results suggested that SGB post-treatment significantly improved the survival rate of rats after hemorrhagic shock.Healthy male Wistar rats（SPF grade,weighing 300±20g）were randomly divided into 6 groups,as follows:Sham group,Sham+SGB group,Shock group,Shock+SGB group,Shock+3-Methyladenine（3-MA,autophagy inhibitor）group and Shock+SGB+rapamycin（RAPA,autophagy agonist）group,each with 6 animals.The awake hemorrhagic shock model of rats was established according to the above method,and the blood loss,resuscitation and SGB post-treatment were performed.During fluid resuscitation,Shock+3-MA group and Shock+SGB+RAPA group were given 3-MA and RAPA treatment respectively.Three hours after resuscitation,the laser speckle dynamic blood flow monitoring system（Peri Cam PSI）was used to observe the blood flow changes in a segment of the ileum（10 cm in length and 3 cm from the ileocecal region）.Then both ends of the ileum were ligated,and FITC-labeled dextran 4(FD4,mg·mL-1)was injected into the intestinal cavity.After 30 minutes,the plasma FD4concentration was measured to evaluate the intestinal permeability.Some of the fresh intestinal tissues were stained with HE to observe the morphological changes under light microscope.The other part is used to compare the wet weight to dry weight ratio of intestinal tissue,namely wet/dry ratio（W/D）.The intestinal villi tissues were collected,and the expression of intestinal barrier proteins（ZO1,Occludin,Claudin-1）and autophagy marker proteins（LC3,Beclin-1,P62）were detected by western blotting technology.The results of blood flow in the loop of intestine showed that the blood flow in the loop of intestine decreased significantly after hemorrhagic shock.SGB or 3-MA significantly increased the blood flow of the intestinal loop in the Shock group,while SGB had no effect on the blood flow of the intestinal loop in the Sham group.RAPA decreased the blood flow in the intestinal loop of the Shock+SGB group.Histological observation showed that the intestinal tissues and villi in the Sham group were intact,with regular morphology and no edema.After hemorrhagic shock,the intestinal villi became shorter and irregular in shape and had obvious histological damage.The height of intestinal villi and the thickness of submucosa and muscular layer were all decreased.Intestinal tissue W/D increased.The intestinal villi height and the thickness of submucosa and muscularis layer were all decreased,and the intestinal tissue W/D increased.the SGB post-treatment or 3-MA treatment all alleviated the morphological damage and edema of intestinal tissue in hemorrhagic shock rats,and improved the intestinal villi height,submucosal thickness and muscle layer thickness.The combined application of RAPA aggravated the intestinal structure damage of rats in the Shock+SGB group.The intestinal permeability results showed that the intestinal permeability of rats in the Shock group was significantly higher than that in the Sham group.The expressions of intestinal barrier tight junction proteins ZO-1,Occludin and Claudin-1 were decreased in the Shock group.The SGB post-treatment or 3-MA treatment significantly restored the permeability of the intestinal barrier in the Shock group,and increased the expression of ZO-1,Occludin and Claudin-1.The combined application of RAPA increased the intestinal permeability of the Shock+SGB group.The results of autophagy marker protein detection showed that the hemorrhagic shock increased the expression of LC3-II and Beclin-1,and decreased the expression of P62 protein in intestinal tissue of rats.The SGB post-treatment and 3-MA treatment significantly down-regulated the expression of LC3-II and Beclin-1and up-regulated the expression of P62in the intestinal tissues of rats in the Shock group.And SGB treatment had no significant effect on the expression of LC3-II,Beclin-1 and P62 in Sham group rats.Compared with the Shock+SGB group,the combined application of RAPA significantly increased the expression of LC3-II and Beclin-1,and decreased the expression of P62 protein.In summary,SGB post-treatment can significantly improve the survival rate of rats after hemorrhagic shock.Both SGB and autophagy inhibitor 3-MA increased intestinal blood flow in hemorrhagic shock rats,reduced intestinal tissue morphological damage,reduced intestinal barrier permeability,and inhibited the activation of autophagy.On the contrary,the beneficial effects of SGB can be reversed by RAPA,a specific agonist of autophagy.These findings indicate that SGB post-treatment can help reduce intestinal barrier damage in rats with hemorrhagic shock,and its mechanism of action is achieved by inhibiting autophagy. |
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