| Exosome is a kind of extracellular vesicle,which can effectively protect its internal material activity and realize the material transport in physiological and pathological state.In recent years,it has been found that exosomes are involved in organ dysfunction and structural damage caused by hemorrhagic shock.The return of the exosome carried by post-hemorrhagic shock mesenteric lymph(PHSML)to the whole body is one of the important mechanisms and key links in the occurrence of acute lung injury(ALI).Therefore,it is of great significance to search for the prevention and treatment of PHSML exosomes to alleviate the tissue and organ injury after hemorrhagic shock.Stellate ganglion block(SGB)has been widely used in the clinical treatment of patients with pain and gradually extended to other diseases.Experimental studies in our laboratory show that SGB can prolong the survival time of rats with severe hemorrhagic shock,improve the function of intestinal barrier and relieve ALI,but the detailed mechanism is not clear.Is the role of SGB in alleviating ALI after hemorrhagic shock related to the exosomes carried by PHSML? There was no report.It is hypothesized that SGB alleviates ALI induced by hemorrhagic shock by affecting the number and intrinsic material activity of exosomes in PHSML.In order to test this hypothesis,based on the establishment of an exosome isolation technique,the current study observed the effect of SGB on the number of exosomes in PHSML,and investigated the direct effect of exosomes in PHSML on lung tissue and endothelium in rats,so as to clarify the role of exosomes in PHSML in alleviating ALI after hemorrhagic shock,and to provide experimental data for prevention and treatment of organ injury caused by hemorrhagic shock.Firstly,we observed the effect of SGB on the number of exosomes in PHSML.SGB method was established through simulating long-term reversibility of ropivacaine injection into stellate ganglion(SG).The exosomes were extracted and identified by using the techniques of particle size analysis and CD63 protein expression,which suggested that the extraction of lymphatic exosomes in this study was successful.Twenty-four male rats were randomly divided into four groups(n=6): Sham group,Sham+SGB group,Shock group and Shock+SGB group.Hemorrhagic shock model was established by routine method,PHSML was drained and exosomes were extracted,the results showed that the number of PHSML was significantly increased after hemorrhagic shock,and SGB decreased the number of PHSML in hemorrhagic shock rats.Further,the expression of Ep CAM protein was detected by Flow cytometry and Western blot.The results showed that each group of rat PHSML expressed specific protein in intestinal epithelial cells,and the exosomes in the lymph fluid came from the intestinal epithelial cells.Secondly,we observed the effect of exosomes on pulmonary tissue structure in rats treated by SGB,36 male rats were randomly divided into Sham group(n=12),Sham+SGB group(n=6),Shock group(n=12)and Shock+SGB group(n=6).Subsequently,twenty four normal rats were randomly divided into fourgroups(n=6),exosomes extracted from PHSML after different treatments were re-infused into each group: Exo-Sham,Exo-(Sham+SGB),Exo-Shock,Exo-(Shock+SGB),twelve normal rats were randomly divided into two groups(n=6),the model of hemorrhagic Shock was established and SGB+Shock was administered to the exosomes(Shock+SGB+Exo-Sham)and the exosomes(Shock+SGB+Exo-Shock)respectively.After hypotension for 60 minutes,fluid resuscitation and reinfusion of exosomes in all rats,lung tissue was taken for observation of morphological changes and lung tissue wet / dry ratio(W/D),to reflect the effect of SGB on the activity of exosomes in PHSML.The results showed that there was no significant difference in histological score and W/D between Exo-Sham group and Exo-(Sham+SGB)group,the histological score and W/D of Exo-Shock group were significantly higher than that of Exo-Sham group,and the histological score and W/D of Exo-(Shock+SGB)group were significantly lower than that of Exo-Shock group.The pulmonary histological score and W/D in Shock+SGB+Exo-Shock group were significantly higher than that in Shock+SGB+Exo-Sham group.Finally,the effects of SGB on exosome in PHSML damaging pulmonary endothelium in rats were observed.First,primary PMVECs were cultured and expression of CD31 was tested by Flow cytometry.Then,Lymph-Sham and exosomes(Exo-Sham),Lymph-Shock and exosomes(Exo-Shock)were applied to PMVECs,and the proliferation activity of PMVECs was measured by CCK8.The results showed that Lymph-Shock and Exo-Shock significantly decreased the proliferation activity of PMVECs.Exo-Sham,Exo-(Sham+SGB),Exo-Shock and Exo-(Shock+ SGB)were used on PMVECs to observe the changes of proliferation activity of PMVECs.The results showed that the proliferation activity of PMVECs in Exo-Shock group was significantly lower than that in the Exo-Sham group.Meanwhile,the proliferative activity of PMVECs in Exo-Shock+SGB group was significantly higher than that in Exo-Shock Group.The results showed that SGB significantly reduced the number and toxicity of exosomes in PHSML,which are important components of SGB in alleviating lung injury induced by hemorrhagic shock. |
