Role Of Intestinal Flora Remodeling In Stellate Ganglion Block Reducing PHSML-Mediated Lung Injury

Hemorrhagic shock causes uncontrol systemic inflammatory response,leading to multiple organ dysfunction.Acute lung injury(ALI)is one of the main causes of death in critically ill patients.Proinflammatory factors or toxic substances produced by ischemic intestine enter the systemic circulation through the mesenteric lymphatic duct.Post-hemorrhagic shock mesenteric lymph(PHSML)return is an important reason of ALI following hemorrhagic shock attack.Hemorrhagic shock impairs the contractility and barrier of intestine,thereby bacteria and their metabolites contained in the injured intestine migrate to systemic circulation,leading to gut derived infection.Studies have shown that stellate ganglion block(SGB)reduced intestinal barrier dysfunction and structural damage by inhibiting autophagy,thereby reducing hemorrhagic shock-induced ALI,which was related to PHSML return.However,it remains unclear whether excessive autophagy occurred in intestinal smooth muscle cells after hemorrhagic shock,and if so,whether SGB improves the intestinal motility and restores the homeostasis of intestinal flora by inhibiting intestinal smooth muscle autophagy.Therefore,the aim of present study is to confirm whether SGB improves intestinal smooth muscle contractility by inhibiting autophagy in conscious rats with hemorrhagic shock,to clarify whether SGB modulates the dyshomeostasis of intestinal microflora by detecting intestinal microflora and its metabolites in gut and PHSML of rats subjected to hemorrhagic shock,to determine the role of SGB alleviating intestinal microflora metabolites return to systemic circulation via mesenteric lymphatic duct pathway in the process of ALI following hemorrhagic shock.Firstly,hemorrhagic shock was established in conscious rats,and SGB was performed before operation.The motility of intestinal segment ex vivo,the immunofluorescence and western blotting was used to assess the influence of SGB,autophagy inhibitor 3-methyladenine(3-MA)and autophagy agonist rapamycin(RAPA)on the motility of intestinal segment,contractile protein p-MLC20 and autophagy protein LC3-II/I and Beclin-1in the episode of hemorrhagic shock.The results showed that the hemorrhagic shock significantly decreased the intestinal motility and p-MLC20 expression,and increased the LC3-II/I and Beclin-1 expression.SGB and 3-MA significantly increased intestinal motility and p-MLC20 expression and decreased LC3-II/I and Beclin-1 expressions in rats following hemorrhagic shock.RAPA reversed the beneficial effects of SGB on the above indices of hemorrhagic shock.Subsequently,the intestinal microflora and its metabolites in intestinal contents of conscious rats with hemorrhagic shock were observed to assess the influence of SGB on intestinal microflora and related metabolites.The results showed that hemorrhagic shock decreased the intestinal microflora diversity,which was reversed by SGB.At the level of genus,hemorrhagic shock enhanced the relative abundance of gram-positive bacteria Lactobacillus,Bifidobacterium,and Streptococcus,and decreased the relative abundance of CF231,Phascolarctobacterium,SMB53,p-75-a5 and Dolella,which were reversed by SGB treatment.The result of metabolomics showed that SGB increased the levels of creatinine,deoxyribose,4-aminophenol,2-methylserine,epigenestone,trans-trans-acacia alcohol,decreased the levels of tetrandrine,carbamoylazide,N-acetyllactamine,4-guanidine,butyric acid in the intestinal contents of rats with hemorrhagic shock.Furthermore,hemorrhagic shock was established in anesthetized rat,the NML,PHSML and SGB treated PHSML were harvested to study the changes of intestinal microflora and related metabolites in mesenteric lymph.The intestinal microflora and related metabolites in mesenteric lymph has the same varying tendency with that of intestinal contents was selected to further assay.It has been indicated that SGB reduced prostaglandin G2,deoxyuracil,nucleoside,9-Oxo ODE,m-hydroxyphenylacetic acid,o-phosphoethanolamine,l-leucine content in mesenteric lymph.The results from three combined omics analysis indicated that D-glucuronic acid(DGA)was a metabolite whose changes in PHSML and intestinal contents were consistent with those of intestinal miroflora metabolites.Therefore,DGA was selected for the further experiments.Finally,at in vitro experiment,PHSML,PHSML-SGB and DGA was co-cultured with pulmonary microvascular endothelial cells(PMVECs)to observe the proliferative activity and autophagy marker protein expression.The results showed that DGA alone did not significantly affect the proliferative activity and autophagy levels of PMVECs.PHSML significantly decreased the proliferative activity of PMVECs and increased the expression of autophagy protein LC3-II/I and Beclin-1.Compared with that of PHSML,the proliferative activity of PMVECs was increased after PHSML-SGB treatment,and the expressions of LC3-II/I and Beclin-1 were decreased.More importantly,compared to that of the PHSML-SGB group,the proliferative activity of PMVECs was decreased and the expressions of LC3-II/I and Beclin-1 were increased after PHSML-SGB plus DGA treatments.These results indicate that the influence of hemorrhagic shock on intestinal contractile function is related to excessive autophagy,the reduced intestinal motility further leads to intestinal microflora imbalance and the increase of intestinal microflora related metabolites DGA,which is transferred to the lung via PHSML and leads to increased autophagy of PMVECs and subsequent ALI.SGB improves intestinal motility by inhibiting intestinal smooth muscle cell autophagy,reversing the intestinal microflora and related metabolites levels,especially the DGA,finally alleviating the autophagy level of PMVECs mediated by PHSML.The results suggest that intestinal microflora reprogram plays an important role in SGB reducing PHSML-mediated lung injury.