| Sepsis is a life-threatening organ dysfunction syndrome caused by the host’s dysfunctional response to infection,which is one of the common causes of death of inpatients in intensive care unit.In the occurrence and development of sepsis,the gut has long been considered to play a crucial role,and is the "motor" that causes multiple organ dysfunction syndrome.The gut acts as a defensive barrier between the body and the environment,preventing toxic agents and pathogens from circulating in the gut.Damage to the intestinal barrier during sepsis dispositions bacteria and endotoxins and adversely affects distal organ structure and function,leading to multiple organ dysfunction syndrome.Dexmedetomidine is a highly selective α2 adrenergic receptor agonist,which has good sedation,easy awakening without causing obvious respiratory depression,and can significantly reduce postoperative delirium in patients.Dexmedetomidine is widely used in severe and perioperative patients.In recent years,the protective effect of dexmedetomidine on organ function in sepsis has become a hot topic of research.Some studies have shown that dexmedetomidine has a protective effect on intestinal barrier damage caused by sepsis.However,what is the mechanism of its protective effect on intestinal barrier function in sepsis?It remains unclear.Macrophages are an important component of the intestinal immune barrier,initiating and coordinating an effective immune response to bacteria that breach the intestinal epithelial barrier.Most resident intestinal macrophages are replenished by blood monocytes.Under physiological conditions,blood-derived monocytes gradually acquire the tissue-resident phenotype of anti-inflammatory macrophages in the intestine,namely M2-type macrophages,which secrete high levels of anti-inflammatory factors such as IL-10.During sepsis,when the body is in an inflammatory state,blood-derived monocytes acquire more pro-inflammatory phenotypes in the intestine,namely M1 macrophages,secreting TNF-α,IL-1β and other pro-inflammatory cytokines,affecting the expression and localization of intestinal tight junction protein.It has been demonstrated that M2-type macrophages promote intestinal epithelial barrier tightness,while pro-inflammatory M1-type macrophages induce increased intestinal barrier permeability.Therefore,the polarization phenotype of macrophages is closely related to intestinal barrier function in sepsis.Does dexmedetomidine exert a protective effect on gut barrier function in sepsis by regulating macrophage polarization phenotype? unclear.Therefore,in this study,the sepsis model was established by cecal ligation and puncture,lipopolysaccharide,Intestinal epithelial cell and RAW264.7 cell models treated with LPS were used to research the effect and mechanism of dexmedetomidine on intestinal barrier function in sepsis rats at the in vitro and in vivo levels,respectively.The studies included the following two parts:(1)The protective effect of dexmedetomidine on intestinal barrier function in sepsis rats;(2)The mechanism of dexmedetomidine regulating macrophage polarization to play a role in intestinal barrier protection.Research content:Part I:Protective effect of dexmedetomidine on intestinal barrier function in septic rats1.Effect of dexmedetomidine on intestinal permeability in septic ratsTo investigate the effect of dexmedetomidine on intestinal permeability,we observed leakage of evans blue from the intestine,the content of d-lactic acid and diamine oxidase,the level of TNF-α and IL-1β and the pathological structure of intestinal tissue.2.Effect of dexmedetomidine on the permeability of intestinal epithelial cells in sepsisThe intestinal epithelial cells were stimulated with lipopolysaccharide to replicate the sepsis model.We measured the expression of ZO-1 and Occludin in intestinal epithelial cells by immunofluorescence and western blot.3.Effect of dexmedetomidine on organ function in septic ratsThe results of dexmedetomidine on liver function(ALT,AST),renal function(creatinine),myocardial function(CK-MB),arterial blood pressure and blood gas were observed.4.Effect of dexmedetomidine on survival time and survival rate of sepsis ratsThe rat model of sepsis was established to observe the effect of dexmedetomidine on survival time and survival rate in sepsis rats.Part II:The mechanism of dexmedetomidine regulating macrophage polarization and exerting intestinal barrier protection1.Effect of dexmedetomidine on intestinal macrophage polarization in sepsis ratsWe uesd flow cytometry to separate macrophage subtypes from single cell suspension of intestinal tissue(CD86 labeled M1 macrophages and CD206 labeled M2 macrophages).And we measured the expression of i NOS in intestinal tissue by immunofluorescence.2.Effect of dexmedetomidine on polarization of RAW264.7 cells stimulated by LPSLPS was used to stimulate RAW264.7 to replicate the sepsis model.The markers of M1 macrophage and M2 macrophage were measured by immunofluorescence,ELISA and western blot.INOS,TNF-α,IL-6,IL-β were labeled M1-type macrophages,and CD206 and Arg1 were labeled M2-type macrophages.3.Metabonomics analysisMetabolomics was used for quantitative analysis of all metabolites in RAW264.7 cells,and KEGG was used for pathway enrichment of differential metabolites.4.The mechanism of dexmedetomidine regulating macrophage polarizationAccording to the metabolomics results,we used ELISA to detect glutathione content in RAW264.7 cells,western blot to detect the expression of x CT and glutathione peroxidase 4,and confocal microscopy to observed ROS and lipid ROS in macrophage.To determine whether dexmedetomidine can regulate the synthesis of glutathione and glutathione peroxidase 4,reduce ROS and lipid ROS,inhibit macrophage ferroptosis,and thereby affect the polarization of macrophages.Results:Part I: Protective effect of dexmedetomidine on intestinal barrier function in septic rats1.Effect of dexmedetomidine on intestinal permeability in septic ratsAfter sepsis,the intestinal permeability of rats were increased,which is manifested by evans blue leakage,blood d-lactic acid and diamine oxidase,and inflammatory factor TNF-αand IL-1 β were significantly increased.Intestinal villi was atrophy and fracture,and the intestinal tract was infiltrated by a great deal of red blood cells and inflammatory cells.After treatment with dexmedetomidine,evans blue leakage,blood d-lactic acid and diamine oxidase,and inflammatory factor TNF-α and IL-1β were significantly decreased.Intestinal villi was significantly increased and the number of red blood cells and inflammatory cells were significantly reduced in this group.2.Effect of dexmedetomidine on permeability of intestinal epithelial cells in sepsisAfter lipopolysaccharide stimulation,the expression of ZO-1 and Occludin were obviously decreased.The results of immunofluorescence showed that the expression of ZO-1was weakened and obviously broken,and the distribution was loose in this group.In dexmedetomidine group,the expression of ZO-1 and Occludin were obviously increased,and immunofluorescence showed that the distribution of ZO-1 was more continuous and clear.3.Effect of dexmedetomidine on organ function in septic ratsIn sepsis group,the function of liver,kidney and myocardium was seriously damaged,the arterial blood pressure was significantly decreased,oxygen partial pressure and p H were obviously decreased,and partial pressure of carbon dioxide was obviously increased.After treatment with dexmedetomidine,the damage of liver,kidney and myocardium were significantly reduced,arterial blood pressure was increased,oxygen partial pressure and p H were obviously increased,and partial pressure of carbon dioxide was obviously decreased.4.Effect of dexmedetomidine on survival time and survival rate of sepsis ratsSurvival rate and survival time of rats in sepsis group were obviously decreased;After dexmedetomidine treatment,the survival rate and time of rats were obviously increased.Part II:The mechanism of dexmedetomidine regulating macrophage polarization and exerting intestinal barrier protection1.Effect of dexmedetomidine on intestinal macrophage polarization in septic ratsIn sepsis group,the number of proinflammatory M1 macrophages were increased,which is manifested by the number of CD86(+)CD206(-)macrophages in single-cell suspension of intestinal tissue was significantly increased,and the expression of i NOS was significantly increased.After treatment with dexmedetomidine,the number of CD86(+)CD206(-)macrophages in intestinal single-cell suspension was significantly decreased,and the number of CD86(-)CD206(+)macrophages was significantly increased,which suggest that dexmedetomidine could inhibit the polarization of macrophages to M1 and promote the polarization of macrophages to M2.2.Effect of dexmedetomidine on polarization of RAW264.7 cells stimulated by LPSAfter LPS stimulation,macrophages were significantly polarized towards M1 type,which showed that the expression of i NOS was significantly increased,and the levels of inflammatory cytokines TNF-α,IL-6 and IL-1β were increased.In dexmedetomidine group,the expression of i NOS was significantly decreased,while the expressions of CD206 and Arg1 were significantly increased,which indicated that the polarization of macrophages towards M1 decreased and that towards M2 type macrophages increased.3.Metabonomics analysisMetabolomics mass spectrometry showed that there were 274 different metabolites between dexmedetomidine group and LPS group,among which cysteine and glutathione were significantly different.KEGG enrichment analysis of the different metabolites showed that cysteine and glutathione were closely related to the ferroptosis pathway.4.The mechanism of dexmedetomidine regulating macrophage polarizationAfter LPS stimulation,the glutathione in RAW264.7 cells was decreased,the expression of x CT and glutathione peroxidase 4 were significantly decreased,the ROS and lipid ROS were significantly increased,which means that lipopolysaccharide promoted ferroptosis in macrophages.In dexmedetomidine group,glutathione was increased,the expression of x CT and glutathione peroxidase 4 were obviously increased,and intracellular ROS and lipid ROS were obviously decreased.This indicated that dexmedetomidine can suppresse ferroptosis in macrophages caused by LPS and inhibit macrophages polarization to M1 type.Conclusion:1.Dexmedetomidine can protect the intestinal barrier function of sepsis rats.By inhibiting inflammatory response,dexmedetomidine can increase the expression of tight junction protein between intestinal epithelial cells,significantly reduce intestinal permeability,and play a protective role in other organ functions of in rats with sepsis,and finally prolong the survival time and improve the survival rate of sepsis rats.2.Dexmedetomidine can significantly reduce the number of intestinal inflammatory M1 macrophages in sepsis rats,promote the polarization of macrophages towards anti-inflammatory M2 type,inhibit the secretion of inflammatory cytokines,and thus play a protective role in the intestinal barrier function of sepsis rats.3.Dexmedetomidine increased the expression of glutathione and glutathione peroxidase4 in macrophages by affecting the transporter x CT,thereby reduced the level of intracellular ROS and Lipid ROS,suppressed ferroptosis in macrophages,and inhibited macrophages polarization to M1 type. |
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