| Sepsis is a serious complication in critically ill patients such as bacterial infections.It is a life-threatening disease and an important cause of death for patients in the Intensive Care Unit(ICU).The definition of sepsis 3.0 was released at the 45th Annual Conference on Critical Care Medicine held in San Francisco in 2014.It is a fatal organ dysfunction caused by an unbalanced response to infection.According to the sepsis diagnostic criteria defined in sepsis 3.0,the mortality of patients with sepsis shock is greater than equal to 40%.According to statistics,about 14,000 people died from sepsis every day in the world.Irrational use of hormones,organ transplantation,human immunodeficiency virus(HIV)infection,tuberculosis,chronic obstructive pulmonary disease(COPD),artificial prosthesis intervention,abuse of antibiotics and advanced age,and cancerare susceptibility factors for sepsis.There are many treatments for sepsis,but there are some limitations and the efficacy is not satisfactory.The most common pathogenic bacteria that cause sepsis are G-bacteria.Several studies have shown that the endotoxin produced after the G-bacterial lysis is the cause of the clinical manifestations of G-bacterial sepsis and is the source of sepsis.Endotoxin is a component of lipopolysaccharide(LPS)on the outer membrane of G-bacterial cells.It releases into the blood after the death of the bacteria,and binds to receptors such as CD 14 on macrophages,resulting in the release of a series of inflammatory cytokines such as tumor necrosis factor(TNF-?),interleukin-1(IL-1),and IL-6,and triggers a series of inflammatory reactions,causing sepsis and sepsis shock.Antibiotic treatment is currently the most important treatment strategy for sepsis.However,a large number of studies have shown that although antibiotics can effectively kill bacteria in the treatment of sepsis caused by G-bacterial infections,they also lead to the release of LPS on the oiter membrane of G-cells,which is bound to cause more serious clinical symptoms of sepsis,and there is currently no treatment for the problem of LPS release after antibiotic treatment.Cyclodextrins(CDs)are a class of cyclic oligosaccharides.They are currently used mainly in the fields of environmental protection,pesticides,foods and medicines and are generally used as pharmaceutical excipients.It has been found that the most prominent structural feature of CDs is the presence of a stereophilic,hydrophobic,and stereospecific cavity in the ring.LPS is an amphiphilic substance with an anionic(phosphoric group)in the hydrophilic region and a hydrophobic region(5 to 7 lipophilic chains).In the natural state,LPS has self-assembly properties.This topic discusses the use of CDs hydrophobic cavity and LPS Lipid A(Lipid A)combined characteristics,blocking the combination of LPS and macrophages play a role in the prevention and treatment of sepsis.The results and conclusions obtained in this study mainly include:1.Effect of CDs on mouse peritoneal macrophage(RAW264.7)activityThe effects of several CDs including a-CD,?-CD,y-CD,and HP-?p-CD on the activity of RAW264.7 cells were investigated using MTT colorimetric method.The study found that at 5mM,10mM,the activity of macrophage was close to 100%,CDs were essentially no cytotoxicity;however,as their concentration increased,the cytotoxicity of CDs gradually increased at 50mM,the cytotoxicity of y-CD and HP-?-CD is significantly lower than that of a-CD and ?-CD.2.CDs inhibit the binding of LPS to macrophages and its inclusion in LPS2.1 The effect of CDs on the binding of LPS to macrophagesThe effects of a-CD,?-CD,y-CD,HP-P-CD and ?-CD-polymers on the binding of LPS to macrophages were investigated using laser confocal fluorescence microscopy.The results showed that the five types of CDs examined showed the effect of inhibiting LPS entry into macrophages at the concentration of 5mM,and the order of inhibition from strong to weak was HP-?-CDyy-CD??-CD>?-CD polymer? ?-CD.2.2 Interaction between CDs and LPSThe characteristics of the binding of LPS and water-soluble fluorescent probe 6-p-toluidine naphthalene-2-naphthalene sulfonate(TNS)to the hydrophobic cavity of CDs were used to determine the interaction of a-CD,?-CD,y-CD,HP-?-CD and ?-CD-polymers with LPS by fluorescence spectrophotometry.In the absence of LPS,TNS binds to five CDs with the highest fluorescence intensity.When LPS was added to the mixed solution of TNS and each CDs,the relative fluorescence intensity in the solution containing HP-?-CD and y-CD decreased the most,and the interaction intensity of the five CDs and LPS was y-CD>HP-?-CD>the other three kinds of CDs.This further proves that the two CDs,y-CD and HP-?-CD,have a stronger interaction with LPS.According to the above experimental results,y-CD and HP-?-CD,which have less cytotoxicity and have a greater binding effect with LPS,are used for further in vivo studies.3.The therapeutic effects of combined use of CDs and antibiotics on sepsis miceThe sepsis model was established using the gold standard-cecal ligation and puncture(CLP)prepared in an animal model of sepsis,and observe the effect of y-CD and HP-?-CD combined with antibiotics and antibiotics alone on the survival rate of CLP-model mice in 7 days.The results showed that compared with the CLP model group,the imipenem group and the two CDs combined with imipenem administration group could significantly improve the survival rate of sepsis mice(P<0.05);compared with the imipenem antibiotic group,y-CD and HP-?-CD combined with imipenem group also significantly increased the survival rate of sepsis mice(P<0.05).And the increase rate of survival rate was 20%and 30%respectively.4.The protective effect of CDs on sepsis miceIntraperitoneal injection of LPS was used to replicate the sepsis injury model in mice.Using HP-?-CD and y-CD to intervene,the levels of extracellular histone H4 and inflammatory cytokines in the blood of mice were measured,and the pathological changes of the viscera and the expression of NF-?B were examined to observe the protective effect of two CDs on sepsis mice.4.1 Effect of CDs on extracellular histone H4 levels in peripheral blood of sepsis miceThe results showed that compared with the normal control group,the content of extracellular histone H4 in peripheral blood of the sepsis model group with LPS replication was significantly higher at 6 h(P<0.05),and it was still at an increasing trend at 12 h and recovered at 24 h;at 6h,compared with the sepsis model group,the content of H4 in the peripheral blood of the y-CD group and the HP-P-CD group was significantly decreased(P<0.05);at 12 hours,there was no significant difference between the groups,but it was significantly lower than that of the model group;at 24h,H4 levels in plasma of LPS control group decreased,but H4 levels of y-CD and LPS control group remained significantly lower(P<0.05),while there was no significant difference between HP-?-CD group and LPS control group.This ind:icates that both y-CD and HP-?-CD significantly reduce the effect of extracellular histones in the plasma of sepsis mice.4.2 Determination of TNF-a levels in mouse plasmaThe results showed that compared with the normal control group,the level of TNF-a in the plasma of the LPS model control group at the three time points examined was significantly higher(P<0.01);compared with the LPS model control group,the concentration of TNF-a in the plasma of the y-CD group and the HP-?-CD group at the three time points examined was significantly lower(P<0.0]);In addition,the TNF-?levels were significantly lower in the y-CD group than in the HP-?-CD group at 12 h(P<0.05).This shows that both y-CD and HP-?-CD have a role in reducing plasma TNF-? levels in sepsis mice,and y-CD is more effective than HP-?-CD.4.3 Determination of IL-6 level in mouse plasmaThe results showed that compared with the normal group,the IL-6 concentration in the plasma of the LPS model group did not change significantly at 6 hours,but increased significantly at 12 hours and 24 hours(P<0.05).Compared with LPS model control group,there was no significant difference in y-CD administration group and HP-?-CD administration group at 6h.At 12 hours,IL-6 concentration in plasma of ?-CD group was significantly lower(P<0.05).At 24 hours,the ?-CD administration group and the HP-?-CD administration group were significantly lower than the LPS control group(P<0.05).In addition,IL-6 levels were significantly lower in the y-CD group than in the HP-P-CD group(P<0.05).The results showed that both y-CD and HP-?-CD reduced plasma IL-6 levels in sepsis mice at 12 and 24 h after LPS was established.Moreover,y-CD reduced plasma IL-6 levels in sepsis mice more significantly than HP-?-CD.4.4 Histopathological examination of sepsis miceThe results of HE staining showed that in the sepsis model group,the alveolar vascular congestation and mononuclear macrophage proliferation were most obvious at 12 h,indicating that the lesions were most severe at 12h.This section also observed the liver and spleen of sepsis mice.The results showed that there were no obvious lesions in the liver and spleen of the sepsis model mice.This indicates that the tissue damage of septic mice reproduced in this experiment is mainly manifested in the lungs.At the three time points of 6h,12h and 24h after modeling,both y-CD and HP-?-CD could effectively reduce alveolar wall thickening,alveolar wall vascular congestion,and mononuclear macrophage proliferation in sepsis mice.The treatment effect of y-CD and HP-?-CD on lung injury in sepsis mice was more obvious at 12 hours,and the therapeutic effect of y-CD was more significant.Immunohistochemistry results showed that NF-KB was expressed in normal control group,LPS model group,and two CDs-administered groups.The above experiments showed that the level of TNF-a in the LPS model group was significantly increased,resulting in a large number of apoptosis of the alveolar epithelial cells,and therefore the positive expression of NF-kB in the LPS group was decreased.The results of this part of the experiment showed that plasma levels of H4,TNF-?a,and IL-6 were significantly elevated in LPS-induced sepsis mice.Both-CD and HP-?-CD can effectively reduce the levels of these inflammatory components and reduce lung injury in sepsis mice.5.Effect of CDs on tissue distribution and excretion of FITC-LPS in miceThe LPS was traced with FITC to study the absorption,distribution and excretion of LPS in mice under the action of CDs.The results showed that LPS was rapidly absorbed into the blood after intraperitoneal injection.After administration for 0.5h,LPS peaked in mouse plasma and then gradually decreased.It was shown that LPS entered the tissues with blood circulation and was then excreted in urine and feces.The presence of y-CD and HP-?-CD both reduced the LPS content in plasma and tissues of mice(compared with the control group,P<0.05),and the effect of y-CD was stronger than that of HP-?-CD.LPS is widely distributed in mice and distributed throughout the investigated tissues,including heart,liver,kidney,lung,and spleen.However,the distribution of tissues is uneven,mainly distributed in the heart,liver and kidneys.After 0.5h of administration,LPS reached the tissues with blood circulation,and the fluorescence intensity in the liver was the highest,indicating that LPS was the first to enter the liver metabolism,and this result was confirmed by the results in feces.The fluorescence intensity in the kidneys followed,indicating that LPS was excreted from the urine through the kidneys with CDs.This result was confirmed by the results in urine.Both CDs could significantly reduce the content of LPS in mouse tissues.From the results of the mouse urine and feces,the fluorescence intensity in the feces is generally lower than in the urine,indicating that LPS is mainly excreted in the form of urine by the kidneys.Both y-CD and HP-P-CD significantly promote the excretion of LPS from urine and feces.6.ConclusionThe interaction of LPS with CDs can block the inflammatory storm triggered by the binding of LPS to macrophages,exert its protective effect on sepsis mice from the source,and promote the excretion of LPS from the body.That is,y-CD or HP-?-CD combined with antibiotics can significantly increase the survival rate of sepsis mice,which is expected to become a new strategy for the clinical treatment of sepsis. |
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