| BackgroundSepsis is a major serious medical condition in Intensive Care unit(ICU). Sepsis is a dysregulated systemic inflammatory reaction caused by bacteria, fungi, virus and other infection factors,which was often followed multiple organ dysfunction syndrome(MODS).However, the acute lung injury(ALI)often occurs earliest in sepsis and can be used as a symbol of deterioration, which is more severe forms called acute respiratory distress syndrome(ARDS).Because the disease has a rapid progression, a complex etiology and pathogenesis and the high mortality rate, it has become the first factor of mortality in ICU. Although the anti-infection and organ function support treatment technology has made remarkable development,the mortality of sepsis associated with ALI is still as high as 50%-70%. So far, there is a lack of definite therapeutic drugs in clinical, therefore,it has very important meaning to research and develop effective drugs for the treatment of sepsis.When sepsis occurs,the innate immune system denfense against microbial invasion.Toll like receptors family(TLRs) is the first line of defense to pathogen invasion.TLRs can identify pathogens, transfer downstream signaling and mediate the generation of inflammatory factors. The TLR4 can specifically identify the major component of the cell wall of Gram-negative bacteria. Subsequently, this triggers several distal events, including the activation of downstream signaling pathways. Among which NF-kappa B(NF-κB) is the critical element in TLR signaling. Once activated, NF-κB regulates a series of cellular responses, including the production of proinflammatory cytokines, such as TNF-α,IL-1β and ICAM-1. These cytokines and mediators are very potent to induce multiple reactions that counteract the growth and dissemination of pathogens, enhancing the entire adaptive immune response.But the persistent inflammation is harmful and even lead to death of the body.In order to avoid inappropriate inflammatory responses, negative regulatory mechanisms on TLR/NF-κB signaling activation can reduce the local inflammation reaction in a certain extent, which may be a new target for clinical control inflammatory reaction.The major feature in sepsis is the cross activation of inflammation,blood coagulation, anti-freezing and fibrinolytic activity decline,which are also the important factors that affect the prognosis.So inhibited the cross-activation of inflammatory and coagulation system may contribute to control sepsis.Based on this, anticoagulant drugs used as anti-inflammatory has become a hot topic in the treatment of sepsis.Unfractionated Heparin(UFH) and Low Molecular Weight Heparin(LMWH) belong to anticoagulant drugs,which is commonly used in clinical.In vitro, heparin and its derivatives can inhibit the inflammatory response through TLR4 pathway.Its derivatives had a capable of anti-inflammation by inhibiting NF-κB signaling through TLR4-Myeloid differentiation factor 88(MyD88) mediated.LMWH can inhibit the expression of NF-κB, which leads to reduce the generation and release of pro-inflammatory factors.Because LMWH has smaller molecular weight, higher bioavailability, greater anticoagulant effect, longer half-life period,fewer side effects, so LMWH is increasingly used in clinical instead of UFH.It is still unclear weather it has protective effect in ALI caused by sepsis.Therefore,we choose LMWH as the research object in this study, to evaluate its effect on ALI induced by sepsis and its possible mechanism, in order to provide a theoretical basis for clinical treatment of LMWH in sepsis.Poly-microbial sepsis induced by cecal ligation and puncture (CLP) is the most frequently used model,because it closely resembles the progression and characteristics of human sepsis.Herein, this study is to observe the influence of low molecular weight heparin on acute lung injury induced by cecal ligation and puncture (CLP), and the morphological studies. Meanwhile, the degree of pulmonary edema, activity of myeloperoxidase(MPO),the expression of inflammation factors in gene and molecule levels and the expression of proteins related to TLR4-NF-κB axis were detected.In the research,we further elucidate the possible effect of LMWH and its mechanism on ALI induced by sepsis.This could provide new ideas for clinical diagnosis and treatment of sepsis in clinical.Part I Study on the protective effect of LMWH on CLP-induced ALI in ratsObjective:The aim of the research is to evaluate the protective effect of LMWH on ALI induced by septic rats.Methods:150 male Wistar rats were randomly divided into the following:sham group; CLP group; LMWH treated with small dose group (LLMWH); LMWH treated with medium dose group (MLMWH)and LMWH with high dose treatment group (LLMWH); Observation and record the behavior of rats and the vital signs of living after operation; Collect the bronchoalveolar lavage fluid (BALF),serum and lung tissue on the time of 6h,12h and 24h after-operation. Arterial blood gas analysis was checked to observe the change of PO2; Lung tissue specimens were used for morphology research by gross specimen observation, HE staining and Masson staining; Evaluation the lung edema through water content;The inflammation cell were counted in BALF; BCA method for the determination of protein concentration in BALF; MPO activity, content of ICAM were measured in lung.The level of TNF-a, IL-1β were detected in blood by radioimmunoassay.Results:1.Effect of LMWH on the general condition of rats:Postoperation 4h,the CLP-rats began to appear arched, reduced activity, reduced food intake, dispirited spirit;over 6h,the rats appear shortness of breath, about 120-150 times/min, cough, arched and forelimb shrinkage;Over 12h,the dyspnea aggravated and breath rate reached to 200-250 times/min; The rats emerge cyanosis in the end of limbs,loss of gloss, the standing hair.The serious one can appear immobility and irregular rhythm breath. Anatomy of the peritoneal cavity shows:The tendency of bleeding is obvious, the proximal bowel were dilated, the ligated cecum was dark.With hepatomegaly and pulmonary congestion.A part of rats exist mesenteric adhesions, with odor or bloody ascites; The situation of dyspnea and activity was significantly reduced in 3 treated groups compared with the CLP group; Liver mild swelling and pulmonary mild congestion can be visible in the abdominal anatomy.Of those, the HLMWH treatment group has significant bleeding tendency.There is no such performance in sham group.2.The changes of respiratory rates on each time point: ① The respiratory rates increased gradually in CLP group, there was significant difference from sham groups on each time point(P<0.01);The most obvious respiratory distress appears from 12h to 24h;The respiratory rates increased significantly On 12h and 24h compared to 6h, the difference was statistically significant (P<0.05),what is more,there was no significance different between 12h and 24h(P>0.05);② After treatment, the respiratory rates had no difference between the LLMWH treatment groups and CLP group on each time point(P>0.05).But the other two treatment groups contrast with CLP group,only on 12h and 24h,the respiratory rates decreased, there were significant difference between the groups (P<0.05);③On the same time point, the different doses of LMWH treatment group had no significant difference in respiratory rate (P>0.05).3.The changes of PO2 on each time point:(DThe PO2 declined obviously in CLP group, there was significant difference from sham groups(P<0.01);The PO2 descends to the lowest on 12h;There were statistically significant between 12h and 6h,12h and 24h (P<0.05);what is more,there was no significance different between 6h and 24h (P>0.05); ②After treatment, the PO2 raised in MLMWH and HLMWH treatment groups compared with CLP group on each time point(P<0.05);There were statistical significance between LLMWH group and CLP group on 12h (P<0.05),but not on 6h and 24h(P>0.05).③On the same time point, the different doses of LMWH treatment group had no significant difference in PO2 (P>0.05).4.Pulmonary pathological changes:4.1 Gross observation on 24h:Sham group:the lung surface were ruddy coated, and has good elasticity.There is no bleeding and oozing;There is no bloody foam and blood coagulation overflow in the lung section. CLP group lung surface appear pale, congestion and edema, more subcapsular point.The gravity dependent pulmonary edema can be visible in part of the rat on lung surface; and there is more bubble liquid overflow along the bronchi section;LLMWH and MLMWH treatment group show: lung surface was pink and edema reduced, with a small amount of bloody fluid overflow from the incision; While the HLMWH treatment group:diffuse bleeding happened on the lung surface in some rats.4.2 HE staining in sham group:The lung tissue structure is clear and complete;The alveolar walls are thin, without inflammatory cells infiltrated.while the CLP model shows:the alveolar wall thickening, neutrophil infiltration, small amounts of red cell exudation, exudation of protein-rich edema,formation of hyaline membrane and desquamation of epithelial cells. The three treatment groupss show:lung lobules structure is partly clear.There is a small amount of exudate in the alveolar cavity,alveolar wall mild thickening.There is attenuated of alveolar exudation and inflammatory cells infiltration in alveolar interstitial.The tracheal epithelial cells are nearly complete.4.3 Masson staining on 24h:The Sham group shows:there is a little green stained collagen deposition,while a large amounts of green stained collagen deposition in the lung interstitiumof CLP group.In the treatment groups the amount of the green stained collagen deposition is between CLP group and Sham group.5.The changes of W/D in different groups:① The W/D increased in CLP group compared with sham group on each time points, there was significant difference (P<0.01);The degree of pulmonary edema raise to the highest on 12h;There were statistically significant between 12h and 6h,12h and 24h (P<0.05); what is more,there was no significance different between 12h and 24h. ② After treatment, the W/D declined in MLMWH group compared with CLP group,there were statistical significance on each time point(P<0.05);While the other two treatment groups,only on 6h and 24h,there were significant difference of W/D compared with CLP group (P<0.05); ③ The W/D had no significant difference in the three LMWH treatment groups on each time piont(P>0.05).6.The inflammatory cell count in BALF:6.1 Total leukocyte count in BALF: ①The leukocyte count increased in CLP group compared with sham group on each time points, there was significant difference (P<0.01); The number of leukocyte count rise to the highest on 12h.There were statistically significances between 6h and 12h,6h and 24h (P<0.01); what is more,there was no significance different between 12h and 24h(P>0.05). ② After treatment, the leukocyte count decreased in 3 treatment groups compared with CLP group on 12h and 24h,there were statistical significance(P<0.01);On 6h,only HLMWH treatment group had significant difference from CLP group in leukocyte count(P<0.05).③The inflammatory cell count in BALF had no significant difference in the three LMWH treatment groups on each time piont(P>0.05).6.2 Total neutrophil count in BALF:①The neutrophil count increased in CLP group compared with sham group on each time points, there was significant difference (P<0.01); The number rise to the highest on 12h.There were statistically significances between 6h and 12h,6h and 24h (P<0.01); The neutrophil count is higher On 24 than that on 6h, there were statistical significance(P<0.01). ② After treatment, the neutrophil count decreased in 3 treatment groups compared with CLP group on 12h and 24h,there were statistical significance(P<0.01);On 6h,only HLMWH treatment group had significant difference from CLP group in neutrophil count(P<0.01).7.The changes of MPO activity in each group: ①The MPO activity increased in CLP group compared with sham group on each time points, there was significant difference (P<0.01); The activity of MPO raise to the top on 12h.There were statistically significance between 6h and 12h(P<0.05); There were no statistical significance between 12h and 24h,6h and 24h(P>0.05). ②After treatment, the MPO activity decreased in 3 treatment groups compared with CLP group on 12h,there were statistical significance(P<0.05);On 6h,only HLMWH treatment group have significant difference from CLP group in MPO activity(P<0.05).On 24h, there were no statistical significance between the treated groups and CLP group(P>0.05).③On 12h,the MPO activity decreased in MLMWH and HLMWH treatment groups compared with LLMWH group, there were significant difference(P<0.05),While there were no difference on 6h and 24h in the three treatment groups(P>0.05).8.The variability of protein content among groups:① The protein content increased in CLP group compared with sham group on each time points, there was significant difference (P<0.01); The protein content raise to the top between 12h and 24h;The protein content is higher on 12h and 24h than that on 6h (P<0.05); There were no statistical significance between 12h and 24h(P>0.05).②After treatment, the protein content decreased in 3 treatment groups compared with CLP group on each time points(P<0.05).On 24h, there were no statistical significance between the treated groups and CLP group(P>0.05);9. Expression levels of cell factors (TNF-α,IL-1β) in plasma:9.1 Expression levels of TNF-α in plasma:① Expression levels of TNF-a in CLP group were significant higher than sham group on each time point(P<0.05), The TNF-a level raise to the top on 12h, and it is higher on 24h than that on 6h in CLP group(P<0.05).②While in three LMWH treatment groups were lower than CLP group on 12h and 24h(P<0.05);On 6h, the HLMWH treatment group were lower than CLP group (P<0.05).9.2 Expression levels of IL-1(3 in plasma:①xpression levels of IL-1β in CLP group were significant higher than sham group on each time points(P<0.05), The IL-1βlevel raise to the top on 12h to 24h,and there is no difference betweenl2h and 24h in CLP group(P>0.05).②While in MLMWH and HLMWH treatment groups, the IL-1β level was lower than CLP group on each time point(P<0.05),LLMWH treatment groups, the IL-1β level was lower than CLP group on 6h and 12h(P<0.05);10. Content determination of ICAM-1:①ICAM-1 in CLP group was higher than sham-operated group on each time point (P<0.05);ICAM-1 in HLMWH was significant lower than CLP group on 6h and 12h (P<0.05).②After treatment, ICAM-1 in MLMWH group and HLMWH group was significant lower than CLP group on each time group (P<0.05);while ICAM-1 in the LLMWH group was significant lower than CLP group only on 6h and 12h(P<0.05).11. The mortality rate in each group:There was no rats deadin Sham group in 72h; While the mortality in CLP -group was 55%;The mortality of LMWH treatment group were:LLMWH(40%),MLMWH(25%), HLMWH(35%).The survival curve showed that, the LMWH treatment group could improve the survival rate of ALI rats.Conclusions:1.The ALI pathophysiological process can successfully reproduced in sepsis models. And its most serious condition of ALI occur on 12h time point in this study,because the degree of pulmonary edema is highest, the inflammatory cell infiltration is most heavy, the expression of inflammatory cytokines in blood and lung tissues is highest.2.Treatment with LMWH markedly improve the inflammation in ALI, the effects seem to be decreased the expression of TNF-α,IL-1β and ICAM-1 in the lung of ALI rats,ameliorated the micro structure change in lung,reduced the lung permeability in ALI,improved the pulmonary gas exchange.Therefore,LMWH administration had protective effect in ALI.3.The Middle dose of LMWH(150U/Kg-1)was better than the other treatment group in the long term effect in ALI,which can also significantly improve the survival of rats.Part Ⅱ Study on the protective mechanism of LMWH in sepsis-induced ALIObjective:To identify the mechanism of the anti-inflammatory effect of LMWH on ALI induced by sepsis in rats,whether through inhibiting TLR4-MyD88-NF-κB signaling pathway.Methods:Rat ALI model was reproduced by cecal ligation and puncture (CLP). All rats were randomly divided into three groups (n=50):Sham group, CLP group, CLP+LMWH treatment group(150 U kg-1). Blood was collected and lung tissue was harvested on 12h for analysis. The lung specimens were harvested for morphological studies. Lung tissue edema was evaluated by tissue water content. Meanwhile, the TNF-α, IL-1β levels in blood and ICAM-1 levels in the lung were studied. The expression of TLR4-MyD88-NF-κB signaling pathway related protein was determined by Western-Blot.Quantitative reverse transcription polymerase chain reaction (qRT-PCR) was used to measured the relative expression of TNF-α, IL-1β and ICAM-1 mRNA in lung tissue.Results:1.Expression levels of cell factors in plasma:Expression levels of TNF-α and IL-1β in CLP group were significant higher than sham-operated group (P<0.05), while in CLP+ LMWH group were lower than CLP group (P<0.05).2.Content determination of ICAM-1:ICAM-1 in CLP group was higher than sham-operated group (P<0.05), while ICAM-1 in CLP+LMWH was significant lower than CLP group (P<0.05)3.Expression levels of inflammation related proteins:Expression level of TLR4, MyD88,p-IKKβ,p-IκBα and NF-κB protein in CLP group was significant higher than Sham-operated group, CLP+ LMWH group (P<0.05, Figure 3).4.Expression levels of TNF-α,IL-1β and ICAM-1 mRNA:Expression levels of each index in CLP group were significant higher than sham-operated group (P<0.05), while in CLP+ LMWH group were lower than CLP group (P<0.05).Conclusions:These data suggest that,The protective effect of LMWH on CLP induced ALI in rats maybe related to inhibit the activation of TLR4-MyD88-NF-κB signaling pathway, downregulate the gene transcription of cytokines and reduce their generation. |
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