Role Of Podoplanin In Septic Lung Injury And Its Monoclonal Antibody SZ168 Study On The Effect And Mechanism Of Septic Lung Injury

Background:Sepsis refers to a series of clinical syndromes caused by a deregulated host response to an infection.It is a common cause of mortality.Among the organs dysfunctioned by sepsis,lung is a frequently involved one.For patients with acute respiratory distress syndrome(ARDS),the damage the disease brings to the body will greatly accelerate depletion of medical resources.As a result,discovery of effective methods and measures to early diagnose and treat septic lung injury is vital for prevention,treatment and improvement of the prognosis of these patients.It has been shown that podoplanin(PDPN)and its ligand CLEC-2 promote tumor cell growth,metastasis and prognosis through platelet aggregation,and influence coagulation.Moreover,they play an important role in regulating the inflammatory response,which does not appear to be entirely platelet dependent as seen in many inflammatory diseases.Moreover,PDPN was expressed in lung,especially in lung macrophages.Thus,it was inferred that PDPN and its ligand CLEC-2 have a key role in septic lung injury through some mechanisms,which also contribute to the good therapeutic effect of PDPN monoclonal antibody on septic lung injury.First,PDPN was verified to be abnormally expressed to a certain extent in serum and lung tissues during the process of septic lung injury.Then,the effect and mechanism of PDPN monoclonal antibody SZ168 in the treatment of septic lung injury were fully investigated through animal experiments and cell culture studies,so as to explore the role of SZ168 in the treatment of septic lung injury,provide new ideas for the development of clinical targeted drugs,and seek safer and more effective treatment options for patients with septic lung injury as soon as possible.Aim:(1)To investigate the clinical application of PDPN in early diagnosis,severity grading,prognosis assessment and selection of possible therapeutic targets for patients with septic lung injury.(2)To observe the intervention effect of PDPN monoclonal antibody at two different doses on lipopolysaccharide(LPS)-induced ARDS in mice,and investigate its possible mechanism of action.Methods:(1)A total of 95(58 male and 37 female)patients with sepsis admitted to the Intensive Care Unit(ICU)of Wuxi People’s Hospital from January 2018 to December 2020 were selected and grouped according to Consensus Definitions for Sepsis and Septic Shock(Sepsis-3)and the Berlin definition of ARDS;if patients were diagnosed with sepsis/septic shock without ARDS,they were put into the sepsis group(40 patients);if patients were diagnosed with sepsis/septic shock and ARDS,they were put into the sepsis-induced ARDS group(55 patients),which,based on the Sepsis-3,were further subdivided into the sepsis+ARDS subgroup(29 cases)and septic shock+ARDS subgroup(26 cases);the sepsis-induced ARDS group was also subdivided into the survival subgroup(40 cases)and the death subgroup(15 cases)according to the 28d final outcome(survival or not);and the control group(30 cases)consisted of healthy patients from the medical examination center of our hospital.The general data of the patients were observed and recorded;the APACHE II score and SOFA score were recorded on the day of admission to ICU;plasma was collected for the determination of PCT,IL-6 and soluble PDPN(sPDPN)in each group;and the 28d prognosis(survival or death)of the patients in each group was observed.(2)A total of 40 experimental mice were divided into 5 groups,with 8 mice in each group.Intraperitoneal injection of LPS was applied to establish sepsis mice models.Grouping:control group(no intervention),blank group(intraperitoneal injection of saline),experimental group(intraperitoneal injection of LPS),low dose group(LPS+SZ168 low)(sepsis+low dose monoclonal antibody intervention group 1),and high dose group(LPS+SZ168 high)(sepsis+high dose monoclonal antibody intervention group 2).At 24h after modeling,blood was collected from each group of mice to measure IL-6,TNF-α and other general inflammatory factors;meanwhile,blood of the mice was collected before they were killed,and specimens were taken from lungs for HE staining and immunohistochemistry;lung permeability-related tests were performed;bronchoalveolar lavage fluid was preserved to detect inflammatory factors IL-6 and TNF-α;and mouse lung macrophages(MHS)were cultured,the phagocytic function was detected by flow cytometry,and their possible influential signaling pathways by WB assay.Another 40 experimental mice were divided into 5 groups as above,and the difference in survival rate of each group was observed.Result:(1)Clinical analysisAnalysis of general data of the patients:There were no differences in gender ratio,mean age,body temperature,creatinine,urea nitrogen,total bilirubin and albumin between the sepsis group and septic lung injury group,sepsis+ARDS subgroup and septic shock+ARDS subgroup,and survival subgroup and death subgroup(P>0.05);while initial lactate and mean arterial pressure were statistically different between the above groups(P<0.05).Association of sPDPN with inflammatory responses and scale scores in patients with sepsis and septic lung injury:sPDPN expression in patients in the sepsis group and sepsis-induced ARDS group showed significant differences compared with the control group;in the sepsis-induced ARDS groups,the expression of sPDPN was most significantly elevated,with statistical differences(P<0.05);while the expressions of CRP,PCT,IL-6 and TNF-α and SOFA and APACHE Ⅱ scores were elevated in both groups compared with the control and blank groups;patients in the sepsis-induced ARDS group showed higher expressions of CRP,PCT,IL-6 and TNF-α,and SOFA and APACHE Ⅱscores,with statistically significant differences(P<0.05);as the plasma expression of sPDPN increased in sepsis patients,the expression of inflammatory factors,extravascular lung water(EVLW)and disease severity increased simultaneously.Patients in the sepsis-induced ARDS group demonstrated a higher correlation,with a statistically significant difference(P<0.05).ELISA test:,IL-6 and TNF-α were significantly increased in the model group,and the expression of these two factors could be decreased in the SZ168 group in a dose-dependent manner,with statistical significance(P<0.05).Association of sPDPN with inflammatory responses and scale scores in patients with different degrees of septic lung injury:The expression of sPDPN also varied among patients with different degrees of sepsis-induced ARDS,and it was observed that sPDPN expression was significantly higher in patients in the septic shock+ARDS subgroup compared with that of the sepsis+ARDS subgroup.In terms of the inflammatory factor and scale scores,the expression concentrations of inflammatory factors were elevated in both the sepsis-induced ARDS and septic shock-induced ARDS subgroups,while patients in the septic shock-induced ARDS subgroup showed higher expressions of CRP,PCT,IL-6,TNF-α and higher EVLW,SOFA,and APACHEII score values;with the increase in intraplasmasPDPN expression in patients with different degrees of sepsis-induced ARDS subgroups,inflammatory factors,extravascular lung water(EVLW),and disease severity all increased significantly,while patients in the septic shock+ARDS subgroup demonstrated a higher correlation,with all of these differences being statistically significant(P<0.05).Evaluation of the prognostic impact of sPDPN on patients with septic lung injury:Patients in the death subgroup showed higher expression levels of sPDPN,CRP,PCT,IL-6,TNF-α,and EVLW,SOFA,and APACHE Ⅱ scores compared with those in the survival subgroup.The increase in sPDPN concentration in serum of patients with sepsis-induced ARDS was accompanied by an increase in the expression of inflammatory factors,extravascular lung water(EVLW)and disease severity,showing a positive correlation.And patients in the sepsis-induced ARDS death subgroup showed a higher correlation in these indicators.To quantify this correlation,the receiver operating characteristic curves(ROC)of sPDPN,PCT,and CRP for prognostic assessment were plotted,with AUC areas of 0.867,0.609,and 0.604,and critical values of 45.33 ng/ml,27.56 ng/L,and 189.74 mg/L for the three factors,respectively.Compared to the other two factors,SPDPN showed a higher correlation,and all of the above differences were also statistically significant(P<0.05).(2)Animal experiments.Survival observation:Mice were active without abnormalities in the control group and blank group during the experiment;24 h after intraperitoneal injection of LPS,mice in the experimental group,in poor conditions,showed arched back and straight hair,cowering and curling,etc.After intraperitoneal injection of LPS solution modeling,they were in poor conditions and death started within 72 hours,and continued to occur;after injection of PDPN monoclonal antibodies SZ168 solution,the status of modeled mice was worse than those in the control group and blank group,but the 7d mortality rate decreased compared to the experimental group,with the decrease in the high dose group being more obvious.ELISA detection of inflammatory factors:Serum inflammatory factors IL-6 and TNF-α were significantly increased in the model group of mice,and the administration of SZ168 was able to reduce the expression of these two factors in a dose-dependent manner,with the reduction being more obvious in the high dose group,and the difference was statistically significant(P<0.05).HE staining analysis of mouse lung specimens:In the control and blank groups,alveolar structure was intact,volume morphology regular and alveolar septum morphology clear;in the experimental group,the alveoli was partially collapsed or poorly inflated,the alveolar wall and alveolar septum significantly widened.The alveolar interstitium,congested and swollen,was infiltrated with a large number of neutrophils and macrophages,which was consistent with sepsis-induced ARDS.After the administration of PDPN monoclonal antibody SZ168,both the low dose group and the high dose group showed reduced swelling and inflammatory cell infiltration compared with the experimental group,and these effects were more significant in the high dose group.WB detection of lung permeability-related factors:The expression of Cav-1 and Occludin was reduced in the LPS group,LPS+SZ168(low)group and LPS+SZ168(high)group;however,compared with the LPS group,the expression of Cav-1 and Occludin was higher in the LPS+SZ168(low)group and LPS+SZ168(high)group,with the expression in the LPS+SZ168(high)group higher of the two,and the difference was statistically significant(P<0.05).Mouse lung macrophage(MH-S cells)-related experiments:MH-S cells grew well and had higher cell density,and the F4/80 immunofluorescence intensity increased in the LPS group,where significant macrophage proliferation was observed;while the F4/80 fluorescence intensity decreased significantly in the LPS+SZ168(low)and LPS+SZ168(high)groups,with the decrease in the LPS+SZ168(high)group more significant of the two,and the difference was statistically significant(P<0.05).Phagocytosis of MH-S cells measured by flow cytometry:Phagocytosis of lung macrophages increased in the LPS group and decreased in the LPS+SZ168(low)and LPS+SZ168(high)groups,with a statistically significant dose dependent difference(P<0.05).WB assays of key proteins related to mouse lung macrophage signaling pathway:Among total cellular proteins,p-ERK,p-JNK,p-p65 and p-IkBa expressions were significantly higher in the LPS group than those in the control group;p-ERK,p-JNK,p-p65,and p-IkBα expressions in the LPS+SZ168(low)group were also significantly lower than those in the LPS group,with expression in the LPS+SZ168(high)group lower of the two.Among the nuclear proteins,the expressions of p-p65,p-IkBa in the LPS group was significantly higher than those in the control group;however,the expressions of p-p65,p-IkBα in the LPS+SZ168(low)group were significantly lower than those in the LPS group,and expressions in the LPS+SZ168(high)group was lower of the two.There was a difference statistically significant(P<0.05).Conclusion:(1)sPDPN was differentially expressed in plasma between patients with different degrees of sepsis-induced ARDS,and between surviving and dead patients.sPDPN expression showed a simultaneous increase with the increased progressive severity of patients with sepsis-induced ARDS,and the 28d mortality rate also gradually increased.There was a major positive correlation between sPDPN and the inflammatory response of the organism and the severity of the disease.Therefore,sPDPN is a highly potential target in early diagnosis,disease and prognosis assessment,and the selection of possible therapeutic targets in sepsis patients with ARDS.(2)PDPN monoclonal antibody(SZ168)exerted a significant inhibitory effect on LPS-induced Septic lung injury,significantly reducing the inflammatory response of lung tissue cells.The mechanism could be explained through reducing the activity of ERK and NF-κB signaling pathways,altering the function of lung macrophages,and improving pulmonary vascular permeability through paracellular and transcellular pathways.The specific pathological regulatory mechanism needs to be further confirmed.